Equine Influenza

EQUINE INFLUENZA
SUMMARY
Equine influenza is an acute respiratory infection of horses, donkeys and mules caused by two
distinct subtypes (H7N7, formerly equi-1, and H3N8, formerly equi-2) of influenza A virus within the
genus Influenzavirus A of the family Orthomyxoviridae. Viruses of the H7N7 subtype have not been
isolated since the late 1970s. Infection of zebras, dogs and humans has also been reported. In fully
susceptible equidae, clinical signs include pyrexia and a harsh dry cough followed by a
mucopurulent nasal discharge; neurological signs have been described as a rare event. In partially
immune vaccinated animals, one or more of these signs may be absent. Characteristically,
influenza spreads rapidly in a susceptible population. While normally confined to equidae, the H3N8
subtype has crossed the species barrier to dogs. Infection normally produces mild fever and
coughing; occasionally fatal pneumonia develops. While equine influenza has not been shown to
cause disease in humans, serological evidence of infection has been described. Diagnosis of
influenza virus infections is based on virus isolation, antigen and /or genome detection from horses
with acute respiratory illness, or on the demonstration of a serological response to infection. Ideally,
both methods are used. The disease is endemic in most countries with substantial equine
populations. In recent years infection has been introduced into Australia and re-introduced into
South Africa and Japan; to date New Zealand and Iceland are reported to be free. Extensive
infection of dogs has been reported in North America as a result of cross species transmission and
subsequent within species transmission.
Identification of the agent: Embryonated hens’ eggs and/or cell cultures can be used for virus
isolation from nasopharyngeal swabs or nasal and tracheal washes. Viral growth is monitored by
haemagglutination (HA) or, in cell cultures, by haemadsorption (HAD) using chicken or guinea-pig
red blood cells. Isolates can be characterised by haemagglutination inhibition (HI) using strainspecific
antisera. Isolates should always be sent immediately to an International Reference
Laboratory (OIE or World Health Organization). Samples that yield negative results should be
repassaged; up to five passages may be necessary to isolate viruses from vaccinated horses.
Infection may also be demonstrated by detection of viral antigen in respiratory secretions using an
enzyme-linked immunosorbent assay.
Serological tests: Diagnosis of influenza virus infections is usually only accomplished by tests on
paired sera; the first sample should be taken as soon as possible after the onset of clinical signs
and the second approximately 2 weeks later. Antibody levels are determined by HI or single radial
haemolysis (SRH).
Requirements for vaccines and diagnostic biologicals: Spread of infection and severity of
disease may be reduced by the use of potent inactivated equine influenza vaccines containing
epidemiologically relevant virus strains. Inactivated equine influenza vaccines contain whole viruses
or their subunits. However, vaccinated infected horses can still shed the virus. The vaccine viruses
are propagated in embryonated hens’ eggs or tissue culture, concentrated, and purified before
inactivation with agents such as formalin or beta-propiolactone. Vaccines provide protection by
inducing humoral antibody to the haemagglutinin protein. Responses are generally short-lived and
multiple doses are required to maintain protective levels of antibody. An adjuvant is usually required
to stimulate durable protective levels of antibody. Live attenuated virus and viral vectored vaccines
have recently been licensed in some countries.
Influenza vaccines are widely available and are routinely used in competition horses in Europe, the
Americas, and Asia. In some countries, vaccination is mandatory for sport horses that are
competing under rules of equestrian organisations. Vaccines are not widely or routinely used on the
Chapter 2.5.7. - Equine influenza
872 OIE Terrestrial Manual 2008
Indian subcontinent, China, Australia or New Zealand, the latter region, having remained free from
infection.
Following a primary course of three doses at intervals of around 0, 1 and 6 months, an annual
booster is the usual minimum requirement. In some regions, repeated vaccinations are given every
3–6 months. Foals born to well vaccinated dams should not commence their vaccination
programme until 6–9 months of age when all maternal immunity has waned; earlier vaccination may
induce tolerance and a long-term poor response to vaccination. Foals with no maternal antibody
may be vaccinated from an early age, particularly in high risk situations.
Immunity stimulated by inactivated and a nonreplicating vectored vaccine is dependent primarily, on
the induction of antibody to the haemagglutinin (HA) and vaccine induced protection correlates well
with levels of circulating antibody to the HA. By contrast immunity induced by infection or
vaccination with a live attenuated vaccine is not dependent on the presence of circulating antibody
and mucosal and cellular responses are likely to contribute.
Vaccine breakdown has been attributed to inadequate vaccine potency, inappropriate vaccination
schedules, and outdated vaccine viruses that have become irrelevant as a result of antigenic drift.
An in-vitro potency test (single radial diffusion) can be used for in-process testing of the antigenic
content of inactivated products before addition of an adjuvant. In process testing of live and
vectored vaccines relies on titration of infectious virus A surveillance programme is underway to
monitor antigenic drift among equine influenza viruses and to provide information on strain selection
for vaccines.
A. INTRODUCTION
Equine influenza is caused by two subtypes: H7N7 (formerly subtype 1) and H3N8 (formerly subtype 2) of
influenza A viruses (genus Influenzavirus A of the family Orthomyxoviridae); however there have been very
few
reports of H7N7 subtype virus infections in the last 20 years (15, 29). Although these are not genuine human
pathogens, humans can become infected with equine influenza virus subtypes. Such infections are unusual
and
subclinical, but may represent a potential biohazard to laboratory personnel (1).
In fully susceptible equidae, clinical signs include pyrexia, and a harsh dry cough; pneumonia in young foals
and
donkeys and encephalitis in horses have been described as rare events (7, 10). Clinical signs associated
with
infection in dogs also include fever and a cough; occasionally infection results in suppurative
bronchopneumonia
and peracute death (4). Characteristically, influenza spreads rapidly in a susceptible population, but in
partially
immune vaccinated animals, one or more of these signs may be absent and spread of the disease within a
vaccinated population may be limited (6). This makes clinical diagnosis of equine influenza more difficult as
other
viral disease, such as equine herpesvirus, may clinically resemble a mild form of influenza. Horses infected
with
equine influenza virus become susceptible to secondary bacterial infection and may develop mucopurulent
nasal
discharge, which can lead to diagnosis of bacterial disease with the underlying cause being overlooked.
Vaccination does not produce sterile immunity; vaccinated horses may shed virus and contribute to the
spread of
EI. Appropriate risk management strategies to deal with this possibility should be developed.
B. DIAGNOSTIC TECHNIQUES
Laboratory diagnosis of influenza virus infections is based on virus isolation from horses with acute
respiratory
illness, or on the demonstration of a serological response to infection. Ideally, both methods are used.
Infection
may also be demonstrated by detection of viral antigen in respiratory secretions using an enzyme-linked
immunosorbent assay (ELISA) or viral genome using polymerase chain reaction (PCR) assays. All influenza
viruses are highly contagious for susceptible hosts, including embryonated hens’ eggs and cell cultures.
Care
must therefore be taken during the handling of infected eggs or cultures to avoid accidental cross-
contamination
(28). Standard strains should not be propagated in the diagnostic laboratory, at least never at the same time
or in
the same place where diagnostic samples are being processed. All working areas must be efficiently
disinfected
before and after virus manipulations, which should preferably be conducted within biohazard containment.
It is important to obtain samples as soon as possible after the onset of clinical signs, preferably within 3–5
days.
These samples include nasopharyngeal swabs and nasal or tracheal washings, the latter taken by
endoscopy.
Swabs may consist of absorbent cotton wool sponge/gauze on wire, and should be long enough to be
passed via
the ventral meatus into the nasopharynx. Swabs should be transferred to a tube containing transport
medium
immediately after use. This medium consists of phosphate buffered saline (PBS) containing 40% glycerol, or
PBS
containing 2% tryptose phosphate broth, 2% antibiotic solution (penicillin [10,000 units], streptomycin
[10,000
OIE Terrestrial Manual 2008 873
units] in sterile distilled water [100 ml]), and 2% fungizone (250 mg/ml stock). If the samples are to be
inoculated
within 1–2 days they may be held at 4°C, but, if kept for longer, they should be stored at –70°C or below.
Preferably, samples should also be transported on ice.
Only one sample is processed at a time. The liquid is expelled from the swab by squeezing with forceps,
which is
then disposed of suitably. Further antibiotics may be added if samples appear to be heavily contaminated
with
bacteria. The remainder of the fluid is stored at –70°C. Samples treated with antibiotics are allowed to stand
on
ice for 30–60 minutes and are then centrifuged at 1500 g for 15 minutes to remove bacteria and debris; the
supernatant fluids are used for inoculation. Filtration of samples is not advised as influenza virus may adsorb
on to
the filter and be lost from the sample.
1. Identification of the agent
Isolation of infectious virus may be carried out in embryonated hens’ eggs or cell cultures and EI nucleic acid
can
be identified by PCR. Traditionally, eggs have been preferred for isolation of equine influenza. Comparison
of
H3N8 viruses isolated in eggs and Madin–Darby canine kidney (MDCK) cells indicated that MDCK cells are
capable of selecting variant viruses that are not representative of the predominant virus in clinical specimens
(11).
However, in recent years some viruses have been successfully isolated in MDCK cells but not in eggs and
selection of variants has occurred as a result of culture in eggs (24), therefore isolation should be attempted
using
both substrates. PCR techniques have been described for the identification of equine influenza virus from
clinical
specimens and for molecular epidemiology (8, 13, 23).
In situations where laboratory facilities for virus isolation are unavailable, influenza virus antigen in nasal
secretions may be detected directly by an antigen-capture ELISA for the H3N8 virus using a monoclonal
antibody
(MAb) against the nucleoprotein (3, 14). Commercial self-contained kits for detecting human influenza are
available and have been shown to detect equine influenza antigen (2). This approach provides a rapid result
on
which management decisions may be based. It should not be used in preference to virus isolation, as it is
essential that new viruses be isolated and sent to reference laboratories for characterisation as part of the
surveillance programme to monitor antigenic drift and emergence of new viruses and to provide isolates for
inclusion in updated vaccines. Positive ELISA results are useful in the selection of samples if resources are
limited
or for the selection of specimens to be sent to a reference laboratory for virus isolation attempts.
a) Virus isolation in embryonated hens’ eggs
Fertile eggs are set in a humid incubator at 37–38°C and turned twice daily; after 10–11 days, they are
examined by candling and live embryonated eggs are selected for use. The area above the air sac is
cleansed with alcohol and a small hole is made through the shell. Several eggs/sample are inoculated
(0.1 ml) in the amniotic cavity with no additional dilution of the sample (sample may also be diluted). The
syringe is withdrawn approximately 1 cm and a further 0.1 ml is inoculated into the allantoic cavity.
Alternatively, the sample may be inoculated into the allantoic cavity alone through a second hole drilled just
below the line of the air sac. The hole(s) is/are sealed with wax or Sellotape, and the eggs are incubated at
34–35°C for 3 days. The embryos that die within 24 hours following inoculation should be discarded. The
eggs that contain embryos that die more that 24 hours after inoculation or contain live embryos after 3 days
are examined for the presence of EI virus.
The eggs are transferred to 4°C for 4 hours or overnight to kill the embryos and to reduce bleeding at
harvest. The shells are disinfected, and the amniotic and/or allantoic fluid is harvested by pipette, each
harvest being kept separate. These are tested for haemagglutination (HA) activity by mixing in equal
volumes (0.025 ml) with chicken red blood cells (RBCs) (0.5% [v/v] packed cells in PBS) in V- or U-bottomed
microtitre plates or 0.4% guinea-pig RBCs (0.4% [v/v] packed cells in PBS) in V- or U-bottomed plates. If
chicken RBCs are used, the plates may be read by tilting to 70so that non-agglutinated cells ‘stream’ to the
bottom of the well. Non-agglutinated guinea-pig cells appear as a button at the bottom of the well and may
take longer to settle. If there is no HA activity, aliquots of each harvest are pooled and passaged into further
eggs. All HA positive samples are divided into aliquots and stored at –70°C; one aliquot is titrated for HA
immediately. If the HA titre is 1/16 or more, the isolate is characterised immediately. If titres are low, positive
samples should be passaged. Care should be taken to avoid generation of defective interfering particles by
prediluting the inoculum 1/10, 1/100, 1/1,000. Positive samples arising from the highest dilution should be
selected as stocks for storage. It may be necessary to undertake as many as five passages to isolate the
virus, particularly from vaccinated horses. If virus has not been recovered by the fifth passage, further
passages are unlikely to be successful.
b) Virus isolation in cell cultures
Cultures of the MDCK cell line (MDCK, ATCC CCL34) may be used to isolate equine influenza viruses. The
cells are grown to confluence in tubes and then infected in triplicate with 0.25–0.5 ml of each sample,
processed as described above. Prior to inoculation, the cell monolayer is washed at least once with tissue
culture medium containing trypsin (2 μg/ml) without serum. The cultures are maintained with serum-free
medium containing 0.5–2 μg/ml trypsin (treated with TPCK [L-1-tosylamine-2-phenylethyl chloromethyl
ketone] to remove chymotrypsin, available pretreated, e.g. from Sigma), and examined daily for evidence of
cytopathic effects (CPE). If positive, or after 7 days in any case, the supernatant fluids are tested for HA.
Fluids with titres of ≥1/16 are characterised immediately. Negative fluids and those with titres <1/16 are
repassaged up to five passages.
Alternatively, the cells are screened for evidence of haemadsorption (HAD). This procedure detects
expression of viral antigens at the cell surface. The medium is removed from the cultures and the tubes are
washed with PBS. One or two drops of a 50% suspension of chicken or guinea-pig RBCs are added, the
tubes are rotated carefully, and kept at room temperature (23°C ±2°C) for 30 minutes. Unbound RBCs are
washed off with PBS, and the cultures are examined microscopically for evidence of HAD.
c) Haemagglutinin typing
The HA subtype of new isolates of equine influenza viruses is best determined by haemagglutination
inhibition (HI; Section B.2.a) using H7N7- and H3N8-specific antisera. Isolates may first be treated with
Tween 80/ether, which destroys viral infectivity and reduces the risk of cross-contamination. In the case of
H3N8 viruses particularly, this treatment enhances the HA activity (12). However, treatment with Tween
80/ether may increase the variability of the results obtained. Standard antigens must be titrated in parallel
with tests to identify viruses and should include H7N7 strains (e.g. A/eq/Prague/56, A/eq/Newmarket/77) and
H3N8 strains (e.g. A/eq/Newmarket/2/93, and A/eq/Kentucky/94). Virus strains may be obtained from OIE
Reference Laboratories (see Table given in Part 3 of this Terrestrial Manual). Additionally, recent isolates
from the same geographical area should be included if available. The standard antigens should be treated
with Tween 80/ether to avoid cross-contamination. Test antigens and standard antigens are always
backtitrated
to confirm their antigen content.
New isolates of equine influenza viruses may be further characterised by HI using strain-specific antisera.
The species in which antibodies are raised will influence the cross-reactivity of the antiserum, with ferrets
providing the most strain-specific antibody (17).
All isolates should be sent immediately to an International Reference Laboratory designated by OIE or the
World Health Organization (WHO) for inclusion in the strain surveillance programme to monitor antigenic
drift
and emergence of new viruses.
d) Neuraminidase typing
Typing of neuraminidase requires specific antisera and no routine technique is available. Typing can be
done
using specific PCR primers.
e) Polymerase chain reaction
PCR assays are increasingly used for the detection of equine influenza genome in nasal secretions.
Realtime
Light Cycler reverse transcription PCR technology has been shown to be more sensitive for the
detection of positive samples than virus culture in eggs or detection of nucleoprotein using ELISA technology
(26). Although genetic sequence of isolates can also be derived from PCR assays it remains essential to
isolate infectious virus in order to examine antigenic properties of new isolates and antigenic drift.
2. Serological tests
Infections are detected by performing serological tests on paired sera to show a rise in antibody. These tests
should be carried out whether virus isolation has been attempted or not. Two simple methods exist, HI and
single
radial haemolysis (SRH), each equally efficient and widely used. The complement fixation (CF) test can also
be
applied, but is not in general use. Both of the paired serum samples should be tested together at the same
time to
minimise variability. The standard antigens are described above (Section B.1.c). If available, isolates from
recent
cases should be included. Freeze-dried post-infection equine antisera to A/eq/Newmarket/77 (H7N7),
A/eq/Newmarket/1/93 (‘American-like’ H3N8) and A/eq/Newmarket/2/93 (‘European-like’ H3N8) and an
influenzanegative
equine serum, are available from the OIE Reference Laboratory, Newmarket (see Table given in Part 3
of this Terrestrial Manual). These sera have been assigned SRH values through an international
collaborative
study and can be used as primary reference sera for this assay.
a) Haemagglutination inhibition test
The antigen is first treated with Tween 80/ether in order to increase the sensitivity of the test, particularly for
H3N8 viruses. The test is best done in microtitre plates using the appropriate dilution equipment. A
macrotest may be used, for which antigen is diluted to a final HA titre of 1/8 per well and the volumes for
PBS, sera and antigen are 0.5 ml. Sera are pretreated to remove nonspecific haemagglutinins, and
inactivated at 56°C for 30 minutes. Pretreatments include the use of one of the following: (a) kaolin and
RBCs absorption, (b) potassium periodate, or (c) Vibrio cholerae receptor-destroying enzyme. Potassium
periodate or V. cholerae receptor-destroying enzyme is the treatment of choice. The treated sera are diluted
in PBS, a standard dose of antigen is added (HA titre of 1/4 per well for microtitration assay), and these are
kept at room temperature (23°C ±2°C) for 30 minutes. After gentle mixing, RBCs are added and the test is
read 30 minutes later. The HI titres are read as the highest dilution of serum giving complete inhibition of
agglutination. Either chicken RBCs (1% [v/v] packed cells) in V-bottomed microtitre plates or guinea-pig
RBCs (0.5% [v/v] packed cells) in V- or U-bottomed plates may be used. If chicken RBCs are used, the
plates may read by tilting to 70so that non-agglutinated cells ‘stream’ to the bottom of the well.
Nonagglutinated
guinea-pig cells appear as a ‘button’ in the bottom of the well and may take longer to settle.
Titre increases of fourfold or more between paired sera indicate recent infection (28).
o Tween 80/ether treatment of the virus
i) To 39.5 ml of infective allantoic fluid, add 0.5 ml of a 10% (v/v) suspension of Tween 80 in PBS to give
a 0.125% (v/v) concentration of Tween 80.
ii) After mixing gently at room temperature for 5 minutes, add 20 ml of diethyl ether to give a final
concentration of 33.3% by volume, and mix the suspension well at 4°C for 15 minutes.
iii) After allowing the layers to separate by standing, remove the aqueous layer containing the disrupted
virus particles to a glass bottle with a loose lid and allow the excess ether to evaporate off overnight
(12).
iv) Store treated virus in aliquots at –70°C.
o Titration of haemagglutination
i) Add 25 μl of PBS to all wells in a row of a microtitre plate.
ii) Add 25 μl of virus to first well (dilution = 1/2) and titrate through, leaving the last well as a control.
iii) Add an extra 25 μl of PBS to all wells.
iv) Add 50 μl of RBCs to all wells. Leave at 22°C (±2°C) for 30 minutes. The HA titre is taken as the last
virus dilution giving partial HA.
o Potassium periodate pretreatment of sera
i) Mix one volume (150 μl) of serum with two volumes (300 μl) of freshly prepared 0.016 M potassium
periodate (0.38 g in 100 ml PBS), and leave at 22°C (±2°C) for 15 minutes.
ii) Add a further one volume of 3% glycerol in PBS to neutralise any excess periodate solution, mix and
leave at room temperature (23°C ±2°C) for 15 minutes.
iii) Inactivate in a 56°C water bath for 30 minutes.
o Test procedure
i) Dispense 25 μl of PBS to all wells of a microtitre plate.
ii) Add serum (25 μl) to the first well of a row of 12, and titrate through, leaving the last well as a control
(1/8 to 1/512, allowing for dilution of 1/4 from treatment of serum).
iii) Dilute the antigen to give a dose of 4 HA units (4 × minimum agglutinating dose, i.e. titre/4).
iv) Add 25 μl to each well, and incubate at 22°C (±2°C) for 30 minutes.
v) Add 50 μl of RBCs to each well. Leave at 22°C (±2°C) for 30 minutes.
vi) The plates may be read by tilting to 70° so that non-agglutinated cells ‘stream’ to the bottom of the well.
No agglutination is recorded as a positive result.
b) Single radial haemolysis
In this test, viral antigens are coupled to fixed RBCs that are suspended in agarose containing guinea-pig
complement (C’). Wells are punched in the agarose and filled with test sera. Influenza antibodies and C’ lyse
the antigen-coated RBCs, resulting in a clear, haemolytic zone around the well; the size of this zone is
directly proportional to the level of strain-specific antibody in the serum sample (16, 25, 27).
Special immunodiffusion plates (MP Biomedical) may be used for the assay, but simple Petri dishes are also
suitable. Sheep RBCs collected into Alsever’s solution are washed three times. The C’ can be obtained
commercially, or normal guinea-pig serum can be used. The antigens are allantoic fluids or purified
preparations; the strains used are the same as for the HI tests. The viruses are coupled to RBCs by
potassium periodate or by chromic chloride. The coupled antigen/RBCs preparations are mixed with C’,
together with a 1% solution of agarose (low melting grade) in PBS. Care must be taken to ensure that the
temperature is not allowed to rise above 42°C at any time. The mixture is poured into plates and left to set.
Wells of 3 mm in diameter and 12 mm apart are punched in the solidified agarose, at least 6 mm from the
edge of the plates. Such plates may be stored at 4°C for 12 weeks. Plates are prepared for each antigen.
Sera are inactivated at 56°C for 30 minutes, but no further treatment is necessary. Paired sera should be
assayed on the same plate. As a minimum, a subtype-specific antiserum should be included as a control
serum in one well on each plate. All sera are tested in a control plate containing all components except virus
to check for nonspecific lysis. Alternatively, an unrelated virus, such as A/PR/8/34 (H1N1), may be used in
the control plate. Sera that show haemolytic activity for sheep RBCs must be pre-absorbed with sheep
RBCs. Zones of lysis should be clear and not hazy or translucent. All clear zones should be measured and
the area of haemolysis calculated.
o Preparation of reagents
i) Saline/HEPES: 0.85% NaCl (4.25 g/500 ml); 0.05 M HEPES (N-2-hydroxyethylpiperazine, N-2-
ethanesulphonic acid; 5.95 g/500 ml); and 0.02% sodium azide. Make to pH 6.5 with NaOH.
ii) Saline/HEPES/BSA: as saline/HEPES with 0.2% (w/v) bovine serum albumin (BSA).
iii) CrCl3 stock solution (2.25 M) 6 g/10 ml: Make fresh 1/400 dilution in 0.85% NaCl for each assay.
iv) PBS (London)/PBS ‘A’: NaCl (10.00 g); KCl (0.25 g); Na2PO4 (1.45 g); KH2PO4 (0.25 g); and Na azide
(0.20 g). Make up to 1 litre with distilled water.
v) Agarose in PBS: Place flask containing PBS ‘A’ on a stirrer. Slowly add 10 g agarose to the stirring
solution. Liquefy in a pressure cooker. Dispense into glass bottles for storage at 22°C (±2°C).
vi) Virus antigen: Allantoic fluid containing infectious virus is harvested and stored at –70°C. A short
titration curve determines the optimum ratio of virus antigen to RBCs to be used when preparing
sensitised sheep RBCs. The H7N7 influenza strains always produce clear zones; the H3N8 strains
sometimes produce hazy zones, in which case it is necessary to concentrate the virus by centrifugation.
vii) Sheep blood: Collect blood into an equal volume of Alsever’s solution and store at 4°C. It may be
necessary to test bleed several sheep, as characteristics of RBCs from individual sheep vary. Keep the
blood for 2 days before use, it may then be usable for up to 3 weeks, providing sterility is maintained.
viii) Complement: Use commercially available guinea-pig complement or collect serum from young
guineapigs
of 300–350 g body weight and store in small volumes at –70°C. For use, thaw in cold water and
hold at 4°C prior to mixing.
ix) Treatment of sera: Use undiluted sera heat inactivated at 56°C for 30 minutes. Avoid repeated freeze–
thaw cycles.
o Test procedure
i) Wash sheep RBCs at least three times in saline/HEPES.
ii) Prepare an appropriate volume of 8% RBCs (v/v packed cells) in saline/HEPES, having first calculated
the number of plates required and allowing 1 ml per 6 × 11 cm immunoplate and 1–2 ml extra.
iii) Add a predetermined volume1 of virus antigen to the 8% RBCs solution. Hold the mixture at 4°C for
10 minutes. Haemagglutination may be observed.
iv) SLOWLY add CrCl3 (1/400 in 0.85% NaCl) at half the total volume of virus/RBCs suspension. Hold at
22°C (±2°C) for 5 minutes with occasional mixing.
v) Sediment the sensitised RBCs by centrifugation at 1500 g for 5 minutes.
vi) Gently resuspend in saline/HEPES/BSA and centrifuge at 1500 g for 5 minutes.
vii) Resuspend RBCs to an 8% suspension in PBS ‘A’.
During the sensitisation process, melt the agarose in a pressure cooker. Shortly before use, pipette 7.8 ml
volumes to Universal bottles and retain at 42°C. Check that the agar has cooled to 42°C before use.
1 Prepare three plates by adding 0.6, 1.2 or 1.8 ml of virus antigen to 2 ml RBCs. Add 1.3, 1.6 and 1.9 ml CrCl 3
respectively
and resuspend to 2 ml in PBS ‘A’. Optimum volume of virus antigen is that which results in the largest and clearest zones
with appropriate reference serum.
o Preparation of plates
i) Add 0.9 ml of virus-sensitised sheep RBCs to 7.8 ml of agarose (42°C). Mix quickly, but gently.
ii) Add 0.3 ml of undiluted guinea-pig serum. Mix again and pour into immunoplates on a levelling table.
Allow to set and air dry without a lid for 5 minutes.
iii) Place lids on plates and store at 4°C in a humid box until used.
iv) Prepare control plates with unsensitised cells or cells sensitised with an unrelated virus. Batches of
prepared plates can be stored for several weeks.
v) Punch 3 mm holes in the set gels to a prepared template, allow for 16 test sera and a positive control
serum. On antigen control plates, prepare five rows of eight wells.
vi) Pipette 10 μl of heat-inactivated (56°C for 30 minutes) test sera and a positive control serum to
appropriate wells. Incubate at 34°C for 20 hours in a humid box.
vii) Measure zone diameters, and calculate areas of haemolysis after the area of the well has been
deducted.
o Interpretation of the results
For results to be valid, positive and negative control sera should give results consistent with those expected
on the basis of prior experience. Areas of haemolysis for the control sera should be clear and intra-
laboratory
variation should be no more than 5% for the control serum. Results may be expressed as mm2 or as a ratio
of the control serum value. Sera giving positive results in the control plate should be adsorbed with sheep
RBCs. For diagnostic purposes, acute and convalescent sera should be tested in duplicate on the same
plate. Increase in zone areas produced by convalescent serum compared with acute serum is evidence of
infection. The increase in area deemed to be significant depends on the reproducibility of the test within the
laboratory, but should be equivalent to a twofold or more increase in antibody concentration. This area can
be calculated from a standard curve generated from a dilution series of a standard antiserum.
C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC
BIOLOGICALS
Equine influenza virus vaccines consist of inactivated whole viruses or their subunits, with or without
adjuvant.
Live attenuated virus and canary pox vectored vaccines have recently become available commercially in
some
countries. Requirements for such vaccines may be anticipated to differ in some particulars from the
following,
which are appropriate to inactivated vaccines.
Immunity generated by inactivated vaccines administered via the intramuscular route is reliant on stimulation
of
circulating antibody to the HA, which neutralises virus; some products have been shown to stimulate
antibody in
respiratory secretions. Critically the integrity and conformation of the HA should be maintained during
inactivation
procedures to ensure that the vaccine stimulates appropriate neutralising antibody. This can be tested by
use of
an immunological assay such as SRD (single radial diffusion), which measures immunologically active HA
capable of reacting with specific anti-HA antibodies. The immunogenicity of the vaccine can be confirmed by
measurement of HA antibody stimulated in small animal models or the target species.
Although canary pox-vectored vaccines, also administered via the intramuscular route, have the potential for
stimulating cytotoxic T cell (CTL) immunity through their ability to present antigen via endogenous
processing,
they currently include only the HA gene, which is not known to be a CTL target. Their action is therefore
reliant on
stimulation of antibody to HA. Infectious titre of the recombinant canary pox virus carrying equine influenza
HA
genes is used as an in vitro measure of potency and immunogenicity is assessed by measurement of
antibody
stimulated in the target species.
Antibody to HA as measured by SRH, stimulated by inactivated whole virus, subunit or canary pox-vectored
vaccine correlates well with protection against infection in an experimental challenge model system (20). In
contrast, a cold-adapted temperature-sensitive mutant used as a live attenuated vaccine replicates in the
upper
respiratory tract and does not stimulate high levels of circulating antibody to HA but nevertheless provides
protection against challenge infection. Immunity is presumed to be mediated through mucosal or cellular
responses rather than circulating antibody. As with the vectored vaccine, in-process control testing is reliant
on
measurement of the infectious virus titre.
Guidelines for the production of veterinary vaccines are given in Chapter 1.1.8 Principles of veterinary
vaccine
production. The guidelines given here and in Chapter 1.1.8 are intended to be general in nature and may be
supplemented by national and regional requirements.
1. Seed management
a) Characteristics of the seed
An ongoing surveillance programme by OIE and WHO Reference Laboratories aimed at providing
information on suitable vaccine strains is being co-ordinated by the OIE Reference Laboratory (Newmarket)
(21). Recommendations on vaccine strains made by the Expert Surveillance Panel will be published
annually
in the OIE Bulletin.
H7N7: Many vaccines still contain an H7N7 strain. However, the Expert Surveillance Panel has
recommended that the H7N7 component should be omitted as there have been few reports of infections with
this subtype have been substantiated during the past 20 years.
H3N8: Antigenic variants of H3N8 viruses co-circulate (5) and it is important to include a strain or strains that
are epidemiologically relevant as recommended by the OIE Expert Surveillance Panel, as published in the
OIE Bulletin.
b) Method of culture
Virus strains may be obtained from OIE Reference Laboratories (see Table given in Part 3 of this Terrestrial
Manual). Viruses selected as vaccine strains should be described in terms of origin and passage history.
The
strains are propagated in the allantoic cavity of 10-day-old embryonated hens’ eggs or cell cultures, such as
MDCK. All manipulations must be conducted separately for each strain. Viral growth is monitored by HA
tests. Passaged virus is identified by serological tests, such as HI or SRH or by PCR using specific primers.
If vaccine virus is grown in cell culture, antigenic studies with ferret sera and MAbs should be undertaken to
ensure that variant viruses have not been selected during passage to prepare master and working seed
viruses. Master and working seed viruses are divided into aliquots and stored in freeze-dried form at –20°C
or at –70°C following testing for extraneous agents. Records of storage conditions should be maintained.
The master seed lot of each vaccine strain selected should be processed at one time to assure a uniform
composition, tested for extraneous agents, and fully characterised. Antisera or MAbs for use in HI tests to
characterise vaccine strains may be obtained from OIE and WHO Reference Laboratories.
Working seed lots are derived from a master seed lot and should be of uniform composition, free from
extraneous agents, and fully characterised. Aliquots of the working seed are used for production of vaccine.
Master and working seed lots should be prepared in specific pathogen free eggs or, as a minimum, in eggs
derived from a healthy flock.
If MDCK cells are used to propagate vaccine virus, master cell lines should be established and stored in
liquid nitrogen, and should be tested for freedom from extraneous agents according to National Control
Authority Guidelines.
Examination of seed viruses for extraneous agents including mycoplasmas and other equine viruses should
be performed by appropriate techniques, including inoculation of susceptible tissue cultures and examination
for cytopathic effect or application of fluorescent antibodies for antigen detection.
The presence of other common equine respiratory pathogens, e.g. equine herpesviruses 1, 2, 4, equine
picornaviruses, equine viral arteritis, and equine adenoviruses, should be specifically excluded.
The absence of bacteria should be confirmed by standard sterility tests and toxicity tests in small animals.
c) Validation as a vaccine
For each vaccine strain, a prototype batch should be prepared to establish its suitability as a vaccine strain,
i.e. purity and safety should be confirmed by standard techniques. The ability of seed-lot viruses to grow to
high titre and generate sufficient antigenic mass to stimulate adequate antibody responses in the target
species, should be confirmed.
Additionally, vaccine virus derived in MDCK cells should be fully characterised to ensure that antigenic
variants have not arisen during the culture process, such that the vaccine virus is no longer representative of
the original isolate.
2. Method of manufacture
Production is based on a seed-lot system that has been validated with respect to the characteristics of the
vaccine
strains. Where eggs are used, each strain of virus is inoculated separately into the allantoic cavity of 9–11-
day-old
embryonated hens’ eggs from a healthy flock. The eggs are incubated at a suitable temperature for 2–3
days, and
the allantoic fluid is collected. Alternatively, each strain is inoculated separately into MDCk cell cultures. The
viral
suspensions of each strain are collected separately and inactivated. If necessary, they may be purified.
Suitable
adjuvants and antimicrobial preservatives may be added.
Monovalent virus pools should be inactivated as soon as possible after their preparation, by a method
approved
by the National Control Authority. If formalin (37% formaldehyde) or beta-propiolactone (2-oxetanone) is
used, the
concentration by volume should not exceed 0.2%. Ideally, pools should be held at 4°C and should be
inactivated
within 5 days of harvest. Inactivation of the vaccine must be demonstrated. A suitable method consists of
inoculating 0.2 ml of undiluted monovalent pool and 1/10 and 1/100 dilutions of the monovalent pool into the
allantoic cavities of groups of fertile eggs (10 eggs in each group), and incubating the eggs at 33–37°C for 3
days.
At least 8 of the 10 eggs should survive at each dosage level. A volume of 0.5 ml of allantoic fluid is
harvested
from each surviving egg. The fluid harvested from each group is pooled, and 0.2 ml of each of the three
pools is
inoculated, undiluted, into a further group of 10 fertile eggs. Haemagglutinin activity should not be detected
in
these new groups of eggs. In some countries, the requirement that 80% of the eggs should survive during
incubation may be impossible to satisfy, in which case the National Control Authority should then specify a
modified requirement to be satisfied. Before inactivation, samples should be collected for bacterial and
fungal
sterility tests.
Monovalent material may be concentrated and purified by high-speed centrifugation or other suitable
methods
approved by the National Control Authority, either before or after the inactivation procedure. It is important to
concentrate and purify the virus under optimum conditions, e.g. temperatures that preserve its antigenic
properties.
The monovalent virus pool shall be shown not to contain viable influenza virus when tested by inoculation of
embryonated hens’ eggs, by a method approved by the National Control Authority. Alternatively, the
satisfactory
inactivation can also be demonstrated by inoculating monolayers of MDCK cells.
3. In-process control
Relevant in-process controls should be applied before and after inactivation and before and after
concentration
and purification.
In-process controls include: (a) identity of virus strains (tested by HI); (b) sterility; (c) virus titre; (d)
haemagglutinin
content (tested by chicken RBCs agglutinating units, CCA [chick cell agglutination]); and (e) immunologically
active HA (tested by SRD or another suitable immunochemical method).
o Single radial diffusion test
SRD is a reliable method for measuring immunologically active HA in terms of μg HA, and is used routinely
for potency testing human influenza vaccines (32).
The potency of inactivated equine influenza vaccine depends on the concentration of immunologically active
haemagglutinin (22, 30, 31).
Assessment of the antigenic content of the vaccine by CCA alone may be misleading, as the sensitivity of
this assay is a reflection of the ability of virus strains to agglutinate RBCs. Disruption of virus may lead to an
apparent increase in HA as measured by CCA. The CCA assay does not provide a measure of the antigenic
properties of the HA (HA may retain its properties to bind to RBCs while losing its ability to stimulate
antibody).
The composition of some equine influenza vaccines is unusual in that products may contain more than one
variant of the H3N8 subtype. In this situation, it is not possible to judge the potency of individual H3N8
components from serological tests performed on sera collected from horses or small animals vaccinated
with
the final product, because of cross-reactivity between the two isolates of the same subtype. Thus, it is
important that a reliable method, such as SRD, be used to measure the potency of individual components
before and after inactivation and prior to mixing and formulation with adjuvant.
In the SRD test, virus preparations are compared with a calibrated reference preparation of known HA
content. Antigens are allowed to diffuse through a gel containing an antiserum specific for a particular HA.
The distance diffused by the antigen before precipitation by the antibody incorporated in the gel is directly
related to the concentration of haemagglutinin in the antigen preparations (19).
Standard reagents for SRD testing are available from the WHO International Laboratory for Biological
Standards2. Reagents for A/eq/Prague/56 (H7N7), and the H3N8 strains A/eq/Miami/63, A/eq/Kentucky/81,
A/eq/Newmarket/1/93 (American lineage) and A/eq/Newmarket/2/93 (European lineage) are currently
available.
4. Batch control
a) Sterility
Tests for sterility and freedom from contamination of biological materials may be found in Chapter 1.1.9.
b) Safety
i) Using no fewer than three horses, each horse is inoculated intramuscularly (at two different sites) with
the dose of vaccine specified by the manufacturer; these inoculations are repeated 2–4 weeks later.
The animals are kept under observation for 10 days after the second set of injections. No abnormal
local or systemic reaction should ensue.
ii) If vaccine is to be used in mares, safety should be demonstrated by giving two doses of vaccine to no
fewer than two pregnant mares at the prescribed interval within the trimester for which the vaccine is
recommended.
c) Potency
Following mixing of viral antigens and adjuvants, aliquots should be potency tested in vivo using horses and
guinea-pigs or a suitable alternative immunochemical assay. Adjuvants cause interference in quantitative
invitro
tests, such as CCA and SRD, although SRD may be used on the final product as a qualitative assay to
demonstrate the presence of antigen for each vaccine strain. For repeated batch tests, only guinea-pigs or a
suitable alternative immunochemical assay are used, subject to agreement of the National Control Authority.
i) Serological responses in horses
For a valid in-vivo potency test, naive seronegative horses must be selected for vaccination. Young
horses or ponies (not less than 6 months old) should be screened for the presence of antibody using
H7N7 and H3N8 viruses including recently isolated viruses relevant to the area in which the horses
were reared. If HI tests are used for screening, H3N8 viruses should be treated with Tween 80/ether to
maximise the sensitivity of the test. Alternatively, SRH may be used to establish the seronegative
status of animals.
To test a vaccine for efficacy in horses, inject a volume corresponding to one vaccine dose by the
recommended route into each of five susceptible seronegative horses. After the period recommended
between the first and second doses, as stated on the label, a volume of vaccine corresponding to the
second dose of vaccine is injected into each horse.
Three blood samples are collected from each animal, the first at the time of the first vaccination, the
second 1 week after the first vaccination, and the third 2 weeks after the second vaccination.
The serological assay used to measure the antibody response to the viruses contained in the vaccine
must be standardised for a valid in-vivo potency test, therefore the SRH assay (see Section B.2.b) is
preferred. Standard sera for the quality control of equine influenza vaccines are available from the
European Pharmacopoeia3. These sera should be tested in parallel with the test sera to ensure that the
test is valid with respect to sensitivity; the values obtained should not vary by more than 20% from the
SRH values assigned in an international collaborative study (18). Due to poor repeatability and
reproducibility of the HI test, no HI titre could be assigned to these sera.
The antibody value measured by SRH should not be less than 150 mm2. This is higher than the value
required in the European Pharmacopoeia Monograph for inactivated equine influenza vaccines
(85 mm2) as this value is not considered to be protective. If the value found for any horse after the first
vaccination indicates that there has been an anamnestic response, the result is not taken into account.
A supplementary test is carried out, as described above, replacing the horses that showed an
anamnestic response with an equal number of new animals.
If the HI test is used, the antibody titre of each serum taken after the second vaccination in each test
should not be less than 1/64 (calculated for the original serum, taking into account the predilution of
1/8). Alternatively, the antibody levels stimulated by the vaccine under test should be shown to be at
2 National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6
3QG,
UK.
3 Serum to A/eq/Newmarket/1/77 (Catalogue number E0850010), A/eq/Newmarket/1/93 (E0850021) and
A/eq/Newmarket/2/93 (E0850022).
least equal to the antibody levels stimulated by a standard vaccine tested in parallel that has been
shown previously to protect horses against challenge infection.
ii) Challenge studies in horses
It may be desirable in certain cases to undertake challenge studies in horses to demonstrate potency,
particularly if vaccines are being assessed for their ability to protect against antigenically dissimilar
viruses. Challenge studies may be carried out by exposing six vaccinated horses/ponies to an aerosol
of virulent influenza virus no fewer than 2 weeks after the second dose of vaccine. Comparisons of
clinical signs, virus excretion and serological responses are made with a group of no fewer than four
unvaccinated control animals challenged at the same time (19, 20). The timing of the challenge
procedure will reflect the claims to be made on the data sheet regarding duration of immunity.
If tests for potency in horses have been carried out with satisfactory results on a representative batch of
vaccine, these tests may be omitted as a routine control on other batches of vaccine prepared using
the same seed-lot system, subject to agreement by the National Control Authority.
iii) Serological responses of guinea-pigs
Inject each of no fewer than five guinea-pigs free from specific antibodies with one vaccine dose.
Collect blood samples 21 days later, and test the serum by SRH or HI (see Sections B.2.a and B.2.b).
Perform the tests of each serum using, respectively, the antigen(s) prepared from the strain(s) used in
the production of the vaccine. The antibody titre of each serum in each test should not be less than the
titre stimulated by a standard vaccine that has been shown to stimulate protective levels of antibody in
horses.
d) Duration of immunity
Where claims for duration of immunity are made on the data sheet, these should be supported with data on
the duration of protective levels of antibody maintained in horses vaccinated according to the recommended
schedule. Antibody levels quoted as protective should be validated in challenge studies (see Section C.4.c.ii)
or by comparison with published reports.
e) Stability
Vaccines should be stored at 5±3°C and protected from light. The shelf life quoted on the data sheet should
be demonstrated by testing the potency of aliquots over time using the guinea-pig potency test (see Section
C.4.c.iii).
f) Preservatives
Preservatives are not normally included.
g) Precautions (hazards)
The contents of each opened vial should be used within 1 hour of opening. Aseptic precautions should be
observed during administration, and only healthy horses should be vaccinated. Occasionally, transient local
and/or general reactions may occur, and rest may be advisable for 24–48 hours after vaccination.
5. Tests on the final product
a) Safety
Tests are performed as described in Section C.4.b.i. Once safety has been demonstrated on a prototype
batch, safety testing in pregnant mares may be omitted for routine testing of subsequent batches of the final
product.
b) Potency
See Section C.4.c.iii. As a minimum, serological testing in guinea-pigs should be performed on each batch of
the final product.
c) Maintaining epidemiologically relevant strains in vaccines
To enable vaccine manufacturers to respond quickly to recommendations from the Expert Surveillance
Panel
to update vaccine strains, the Committee for Veterinary Medicinal Products for the European Agency for the
Evaluation of Medicinal Products has developed a fast-track licensing system to be used when vaccine
strains are updated (9).
REFERENCES
1. ALEXANDER D.J. & BROWN I.H. (2000). Recent zoonoses caused by influenza A viruses. Rev. sci. tech. Off.
Int. Epiz., 19, 197–225.
2. CHAMBERS T.M., SHORTRIDGE K.F., LI P.H., POWELL D.G. & WATKINS K.L. (1994). Rapid diagnosis of equine
influenza by the Directigen FLU-A enzyme immunoassay. Vet. Rec., 135, 275–279.
3. COOK R.F., SINCLAIR R. & MUMFORD J.A. (1988). Detection of influenza nucleoprotein antigen in nasal
secretions from horses infected with A/equine influenza (H3N8) viruses. J. Virol. Methods, 20, 1–12.
4. CRAWFORD P.C., DUBOVI E.J., CASTLEMAN W.L., STEPHENSON I., GIBBS E.P., CHEN L., SMITH C., HILL R.C.,
FERRO P., POMPEY J., BRIGHT R.A., MEDINA M.J., JOHNSON C.M., OLSEN C.W., COX N.J., KLIMOV A.I., KATZ J.M.
& DONIS R.O. (2005). Transmission of equine influenza virus to dogs. Science, 310, 482–485.
5. DALY J.M., LAI A.C.K., BINNS M.M., CHAMBERS T.M., BARRANDEGUY M. & MUMFORD J.A. (1996). Antigenic
and
genetic evolution of equine H3N8 influenza A viruses. J. Gen. Virol., 77, 661–671.
6. DALY J.M., NEWTON J.R. & MUMFORD J.A. (2004). Current perspectives on control of equine influenza. Vet.
Res., 35, 411–423.
7. DALY J.M., WHITWELL K.E., MILLER J., DOWD G., CARDWELL J.M. & SMITH K.C. (2006). Investigation of
equine
influenza cases exhibiting neurological disease: coincidence or association? J. Comp. Pathol., 134 (2–3),
231–135. [PMID: 16527298]
8. DONOFRIO J.C., COONROD J.D. & CHAMBERS T.M. (1994). Diagnosis of equine influenza by the polymerase
chain reaction. J. Vet. Diagn. Invest., 6, 39–43.
9. EUROPEAN AGENCY FOR THE EVALUATION OF MEDICINAL PRODUCTS (EMEA), COMMITTEE FOR VETERINARY
MEDICINAL PRODUCTS (CVMP) (1998). Note for Guidance: Harmonisation of Requirements for Equine
Influenza Vaccines Specific Requirements for Substitution or Addition of a Strain or Strains. Document
EMEA/CVMP/112/98-FINAL, EMEA, London, UK.
10. GERBER H. (1970). Clinical features, sequelae and epidemiology of equine influenza. In: Equine
Infectious
Diseases II, Bryans J.T. & Gerber H., eds. S. Karger, Basel, Switzerland, 63–80.
11. ILOBI C.P., HENFREY R., ROBERTSON J.S., MUMFORD J.A., ERASMUS B.J. & WOOD J.M. (1994). Antigenic and
molecular characterisation of host-cell mediated variants of equine H3N8 influenza viruses. J. Gen. Virol.,
75, 669–673.
12. JOHN T.J. & FULGINITI V.A. (1966). Parainfluenza 2 virus: increase in haemagglutinin titre on treatment
with
Tween-80 and ether. Proc. Soc. Exp. Biol. Med., 121, 109–111.
13. LAI A.C., CHAMBERS T.M., HOLLAND R.E. JR, MORLEY P.S., HAINES D.M., TOWNSEND H.G. & BARRANDEGUY
M.
(2001). Diverged evolution of recent equine-2 influenza (H3N8) virurs in the Western Hemisphere. Arch.
Virol., 149, 1063–1074.
14. LIVESAY G.J., O’NEILL T., HANNANT D, YADAV M.P. & MUMFORD J.A. (1993). The outbreak of equine
influenza
(H3N8) in the United Kingdom in 1989; diagnostic use of an antigen capture ELISA. Vet. Rec., 133, 515–
519.
15. MADIC J., MARTINOVIC S., NAGLIC T., HAJSIG D. & CVETNIC S. (1996). Serological evidence for the presence
of
A/equine-1 influenza virus in unvaccinated horses in Croatia. Vet. Rec., 138, 68.
16. MORLEY P.S., BOGDAN J.R., TOWNSEND H.G.G. & HAINES D.M. (1995). The effect of changing single radial
haemolysis assay method when quantifying influenza A antibodies in serum. Vet. Microbiol., 44, 101–110.
17. MUMFORD J.A. (1992). Progress in the control of equine influenza. In: Equine Infectious Disease VI:
Proceedings of the Sixth International Conference, Plowright W., Rossdale P.D. & Wade J.F., eds.
Newmarket, R & W Publications, UK, 207–217.
18. MUMFORD J. (2000). Collaborative study for the establishment of three European Pharmacopoeia
biological
reference preparations for equine influenza horse antiserum. PHARMEUROPA Special Issue, Bio 2000-1,
5–21.
19. MUMFORD J.A., HANNANT D. & JESSETT D.M. (1990). Experimental infection of ponies with equine
influenza
(H3N8) viruses by intranasal inoculation or exposure to aerosols. Equine Vet. J., 22, 93–98.
20. MUMFORD J.A. & WOOD J. (1993). Establishing an acceptability threshold for equine influenza vaccines.
Dev.
Biol. Stand., 79, 137–146.
21. MUMFORD J.A. & WOOD J. (1993). WHO/OIE Meeting: Consultation on Newly Emerging Strains of Equine
Influenza. Vaccine, 11, 1172–1175.
22. MUMFORD J., WOOD J.M., SCOTT A.M., FOLKERS C. & SCHILD G.C. (1983). Studies with inactivated equine
influenza vaccine 2. Protection against experimental infection with influenza virus A/equine/Newmarket/79
(H3N8). J. Hyg. (Camb.), 90, 385–395.
23. OXBURGH L. & HAGSTROM A.A. (1999). A PCR based method for the identification of equine influenza
virus
from clinical samples. Vet. Microbiol., 67, 161–174.
24. OXBURGH L. & KLINGBORN B. (1999). Cocirculation of two distinct lineages of equine influenza virus
subtype
H3N8. J. Clin. Microbiol., 37, 3005–3009.
25. PLATEAU E. & CRUCIERE C. (1983). Study on radial haemolysis method for the detection of anti influenza
equiantibodies in equine sera: reliability and expression of the results. Zentralbl. Veterinarmed [B], 30, 512–
520.
26. QUINLIVAN M., DEMPSEY E., RYAN F., ARKINS S. & CULLINANE A. (2005). Real-time reverse transcription
PCR for
detection and quantitative analysis of equine influenza virus. J. Clin. Microbiol., 43, 5055–5057.
27. SCHILD G.C., PEREIRA, M.S. & CHAKRAVERTY P. (1975). Single radial haemolysis: a new method for the
assay
of antibody to influenza haemagglutinin. Bull. WHO, 52, 43–50.
28. UNITED STATES DEPARTMENT OF HEALTH AND HUMAN SERVICES (1982). Concepts and Procedures for
Laboratory-Based Influenza Surveillance. Centers for Disease Control, Atlanta, Georgia, USA, 81–835.
29. WEBSTER R.G. (1993). Are equine 1 influenza viruses still present in horses? Equine Vet. J., 25, 537–
538.
30. VAN MAANEN C. & CULLINANE A. (2002) Equine influenza virus infections: an update. Vet. Q, 24 (2), 79–
94.
31. WOOD J.M., MUMFORD J., FOLKERS C., SCOTT A.M. & SCHILD G.C. (1983). Studies with inactivated equine
influenza vaccine 1. Serological responses of ponies to graded doses of vaccine. J. Hyg. (Camb.), 90, 371–
384.
32. WOOD J.M., SCHILD G.C., FOLKERS C., MUMFORD J. & NEWMAN R.W. (1983). The standardisation of
inactivated
equine influenza vaccines by single-radial immunodiffusion. J. Biol. Stand., 11, 133–136.
33. WOOD J.M., SCHILD G.C., NEWMAN R.W. & SEAGROATT V. (1977). An improved single-radial-
immunodiffusion
technique for the assay of influenza haemagglutinin antigen. Application for potency determinations of
inactivated whole virus and subunit vaccines. J. Biol. Stand., 5, 237–247.
*
**
NB: There are OIE Reference Laboratories for Equine influenza Table in Part 3 of this Terrestrial Manual or
consult the OIE Web site for the most up-to-date list: www.oie.int.
C H A P T E R 2 . 5 . 1 1 .
GLANDERS
SUMMARY
Glanders is a contagious and fatal disease of horses, donkeys, and mules, and is caused by
infection with the bacterium Burkholderia mallei (the name recently changed from Pseudomonas
mallei and was previously classified as Pfeifferella, Loefflerella, Malleomyces or Actinobacillus). The
disease causes nodules and ulcerations in the upper respiratory tract and lungs. A skin form also
occurs, known as ‘farcy’. Control of glanders requires testing of suspect clinical cases, screening of
apparently normal equids, and elimination of positive reactors. It is transmitted to humans and all
infected/contaminated or potentially infected/contaminated material must be handled in a laboratory
that meets the requirements for Containment Group 3 pathogens.
Identification of the agent: Smears from fresh material may reveal Gram-negative nonsporulating,
nonencapsulated rods. The presence of a capsule-like cover has been demonstrated by electron
microscopy. The bacteria grow aerobically and prefer media that contain glycerol. Unlike the
Pseudomonas species and the closely related bacterium Burkholderia pseudomallei, Burkholderia
mallei is nonmotile. Guinea-pigs are highly susceptible, and males can be used, if strictly
necessary, to recover the organism from a heavily contaminated sample. Commercially available
biochemical identification kits lack diagnostic sensitivity. Specific monoclonal antibodies and
polymerase chain reaction (PCR) as well as real-time PCR assays are available. The latter have
also been evaluated in recent outbreaks.
Mallein and serological tests: The mallein test is a sensitive and specific clinical test for
hypersensitivity against Burkholderia mallei. Mallein, a water soluble protein fraction of the
organism, is injected subcutaneously, intradermo-palpebrally or given by eyedrop. In infected
animals, the skin or the eyelid swells markedly within 1–2 days. Complement fixation test and
enzyme-linked immunosorbent assays are the most accurate and reliable serological tests for
diagnostic use. A rose bengal plate agglutination test has recently been developed in Russia; it has
been validated in Russia only.
Requirements for vaccines and diagnostic biologicals: There are no vaccines. Mallein purified
protein derivative is currently available commercially from the Central Veterinary Control and
Research Institute, 06020 Etlik, Ankara, Turkey.
A. INTRODUCTION
Glanders is a bacterial disease of perissodactyls or odd-toed ungulates with zoonotic potential known since
ancient times. It is caused by the bacterium Burkholderia mallei (the name recently changed from
Pseudomonas
mallei (45) and has been classified in the past as Pfeifferella, Loefflerella, Malleomyces or Actinobacillus).
Outbreaks of the disease may occur in felines living in the wild or in zoological gardens. Susceptibility to
glanders
has been proved in camels, bears, wolfs and dogs. Carnivores may become infected by eating infected
meat, but
cattle and pigs are resistant (22). Small ruminants may also be infected if kept in close contact to glanderous
horses (42). Glanders in the acute form occurs most frequently in donkeys and mules with high fever and
respiratory signs (swollen nostrils, dyspnoea, and pneumonia); death occurs within a few days. In horses,
glanders generally takes a more chronic course and they may survive for several years. Chronic and
subclinical
‘occult’ cases are dangerous sources of infection due to permanent or intermittent shedding of bacteria (42).
In horses, inflammatory nodules and ulcers develop in the nasal passages and give rise to a sticky yellow
discharge, accompanied by enlarged firm submaxillary lymph nodes. Stellate scarring follows upon healing
of the
ulcers. The formation of nodular abscesses in the lungs is accompanied by progressive debility, febrile
episodes,
Chapter 2.5.11. - Glanders
coughing and dyspnoea. Diarrhoea and polyuria can also occur. In the skin form (‘farcy’), the lymphatics are
enlarged and nodular abscesses (‘buds’) of 0.5–2.5 cm develop, which ulcerate and discharge yellow oily
pus.
Nodules are regularly found in the liver and spleen. Discharges from the respiratory tract and skin are
infective,
and transmission between animals, which is facilitated by close contact, by inhalation, ingestion of
contaminated
material (e.g. from infected feed and water troughs), or by inoculation (e.g. via a harness) is common. The
incubation period can range from a few days to many months (23, 42).
Glanders is transmissible to humans by direct contact with diseased animals or infected/contaminated
material. In
the untreated acute disease. 95% mortality can occur within 3 weeks (25). However, survival is possible if
the
infected person is treated early and aggressively with multiple systemic antibiotic therapies (17, 33). A
chronic
form with abscessation also occurs (25). When handling suspect or known infected animals or fomites,
stringent
precautions should be taken to prevent self-infection or transmission of the bacterium to other equids.
Laboratory
samples should be securely packaged, kept cool and shipped as outlined in Chapter 1.1.1 Collection and
shipment of diagnostic specimens. All manipulations with potentially infected/contaminated material must be
performed in a laboratory that meets the requirements for Containment Group 3 pathogens as outlined in
Chapter
1.1.2 Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities.
Glanders has been eradicated from many countries by statutory testing, elimination of infected animals, and
import restrictions. It persists in some Asian, African and South American countries. It can be considered a
reemerging
disease and may be imported by pet or racing equids into glanders free areas (26).
Cases for specific glanders investigation should be differentiated on clinical grounds from other chronic
infections
of the nasal mucosae or sinuses, and from strangles (Streptococcus equi infection), ulcerative lymphangitis
(Corynebacterium pseudotuberculosis), pseudotuberculosis (Yersinia pseudotuberculosis) and
sporotrichosis
(Sporotrichium spp.). Glanders should be excluded positively from suspected cases of epizootic
lymphangitis
(caused by Histoplasma farciminosum), with which it has many clinical similarities. In humans in particular,
glanders should be distinguished from melioidosis (B. pseudomallei infection), which is caused by an
organism
with close similarities to B. mallei (22).
a) Morphology of Burkholderia mallei
The organisms are fairly numerous in smears from fresh lesions, but in older lesions they are scanty (41).
They should be stained by methylene blue or Gram stain. The organisms are mainly extracellular, fairly
straight Gram-negative rods with rounded ends, 2–5 μm long and 0.3–0.8 μm wide with granular inclusions
of various size. They often stain irregularly and do not have a readily visible capsule, under the light
microscope, or form spores. The presence of a capsule-like cover has been established by electron
microscopy. This capsule is composed of neutral carbohydrates and serves to protect the cell from
unfavourable environmental factors. Unlike other organisms in the Pseudomonas group and its close relative
Burkholderia pseudomallei, Burkholderia mallei has no flagellae and are therefore nonmotile (19, 31). The
organisms are difficult to demonstrate in tissue sections, where they may have a beaded appearance (21).
In
culture media, they vary in appearance depending on the age of the culture and type of medium. In older
cultures, there is much pleomorphism. Branching filaments form on the surface of broth cultures (26).
b) Cultural characteristics
It is preferable to attempt isolation from unopened uncontaminated lesions (21). The organism is aerobic
and
facultatively anaerobic only in the presence of nitrate (8, 19), growing optimally at 37°C (20). It grows well,
but slowly, on ordinary culture media, 72-hour incubation of cultures is recommended; glycerol enrichment is
particularly useful. After a few days on glycerol agar, there is a confluent, slightly cream-coloured growth that
is smooth, moist, and viscuous. With continued incubation, the growth thickens and becomes dark brown
and tough. It also grows well on glycerol potato agar and in glycerol broth, on which a slimy pellicle forms.
On plain nutrient agar, the growth is much less luxuriant, and growth is poor on gelatin (34). In samples not
obtained under sterile conditions B. mallei is regularly overgrown by other bacteria.
Alterations to characteristics may occur in vitro, so fresh isolates should be used for identification reactions.
Litmus milk is slightly acidified by B. mallei, and coagulation may occur after long incubation. The organism
reduces nitrates. Although some workers have claimed that glucose is the only carbohydrate that is
fermented (slowly and inconstantly), other workers have shown that if an appropriate medium and indicator
are used, glucose and other carbohydrates, such as arabinose, fructose, galactose and mannose, are
consistently fermented by B. mallei (6). Indole is not produced, horse blood is not haemolysed and no
diffusible pigments are produced in cultures (19). A commercial laboratory test kit (e.g. API [Analytical Profile
Index] system: Analytab Products, BioMerieux or Biolog [Hayward, California]) can be used for easy
confirmation that an organism belongs to the Pseudomonas group. In general, commercially available
systems are not suited to unambiguously identifying members of the steadily growing number of species of
the genus Burkholderia (9). Lack of motility is then of special relevance. A bacteriophage specific for
B. mallei is available (43).
All culture media prepared should be subjected to quality control and must support growth of the suspect
organism from a small inoculum. The reference strain should be cultured in parallel with the suspicious
samples to ensure that the tests are working correctly.
In contaminated samples, supplementation of media with substances that inhibit the growth of Gram-positive
organisms (e.g. crystal violet, proflavine) has proven to be of use, as has pretreatment with penicillin (1,000
units/ml for 3 hours at 37°C) (22). A selective medium has been developed (44) composed of polymyxin E
(1,000 units), bacitracin (250 units), and actidione (0.25 mg) incorporated into nutrient agar (100 ml)
containing glycerine (4%), donkey or horse serum (10%), and ovine haemoglobin or tryptone agar (0.1%).
Outside the body, the organism has little resistance to drying, heat, light or chemicals, so that survival
beyond 2 weeks is unlikely (25). Under favourable conditions, however, it can probably survive a few
months. Burkholderia mallei can remain viable in tap water for at least 1 month (34). For disinfection,
benzalkonium chloride or ‘roccal’ (1/2,000), sodium hypochlorite (500 ppm available chlorine), iodine,
mercuric chloride in alcohol, and potassium permanganate have been shown to be highly effective against
B.
mallei (20). Phenolic disinfectants are less effective.
c) Laboratory animal inoculation
Guinea-pigs, hamsters and cats have been used for diagnosis when necessary. If isolation in a laboratory
animal is considered unavoidable, suspected material is inoculated intraperitoneally into a male guinea-pig.
As this technique has a sensitivity of only 20%, the inoculation of at least five animals is recommended (25).
Positive material will cause a severe localised peritonitis and orchitis (the Strauss reaction). The number of
organisms and their virulence determines the severity of the lesions. Additional steps are used when the test
material is heavily contaminated (11). The Strauss reaction is not specific for glanders, and other organisms
can elicit it. Bacteriological examination of infected testes should confirm the specificity of the response
obtained.
d) Confirmation by polymerase chain reaction and real-time PCR
In the past few years, several PCR and real-time PCR assays for the identification of Burkholderia mallei
have been developed (2, 13, 32, 36, 38, 39), but only a PCR and a real-time PCR assay were evaluated
using samples from a recent outbreak of glanders in horses (30, 37). These two assays will be described in
more detail here. However, the robustness of these assays will have to be demonstrated in the future by
interlaboratory studies. The guidelines and precautions outlined in Chapter 1.1.5 Validation and quality
control of polymerase chain reaction methods used for the diagnosis of infectious diseases, have to be
taken
into account.
o DNA preparation
Single colonies are transferred from an agar plate to 200 μl lysis buffer (5× buffer D [PCR Optimation Kit,
Invitrogen, DeShelp, The Netherlands, 1/5 diluted in ultra-pure water]; 0.5% Tween 20 [ICI, American
Limited, Merck, Hohenbrunn, Germany]; 2 mg/ml proteinase K [Roche Diagnostics, Mannheim, Germany]).
After incubation at 56°C for 1 hour and inactivation for 10 minutes at 95°C, 2 and 4 μl of the cleared lysate
are used as template in the PCR or the real-time PCR assay, respectively.
Tissue samples of horses (skin, liver, spleen, lung, and conchae) inactivated and preserved in formalin
(48 hours, 10% v/v) are cut with a scalpel into pieces of 0.5 × 0.5 cm (approximately 500 mg). The
specimens are washed twice in deionised water (10 ml), incubated over night in sterile saline at 4°C, and
minced using liquid nitrogen, a mortar and a pestle. Total DNA is prepared from 50 mg tissue using the
QIAamp Tissue KitTM according to the manufacturer’s instructions (Qiagen, Hilden, Germany). DNA is eluted
with 80 μl dH2O of which 4 μl are used as template.
o PCR assay (30)
The assay may have to be adapted to the PCR instrument used with minor modifications to the cycle
conditions and the concentration of the chemicals used.
The oligonucleotides used in ref. 30 are designed based on differences of the fliP sequences from B. mallei
ATCC 23344T (accession numbers NC_006350, NC_006351) and B. pseudomallei K96243 (accession
numbers NC_006348, NC_006349). Primers Bma-IS407-flip-f (5’-TCA-GGT-TTG-TAT-GTC-GCT-CGG-3’)
and Bma- IS407-flip-r (5’-CTA-GGT-GAA-GCT-CTG-CGC-GAG-3’) are used to amplify a 989 bp fragment.
PCR is done using 50 μl ready-to-go mastermix (Eppendorf, Hamburg, Germany) and 15 pmol of each
primer. Thermal cycling conditions are 94°C for 30 seconds; 65°C for 30 seconds and 72°C for 60 seconds.
This cycle is repeated for 35 times. A final elongation step is added (72°C for 7 minutes). Visualisation of the
products is done by UV light after agarose gel (1% w/v in TAE buffer) electrophoresis and staining with
ethidium bromide. No template controls containing PCR-grade water instead of template and positive
controls containing DNA of B. mallei have to be included in each run to detect contamination by amplicons of
former runs or amplification failure.
The lower detection limit is 10 fg or 2 genome equivalents.
o Real-time PCR assay (37)
The assay has to be adapted to the real-time PCR instrument used with minor modifications, e.g. the cycling
vials have to be chosen according to the manufacturer’s recommendations and the concentration of the
oligonucleotides may have to be doubled or the labelling of the probes has to be changed. The authors used
a MX3000PTM real-time PCR system (Stratagene, Amsterdam, The Netherlands) and 96-well plates
(ThermoFast 96 ABGeneTM, Rapidozym, Berlin, Germany).
The oligonucleotides used in ref. 37 were designed based on differences of the fliP sequences from B.
mallei
ATCC 23344T (accession numbers NC_006350, NC_006351) and B. pseudomallei K96243 (accession
numbers NC_006348, NC_006349). The fluorogenic probe is synthesised with 6-carboxy-fluorescein (FAM)
at the 5’-end and black hole quencher 1 (BHQ1) at the 3’-end. Oligonucleotides used were Bma-flip-f (5’-
CCC-ATT-GGC-CCT-ATC-GAA-G-3’), Bma-flip-r (5’-GCC-CGA-CGA-GCA-CCT-GAT-T-3’) and probe
Bmaflip
(5’-6FAM-CAG-GTC-AAC-GAG-CTT-CAC-GCG-GAT-C-BHQ1-3’).
The 25 μl reaction mixture consists of 12.5 μl 2× TaqManTM Universal MasterMix (Applied Biosystems,
Foster City, USA), 0.1 μl of each primer (10 pmol/μl), 0.1 μl of the probe (10 pmol/μl) and 4 μl sample.
Thermal cycling conditions are 50°C for 2 minutes; 95°C for 10 minutes; 45 (50) cycles at 95°C for
25 seconds and 63°C for 1 minute. Possible contaminations with amplification products from former
reactions are inactivated by an initial incubation step using uracil N’-glycosilase.
The authors suggest to include an internal inhibition control based on a bacteriophage lambda gene target
(Lambda-F [5’-ATG-CCA-CGT-AAG-CGA-AAC-A-3] Lambda-R [5’-GCA-TAA-ACG-AAG-CAG-TCG-AGT-3’],
Lam-YAK [5’-YAK-ACC-TTA-CCG-AAA-TCG-GTA-CGG-ATA-CCG-C-DB-3’]), which can be titrated to give
reproducible cycle threshold values. However, depending on the sample material a house keeping gene
targeting PCR may be used additionally or as an alternative. No template controls containing 4 μl of
PCRgrade
water instead and positive controls containing DNA of B. mallei have to be included in each run to
detect amplicon contamination or amplification failure.
The linear range of the assay was determined to cover concentrations from 240 pg to 70 fg bacterial
DNA/reaction. The lower limit of detection defined as the lowest amount of DNA that was consistently
detectable in three runs with eight measurements each is 60 fg DNA or four genome equivalents (95%
probability). The intra-assay variability of the fliP PCR assay for 35 pg DNA/reaction is 0.68 % (based on Ct
values) and for 875 fg 1.34%, respectively. The inter-assay variability for 35 pg DNA/reaction is 0.89%
(based on Ct values) and for 875 fg DNA 2.76 %, respectively.
e) Other methods
The genome of the Burkholderia mallei type strain ATCC 23344T was sequenced in 2004 (27). Several
genomes of other isolates followed and revealed a wide genomic plasticity. Passages in different host
species or culture media may provoke considerable sequence alterations (29). The loss of the ability to
produce LPS and/or capsule polysaccharide upon ongoing culture due to mutation is a well known fact and
results in reduced or absent virulence and influences serologic tests (26). Several molecular typing
techniques have successfully been introduced. Simple molecular techniques like PCR-restriction fragment
length polymorphism (35) and pulsed field gel electrophoresis (5) can be used for further discrimination of
isolates. Ribotyping using restriction enzymes PstI and EcoRI in combination with an E. coli 18-mer rDNA
probe produced 17 distinct ribotypes within 25 B. mallei isolates (12). These techniques are still the in-house
tests of specialised laboratories as an extensive strain collection is necessary. Multilocus sequence typing
(MLST) can be done with purified DNA so there is no need for excessive cultivation of the agent or the
keeping of strain collections. Web-based analysis might even enhance diagnostics (10). No specific
histopathology features can be described for lesions caused by B. mallei. For immunuhistochemical
analysis,
B. mallei specific hyperimmune sera of rabbits can be used.
2. Mallein and serological tests
a) The mallein test (a prescribed test for international trade)
The mallein purified protein derivative (PPD), which is available commercially, is a solution of water-soluble
protein fractions of heat-treated B. mallei. The test depends on infected horses being hypersensitive to
mallein. Advanced clinical cases in horses and acute cases in donkeys and mules may give inconclusive
results requiring additional methods of diagnosis to be employed (1).
o The intradermo-palpebral test
This is the most sensitive, reliable and specific test for detecting infected perissodactyls or odd-toed
ungulates, and has largely displaced the ophthalmic and subcutaneous tests (3): 0.1 ml of concentrated
mallein PPD is injected intradermally into the lower eyelid and the test is read at 24 and 48 hours. A positive
reaction is characterised by marked oedematous swelling of the eyelid, and there may be a purulent
discharge from the inner canthus or conjunctiva. This is usually accompanied by a rise in temperature. With
a negative response, there is usually no reaction or only a little swelling of the lower lid.
o The ophthalmic test
This is less reliable than the intradermo-palpebral test. A few drops of mallein are instilled into the eye at the
canthus. In an infected animal, the eyelids, and sometimes the side of the face, become swollen and there
may be a little discharge from the eye. The reaction may also occur to a lesser extent in the opposite eye.
o The subcutaneous test
This test interferes with subsequent serological diagnosis and so the other two mallein tests are preferred.
Also, the test may not be acceptable in some countries. The horse’s temperature has to be under 102°F
(38.8°C) on the day before the test, at the time of the injection, and at 9, 12 and 15 hours after the injection.
A 10 cm square skin patch in the middle of the neck is clipped and disinfected; 2.5 ml of dilute mallein are
injected subcutaneously into the centre of the patch. With a positive test, the horse develops a pyrexia of
104°F (40.0°C) or over during the first 15 hours, and a firm painful swelling with raised edges develops
within
24 hours at the injection site. In nonglandered horses, there is no, or minimal, transient local swelling.
Doubtful reactors may be retested after 14 days using a double dose of mallein.
b) Complement fixation test (a prescribed test for international trade)
Although not as sensitive as the mallein test, the CF test is an accurate serological test that has been used
for glanders diagnosis for many years (3). It is reported to be 90–95% accurate, serum being positive within
1 week of infection and remaining positive in the case of exacerbation of the chronic process (34). Recently,
however, the specificity of CF testing has been questioned (26).
o Antigen preparation (16)
i) Flasks of beef infusion broth with 3% glycerol are inoculated with log-phase growth B. mallei and
incubated at 37°C for 8–12 weeks.
ii) The cultures are inactivated by exposing the flasks to flowing steam (100°C) for 60 minutes.
iii) The clear supernatant is decanted and filtered. The filtrate is heated again by exposure to live steam for
75 minutes on 3 consecutive days, and clarified by centrifugation.
iv) The clarified product is concentrated to one-tenth the original volume by evaporation on a steam or hot
water bath.
v) Concentrated antigen is bottled in brown-glass bottles to protect from light and stored at 4°C. Antigen
has been shown to be stable for at least 10 years in this concentrated state.
vi) Lots of antigen are prepared by diluting the concentrated antigen 1/20 with sterile physiological saline
with 0.5% phenol. The diluted antigen is dispensed into brown-glass vials and store at 4°C. The final
working dilution is determined by a block titration. The final working dilution for CF test use is made at
the time the CF test is performed.
The resulting antigen is primarily lipopolysaccharide. An alternative procedure is to use young cultures by
growing the organism on glycerol–agar slopes for 12 hours and washing off with normal saline. A
suspension
of the culture is heated for 1 hour at 70°C and the heat-treated bacterial suspension is used as antigen. The
disadvantage of this antigen preparation method is that the antigen contains all the bacterial cell
components. The antigen should be safety tested by inoculating blood agar plates.
o Test procedure (24)
i) Serum is diluted 1/5 in veronal (barbiturate) buffered saline containing 0.1% gelatin (VBSG) or CFD
(complement fixation diluent – available as tablets) without gelatine.
ii) Diluted serum is inactivated for 30 minutes at 56°C. The USDA complement fixation protocol calls for
inactivation for 35 minutes (24). (Serum of equidae other than horses should be inactivated at 63°C for
30 minutes.)
iii) Twofold dilutions of the sera are prepared in 96-well round-bottom microtitre plates.
iv) Guinea-pig complement is diluted in the chosen buffer and 5 (or optionally 4) complement haemolytic
units-50% (CH50) are used.
v) Sera, complement and antigen are reacted in the plates and incubated for 1 hour at 37°C. (An alternate
acceptable procedure is overnight incubation at 4°C.)
vi) A 2% suspension of sensitised washed sheep red blood cells is added. The USDA protocol calls for
confirmation of positive reactions in a tube test using 3% sheep red blood cells (24).
vii) Plates are incubated for 45 minutes at 37°C, and then centrifuged for 5 minutes at 600 g.
A sample that produces 100% haemolysis at the 1/5 dilution is negative, 25–75% haemolysis is suspicious,
and no haemolysis (100% fixation) is positive. Unfortunately, false-positive results can occur, and
B. pseudomallei and B. mallei cross react and cannot be differentiated by serology (3, 25). Also healthy
horses can have a false positive CF reaction for a variable period following a mallein intradermal test.
c) Enzyme-linked immunosorbent assays
Both plate and membrane (blot) enzyme-linked immunosorbent assays (ELISAs) have been reported for the
serodiagnosis of glanders, but none of these procedures has been shown to differentiate serologically
between B. mallei and B. pseudomallei. Blotting approaches have involved both dipstick dot-blot and
electrophoretically separated and transferred western blot methods (14, 40). A competitive ELISA that uses
an uncharacterised anti-lipopolysaccharide monoclonal antibody has also been developed and found to be
similar to the CF test in performance (15). Continuing development of monoclonal antibody reagents specific
for B. mallei antigenic components offers the potential for more specific ELISAs in the foreseeable future
that
will help resolve questionable test results of quarantined imported horses (4, 7, 18, 25). At this time, none of
these tests has been validated.
d) Other serological tests
The avidin–biotin dot ELISA has been described (40), but has not yet been widely used or validated. The
antigen is heat-inactivated bacterial culture that has been concentrated and purified. A dot of this antigen is
placed on a nitrocellulose dipstick that is then used to test for antibody against B. mallei in equine serum.
Using antigen-dotted, preblocked dipsticks, the test can be completed in approximately 1 hour. Serum or
whole blood can be used for the test, and partial haemolysis does not impart any background colour to the
antigen-coated area on the nitrocellulose. Recently, polysaccharide microarray technology has offered a
new
promising approach to improve sensitivity in serology (28).
The rose bengal plate agglutination test (RBT) has been described for the diagnosis of glanders in horses
and other susceptible animals; this test has been validated in Russia only. The antigen is a heat-inactivated
bacterial suspension coloured with rose bengal, which is used in a plate agglutination test.
The accuracy of other agglutination tests and precipitin is unsatisfactory for use in control programmes.
Horses with chronic glanders and those in a debilitated condition give negative or inconclusive results.
BIOLOGICALS
No vaccines are available.
Mallein PPD for use in performing the intradermo-palpebral and ophthalmic tests is produced commercially
by the
Central Veterinary Control and Research Institute, 06020 Etlik, Ankara, Turkey.
The ID-Lelystad has provided the following information on requirements for mallein PPD.
1. Seed management
Three strains of Burkholderia mallei are employed in the production of mallein PPD, namely Bogor strain
(originating from Indonesia), Mukteswar strain (India) and Zagreb strain (Yugoslavia). The seed material is
kept as
a stock of freeze-dried cultures. The strains are subcultured on to glycerol agar at 37°C for 1–2 days. For
maintaining virulence and antigenicity, the strains may be passaged through guinea-pigs.
Dorset−Henley medium, enriched by the addition of trace elements, is used for production of mallein PPD.
The
liquid medium is inoculated with a thick saline suspension of B. mallei, grown on glycerol agar. The
production
medium is incubated at 37°C for about 10 weeks. The bacteria are then killed by steaming for 3 hours in a
Koch’s
steriliser. The fluid is then passed through a layer of cotton wool to remove coarse bacterial clumps. The
resulting
turbid fluid is cleared by membrane filtration, and one part trichloroacetic acid 40% is immediately added to
nine
parts culture filtrate. The mixture is allowed to stand overnight and a light brownish to greyish precipitate
settles.
The supernatant fluid is pipetted off and discarded. The precipitate is centrifuged for 15 minutes at 2500 g
and the
layer of precipitate is washed three or more times in a solution of 5% NaCl, pH 3, until the pH is 2.7. The
washed
precipitate is dissolved by stirring with a minimum of an alkaline solvent. The fluid is dark brown and a pH of
6.7
will be obtained. This mallein concentrate has to be centrifuged thoroughly and the supernatant is diluted
with an
equal amount of a glucose buffer solution. The protein content of this product is estimated by the Kjeldahl
method
and freeze-dried after it has been dispensed into ampoules.
During the period of incubation, the flasks are inspected frequently for any signs of contamination, and
suspect
flasks are discarded. A typical growth of the B. mallei cultures comprises turbidity, sedimentation, some
surface
growth with a tendency towards sinking, and the formation of a conspicuous slightly orange-coloured ring
along
the margin of the surface of the medium.
4. Batch control
Each batch of mallein PPD is tested for sterility, safety, preservatives, protein content and potency.
Sterility testing is performed according to the European Pharmacopoeia guidelines.
The examination for safety is conducted on from five to ten normal healthy horses by carrying out the
intradermopalpebral
test. The resulting swelling should be, at most, barely detectable and transient, without any signs of
conjunctival discharge.
Preparations containing phenol as a preservative should not contain more than 0.5% (w/v) phenol. The
protein
content should be not less than 0.95 mg/ml and not more than 1.05 mg/ml.
Potency testing is performed in guinea-pigs and horses. The animals are sensitised by subcutaneous
inoculation
with a concentrated suspension of heat-killed B. mallei in paraffin oil or incomplete Freund’s adjuvant. Cattle
can
also be used instead of horses. The production batch is bioassayed against a standard mallein PPD by
intradermal injection in 0.1 ml doses in such a way that complete randomisation is obtained.
In guinea-pigs, the different areas of erythema are measured after 24 hours, and in horses the increase in
skin
thickness is measured by calipers. The results are statistically evaluated, using standard statistical methods
for
parallel-line assays.
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42. WITTIG M.B., WOHLSEIN P., HAGEN R.M., AL DAHOUK S., TOMASO H., SCHOLZ H.C., NIKOLAOU K., WERNERY
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WERNERY U., KINNE J., ELSCHNER M. & NEUBAUER H. (2006). Glanders--a comprehensive review. Dtsch.
Tierarztl. Wochenschr., 113,323–230.
43. WOODS D.E., JEDDELOH J.A., FRITZ D.L. & DESHAZER D. (2002). Burkholderia thailandensis E125 harbors
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44. XIE X., XU F., XU B., DUAN X. & GONG R. (1980). A New Selective Medium for Isolation of Glanders Bacilli.
Collected papers of veterinary research. Control Institute of Veterinary Biologics, Ministry of Agriculture,
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45. YABUUCHI E., KOSAKO Y., OYAIZU H., YANO I., HOTTA H., HASHIMOTO Y., EZAKI T. & ARAKAWA M. (1992).
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nov. Microbiol. Immunol., 36, 1251–1275.
*
**
NB: There is an OIE Reference Laboratory for Glanders Table in Part 3 of this Terrestrial Manual or consult
the
OIE Web site for the most up-to-date list: www.oie.int.
C H A P T E R 2 . 5 . 8 .
EQUINE PIROPLASMOSIS
SUMMARY
Equine piroplasmosis is a tick-borne protozoal disease of horses, mules, donkeys and zebra. The
aetiological agents are blood parasites named Theileria equi and Babesia caballi. Theileria equi
was previously designated as Babesia equi. Infected animals may remain carriers of these
parasites for long periods and act as sources of infection for ticks, which act as vectors. The
parasites are found inside the red blood cells of the infected animals.
The introduction of carrier animals into areas where tick vectors are prevalent can lead to an
epizootic spread of the disease.
Identification of the agent: Infected horses can be identified by demonstrating the parasites in
stained blood or organ smears during the acute phase of the disease. Romanovsky-type staining
methods, such as Giemsa, give the best results. In carrier animals, low parasitaemias make it
extremely difficult to detect parasites, especially in the case of B. caballi infections, although they
may sometimes be demonstrated by using a thick blood smear technique.
Paired merozoites joined at their posterior ends are a diagnostic feature of B. caballi infection. The
parasites in the erythrocytes measure 2 × 5 μm. The merozoites of T. equi are less than 2–3 μm
long, and are pyriform, round or ovoid. A characteristic of T. equi is the arrangement of four pearshaped
merozoites forming a tetrad known as a ‘Maltese cross’.
Molecular techniques for the detection of T. equi and B. caballi based on species-specific
polymerase chain reaction (PCR) assays, targeting the 18S rRNA gene, have been developed and
continue to expand. These tests have been shown to be highly specific and sensitive and promise
to play an increasing role in the diagnosis of infections.
Serological tests: Infections in carrier animals are best demonstrated by testing their sera for the
presence of specific antibodies.
Currently, the indirect fluorescent antibody (IFA) test and the competitive enzyme-linked
immunosorbent assay (C-ELISA) are the primary tests used for qualifying horses for importation.
The complement fixation (CF) test, for many years the primary test, has been replaced by the IFA
and C-ELISA. These tests have proven to be more effective at detecting long-term infected animals
and animals treated with antiparasitic drugs; these animals may be CF negative but still be infected.
The IFA test and C-ELISA have been shown to be highly specific for each of the two species of
piroplasmosis agents involved. Indirect ELISAs may also be used to detect antibodies to both
species in infected horses, although cross-reactions between T. equi and B. caballi occur.
Application of recombinant T. equi and B. caballi merozoite proteins in diagnostic assays appear to
be very promising in the accurate determination of equine piroplasmosis infection.
Requirements for vaccines and diagnostic biologicals: There are no biological products
available.
A. INTRODUCTION
Equine piroplasmosis is a tick-borne protozoal disease of horses, mules, donkeys and zebra. The
aetiological
agents of equine piroplasmosis are Theileria equi and Babesia caballi. Twelve species of Ixodid ticks in the
genera Dermacentor, Rhipicephalus and Hyalomma have been identified as transstadial vectors of B. caballi
and
T. equi, while eight of these species were also able to transmit B. caballi infections transovarially (7, 36, 44,
45).
Infected animals may remain carriers of these blood parasites for long periods and act as sources of
infection for
tick vectors.
Chapter 2.5.8. – Equine piroplasmosis
The parasites occur in southern Europe, Asia, countries of the Commonwealth of Independent States, Africa,
Cuba, South and Central America, and certain parts of the southern United States of America. Theileria equi
has
also been reported from Australia (but, apparently never established itself in this region), and is now
believed to
have a wider general distribution than B. caballi.
During the life cycle of Babesia, initially the sporozoites invade red blood cells (RBCs) where they transform
into
trophozoites (10, 37). In this situation the trophozoites grow and divide into two round, oval or pear-shaped
merozoites. The mature merozoites are now capable of infecting new RBCs and the division process is then
repeated.
Babesia caballi: the merozoites in the RBCs are pear-shaped, 2–5 μm long and 1.3–3.0 μm in diameter (27).
The
paired merozoites joined at their posterior ends are considered to be a diagnostic feature of B. caballi
infection
(32).
Theileria equi: the merozoites of this organism are relatively small, less than 2–3 μm long (27), and are
pyriform,
round or ovoid. A characteristic of T. equi is the arrangement of four pear-shaped merozoites, measuring
about
2 μm in length, forming a tetrad known as the ‘Maltese cross’ arrangement (14).
In T. equi infection it has been shown that sporozoites inoculated into horses via a tick bite invade the
lymphocytes (41). The sporozoites undergo development in the cytoplasm of these lymphocytes and
eventually
form Theileria-like schizonts. Merozoites released from these schizonts enter RBCs. The taxonomic position
of
T. equi has been controversial and only relatively recently has it been redescribed as a Theileria (33).
Further
support for the close relation with Theileria spp. also comes from the homology found between 30 and 34
kDa
T. equi surface proteins and similar sized proteins of various Theileria spp. (22, 24). However, the position of
T. equi in phylogenetic trees based on the small subunit ribosomal RNA genes is variable and mostly appear
as a
sister clade of the Theilerids (6, 35) leading some to suggest that T. equi is ancestral to the Theilerids (6) or
a
different group altogether (3).The clinical signs of equine piroplasmosis are often nonspecific, and the
disease can
easily be confused with other conditions.
Piroplasmosis can occur in peracute, acute and chronic forms. The acute cases are more common, and are
characterised by fever that usually exceeds 40°C, reduced appetite and malaise, elevated respiratory and
pulse
rates, congestion of mucous membranes, and faecal balls that are smaller and drier than normal.
Clinical signs in subacute cases are similar. In addition, affected animals show loss of weight, and fever is
sometimes intermittent. The mucous membranes vary from pale pink to pink, or pale yellow to bright yellow.
Petechiae and/or ecchymoses may also be visible on the mucous membranes. Normal bowel movements
may be
slightly depressed and the animals may show signs of mild colic. Mild oedematous swelling of the distal part
of
the limbs sometimes occurs.
Chronic cases usually present nonspecific clinical signs such as mild inappetence, poor performance and a
drop
in body mass. The spleen is usually found to be enlarged on rectal examination.
A rare peracute form where horses are found either dead or moribund has been reported (28).
Infected horses may be identified by demonstrating the parasites in stained blood, optimally collected from
superficial skin capillaries, or organ smears during the acute phase of the disease. Romanovsky-type
staining
methods, such as the Giemsa method, usually give the best results (43). However, even in acute clinical
cases of
B. caballi infection, the parasitaemia is very low and difficult to detect. Experienced workers sometimes use
a
thick blood smear technique (30) to detect very low parasitaemia. Thick films are made by placing a small
drop
(approximately 50 μl) of blood on to a clean glass slide which is then air-dried, heat fixed at 80°C for 5
minutes,
and stained in 5% Giemsa for 20–30 minutes.
An accurate identification of the species of the parasite is sometimes desirable, as mixed infections of T.
equi and
B. caballi probably occur frequently.
Identification of equine piroplasmosis in carrier animals by means of blood smear examination is not only
very
difficult but also inaccurate and therefore serological methods are preferred for this (see below). However,
falsenegative
or false-positive reactions may be encountered in the course of serological tests (8, 9, 49). In such
cases, the passage of whole blood, although a cumbersome and expensive exercise, is a very useful
technique to
determine the true position. Large quantities of whole blood (500 ml) are transfused into a susceptible,
preferably
splenectomised, horse. This animal is then kept under close observation for clinical signs of disease.
Diagnosis is
confirmed by the presence of parasites in its RBCs.
In an additional technique, a specific uninfected tick vector is fed on a suspect animal, and the organism can
then
either be identified in the tick itself or through the transmission of the organism by the tick vector to another
susceptible animal.
Success in the establishment of in-vitro cultures of T. equi and B. caballi may be one alternative to
supplement
the methods described above, in order to identify carriers of the parasites (15, 16, 53, 54). Babesia caballi
parasites were successfully cultured from the blood of two horses that tested negative by the complement
fixation
(CF) test (16). Similarly, T. equi could be cultured from horses that did not show any patent parasitaemia at
the
time of the initiation of the cultures (53, 54).
Molecular techniques for the detection of T. equi and B. caballi have been described. These methods are
based
on species-specific polymerase chain reaction (PCR) assays, which mainly target the 18S rRNA gene (4, 6,
40).
Further refinements to the technique includes nested PCR (38), loop-mediated isothermal amplification
(LAMP)
(1) with reported increased sensitivity; a highly sensitive reverse line blot assay (RLB) and multiplex PCR for
simultaneous detection and identification of Theileria and Babesia species in horses (2, 35).
It is extremely difficult to diagnose the organisms in carrier animals by means of the microscopic
examination of
blood smears. Furthermore, it is by no means practical on a large scale. The serological testing of animals is
therefore recommended as a preferred method of diagnosis, especially when horses are destined to be
imported
into countries where the disease does not occur, but the vector is present.
Sera should be collected and dispatched to diagnostic laboratories in accordance with the specifications of
that
laboratory. Horses for export that have been subjected to serological tests and shown to be free from
infection,
should be kept free of ticks to prevent accidental infections.
A number of serological techniques have been used in the diagnosis of piroplasmosis, such as the CF test,
the
indirect fluorescent antibody (IFA) test and the enzyme-linked immunosorbent assay (ELISA). In addition, a
simple and rapid immunochromatographic test for T. equi has also recently been described and might be a
very
useful test for the mass screening of serum samples (17).
a) Indirect fluorescent antibody test (a prescribed test for international
trade)
The IFA test has been successfully applied to the differential diagnosis of T. equi and B. caballi infections
(29). The recognition of a strong positive reaction is relatively simple, but any differentiation between weak
positive and negative reactions requires considerable experience in interpretation. A detailed description of
the protocol of the IFA test has been given (29, 34). An example of an IFA protocol is given below.
−Antigen production
Blood for antigen is obtained from horses with a rising parasitaemia, ideally 2–5%. Carrier animals that have
already produced antibodies are not suitable for antigen production. Blood (about 15 ml) is collected into
235 ml of phosphate buffered saline (PBS), pH 7.2. The RBCs are washed three times in cold PBS (1000 g
for 10 minutes at 4°C). The supernatant fluid and the white cell layer are removed after each wash. After the
last wash, the packed RBCs are reconstituted to the initial volume with 4% bovine serum albumin fraction V
made up in PBS, i.e. the original packed cell volume = 30%, so that one-third consists of RBCs. If the
original RBC volume is 15 ml, then 5 ml of packed RBCs + 10 ml of 4% bovine albumin in PBS constitutes
the antigen. After thorough mixing, the antigen is placed on to prepared wells on a glass slide using a
template or a syringe (34). Alternatively, the cells can be spread smoothly on to microscope slides, covering
the entire slide with an even, moderately thick film. These slides are allowed to dry, wrapped in soft paper
and sealed in plastic bags or wrapped in aluminium foil, and stored at –20°C for up to 1 year.
−Test procedure
i) Each sample of serum is tested against an antigen of B. caballi and of T. equi.
ii) Prior to use, the frozen antigen slides are removed from storage at –20°C and incubated at 37°C for
10 minutes.
iii) The antigen smears are then removed from their protective covering and fixed in cold dry acetone
(–20°C) for 1 minute. Commercially produced slides are available that are pre-fixed.
iv) If smears were prepared on the whole slide surface, squares (14–21 in number, i.e. 2–3 rows of
7 each) are formed on the antigen smears with nail varnish or rapidly drying mounting medium (i.e.
Cystoseal).
v) Test, positive and negative control sera are diluted from 1/80 to 1/1280 in PBS. Negative and positive
control sera are included in each test.
vi) Sera are applied (10 μl each) at appropriate dilutions to the different wells or squares on the antigen
smear, incubated at 37°C for 30 minutes, and washed several times in PBS and once in water.
vii) An anti-horse immunoglobulin prepared in rabbits and conjugated with fluorescein isothiocyanate (this
conjugate is available commercially) is diluted in PBS and applied to the smear, which is then
incubated and washed as before.
viii) After the final wash, two drops of a solution containing equal parts of glycerin and PBS are placed on
each smears and mounted with a cover-slip.
ix) The smear is then examined under the microscope for the fluorescing parasites. Sera diluted 1/80 or
more that show strong fluorescence are usually considered to be positive, although due consideration
is also given to the patterns of fluorescence of the positive and negative controls.
b) Enzyme-linked immunosorbent assay (a prescribed test for international
trade)
In recent years a number of recombinant antigens for the use in ELISAs have been described. Recombinant
T. equi (EMA-1; EMA-2; Be82 and Be158) and B. caballi proteins (RAP-1; Bc48; Bc134) have been
produced in Escherichia coli (12, 13, 18, 20, 21, 25, 46) or in insect cells by baculovirus (52, 47).
Recombinant antigens produced in E. coli or by baculovirus have the obvious advantage of avoiding the
need to infect horses for antigen production, and of eliminating the cross-reactions that have been
experienced in the past with the crude ELISA antigens. They also provide a consistent source of antigen for
international distribution and standardisation.
Indirect ELISAs using EMA-2 and BC48 have shown high sensitivity and specificity in detecting antibodies in
infected horses (18, 19, 47). Initial results from these tests are promising and further validation of the assays
is underway.
A competitive inhibition ELISA (C-ELISA) using EMA-1 protein and a specific monoclonal antibody (MAb)
that defines this merozoite surface protein epitope, have been used in a C-ELISA for T. equi (25). This
CELISA
overcomes the problem of antigen purity, as the specificity of this assay depends only on the
specificity defined by the MAb T. equi epitope. A 94% correlation was shown between the C-ELISA and the
CF test in detecting antibodies to T. equi. Sera that gave discrepant results were evaluated for their ability to
immunoprecipitate 35S-methionine-labelled in-vitro translated products of T. equi merozoite mRNA. Samples
that were C-ELISA positive and CF test negative clearly precipitated multiple T. equi proteins. However,
immunoprecipitation results with serum samples that were C-ELISA negative and CF test positive were
inconclusive (26). This C-ELISA for T. equi was also recently validated in Morocco and Israel, giving a
concordance of 91% and 95.7% with the IFA test, respectively (39, 42). A similar C-ELISA has been
developed using the recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and an MAb reactive with a
peptide epitope of a 60 kDa B. caballi antigen (21). The results of 302 serum samples tested with this
CELISA
and the CF test showed a 73% concordance. Of the 72 samples that were CF test negative and CELISA
positive, 48 (67%) were shown to be positive on the IFA test, while four of the five samples that
tested CF test positive and C-ELISA negative were positive on the IFA test (21).
A test protocol for an equine piroplasmosis C-ELISA has been described and used for additional validation
studies (23, 51). The apparent specificity of the T. equi and B. caballi C-ELISAs lay between 99.2% and
99.5% using sera from 1000 horses presumed to be piroplasmosis-free. One thousand foreign-origin horses
of unknown infection status were tested by the C-ELISA and the CF test with an apparent greater sensitivity
of the C-ELISA. The results were 1.1% (T. equi) and 1.3% (B. caballi) more seropositive animals detected by
C-ELISA than by the CF test; the additional positive results were confirmed by IFA testing. A similar study of
645 foreign-origin horses tested for import and pre-import purposes used heat-treated sera (58°C for
30 minutes), and resulted in 3.6% (T. equi) and 2.1% (B. caballi) more seropositive animals detected by the
C-ELISA than by the CF test. Both C-ELISAs were highly reproducible well-to-well, plate-to-plate, and dayto-
day, with overall variances of ± 1.2% and ±1.6% for the T. equi and B. caballi tests, respectively.
The C-ELISA protocol is given below.
A detailed description of antigen production and a test protocol has been given by the National Veterinary
Services Laboratories of the United States Department of Agriculture (USDA) (51). A commercial kit is now
available that is based on the same antigens and monoclonal antibodies).
−Solutions
Antigen coating buffer: prepare the volume of antigen coating buffer required using the following amounts of
ingredients per litre: 2.93 g sodium bicarbonate; 1.59 g sodium carbonate; sufficient ultra-pure water to
dissolve, and make up to 1 litre with ultra-pure water. Adjust to pH 9.6.
C-ELISA wash solution (high salt diluent): prepare the volume of C-ELISA wash solution required by using
the following amounts of ingredients per litre: 29.5 g sodium chloride; 0.22 g monobasic sodium phosphate;
1.19 g dibasic sodium phosphate; 2.0 ml Tween 20; sufficient ultra-pure water to dissolve, and make up to
1 litre with ultra-pure water. Mix well. Adjust pH to 7.4. Sterilise by autoclaving at 121°C.
Frozen transformed E. coli culture is inoculated at a 1/10,000 dilution into any standard non-selective
bacterial growth broth (e.g. Luria broth) containing added carbenicillin (100 μg/ml) and
isopropylthiogalactoside
(IPTG, 1 mM). Cultures are incubated on an orbital shaker set at 200 rpm at 37°C overnight.
Cells grown overnight are harvested by centrifugation (5000 g for 10 minutes), washed in 50 mM Tris/HCl
and 5 mM ethylene diamine tetra-acetic acid (EDTA) buffer, pH 8.0, and harvested again as before. (Antigen
is available from the National Veterinary Services Laboratories, P.O. Box 844, Ames, Iowa 50010, USA.)
Cells are resuspended to 10% of the original volume in the Tris/EDTA buffer to which 1 mg/ml of lysozyme
has been added, and incubated on ice for 20 minutes. Nonidet P-40 detergent (NP-40) is then added to a
final 1% concentration (v/v), vortexed, and the mixture is incubated on ice for 10 minutes. The material is
next sonicated four times for 30 seconds each time at 100 watts, on ice, allowing 2 minutes between
sonications for the material to remain cool. The sonicate is centrifuged at 10,000 g for 20 minutes. The
resulting supernatant is dispensed in 0.5 ml aliquots in microcentrifuge tubes and may then be stored at
–70°C for several years. The presence of heterologous host bacterial antigens does not interfere with the
binding of specific equine anti-piroplasma antibodies or the binding of the paired MAbs to their respective
expressed recombinant antigen epitopes and is confirmed by the following procedures. The
antigencontaining
supernatants are quality controlled by titrating them with their paired MAbs and with reference
monospecific equine antisera to verify both an adequate level of expression and complete specificity for the
homologous species of piroplasmosis agent. Normal serum (negative serum) controls must not interfere with
binding of the MAbs or positive equine reference sera to the expressed antigen preparation.
i) Microtitration plates are prepared by coating the wells with 50 μl of either T. equi antigen or B. caballi
antigen diluted in antigen-coating buffer. The dilution used is determined by standard serological
titration techniques. The plate is sealed with sealing tape, stored overnight at 4°C, and frozen at
–70°C. Plates can be stored at –70 °C for up to 6 months.
ii) The primary anti-T. equi or anti-B. caballi MAb and secondary antibody-peroxidase conjugate is diluted
as directed by the manufacturer at the time of use in the C-ELISA, with antibody-diluting buffer
(supplied with the test kit).
iii) Plates are thawed at room temperature, the coating solution is decanted, and the plates are washed
twice with C-ELISA wash solution.
iv) The serum controls and test serum samples are diluted 1/2 with serum-diluting buffer before 50 μl of
sera is added to wells. Each unknown serum sample is tested in single or duplicate wells. Positive
control sera and blanks are tested in duplicate while negative controls are tested in triplicate on
different parts of the plate. Plates are incubated covered, at room temperature (21–25°C) for
30 minutes in a humid chamber, and then washed three times in C-ELISA wash solution.
v) All wells then receive 50 μl/well of diluted primary anti-T. equi or anti-B. caballi MAb. (The MAb is
produced in a cell culture bioreactor and is available from the National Veterinary Services
Laboratories, P.O. Box 844, Ames, Iowa 50010, USA.) Plates are incubated covered for 30 minutes at
room temperature (21–25°C) in a humid chamber, and then washed three times in C-ELISA wash
solution.
vi) Diluted secondary peroxidase anti-murine IgG (50 μl/well) conjugate is added to each well. Plates are
incubated covered for 30 minutes at room temperature (21–25°C) in a humid chamber, and then
washed three times in C-ELISA wash solution.
vii) Chromogenic enzyme substrate (50 μl/well) is added to all wells, and plates are incubated for
15 minutes at room temperature (21–25°C) during colour development.
viii) The colour development is stopped by adding 50 μl of stop solution to all wells and the plates are read
immediately on a plate reader.
ix) The plates are read at 620, 630 or 650 nm wavelength (OD). The average OD is calculated for the
duplicate wells for all control sera and blank wells. For a valid test, the mean of the negative controls
must produce an OD >0.300 and <2.000. The mean positive control sera must produce an inhibition of
≥40%.
x) Per cent inhibition [%I] is calculated as follows: %I = 100 – [(Sample OD × 100) ÷ (Mean negative
control OD)].
xi) If a test samples produces ≥40% inhibition it is considered positive. If the test sample produces <40%
inhibition it is considered negative.
c) Complement fixation
The CF test has been used in the past by some countries to qualify horses for importation (48). A detailed
description of antigen production and a test protocol has been given, for example by the USDA (7, 9, 50).
The CF test may not identify all infected animals, especially those that have been drug-treated or that
produce anti-complementary reactions, or because of the inability of IgG(T) (the major immunoglobulin
isotype of equids) to fix guinea-pig complement (31). Therefore the IFA test and C-ELISA have replaced the
CF as the prescribed tests for international trade.
An example of a CF test protocol is given below.
−Solutions
Alsever’s solution: prepare 1 litre of Alsever’s solution by dissolving 20.5 g glucose; 8.0 g sodium citrate;
4.2 g sodium chloride in sufficient distilled water. Adjust to pH 6.1 using citric acid, and make up the volume
to 1 litre with distilled water. Sterilise by filtration.
Stock veronal buffer (5×): dissolve the following in 1 litre of distilled water: 85.0 g sodium chloride; 3.75 g
sodium 5,5 diethyl barbituric; 1.68 g magnesium chloride (MgCl2.6H2O); 0.28 g calcium chloride. Dissolve
5.75 g of 5,5 diethyl barbituric acid in 0.5 litre hot (near boiling) distilled water. Cool this acid solution and
add to the salt solution. Make up to 2 litres with distilled water and store at 4°C. To prepare a working
dilution, add one part stock solution to four parts distilled water. The final pH should be from 7.4 to 7.6.
Blood is obtained from horses with a high parasitaemia (e.g. 3–7% parasitaemia for B. caballi and 60–85%
for T. equi), and mixed with equal volumes of Alsever’s solution as an anticoagulant. The plasma/Alsever’s
supernatant and buffy coat are removed when the RBCs have settled to the bottom of the flask. The RBCs
are washed several times with cold veronal buffer and then disrupted. The antigen is recovered from the
lysate by centrifugation at 30,900 g for 30 minutes.
The recovered antigen is washed several times in cold veronal buffer by centrifugation at 20,000 g for
15 minutes. Polyvinyl pyrrolidone 40,000 (1–5%[ w/v]) is added as a stabiliser and the preparation is mixed
on a magnetic stirrer for 30 minutes, strained through two thicknesses of sterile gauze, dispensed into 2 ml
volumes and freeze-dried. The antigen can then be stored at below –50°C for several years.
i) The specificity and potency of each batch of antigen should be checked against standard antisera of
known specificity and potency. Optimal antigen dilutions are also determined in a preliminary
checkerboard titration.
ii) Test sera are inactivated for 30 minutes at 58°C (donkey and mule sera are inactivated at 62.5°C for
35 minutes) and tested in dilutions of 1/5 to 1/5120. Veronal buffer is used for all dilutions.
iii) Complement is prepared and titrated spectrophotometrically to determine the 50% haemolytic dose
(C’H50) (45) and used in the test at five times C’H50. The haemolytic system consists of equal parts of a
2% sheep (RBC) suspension and veronal buffer with 5 minimum haemolytic doses (MHDs) of
haemolysin (amboceptor) (50). Some laboratories use twice the 100% haemolytic dose, which gives
equivalent sensitivity.
iv) The test has been adapted to microtitration plates (11). The total volume of the test is 0.125 ml, made
up of equal portions (0.025 ml) of antigen, complement (five times C’H50) and diluted serum. Incubation
is performed for 1 hour at 37°C.
v) A double portion (0.05 ml) of the haemolytic system is added and the plates are incubated for a further
45 minutes at 37°C with shaking after 20 minutes.
vi) The plates are centrifuged for 1 minute at 200 g before being read over a mirror.
vii) A lysis of 50% is recorded as positive, with the titre being the greatest serum dilution giving 50% lysis.
A titre of 1/5 is regarded as positive. A full set of controls (positive and negative sera) must be included
in each test as well as control antigen prepared from normal (uninfected) horse RBCs.
Anticomplementary samples are examined by the IFA test. Donkey sera are frequently anticomplementary.
BIOLOGICALS
No biological products are available currently.
REFERENCES
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IGARASHI I.
(2005). Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia
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12. HIRATA H., IKADAI H., YOKOYAMA N., XUAN X., FUJISAKI K., SUZUKI N., MIKAMI T. & IGARASHI I. (2002). Cloning
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338.
14. HOLBROOK A.A., JOHNSON A.J. & MADDEN B.S. (1968). Equine piroplasmosis: Intraerythrocytic
development of
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17. HUANG X, XUAN X, XU L, ZHANG S, YOKOYAMA N, SUZUKI N, & IGARASHI I. (2004). Development of an
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18. HUANG X., XUAN X., YOKOYAMA N., XU L., SUZUKI H., SUGIMOTO C., NAGASAWA H., FUJISAKI K. & IGARASHI I.
(2003). High-level expression and purification of a truncated merozoite antigen-2 of Babesia equi in
Escherichia coli and its potential for immunodiagnosis. J. Clin. Microbiol., 41, 1147–1151.
19. IKADAI H., OSORIO C.R., XUAN X., IGARASHI I., KANEMARU T., NAGASAWA H., FUJISAKI K., SUZUKI N. & MIKAMI
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20. IKADAI H., XUAN X., IGARASHI I., TANAKA S., KANEMARU T., NAGASAWA H., FUJISAKI K., SUZUKI N. & MIKAMI T.
(1999). Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential
use of the recombinant antigen in an enzyme-linked immunosorbent assay. J. Clin. Microbiol., 37, 3475–
3480.
21. KAPPMEYER L.S., PERRYMAN L.E., HINES S.A., BASZLER T.V., KATZ J.B., HENNAGER S.G. & KNOWLES D.P.
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competitive-inhibition enzyme-linked immunosorbent assay. J. Clin. Microbiol., 37, 2285–2290.
22. KAPPMEYER L.S., PERRYMAN L.E. & KNOWLES D.P (1993). A Babesia equi gene encodes a surface protein
with
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23. KATZ J., DEWALD R. & NICHOLSON J. (2000). Procedurally similar competitive immunoassay systems for
the
serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum and Burkholderia mallei infection
in horses. J. Vet. Diagn. Invest., 12, 46–50.
24. KNOWLES D.P. KAPPMEYER, L.S. & PERRYMAN L.E. (1997). Genetic and biochemical analysis of
erythrocytestage
surface antigens belonging to a family of highly conserved proteins of Babesia equi and Theileria
species. Mol. Biochem. Parasitol., 90, 69–79.
25. KNOWLES D.P., KAPPMEYER, L.S., STILLER D., HENNAGER S.G. & PERRYMAN L.E. (1992). Antibody to a
recombinant merozoite protein epitope identifies horses infected with Babesia equi. J. Clin. Microbiol., 30,
3122–3126.
26. KNOWLES D.P., PERRYMAN L.E. & KAPPMEYER L.S. (1991). Detection of equine antibody to Babesia equi
merozoite proteins by a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent
assay. J. Clin. Microbiol., 29, 2056–2058.
27. LEVINE N.D. (1985). Veterinary protozoology. Iowa State University Press, Ames, Iowa, USA.
28. LITTLEJOHN A. (1963). Babesiosis. In: Equine Medicine and Surgery, Bone J.F., Catcott E.J., Gabel A.A.,
Johnson L.E. & Riley W.F., eds. American Veterinary Publications, California, USA, 211–220.
29. MADDEN P.A. & HOLBROOK A.A. (1968). Equine piroplasmosis: Indirect fluorescent antibody test for
Babesia
caballi. Am. J. Vet. Res., 29, 117–123.
30. MAHONEY D.F. & SAAL J.R. (1961). Bovine babesiosis: Thick blood films for the detection of parasitaemia.
Aust. Vet. J., 37, 44–47.
31. MCGUIRE T.C., VAN HOOSIER G.L. JR & HENSON J.B. (1971). The complement-fixation reaction in equine
infectious anemia: demonstration of inhibition by IgG (T). J. Immunol., 107, 1738–1744.
32. MEHLHORN H. & SCHEIN E. (1984). The piroplasms: Life cycle and sexual stages. Adv. Parasitol., 23, 37–
103.
33. MELHORN H. & SCHEIN E. (1998). Redescription of Babesia equi Laveran, 1901 as Theileria equi.
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34. MORZARIA S.P., BROCKLESBY D.W. & HARRADINE D.L. (1977). Evaluation of the indirect fluorescent
antibody
test for Babesia major and Theileria mutans in Britain. Vet. Rec., 100, 484–487.
35. NAGORE D., GARCIA-SANMARTIN J., GARCIA-PEREZ A.L., JUSTE R.A. & HURTADO A. (2004). Detection and
identification of equine Theileria and Babesia species by reverse line blotting: epidemiological survey and
phylogenetic analysis. Vet. Parasitol., 123, 41–54.
36. NEITZ W.O. (1956). Classification, transmission and biology of piroplasms of domestic animals. Ann. N.Y.
Acad. Sci., 64, 56–111.
37. POTGIETER F.T. & ELS H.J. (1977). The fine structure of intra-erythrocytic stages of Babesia bigemina.
Onderstepoort J. Vet. Res., 44, 157–168.
38. RAMPERSAD J., CESAR E., CAMPBELL M.D., SAMLAL M., & AMMONS, D. (2003). A field evaluation of PCR for
the
routine detection of Babesia equi in horses. Vet. Parasitol., 114, 81–87.
39. RHALEM A., SAHIBI H., LASRI S., JOHNSON W.C., KAPPMEYER L.S., HAMIDOUCH A., KNOWLES D.P. & GOFF
W.L.
(2001). Validation of a competitive enzyme-linked immunosorbent assay for diagnosing Babesia equi
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Diagn. Invest., 13, 249–251.
40. SAHAGUN-RUIZ A., WAGNELA S.D., HOLMAN P.J., CHIEVES L.P. & WAGNER G.G. (1997). Biotin-labeled DNA
probe in a PCR-based assay increases detection sensitivity for the equine hemoparasite Babesia caballi.
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41. SCHEIN E., REHBEIN G., VOIGT W.P. & ZWEYGARTH E. (1981). Babesia equi (Leveran, 1901). Development
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42. SHKAP V., COHEN I., LEIBOVITZ B., SAVITSKY, PIPANO E., AVNI G., SHOFER S., GIGER U., KAPPMEYER L. &
KNOWLES
D. (1998). Seroprevalence of Babesia equi among horses in Israel using competitive inhibition ELISA and
IFA assays. Vet. Parasitol., 76, 251–259.
43. SHUTE P.G. (1966). The staining of malaria parasites. Trans. R. Soc. Trop. Med. Hyg., 60, 412–416.
44. STILLER D. & FRERICH S.W.M. (1979). Experimental transmission of Babesia caballi to equids by different
stages of the tropical horse tick, Anocentor nitens. Recent Adv. Acarol., 2, 262–268.
45. STILLER D., FRERICHS W.M., LEATCH G. & KUTTLER K.L. (1980). Transmission of equine babesiosis and
bovine
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46. TAMAKI Y., HIRATA H., TAKABATAKE N., BORK S., YOKOYAMA N., XUAN X., FUJISAKI K. & IGARASHI I. (2004).
Molecular cloning of a Babesia caballi gene encoding the 134-kilodalton protein and evaluation of its
diagnostic potential in an enzyme-linked immunosorbent assay. Clin. Diagn. Lab. Immunol., 11, 211–215.
47. TANAKA T., XUAN X., IKADAI H., IGARASHI I., NAGASAWA H., FUJISAKI K., MIKAMI T. & SUZUKI N. (1999).
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50. UNITED STATES DEPARTMENT OF AGRICULTURE (USDA) ANIMAL AND PLANT HEALTH INSPECTION SERVICE,
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51. UNITED STATES DEPARTMENT OF AGRICULTURE (USDA) ANIMAL AND PLANT HEALTH INSPECTION SERVICE,
VETERINARY SERVICES (2005). Competitive ELISA for Serodiagnosis of Equine Piroplasmosis (Babesia equi
and Babesia caballi), USDA, National Veterinary Services Laboratories, Ames, Iowa, USA.
52. XUAN X., LARSEN A., IDADAI H., TNANKA T., IGARASHI I., NAGASAWA H., FUJISAKI K., TOYODA Y., SUZUKI N. &
MIKAMI T. (2001). Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus
and evaluation of its diagnostic potential in an enzyme-linked immunosorbant assay. J. Clin. Microbiol., 39,
705–709.
53. ZWEYGARTH E., JUST M.C. & DE WAAL D.T. (1995). Continuous in vitro cultivation of erythrocytic stages of
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54. ZWEYGARTH E., JUST M.C. & DE WAAL D.T. (1997). In vitro cultivation of Babesia equi: detection of carrier
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*
**
NB: There is an OIE Reference Laboratories for Equine piroplasmosis (see Table in Part 3 of this Terrestrial
Manual or consult the OIE Web site for the most up-to-date list: www.oie.int).
C H A P T E R 2 . 5 . 4 .
EPIZOOTIC LYMPHANGITIS
SUMMARY
Epizootic lymphangitis is a contagious, chronic disease of horses and other Equidae characterised
clinically by a spreading, suppurative, ulcerating pyogranulomatous dermatitis and lymphangitis.
This is seen particularly in the neck, legs and chest. It can also present as an ulcerating
conjunctivitis, or rarely a multifocal pneumonia. Transmission is by contact of infected material with
traumatised skin, by biting flies, ticks or inhalation. The causative agent, Histoplasma capsulatum
var. farciminosum, is a thermally dimorphic, fungal soil saprophyte. Differential diagnoses include
glanders (farcy), caused by Burkholderia mallei, ulcerative lymphangitis due to Corynebacterium
pseudotuberculosis, sporotrichosis caused by Sporothrix schenckii, and the skin lesions of
histoplasmosis caused by H. capsulatum var. capsulatum. Amphotericin B injection with local
wound drainage and inorganic iodides are used to treat early cases.
Identification of the agent: Identification of the agent is made by its appearance in smears of the
exudate or in histological sections of the lesion material. The yeast form of the organism is present
in large numbers in well established lesions, and appears as pleomorphic ovoid to globose
structures, approximately 2–5 μm in diameter, located both extracellularly and intracellularly in
macrophages and giant cells. Organisms are usually surrounded by a ‘halo’ when stained with
Gram stain, haematoxylin and eosin, Periodic acid–Schiff reaction or Gomori methenamine–silver
stain. The mycelial form of the organism grows slowly under aerobic conditions at 25–30°C on a
variety of media, including Mycobiotic agar, enriched Sabouraud’s dextrose agar, brain–heart
infusion agar, and pleuropneumonia-like organism nutrient agar. Conversion to the yeast phase at
37°C must be demonstrated.
Serological and other tests: Antibodies to H. capsulatum var. farciminosum develop at or before
the onset of clinical signs. Assays reported for detection of antibody include fluorescent antibody,
enzyme-linked immunosorbent assay, and passive haemagglutination tests. In addition, a skin
hypersensitivity test has been described.
Requirements for vaccines and diagnostic biologicals: Killed and live vaccines have been used
on a limited scale in endemic areas, but they are not readily available.
A. INTRODUCTION
Epizootic lymphangitis is a contagious, chronic disease of horses, mules and donkeys. The disease is
characterised clinically by a suppurative, ulcerating, and spreading pyogranulomatous, multifocal dermatitis
and
lymphangitis. It is seen most commonly in the extremities, chest wall and the neck, but it can also be present
as
an ulcerating conjunctivitis of the palpebral conjunctiva, or rarely as a multifocal pneumonia. The organism
may
also invade open lesions including ruptured strangles abscesses and castration wounds. It has also been
called
pseudofarcy or pseudoglanders. Another synonym is equine histoplasmosis, which may be a more accurate
name
for the disease, as not all clinical cases present obvious lymphangitis. The form that the disease takes
seems to
depend primarily on the route of entry (17). The traumatised skin is either infected directly by infected pus,
nasal
or ocular excretions or indirectly by soil or contaminated harnesses, grooming equipment, feeding and
watering
utensils, wound dressings or flies. It is also believed that ticks may play a role in the transmission of this
agent (4).
The conjunctival form of the disease is believed to be spread by flies of the Musca or Stomoxys genera (17).
The
pulmonary form of the disease is infrequent and is presumed to occur after inhalation of the organism. The
incubation period is from about 3 weeks to 2 months (3). In all cases, the lesions are nodular and
granulomatous
in character, and the organism, once established, spreads locally by invasion and then via the lymphatics.
There
is often thickening, or ‘cording’, of lymphatics, with the formation of pyogranulomatous nodules. Regional
lymph
nodes may by enlarged and inflamed. Lesions usually heal spontaneously after 2–3 months, resulting in
stellate
scar formation. However, extensive lesions with high mortality rates can occur in areas where there is poor
veterinary care and nutrition (3).
Chapter 2.5.4. - Epizootic lymphangitis
The causative agent, Histoplasma capsulatum var. farciminosum, is a thermally dimorphic fungus. The
mycelial
form is present in soil; the yeast form is usually found in lesions. Histoplasma farciminosum was formerly
described as an independent species, but this assessment has been changed and it is now considered to be
a
variety of H. capsulatum due to the close morphological similarities of both the mycelial and yeast forms
(19).
Antigenically, H. capsulatum var. farciminosum and H. capsulatum var. capsulatum are indistinguishable,
however
the latter is the cause of disseminated histoplasmosis, is endemic in North America and has a wide host
range
(16). DNA sequences of four protein-coding genes have been analysed to elucidate the evolutionary
relationships
of H. capsulatum varieties. This indicated that H. capsulatum var. farciminosum is deeply buried in the
branch of
SAm Hcc group A, (H60 to -64, -67, -71, -74 and -76), looking as if it were an isolate of South American
H. capsulatum var. capsulatum (14).
The cutaneous form of the disease may be confused with farcy (the skin form of glanders), which is caused
by
Burkholderia mallei, ulcerative lymphangitis, which is caused by Corynebacterium pseudotuberculosis,
indolent
ulcers acaused by Rhodococcus equi, sporotrichosis caused by Sporothrix schenckii, and histoplasmosis
caused
by H. capsulatum var. capsulatum, cryptococcosis, strangles. sarcoids and cutaneous lymphosarcomas (13,
15).
The disease is more common in the tropics and subtropics and is endemic in north, east and north-east
Africa,
and some parts of Asia, including some countries bordering the Mediterranean Sea, India, Pakistan and
Japan.
The disease is common in Ethiopia, especially in cart horses, affecting an average of 18.8% of horses in
warm,
humid areas between 1500 and 2300 metres above sea level (3, 4). Reports from other parts of the world
are
sporadic and all cases must be verified by laboratory testing. The prevalence of the disease increases with
assembling of animals; it was much more common, historically, when large numbers of horses were stabled
together for cavalry and other transportation needs. Mainly, it is horses, mules, and donkeys that are
affected by
the disease, although infection may occur in camels, cattle and dogs (19). Experimentally, other animals are
refractory to infection subsequent to inoculation, with the exception of certain laboratory animal species such
as
mice, guinea-pigs and rabbits (12, 17). Infection in humans has also been reported (2, 6, 11).
The disease is eradicated by the humane slaughter of infected horses, disinfection of infected premises and
restricting the movement of equids from infected premises. In endemic areas where eradication is not
possible,
inorganic iodides can be used for therapy in early cases (1). Localised nodules can also be lanced, the pus
drained and the nodules packed with a 7% tincture of iodine. If affordable, Amphotericin B can be used.
As the clinical signs of epizootic lymphangitis can be confused with those of other diseases in the field,
definitive
diagnosis rests on laboratory confirmation.
Material should be collected directly from unruptured nodules. For microbiological isolation, the material
should be
placed in a liquid nutrient medium with antibacterials and kept refrigerated until culturing, which should be
attempted as soon as possible. For direct examination, swabs of lesion material can be smeared on glass
slides
and fixed immediately. For histopathology, sections of lesion material, including both viable and nonviable
tissue,
should be placed in 10% neutral buffered formalin. Confirmation of the disease is dependent on the
demonstration
of H. capsulatum var. farciminosum.
a) Direct microscopic examination
o Gram-stained smears
Smears can be stained directly with Gram’s stain and examined for the typical yeast form of the organism,
which will appear as Gram-positive, pleomorphic, ovoid to globose structures, approximately 2–5 μm in
diameter (2). They may occur singly or in groups, and may be found either extracellularly or within
macrophages. A halo around the organisms (unstained capsule) is frequently observed.
o Histopathology
In haematoxylin and eosin (H&E)-stained histological sections, the appearance of the lesion is quite
characteristic and consists of pyogranulomatous inflammation with fibroplasia. Langhans giant cells are
common. The presence of numerous organisms, both extracellularly and intracellularly within macrophages
or multinucleated giant cells in tissue sections stained with H&E, Periodic acid–Schiff reaction and Gomori
methenamine–silver stain are observed (16). There is some indication that the number of organisms
increases with chronicity. The organisms are pleomorphic, often described as slightly lemon-shaped
basophilic masses, varying from 2 to 5 μm in diameter, that are surrounded by a ‘halo’ when stained with
H&E or Gram’s stain (1).
o Electron microscopy
Electron microscopy has been applied to skin biopsy samples of 1.5–2.0 mm immediately prefixed in
phosphate buffered 2% glutaraldehyde solution at 4°C and post-fixed in 1% osmium tetroxide. Ultra-thin
sections were cut and stained with uranyl acetate and lead citrate. Examination demonstrated the fine
internal structure of the organism, H. capsulatum var. farciminosum, including the cell envelope, plasma
membrane, cell wall, capsule and inner cell structures (1).
b) Culture
The mycelial form of H. capsulatum var. farciminosum grows slowly on laboratory media (2–8 weeks at
26°C). Media that can be used include Mycobiotic agar (2), Sabouraud’s dextrose agar agar enriched with
2.5% glycerol, brain–heart infusion agar supplemented with 10% horse blood, and pleuropneumonia-like
organism (PPLO) nutrient agar enriched with 2% dextrose and 2.5% glycerol, pH 7.8 (11, 16). The addition
of antibiotics to the media is recommended: cycloheximide (0.5 g/litre) and chloramphenicol (0.5 g/litre).
Broad-spectrum antibacterial activity is obtained if gentamicin (50 mg/litre) and penicillin G (6 ×
106 units/litre) are used instead of chloramphenicol. Colonies appear in 2–8 weeks as dry, grey-white,
granular, wrinkled mycelia. The colonies become brown with aging. Aerial forms occur, but are rare. The
mycelial form produces a variety of conidia, including chlamydospores, arthroconidia and some
blastoconidia. However, the large round double-walled macroconidia that are often observed in
H. capsulatum var. capsulatum are lacking.
As a confirmatory test the yeast form of H. capsulatum var. farciminosum can be induced by subculturing
some of the mycelium into brain–heart infusion agar containing 5% horse blood or by using Pine’s medium
alone at 35–37°C in 5 % CO2. Yeast colonies are flat, raised, wrinkled, white to greyish brown, and pasty in
consistency (16). However, complete conversion to the yeast phase may only be achieved after four to five
repeated serial transfers on to fresh media every 8 days.
c) Animal inoculation
Experimental transmission of H. capsulatum var. farciminosum has been attempted in mice, guinea-pigs and
rabbits. Immunosuppressed mice are highly susceptible to experimental infection and can be used for
diagnostic purposes (1).
There are published reports of various tests to detect antibodies as well as a skin hypersensitivity test for
detection of cell-mediated immunity. Antibodies usually develop at or just after the onset of clinical signs.
a) Fluorescent antibody tests
o Indirect fluorescent antibody test
The following non-quantitative procedure is as described by Fawi (7).
i) Slides containing the organisms are made by smearing the lesion contents on to a glass slide or by
emulsifying the cultured yeast phase of the organism in a saline solution and creating a thin film on a
glass slide.
ii) The slides are heat-fixed by passing the slide through a flame.
iii) The slides are then washed in phosphate buffered saline (PBS) for 1 minute.
iv) Undiluted test sera are placed on the slides, which are then incubated for 30 minutes at 37°C.
v) The slides are washed in PBS three times for 10 minutes each.
vi) Fluorescein isothiocyanate (FITC)-conjugated anti-horse antibody at an appropriate dilution is flooded
over the slides, which are then incubated for 30 minutes at 37°C.
vii) Washing in PBS is repeated three times for 10 minutes each.
viii) The slides are examined using fluorescence microscopy.
o Direct fluorescent antibody test
The following procedure is as described by Gabal et al. (8).
i) The globulin fraction of the test serum is precipitated, and then re-suspended to its original serum
volume in saline. The serum is then conjugated to FITC.
ii) Small colony particles of the cultured mycelial form of the organism are suspended in 1–2 drops of
saline on a glass slide. With a second slide, the colony particles are crushed and the solution is
dragged across the slide to create a thin film.
iii) The smears are heat-fixed.
iv) The slides are washed in PBS for 1 minute.
v) The slides are incubated with dilutions of conjugated serum for 60 minutes at 37°C.
vi) The slides are washed in PBS three times for 5 minutes each.
vii) The slides are examined using fluorescence microscopy.
b) Indirect Enzyme-linked immunosorbent assay
The following procedure is as described by Gabal & Mohammed (10).
i) The mycelial form of the organism is produced on Sabouraud’s dextrose agar in tubes, and incubated
for 4 weeks at 26°C. Three colonies are ground in 50 ml of sterile PBS. The suspension is diluted 1/100
and the 96-well microtitre plates are coated with 100 μl/well.
ii) The plates are incubated at 4°C overnight.
iii) The plates are washed with PBS containing Tween 20 (0.5 ml/litre) (PBS-T) three times for 3 minutes
each.
iv) The plates are incubated with 5% bovine serum albumin, 100 μl/well, at 23–25°C for 30 minutes, with
shaking.
v) The plates are washed with PBS-T three times for 3 minutes each.
vi) The sera are serially diluted using twofold dilution in duplicate in PBS-T, starting with a 1/50 dilution and
incubated for 30 minutes at 23–25°C.
vii) The plates are washed with PBS-T three times for 3 minutes each.
viii) Peroxidase-labelled goat anti-horse IgG is diluted 1/800 and used at 100 μl/well, with incubation for
30 minutes at 23–25°C, with shaking.
ix) The plates are washed with PBS-T three times for 3 minutes each.
x) Finally, 100 μl/well of hydrogen peroxide and ABTS (2,2’-Azino-di-[3-ethyl-benzthiazoline]-6-sulphonic
acid) in a citric acid buffer, pH 4, is added.
xi) The plates are read at 60 minutes in a spectrophotometer at wavelength 405 nm.
xii) The absorbance values are obtained twice from each serum dilution and the standard deviation and
average percentage of the absorbance values of the different serum samples are considered in the
interpretation of the results.
c) Passive haemagglutination test
The following procedure is as described by Gabal & Khalifa (9).
i) The organism is propagated for 8 weeks on Sabouraud’s dextrose agar. Five colonies are scraped,
ground, suspended in 200 ml of saline, and sonicated for 20 minutes. The remaining mycelial elements
are filtered out, and the filtrate is diluted 1/160.
ii) Normal sheep red blood cells (RBCs) are washed, treated with tannic acid, washed, and re-suspended
as a 1% cell suspension.
iii) Different dilutions of the antigen preparation are mixed with the tanned RBCs and incubated in a water
bath at 37°C for 1 hour. The RBCs are collected by centrifugation, washed three times in buffered
saline and re-suspended to make a 1% cell suspension.
iv) Test sera are inactivated by heating at 56°C for 30 minutes and then absorbed with an equal volume of
washed RBCs.
v) Dilutions of serum (0.5 ml) are placed in test tubes with 0.05 ml of antigen-coated tanned RBCs.
vi) Agglutination is recorded at 2 and 12 hours.
vii) Agglutination is detected when the RBCs form a uniform mat on the bottom of the tube. A negative test
is indicated by the formation of a ‘button’ of RBCs at the bottom of the tube.
d) Skin hypersensitivity tests
Two skin hypersensitivity tests for the diagnosis of epizootic lymphangitis have been described. The first test
was described by Gabal & Khalifa and adapted by Armeni et al. (5, 9).
i) A pure culture of H. farcimonsum is propagated for 8 weeks on Sabouraud’s dextrose agar containing
2.5% glycerol. Five colonies are scraped, ground, suspended in 200 ml of saline, undergo five freeze–
thaw cycles and are sonicated at an amplitude of 40° for 20 minutes. The remaining mycelial elements
are removed by centrifugation at 1006 g at 4°C for 11 minutes. Sterility of the preparation is verified by
incubating an aliquot on Sabouraud’s dextrose agar at 26°C for 4 weeks.
ii) Animals are inoculated intradermally with 0.1 ml containing 0.2 mg/ml protein in the neck.
iii) The inoculation site is examined for the presence of a local indurated and elevated area at 24–48 hours
post-injection. An increase in skin thickness of > 4 mm is considered to be positive.
Alternatively, a ‘histofarcin’ test has been described by Soliman et al. (18).
i) The mycelial form of the organism is grown on polystyrene discs floating on 250 ml of PPLO media
containing 2% glucose and 2.5% glycerine at 23–25°C for 4 months.
ii) The fungus-free culture filtrate is mixed with acetone (2/1) and held at 4°C for 48 hours.
iii) The supernatant is decanted and the acetone is allowed to evaporate.
iv) Precipitate is suspended to 1/10 original volume in distilled water.
v) Animals are inoculated intradermally with 0.1 ml of antigen in the neck.
vi) The inoculation site is examined for the presence of a local indurated and elevated area at 24, 48 and
72 hours post-injection.
BIOLOGICALS
Control of the disease is usually through elimination of the infection. This is achieved by culling infected
horses
and application of strict hygiene practices to prevent spread of the organism. There are published reports on
the
use of killed (2) and live attenuated vaccines (20) in areas where epizootic lymphangitis is endemic,
apparently
with relatively good results.
The antigens used for skin hypersensitivity testing are described in the previous section.
REFERENCES
1. AL-ANI F.K. (1999). Epizootic lymphangitis in horses: a review of the literature. Rev. sci. tech. Off. int.
Epiz.,
18, 691–699.
2. AL-ANI F.K., ALI A.H. & BANNA H.B. (1998). Histoplasma farciminosum infection of horses in Iraq.
Veterinarski
Arhiv., 68, 101–107.
3. AMENI G. (2006). Preliminary trial on the reproducibility of epizootic lymphangitis through experimental
infection of two horses. Short Communication. Veterinary J., 172 (3), 553–555.
4. AMENI G. & TEREFE W. (2004). A cross-sectional study of epizootic lymphangitis in cart-mules in western
Ethiopia. Preventive Vet. Med., 66, 93–99.
5. AMENI G. TEREFE W. & HAILU A. (2006). Histofarcin test for the diagnosis of epizootic lymphanigitis in
Ethiopia:
development, optimisation and validation in the field. Veterinary J., 171, 358–362.
6. CHANDLER F.W., KAPLAN W. & AJELLO L. (1980). Histopathology of Mycotic Diseases. Year Book Medical
Publishers, Chicago, USA, 70–72 and 216–217.
7. FAWI M.T. (1969). Fluorescent antibody test for the serodiagnosis of Histoplasma farciminosum infections
in
Equidae. Br. Vet. J., 125, 231–234.
8. GABAL M.A., BANA A.A. & GENDI M.E. (1983). The fluorescent antibody technique for diagnosis of equine
histoplasmosis (epizootic lymphangitis). Zentralbl. Veterinarmed. [B], 30, 283–287.
9. GABAL M.A. & KHALIFA K. (1983). Study on the immune response and serological diagnosis of equine
histoplasmosis (epizootic lymphangitis). Zentralbl. Veterinarmed. [B], 30, 317–321.
10. GABAL M.A. & MOHAMMED K.A. (1985). Use of enzyme-linked immunosorbent assay for the diagnosis of
equine Histoplasma farciminosi (epizootic lymphangitis). Mycopathologia, 91, 35–37.
11. GUERIN C., ABEBE S. & TOUATI F. (1992). Epizootic lymphangitis in horses in Ethiopia. J. Mycol. Med., 2,
1–5.
12. HERVE V., LE GALL-CAMPODONICO P., BLANC F., IMPROVISI, L., DUPONT, B, MATHIOT C. & LE GALL F. (1994).
Histoplasmose a Histoplasma farciminosum chez un cheval africain. J. Mycologie Med., 4, 54.
13. JUNGERMAN P.F. & SCHWARTZMAN R.M. (1972). Veterinary Medical Mycology. Lea & Febiger. Philadelphia,
USA.
14. KASUGA T., TAYLOR T.W. & WHITE T.J. (1999). Phylogenetic relationships of varieties and geographical
groups
of the human pathogenic fungus Histoplasma capsulatum darling. J. Clin. Microbiol., 37, 653–663.
15. LEHMANN P.F. HOWARD D.H. & MILLER J.D. (1996). Veterinary Mycology. Springer-Verlag, Berlin, Germany,
96, 251–263.
16. ROBINSON W.F. & MAXIE M.G. (1993). The cardiovascular system. In: Pathology of Domestic Animals, Vol.
3.
Academic Press, New York, USA, 82–84.
17. SINGH T. (1965). Studies on epizootic lymphangitis. I. Modes of infection and transmission of equine
histoplasmosis (epizootic lymphangitis). Indian J. Vet. Sci., 35, 102–110.
18. SOLIMAN R., SAAD M.A. & REFAI M. (1985). Studies on histoplasmosis farciminosii (epizootic lymphangitis)
in
Egypt. III. Application of a skin test (‘histofarcin’) in the diagnosis of epizootic lymphangitis in horses.
Mykosen, 28, 457–461.
19. UEDA Y., SANO A. TAMURA M., INOMATA T., KAMEI K., YOKOYAMA K., KISHI F., ITO J., Y., MIYAJI M. & NISHIMURA
K.
(2003). Diagnosis of histoplasmosis by detection of the internal transcribed spacer region of fungal rRNA
gene from a paraffin-embedded skin sample from a dog in Japan. Vet. Microbiol., 94, 219–224.
20. ZHANG W.T., WANG Z.R., LIU Y.P., ZHANG D.L., LIANG P.Q., FANG Y.Z., HUANG Y.J. & GAO S.D. (1986).
Attenuated vaccine against epizootic lymphangitis in horses. Chinese J. Vet. Sci. Tech., 7, 3–5.
*
**
858 OIE Manual Terrestrial 2008
C H A P T E R 2 . 5 . 5 .
EQUINE ENCEPHALOMYELITIS
(Eastern and Western)
SUMMARY
Eastern and Western equine encephalomyelitis viruses belong to the genus Alphavirus of the family
Togaviridae. Alternate infection of birds and mosquitoes maintain these viruses in nature. The
disease occurs sporadically in horses and humans from mid-summer to late autumn. Horses and
humans are tangential dead-end hosts. The disease in horses is characterised by fever, anorexia,
and severe depression. In severe cases, the disease in horses progresses to hyperexcitability,
blindness, ataxia, severe mental depression, recumbency, convulsions, and death. Eastern equine
encephalomyelitis (EEE) virus infection in horses is often fatal, while Western equine
encephalomyelitis (WEE) virus can cause a subclinical or mild disease with less than 30% mortality.
EEE and WEE have been reported to cause disease in poultry, game birds and ratites. Sporadic
cases of EEE have been reported in cows, sheep, pigs, deer, and dogs.
Identification of the agent: A presumptive diagnosis of EEE or WEE can be made when
susceptible horses display the characteristic somnolence and other signs of neurological disease in
areas where haematophagous insects are active. There are no characteristic gross lesions.
Histopathological lesions can provide a presumptive diagnosis. EEE virus can usually be isolated
from the brain and sometimes other tissues of dead horses, however WEE virus is rarely isolated.
EEE and WEE viruses can be isolated from field specimens by inoculating newborn mice,
embryonating chicken eggs, cell cultures, or newly hatched chickens. The virus is identified by
complement fixation (CF), immunofluorescence, or plaque reduction neutralisation (PRN) tests.
EEE and WEE viral RNA may also be detected by reverse-transcription polymerase chain reaction
methods.
Serological tests: Antibody can be identified by PRN, haemagglutination inhibition (HI), CF tests,
or IgM capture enzyme-linked immunosorbent assay.
Requirements for vaccines and diagnostic biologicals: EEE and WEE vaccines are safe and
immunogenic. They are produced in cell culture and inactivated with formalin.
A. INTRODUCTION
Eastern equine encephalomyelitis (EEE) and Western equine encephalomyelitis (WEE) viruses are
members of
the genus Alphavirus of the family Togaviridae. The natural ecology for virus maintenance occurs via
alternate
infection of birds and ornithophilic mosquitoes. Clinical disease may be observed in humans and horses,
both of
which are dead-end hosts for these agents. EEE has been diagnosed in Quebec and Ontario in Canada,
central
and eastern regions of the United States of America (USA), the Caribbean Islands, Mexico, and Central and
South America. Disease caused by the WEE virus has been reported in the western USA and Canada,
Mexico,
and Central and South America (13, 16, 24). Highlands J virus, antigenically related to WEE virus, has been
isolated in eastern USA. Although Highlands J virus is generally believed not to cause disease in mammals,
it has
been isolated from the brain of a horse dying of encephalitis in Florida (7).
Even though the mortality is lower for WEE, the clinical signs of EEE and WEE can be identical. The disease
caused by either virus is also known as sleeping sickness. Following an incubation period of 5–14 days,
clinical
signs include fever, anorexia, and depression. A presumptive diagnosis of EEE or WEE virus infection in
unvaccinated horses can be made if the characteristic somnolence is observed during the summer in
temperate
climates or the wet season in tropical and subtropical climates, when the mosquito vector is plentiful.
However, a
number of other diseases, such as West Nile virus and Venezuelan equine encephalomyelitis (chapters
2.1.20
and 2.5.14, respectively), produce similar clinical signs and the diagnosis must be confirmed by the
described
diagnostic test methods. WEE virus infection in horses is often observed over a wide geographical area, e.g.
sporadic cases over 1000 square miles. EEE virus infections are usually observed in limited geographical
areas.
Chapter 2.5.5. - Equine encephalomyelitis (Eastern and Western)
Isolated events of high mortality in captive-raised game birds, primarily pheasants, chukars, aquarium
penguins,
and quail have been traced to WEE, EEE, or Highlands J virus infection (13, 16, 19). Most encephalomyelitis
infections in domestic fowl are caused by EEE virus and occur on the east coast states of the USA. The
virus is
introduced by mosquitoes, but transmission within the flocks is primarily by feather picking and cannibalism.
Both
EEE and WEE viruses have caused a fatal disease in ratites. Haemorrhagic enteritis has been observed in
emus
infected with EEE and WEE viruses, and morbidity and mortality rates may be greater than 85%. Highlands
J and
EEE viruses have been found to produce depression, somnolence, decreased egg production, and
increased
mortality in turkeys (5). EEE virus has been reported to cause disease in cows (11, 14), sheep (1), pigs (3),
whitetailed
deer (18), and dogs (4).
EEE virus causes severe disease in humans with a mortality rate of 30–70% and a high frequency of
permanent
sequelae in patients who survive. WEE is usually mild in adult humans, but can be a severe disease in
children.
The fatality rate is between 3 and 14%. Severe infection and death caused by EEE and WEE viruses have
been
reported in laboratory workers; therefore, any work with these viruses must be performed at containment
level 3
(see Chapter 1.1.2 Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities).
It is
recommended that personnel be immunised against EEE and WEE viruses (22). Precautions should also be
taken to prevent human infection when performing post-mortem examinations on horses suspected of being
infected with the equine encephalomyelitis viruses.
The most definitive method for diagnosis of EEE or WEE is the isolation of the viruses. EEE virus can
usually be
isolated from the brains of horses, unless more than 5 days have elapsed between the appearance of
clinical
signs and the death of the horse. EEE virus can frequently be isolated from brain tissue even in the
presence of a
high serum antibody titre. WEE virus is rarely isolated from tissues of infected horses. Brain is the tissue of
choice
for virus isolation, but the virus has been isolated from other tissues, such as the liver and spleen. It is
recommended that a complete set of these tissues be collected in duplicate, one set for virus isolation and
the
other set in formalin for histopathological examination. Specimens for virus isolation should be sent
refrigerated if
they can be received in the laboratory within 48 hours of collection; otherwise, they should be frozen and
sent with
dry ice. A complete set of tissues will allow the performance of diagnostic techniques for other diseases. For
isolation, a 10% suspension of tissue is prepared in phosphate buffered saline (PBS), pH 7.8, containing
bovine
serum albumin (BSA) (fraction V; 0.75%), penicillin (100 units/ml), and streptomycin (100 μg/ml). The
suspension
is clarified by centrifugation at 1500 g for 30 minutes.
EEE and WEE viruses can be isolated in a number of cell culture systems. The most commonly used cell
cultures
are primary chicken or duck embryo fibroblasts, continuous cell lines of African green monkey kidney (Vero),
rabbit kidney (RK-13), or baby hamster kidney (BHK-21). Isolation is usually attempted in 25 cm2 cell culture
flasks. Confluent cells are inoculated with 1.0 ml of tissue suspension. Following a 1–2-hour absorption
period,
maintenance medium is added. Cultures are incubated for 6–8 days, and one blind passage is made. EEE
and
WEE viruses will produce a cytopathic change in cell culture. Cultures that appear to be infected are frozen.
The
fluid from the thawed cultures is used for virus identification.
The newborn mouse is also considered to be a sensitive host system. Inoculate intracranially one or two
litters of
1–4-day-old mice with 0.02 ml of inoculum using a 26-gauge 3/8 inch (9.3 mm) needle attached to a 1 ml
tuberculin syringe. The inoculation site is just lateral to the midline into the midportion of one lateral
hemisphere.
Mice are observed for 10 days. Mice that die within 24 hours of inoculation are discarded. From 2 to 10 days
postinoculation, dead mice are collected daily and frozen at –70°C. Mouse brains are harvested for virus
identification by aspiration using a 20-gauge 1 inch (2.5 cm) needle attached to a 1 ml tuberculin syringe. A
second passage is made only if virus cannot be identified from mice that die following inoculation.
The chicken embryo is considered to be less sensitive than newborn mice when used for primary isolation of
EEE
and WEE viruses. Tissue suspensions can be inoculated by the yolk-sac route into 6–8-day-old
embryonating
chicken eggs. There are no diagnostic signs or lesions in the embryos infected with these viruses.
Inoculated
embryos should be incubated for 7 days, but deaths usually occur between 2 and 4 days post-inoculation.
Usually
only one passage is made unless there are dead embryos from which virus cannot be isolated. Newly
hatched
chickens are susceptible and have been used for virus isolation. If this method is used, precautions must be
taken
to prevent aerosol exposure of laboratory personnel, as infected birds can shed highly infectious virus.
EEE or WEE viruses can be identified in infected mouse or chicken brains, cell culture fluid, or amnionic-
allantoic
fluid by complement fixation. A 10% brain suspension is prepared in veronal (barbitone) buffer; egg and cell
culture fluids are used undiluted or diluted 1/10 in veronal buffer. The fluid or suspension is centrifuged at
9000 g
for 30 minutes, and the supernatant fluid is tested against hyperimmune serum or mouse ascitic fluid
prepared
against EEE and WEE viruses using a standard CF procedure (21). The CF test requires the overnight
incubation
at 4°C of serum-antigen with 7 units of complement. Virus can be identified in cell culture by direct
immunofluorescent staining. The less commonly used method of virus identification is the neutralisation test,
as
outlined below.
Reverse-transcription polymerase chain reaction (RT-PCR) methods to detect EEE, WEE and VEE viral
nucleic
acid in mosquitoes and vertebrate tissues have been described, although few have been extensively
validated for
mammalian samples (8, 9, 12, 23). A multiplex PCR method was developed to expedite differential diagnosis
in
cases of suspected EEE or West Nile arboviral encephalomyelitis in horses (6). The assay has enhanced
speed
and sensitivity compared to cell culture virus isolation and has been used effectively in the USA during
several
recent arbovirus seasons. Recently, a combination of an RT-PCR with an enzyme-linked immunosorbent
assay
(ELISA: RT-PCR-ELISA) was reported as a method to identify alpha-viruses that are pathogenic to humans
(25).
Antigen-capture ELISA has been developed for EEE surveillance in mosquitoes. This can be used in
countries
that do not have facilities for virus isolation or PCR (2). Immunohistochemical procedures for diagnosis of
EEE
have also been described (15).
Serological confirmation of EEE or WEE virus infection requires a four-fold or greater increase or decrease
in
antibody titre in paired serum samples collected 10–14 days apart. Most horses infected with EEE or WEE
virus
have a high antibody titre when clinical disease is observed. Consequently, a presumptive diagnosis can be
made
if an unvaccinated horse with appropriate clinical signs has antibody against only EEE or WEE virus. The
detection of IgM antibody by the ELISA can also provide a presumptive diagnosis of acute infection (17).
The
plaque reduction neutralisation (PRN) test or, preferably, a combination of PRN and haemagglutination
inhibition
(HI) tests is the procedure most commonly used for the detection of antibody against EEE and WEE viruses.
There may be cross-reactions between antibody against EEE and WEE virus in the CF and HI tests. CF
antibody
against both EEE and WEE viruses appears later and does not persist; consequently, it is less useful for the
serological diagnosis of disease.
a) Complement fixation
The CF test is frequently used for the demonstration of antibodies, although the antibodies detected by the
CF test may not persist for as long as those detected by the HI or PRN tests. A sucrose/acetone mouse
brain extract is commonly used as antigen. The positive antigen is inactivated by treatment with 0.1%
betapropiolactone.
In the absence of an international standard serum, the antigen should be titrated against a locally prepared
positive control serum. The normal antigen, or control antigen, is mouse brain from uninoculated mice
similarly extracted and diluted.
Sera are diluted 1/4 in veronal buffered saline containing 1% gelatin (VBSG), and inactivated at 56°C for
30 minutes. Titrations of positive sera may be performed using additional twofold dilutions. The CF antigens
and control antigen (normal mouse brain) are diluted in VBSG to their optimal amount of fixation as
determined by titration against the positive sera; guinea-pig complement is diluted in VBSG to contain
5 complement haemolytic units-50% (CH50). Sera, antigen, and complement are reacted in 96-well
roundbottom
microtitre plates at 4°C for 18 hours. The sheep red blood cells (SRBCs) are standardised to 2.8%
concentration. Haemolysin is titrated to determine the optimal dilution for the lot of complement used.
Haemolysin is used to sensitise 2.8% SRBCs and the sensitised cells are added to all wells on the microtitre
plate. The test is incubated for 30 minutes at 37°C. The plates are then centrifuged (200 g), and the wells
are scored for the presence of haemolysis. The following controls are used: (a) serum and control serum
each with 5 CH50 and 2.5 CH50 of complement; (b) CF antigen and control antigen each with 5 CH50, and
2.5 CH50 of complement; (c) complement dilutions of 5 CH50, 2.5 CH50, and 1.25 CH50; and (d) cell control
wells with only SRBCs and VBSG diluent. These controls test for anticomplementary serum and
anticomplementary antigen, activity of complement used in the test, and integrity of the SRBC indicator
system in the absence of complement, respectively.
To avoid anticomplementary effects, sera should be separated from the blood as soon as possible. Positive
and negative control sera should be used in the test.
b) Haemagglutination inhibition
The antigen for the HI test is the same as described above for the CF test. The antigen is diluted so that the
amount used in each haemagglutinating unit (HAU) is from four to eight times that which agglutinates 50%
of
the RBCs in the test system. The haemagglutination titre and optimum pH for each antigen are determined
with goose RBCs diluted in pH solutions ranging from pH 5.8 to pH 6.6, at 0.2 intervals.
Sera are diluted 1/10 in borate saline, pH 9.0, and then inactivated at 56°C for 30 minutes. Kaolin treatment
is used to remove nonspecific serum inhibitors. Alternatively, nonspecific inhibitors may be removed by
acetone treatment of serum diluted 1/10 in PBS followed by reconstitution in borate saline. Sera should be
absorbed before use by incubation with a 0.05 ml volume of washed packed goose RBCs for 20 minutes at
4°C.
Following heat inactivation, kaolin treatment and absorption, twofold dilutions of the treated serum are
prepared in borate saline, pH 9.0 with 0.4% bovalbumin. Serum dilutions (0.025 ml/well) are prepared in a
96-well round-bottom microtitre plate in twofold dilutions in borate saline, pH 9.0, with 0.4% bovalbumin.
Antigen (0.025 ml/well) is added to the serum. Plates are incubated at 4°C overnight. RBCs are derived from
normal white male geese1 and washed three times in dextrose/gelatin/veronal (DGV), and a 7.0%
suspension is prepared in DGV. The 7.0% suspension is then diluted 1/24 in the appropriate pH solution,
and 0.05 ml per well is added immediately to the plates. Plates are incubated for 30 minutes at 37°C.
Positive and negative control sera are incorporated into each test. A test is considered to be valid only if the
control sera give the expected results. Titres of 1/10 and 1/20 are suspect, and titres of 1/40 and above are
positive.
c) Enzyme-linked immunosorbent assay
The ELISA is performed by coating flat-bottomed plates with anti-equine IgM capture antibody (17). The
antibody is diluted according to the manufacture’s recommendations in 0.5 M carbonate buffer, pH 9.6, and
50 μl is added to each well. The plates are incubated at 37°C for 1 hour, and then at 4°C overnight. Prior to
use, the coated plates are washed three times with 200–300 μl/well of 0.01 M PBS containing 0.05%
Tween 20. After the second wash, 200 μl/well of PBS/Tween/5% nonfat dried milk is added and the plates
are incubated at room temperature for 1 hour. Following incubation, the plates are washed again three times
with PBS/Tween. Test and control sera are diluted 1/400 in 0.01 M PBS, pH 7.2, containing 0.05%
Tween 20, and 50 μl is added to each well. The plates are incubated at 37°C for 90 minutes and then
washed three times. Next, 50 μl of viral antigen is added to all wells. (The dilution of the antigen will depend
on the source and should be empirically determined.) The plates are incubated overnight at 4°C, and
washed three times. Then, 50 μl of horseradish-peroxidase-conjugated monoclonal antibody (MAb) to
encephalitis virus2 is added. The plates are incubated for 90 minutes at 37°C and then washed six times.
Finally, 50 μl of freshly prepared ABTS (2,2’-Azino-bis-[3-ethylbenzo-thiazoline-6-sulphonic acid]) substrate
+
hydrogen peroxidase is added, and the plates are incubated at room temperature for 15–40 minutes The
absorbance of the test serum is measured at 405 nm. A test sample is considered to be positive if the
absorbance of the test sample in wells containing virus antigen is at least twice the absorbance of negative
control serum in wells containing virus antigen and at least twice the absorbance of the sample tested in
parallel in wells containing normal antigen.
d) Plaque reduction neutralisation
The PRN test is very specific and can be used to differentiate between EEE and WEE virus infections. The
PRN test is performed in duck embryo fibroblast, Vero, or BHK-21 cell cultures in 25 cm2 flasks or six-well
plates. Volumes listed are for flasks; the volume should be halved for wells in six-well plates. The sera can
be screened at a 1/10 and 1/100 final dilution. Endpoints can be established using the PRN or HI test.
Serum used in the PRN assay is tested against 100 plaque-forming units (PFU) of virus (50 PFU for six-well
plates). The virus/serum mixture is incubated at 37°C for 75 minutes before inoculation on to confluent cell
culture monolayers in 25 cm2 flasks. The inoculum is adsorbed for 1 hour, followed by the addition of 6 ml of
overlay medium. The overlay medium consists of two solutions that are prepared separately. Solution I
contains 2 × Earle’s Basic Salts Solution without phenol red, 4% fetal bovine serum, 100 μg/ml gentamicin,
200 μg/ml nystatin, 0.45% solution of sodium bicarbonate, and 0.002% neutral red. When duck embryo
fibroblasts are used, Solution 1 also contains 6.6% yeast extract lactalbumin hydrolysate. Solution II consists
of 2% Noble agar that is sterilised and maintained at 47°C. Equal volumes of solutions I and II are adjusted
to 47°C and mixed together just before use. The test is incubated for 48–72 hours, and endpoints are based
on a 90% reduction in the number of plaques compared with the virus control flasks, which should have
about 100 plaques.
BIOLOGICALS
Inactivated vaccines against EEE and WEE viruses are available commercially. Attenuated EEE and WEE
virus
vaccines have not proven satisfactory. The vaccines licensed for use in the USA are prepared using the
following
combinations: EEE and WEE; EEE, WEE, and Venezuelan equine encephalomyelitis (VEE); and EEE and
VEE.
In addition, tetanus toxoid, inactivated influenza virus, and inactivated West Nile virus have been combined
with
1 RBCs from adult domestic white male geese are preferred, but RBCs from other male geese can be used. If cells from
female geese are used, there may be more test variability. It has been reported that rooster RBCs cause a decrease in
the
sensitivity of the test.
2 Available from: Centers for Disease Control and Prevention, Biological Reference Reagents, 1600 Clifton Road NE, Mail
Stop C21, Atlanta, Georgia 30333, United States of America.
EEE and WEE or EEE, WEE, and VEE. Early vaccines were produced from virus propagated in
embryonating
chicken eggs and inactivated with formalin. Current vaccines are prepared from virus propagated in cell
culture,
and inactivated with formalin (10) or monoethylamine.
1. Seed management
Standard strains of EEE and WEE viruses that were isolated over 20 years ago have been used for vaccine
production and have been proven to produce a protective immunity. Strains of EEE virus that differ
antigenically and in molecular structure have been identified from different geographical regions. However,
the North American and Caribbean isolates appear to be similar (26). Strains of WEE virus isolated from
different countries have been found to be similar both by MAb testing and RNA oligonucleotide fingerprinting
analysis (16). A recent well-characterised isolate from the country where the vaccine is to be used would be
advantageous. Viruses that are selected must be immunogenic and replicate to high titres in cell culture.
Primary chicken embryo fibroblasts and Vero cells have been used for propagation of viruses used for
vaccine production. The fibroblasts should be prepared from specific pathogen free embryos. Other
susceptible cell lines could also be used.
If a cell line is used, the master cell stock is tested to confirm the identity of the cell line, species of origin,
and freedom from extraneous agents. If primary cell cultures are used, a monolayer from each batch of each
subculture should be tested for extraneous agents including bacteria, fungi, mycoplasma, and viruses. The
master seed virus should also be tested to ensure freedom from bacteria, fungi, mycoplasma, and
extraneous viruses (see Chapter 1.1.9 Tests for sterility and freedom from contamination of biological
materials for a description of the method).
The vaccines are administered by the intramuscular (in most cases) or intradermal route in the cervical
region in two doses given 2–4 weeks apart. Annual revaccination is recommended. All foals vaccinated
before 1 year of age should be revaccinated before the next vector season.
Details of the manufacture of vaccines currently on the market are not available. Consequently, the
information
provided here is intended only as background reference material on the vaccines and not as a method of
manufacture. The virus and cell culture system should be selected so that a high virus titre, −106 TCID50
(50%
tissue culture infective dose) per ml, is obtained in under 48 hours. Virus for vaccine production can be
prepared
from the supernatant fluid from infected cell cultures. The fluid is harvested when 70–100% of the
monolayers
have the characteristic cytopathic changes. The virus titre is determined by titration in cell culture or mice.
The
fluid is clarified by low speed centrifugation and filtered through gauze. The virus is inactivated by adding
formalin
to a final concentration of 1/2000 (0.05%) and holding at 37°C for 24 hours. Residual formaldehyde is
neutralised
by sodium bisulphite (10). The residual free formalin content in the inactivated vaccine should not exceed
0.2%
formaldehyde.
Cultures should be examined daily for virus-induced cytopathic effect. The harvested virus should be tested
for
microbial contamination. The efficacy of the inactivation process should be checked by testing for viable
virus.
4. Batch control
a) Sterility
b) Safety
The inactivated vaccine is safety tested by inoculating subcutaneously at least ten 6–12-hour-old chickens
with 0.5 ml of the vaccine. The chickens are observed each day for 10 days for unfavourable reactions that
are attributable to the vaccine (20). Safety testing can also be carried out by inoculating intracerebrally at
least eight 1–4-day-old mice with 0.02 ml of the vaccine, and observing for 7 days. It is critical that safety
tests be conducted on each lot of vaccine to insure that there is no residual virulent virus present.
c) Potency
Potency testing is performed by inoculating each of ten guinea pigs with either EEE or WEE virus, using
onehalf
the horse dose on two occasions, 14–21 days apart, by the route recommended for the horse. Serum
samples from each vaccinate and each control are tested 14–21 days after the second dose using the PRN
test. The EEE titres should be −1/40, and the WEE titres should be −1/40 (20), using Vero cells. If duck
embryo fibroblasts are used in the PRN test, the titres will be lower. An alternative potency test is to use
intracerebral challenge, 14–21 days after the second vaccination. Each guinea pig is inoculated with 0.1 ml
of virus containing 100 LD50 (50% lethal dose). Simultaneous titration is carried out. In order for the vaccine
to be approved, 80% of the guinea pigs must survive both viruses.
Comprehensive studies on duration of immunity are not available. An annual revaccination is recommended.
Foals that are vaccinated before 1 year of age should be revaccinated before the next vector season.
e) Stability
The lyophilised vaccine is stable and immunogenic for 3 years if kept refrigerated at 2–7°C. After 3 years,
vaccine should be discarded. The vaccines should be used immediately after reconstitution.
f) Preservatives
The preservatives used are thimerosal at a 1/10,000 dilution and antibiotics (neomycin, polymyxin
amphotercin B and gentamicin).
Severe infection and death caused by EEE and WEE viruses have been reported in laboratory workers;
therefore, any work with these viruses must be carried out at least in a containment level 3 laboratory (see
Chapter 1.1.2) using biological safety cabinets, and it is recommended that personnel be immunised against
EEE and WEE viruses (22).
Pregnant mares and foals under 2 weeks old should not be vaccinated.
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.
REFERENCES
1. BAUER R.W., GILL M.S., POSTON R.B. & KIM D. Y. (2005). Naturally occurring eastern equine encephalitis in
a
Hampshire wether. J. Vet. Diagn. Invest., 17, 281–285.
2. BROWN T.M., MITCHELL C.J., NASCI R.S., SMITH G.C. & ROEHRIG J.T. (2001). Detection of eastern equine
encephalitis virus in infected mosquitoes using a monoclonal antibody-based antigen-capture enzyme-linked
immunosorbent assay. Am. J. Trop. Med. Hyg., 65, 208–213.
3. ELVINGER F., BALDWIN C.A., LIGGETT A.D., TANK K.N., & STALLKNECHT D.E. (1996). Prevalence of exposure
to
eastern equine encephalomyelitis virus in domestic and feral swine in Georgia. J. Vet. Diagn. Invest., 8,
481–
484.
4. FARRAR M.D., MILLER D. L., BALDWIN C. A., STIVER S. L. & HALL C. L. (2005). Eastern equine encephalitis in
dogs. J. Vet. Diagn. Invest., 17, 614–617.
5. GUY J.S. (1997). Arbovirus Infections. In: Diseases of Poultry, Calnek B.W., Barnes H.J., Beard C.W.,
McDougald L.R., & Saif Y.M., ed. Iowa State University Press, Ames, Iowa, USA, 765–772.
6. JOHNSON D.J., OSTLUND E.N. & SCHMITT B.J. (2003). Nested multiplex RT-PCR for detection and
differentiation of West Nile virus and eastern equine encephalomyelitis virus in brain tissues. J. Vet. Diagn.
Invest., 15, 488–493.
7. KARABATSOS N., LEWIS A.L., CALISHER C.H., HUNT A.R. & ROEHRIG J.T. (1988). Identification of Highland J
virus from a Florida horse. Am. J. Trop. Med. Hyg., 39, 603–606.
8. LAMBERT A.J., MARTIN D.A. & LANCIOTTI R.S. (2003). Detection of North American eastern and western
equine
encephalitis viruses by nucleic acid amplification assays. J. Clin. Microbiol., 41, 379–385.
9. LINSSEN B., KINNEY R.M., AGUILAR P., RUSSELL K.L., WATTS D.M., KAADEN O.R. & PFEFFER M. (2000).
Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses. J.
Clin. Microbiol., 38, 527–535.
10. MAIRE L.F. III, MCKINNEY R.W. & COLE F.E. Jr (1970). An inactivated eastern equine encephalomyelitis
vaccine propagated in chick-embryo cell culture. I. Production and testing. Am. J. Trop. Med. Hyg., 19, 119–
122.
11. MCGEE E.D., LITTLETON C.H., MAPP J.B. & BROWN R.J. (1992). Eastern equine encephalomyelitis in an
adult
cow. Vet. Pathol., 29, 361–363.
12. MONROY A.M., SCOTT T.W. & WEBB B.A. (1996). Evaluation of reverse transcriptase polymerase chain
reaction for the detection of eastern equine encephalomyelitis virus during vector surveillance. J. Med.
Entomol., 33, 449–457.
13. MORRIS C.D. (1989). Eastern equine encephalomyelitis. In: The Arboviruses: Epidemiology and Ecology,
Vol.
3, Monath T.P., ed. CRC Press, Boca Raton, Florida, USA, 1–12.
14. PURSELL A.R., MITCHELL F.E. & SEIBOLD H.R. (1976). Naturally occurring and experimentally induced
eastern
encephalomyelitis in calves. J. Am. Vet. Med. Assoc., 169, 1101–1103.
15. PATTERSON J.S., MAES R.K., MULLANEY T.P., & BENSON C.L. (1996). Immunohistochemical Diagnosis of
Eastern Equine Encephalomyelitis. J. Vet. Diagn. Invest., 8, 156–160.
16. REISEN W.K. & MONATH T.P. (1989). Western equine encephalomyelitis. In: The Arboviruses:
Epidemiology
and Ecology, Vol. 5, Monath T.P., ed. CRC Press, Boca Raton, Florida, USA, 89–137.
17. SAHU S.P., ALSTAD A.D., PEDERSEN D.D. & PEARSON J.E. (1994). Diagnosis of eastern equine
encephalomyelitis virus infection in horses by immunoglobulin M and G capture enzyme-linked
immunosorbent assay. J. Vet. Diagn. Invest., 6, 34–38.
18. TATE C.M., HOWERTH E.W., STALLKNECHT D.E., ALLISTION, A.B., FISHER J.R. & MEAD D.G. (2005). Eastern
equine encephalitis in a free-ranging white-tailed deer (Odocoileus virginianus). J. Wildl. Dis., 41, 241–245.
19. TUTTLE A.D., ANDREADIS T.G., FRASCA S. JR & DUNN J.L. (2005). Eastern equine encephalitis in a flock of
African penguins maintained at an aquarium. J. Am. Vet. Med. Assoc., 226, 2059–2062.
20. UNITED STATES CODE OF FEDERAL REGULATIONS (2000). Encephalomyelitis vaccine: Eastern and Western
killed virus. Title 9, Part 113, Section 113.207. US Government Printing Office, Washington DC, USA, 601–
602.
21. UNITED STATES DEPARTMENT OF HEALTH, EDUCATION AND WELFARE (1974). A Guide to the Performance of
the
Standardised Complement Fixation Method and Adaptation to Micro Test. Centers for Disease Control,
Atlanta, Georgia, USA.
22. UNITED STATES DEPARTMENT OF HEALTH AND HUMAN SERVICES (1999). Biosafety in Microbiological and
Biomedical Laboratories. US Government Printing Office, Washington DC, USA, 183–184.
23. VODKIN M.H., MCLAUGHLIN G.L., DAY J.F., SHOPE R.E. & NOVAK R.J. (1993). A rapid diagnostic assay for
eastern equine encephalomyelitis viral-RNA. Am. J. Trop. Med. Hyg., 49, 772–776.
24. WALTON T.E. (1981). Venezuelan, eastern, and western encephalomyelitis. In: Virus Diseases of Food
Animals. A World Geography of Epidemiology and Control. Disease Monographs, Vol. 2, Gibbs E.P.J., ed.
Academic Press, New York, USA, 587–625.
25. WANG E., PAESSLER S., AGUILAR P.V., CARRARA A.S., NI H., GREENE I.P. & WEAVER S.C. (2006). Reverse
transcription-PCR-enzyme-linked immunosorbent assay for rapid detection and differentiation of alphavirus
infections. J. Clin. Microbiol., 44, 4000–4008.
26. WEAVER S.C., HAGENBAUGH A., BELLEW L.A., GOUSSET L., MALLAMPALLI V., HOLLAND J.J. & SCOTT T.W.
(1994).
Evolution of Alphaviruses in the eastern equine encephalomyelitis complex. J. Virol., 68, 158–169.
*
**
NB: There is an OIE Reference Laboratory for Equine encephalomyelitis (Eastern and Western) (see Table
in
Part 3 of this Terrestrial Manual or consult the OIE Web site for the most up-to-date list: www.oie.int).
C H A P T E R 2 . 5 . 6 .
EQUINE INFECTIOUS ANAEMIA
SUMMARY
Equine infectious anaemia (EIA) is a persistent viral infection of equids. The causative agent, EIA
virus (EIAV) is a lentivirus in the family Retroviridae, sSubfamily Orthoretrovirinae. Other members
of the lentivirus genus include: bovine immunodeficiency virus; caprine arthritis encephalitis virus;
feline immunodeficiency virus; human immunodeficiency virus 1; human immunodeficiency virus 2;
and maedi/visna virus. EIA can be diagnosed on the basis of clinical signs, pathological lesions,
serology and molecular methods. Infected horses remain viraemic carriers for life and, with very
rare exceptions, yield a positive serological test result. Antibody response usually persists and
antibody-positive animals, older than 6–8 months, are identified as virus carriers (below 6–8 months
of age, serological reactions can be due to maternal antibodies; status can be confirmed by
molecular techniques). Infected equids are potential virus reservoirs. Biting flies are mechanical
vectors for the virus in nature.
Identification of the agent: Virus from a horse can be isolated by inoculating suspect blood into a
susceptible horse or on to leukocyte cultures prepared from susceptible horses. Recognition of
infection in horses that have been inoculated experimentally may be made on the basis of clinical
signs, haematological changes and a positive antibody response determined by an immunodiffusion
test or enzyme-linked immunosorbent assay (ELISA) or by molecular techniques. Successful virus
isolation in horse leukocyte cultures is confirmed by the detection of specific EIA antigen, by
immunofluorescence assay, polymerase chain reaction, reverse-transcriptase assay, or by the
inoculation of culture fluids into susceptible horses. Virus isolation is rarely attempted because of
the time, difficulty and expense involved.
Serological tests: Agar gel immunodiffusion (AGID) tests and ELISAs are simple, reliable tests for
the demonstration of EIAV infection. When ELISAs are positive they should be confirmed using the
AGID test. EIA antigens can be prepared from infected tissue cultures or by using recombinant
DNA technology.
Requirements for vaccines and diagnostic biologicals: There are no biological products
currently available.
A. INTRODUCTION
Equine infectious anaemia (EIA) occurs world-wide. The infection, formerly known as swamp fever, is limited
to
equids. The disease is characterised by recurrent febrile episodes, thromboctopenia, anaemia, rapid loss of
weight and oedema of the lower parts of the body. If death does not result from one of the acute clinical
attacks, a
chronic stage develops and the infection tends to become inapparent. The incubation period is normally 1–
3 weeks, but may be as long as 3 months. In acute cases, lymph nodes, spleen and liver are hyperaemic
and
enlarged. Histologically these organs are infiltrated with nests of immature lymphocytes and plasma cells.
Kupffer
cells in the liver often contain haemosiderin or erythrocytes. The enlarged spleen may be felt on rectal
examination. Differential diagnoses include equine viral arteritis (Chapter 2.5.10), and other causes of
oedema,
fever, or anemia.
Once a horse is infected with EIA virus (EIAV), its blood remains infectious for the remainder of its life. This
means that the horse is a viraemic carrier and can potentially transmit the infection to other horses (4).
Transmission occurs by transfer of blood from an infected horse. In nature, spread of the virus is most likely
via
interrupted feeding of bloodsucking horseflies on a clinically ill horse and then on susceptible horses.
Transmission can also occur by the iatrogenic transfer of blood through the use of contaminated needles. In
utero
infection of the fetus may occur (9). The virus titre is higher in horses with clinical signs and the risk of
transmission is higher from these animals than the carrier animals with a lower virus titre.
Chapter 2.5.6. - Equine infectious anaemia
Agar gel immunodiffusion (AGID) tests (7) and enzyme-linked immunosorbent assays (ELISAs) (17) are
accurate,
reliable tests for the detection of EIA in horses, except for animals in the early stages of infection and foals of
infected dams. In other rare circumstances, misleading results may occur when the level of virus circulating
in the
blood during an acute episode of the disease is sufficient to bind available antibody, and if initial antibody
levels
never rise high enough to be detectable (18). Although the ELISA will detect antibodies somewhat earlier
and at
lower concentrations than the AGID test, positive ELISAs are confirmed using the AGID test. This is because
false-positive results have been noted with ELISAs. The AGID test also has the advantage of distinguishing
between EIA and non-EIA antigen–antibody reactions by lines of identity.
The EIAV is a lentivirus in the family Retroviridae, subfamily Orthoretrovirinae. Other members of the
lentivirus
genus include: bovine immunodeficiency virus; caprine arthritis encephalitis virus; feline immunodeficiency
virus;
human immunodeficiency virus 1; human immunodeficiency virus 2; and maedi/visna virus. Nucleic acid
sequence
comparisons have demonstrated a marked relatedness among these viruses.
a) Virus isolation and identification
Virus isolation is usually not necessary to make a diagnosis.
Isolation of the virus from suspect horses may be made by inoculating their blood on to leukocyte cultures
prepared from horses free of infection. Virus production in cultures can be confirmed by detection of specific
EIA antigen by ELISA (16), by imunofluorescence assay (20), by molecular tests or by subinoculation into
susceptible horses. Virus isolation is rarely attempted because of the difficulty of growing horse leukocyte
cultures.
When the exact status of infection of a horse cannot be ascertained, the inoculation of a susceptible horse
with suspect blood should be employed. In this case a horse that has previously been tested for antibody
and shown to be negative is given an immediate blood transfusion from the suspect horse, and its antibody
status and clinical condition are monitored for at least 45 days. Usually, 1–25 ml of whole blood given
intravenously is sufficient to demonstrate infection, but in rare cases it may be necessary to use a larger
volume of blood (250 ml) or washed leukocytes from such a volume (5).
b) Polymerase chain reaction
A nested polymerase chain reaction (PCR) assay to detect EIA proviral DNA from the peripheral blood of
horses has been described (13). The nested PCR method is based on primer sequences from the gag
region of the proviral genome. It has proven to be a sensitive technique to detect field strains of EAV in white
blood cells of EIA infected horses; the lower limit of detection is typically around 10 genomic copies of the
target DNA (13, 14). A real-time reverse-transcriptase PCR assay has also been described (8). To confirm
the results of these very sensitive assays, it is recommended that duplicate samples of each diagnostic
specimen be processed. Because of the risk of cross contamination, it is also important that proper
procedures are followed. Methods to insure the validity of PCR testing are discussed in detail in Chapter
1.1.5 Validation and quality control of polymerase chain reaction methods used for the diagnosis of
infectious
diseases.
The following are some of the circumstances where the PCR assay maybe used for the detection of EIAV
infection in horses:
• Conflicting results on serologic tests;
• Suspected infection but negative or questionable serologic results;
• Complementary test to serology for the confirmation of positive results;
• Confirmation of early infection, before serum antibodies to EIAV develop;
• Ensuring that horses that are to be used for antiserum or vaccine production or as blood donors are
free of EIAV;
• Confirmation of the status of a foal from an infected mare.
Due to the persistence of EIAV in infected equids, detection of serum antibody to EIAV confirms the
diagnosis of
EIAV infection.
a) Agar gel immunodiffusion test (the prescribed test for international
trade)
Precipitating antibody is rapidly produced as a result of EIA infection, and can be detected by the AGID test.
Specific reactions are indicated by precipitin lines between the EIA antigen and the test serum and
confirmed
by their identity with the reaction between the antigen and the positive standard serum. Horses in the first 2–
3 weeks after infection will usually give negative serological reactions. In rare cases the post-infection time
prior to the appearance of detectable antibody may extend up to 60 days.
Reagents for AGID are available commercially from several companies. Alternately, AGID antigen and
reference serum may be prepared as described below.
o Preparation of antigen
Specific EIA antigen may be prepared from the spleen of acutely infected horses (6), from infected equine
tissue culture (11), from a persistently infected canine thymus cell line (3), or from proteins expressed in
bacteria or baculovirus using the recombinant DNA technique (2, 10). Preparation from infected cultures or
from recombinant DNA techniques gives a more uniform result than the use of spleen cells and allows for
better standardisation of reagents.
To obtain a satisfactory antigen from spleen, a horse must be infected with a highly virulent strain of EIAV.
The resulting incubation period should be 5–7 days, and the spleen should be collected 9 days after
inoculation, when the virus titre is at its peak and before any detectable amount of precipitating antibody is
produced. Undiluted spleen pulp is used in the immunodiffusion test as antigen (6). Extraction of antigen
from the spleen with a saline solution and concentration with ammonium sulphate does not give as
satisfactory an antigen as selection of a spleen with a very high titre of EIA antigen.
Alternatively, equine fetal kidney or dermal cells or canine thymus cells are infected with a strain of EIAV
adapted to grow in tissue culture (American Type Culture Collection). Virus is collected from cultures by
precipitation with 8% polyethylene glycol or by pelleting by ultracentrifugation. The diagnostic antigen, p26,
is
released from the virus by treatment with detergent or ether (11). EIAV core proteins, expressed in bacteria
or baculovirus, are commercially available and find practical use as high quality antigens for serological
diagnosis.
The p26 is an internal structural protein of the virus that is coded for by the gag gene. The p26 is more
antigenically stable among EIAV strains than the virion glycoproteins gp45 and gp90 (12). There is evidence
of strain variation in the p26 amino acid sequence; however there is no evidence to indicate that this
variation influences any of the serological diagnostic tests (21).
o Preparation of standard antiserum
A known positive antiserum may be collected from a horse previously infected with EIAV. This serum should
yield a single dense precipitation line that is specific for EIA, as demonstrated by a reaction of identity with a
known standard serum. It is essential to balance the antigen and antibody concentrations in order to ensure
the optimal sensitivity of the test. Reagent concentrations should be adjusted to form a narrow precipitation
line approximately equidistant between the two wells containing antigen and serum.
o Test procedure (1, 6, 15)
i) Immunodiffusion reactions are carried out in a layer of agar in Petri dishes. For Petri dishes that are
100 mm in diameter, 15–17 ml of 1% Noble agar in 0.145 M borate buffer (9 g H3BO3, plus 2 g NaOH
per litre), pH 8.6 (± 0.2) is used. Six wells are punched out of the agar surrounding a centre well of the
same diameter. The wells are 5.3 mm in diameter and 2.4 mm apart. Each well must contain the same
volume of reagent.
ii) The antigen is placed in the central well and the standard antiserum is placed in alternate exterior wells.
Serum samples for testing are placed in the remaining three wells.
iii) The dishes are maintained at room temperature in a humid environment.
iv) After 24–48 hours the precipitation reactions are examined over a narrow beam of intense, oblique light
and against a black background. The reference lines should be clearly visible at 24 hours, and at that
time any test sera that are strongly positive may also have formed lines of identity with those between
the standard reagents. A weakly positive reaction may take 48 hours to form and is indicated by a slight
bending of the standard serum precipitation line between the antigen well and the test serum well. Sera
with high precipitating antibody titres may form broader precipitin bands that tend to be diffuse. Such
reactions can be confirmed as specific for EIA by dilution at 1/2 or 1/4 prior to retesting; these then give
a more distinct line of identity. Sera devoid of EIA antibody will not form precipitation lines and will have
no effect on the reaction lines of the standard reagents.
v) Interpretation of the results: Horses that are in the early stages of an infection may not give a positive
serological reaction in an AGID test. Such animals should be bled again after 3–4 weeks. In order to
make a diagnosis in a young foal, it may be necessary to determine the antibody status of the dam. If
the mare passes EIA antibody to the foal through colostrum, then a period of 6 months or longer after
birth must be allowed for the maternal antibody to wane. Sequential testing of the foal at monthly
intervals may be useful to observe the decline in maternal antibody. To conclude that the foal is not
infected, a negative result must be obtained (following an initial positive result) at least 2 months after
separating the foal from contact with the EIA positive mare or any other positive horse. Alternatively
PCR could be performed on the blood of the foal to determine the presence/absence of EIA proivirus.
b) Enzyme-linked immunosorbent assay
There are four ELISAs that are approved by the United States Department of Agriculture for the diagnosis of
equine infectious anaemia and are available internationally; a competitive ELISA and three non-competitive
ELISAs. The competitive ELISA and two non-competitive ELISAs detect antibody produced against the p26
core protein antigen. The third non-competitive ELISA incorporates both p26 core protein and gp45 (viral
transmembrane protein) antigens. Typical ELISA protocols are used in all tests. If commercial ELISA
materials are not available, a non-competitive ELISA using p26 antigen purified from cell culture material
may be employed (16).
A positive test result by ELISA should be retested using the AGID test to confirm the diagnosis because
some false-positive results have been noted with the ELISA. The results can also be confirmed by the
immunoblot technique. A standard antiserum for immunodiffusion, which contains the minimum amount of
antibody that should be detected by laboratories, is available from the OIE Reference Laboratories (see
Table given in Part 3 of this Terrestrial Manual). Uniform methods for EIA control have been published (19).
BIOLOGICALS
No biological products are available currently.
REFERENCES
1. ASSOCIATION FRANÇAISE DE NORMALISATION (AFNOR) (2000). Animal Health Analysis Methods. Detection of
Antibodies against Equine Infectious Anaemia by the Agar Gel Immunodiffusion Test. NF U 47-002. AFNOR,
11 avenue Francis de Pressensé, 93571 Saint-Denis La Plaine Cedex, France.
2. ARCHAMBAULT D., WANG Z., LACAL J.C., GAZIT A., YANIV A., DAHLBERG J.E. & TRONICK S.R. (1989).
Development of an enzyme-linked immunosorbent assay for equine infectious anaemia virus detection using
recombinant Pr55gag. J. Clin. Microbiol., 27, 1167–1173.
3. BOUILLANT A.M.P., NELSON K., RUCKERBAUER C.M., SAMAGH B.S. & HARE W.C.D. (1986). The persistent
infection of a canine thymus cell line by equine infectious anaemia virus and preliminary data on the
production of viral antigens. J. Virol. Methods, 13, 309–321.
4. CHEEVERS W.M. & MCGUIRE T.C. (1985). Equine infectious anaemia virus; immunopathogenesis and
persistence. Rev. Infect. Dis., 7, 83–88.
5. COGGINS L. & KEMEN M.J. (1976). Inapparent carriers of equine infectious anaemia (EIA) virus. In:
Proceedings of the IVth International Conference on Equine Infectious Diseases. University of Kentucky,
Lexington, Kentucky, USA, 14–22.
6. COGGINS L., NORCROSS N.L. & KEMEN M.J. (1973). The technique and application of the immunodiffusion
test
for equine infectious anaemia. Equine Infect. Dis., III, 177–186.
7. COGGINS L., NORCROSS N.L. & NUSBAUM S.R. (1972). Diagnosis of equine infectious anaemia by
immunodiffusion test. Am. J. Vet. Res., 33, 11–18.
8. COOK R.F., COOK S.J., LI F.L., MONTELARO R.C., & ISSEL C.J. (2002). Development of a multiplex real-time
reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV). Virol Methods,
105, 171–179.
9. KEMEN M.J. & COGGINS L. (1972). Equine infectious anaemia: transmission from infected mares to foals. J.
Am. Vet. Med. Assoc., 161, 496–499.
10 KONG X. K., PANG H., SUGIURA T., SENTSUI H., ONODERA T., MATSUMOTO Y. & AKASHI H. (1997). Application
of
equine infectious anaemia virus core proteins produced in a Baculovirus expression system, to serological
diagnosis. Microbiol. Immunol., 41, 975–980.
11 MALMQUIST W.A., BARNETT D. & BECVAR C.S. (1973). Production of equine infectious anaemia antigen in a
persistently infected cell line. Arch. Gesamte Virusforsch., 42, 361–370.
12. MONTELARO R.C., PAREKH B., ORREGO A. & ISSEL C.J. (1984). Antigenic variation during persistent
infection by
equine infectious anaemia, a retrovirus. J. Biol. Chem., 16, 10539–10544.
13. NAGARAJAN M.M. & SIMARD C. (2001). Detection of horses infected naturally with equine infectious
anemia
virus by nested polymerase chain reaction. J. Virol. Methods, 94, 97–109.
14. NAGARAJAN M.M. & SIMARD C. (2007). Gag genomic heterogeneity of equine infectious anemia virus
(EIAV) in
naturally infected horses in Canada: implication on EIA diagnosis and peptide-based vaccine development.
Virus Res., 129, 228–235.
15. PEARSON J.E. & COGGINS L. (1979). Protocol for the immunodiffusion (Coggins) test for equine infectious
anaemia. Proc. Am. Assoc. Vet. Lab. Diagnosticians, 22, 449–462.
16. SHANE B.S., ISSEL C.J. & MONTELARO R.C. (1984). Enzyme-linked immunosorbent assay for detection of
equine infectious anemia virus p26 antigen and antibody. J. Clin. Microbiol., 19, 351–355.
17. SUZUKI T., UEDA S. & SAMEJINA T. (1982). Enzyme-linked immunosorbent assay for diagnosis of equine
infectious anaemia. Vet. Microbiol., 7, 307–316.
18. TOMA B. (1980). Réponse sérologique négative persistante chez une jument infectée. Rec. Med. Vet.,
156,
55–63.
19. UNITED STATES DEPARTMENT OF AGRICULTURE ANIMAL AND PLANT HEALTH INSPECTION SERVICE (2002). Equine
Infectious Anemia Uniform Methods and Rules. http://www.aphis.usda.gov/oa/pubs/eiaumr.pdf
20. WEILAND F., MATHEKA H.D. & BOHM H.O. (1982). Equine infectious anaemia: detection of antibodies using
an
immunofluorescence test. Res. Vet. Sci., 33, 347–350.
21. ZHANG W., AUYONG D.B., OAKS J.L. & MCGUIRE T.C. (1999). Natural variation of equine infectious anemia
virus Gag-protein cytotoxic T lymphocyte epitopes. Virology, 261, 242–252.
*
**
NB: There are OIE Reference Laboratories for Equine infectious anaemia (see Table in Part 3 of this
Terrestrial
C H A P T E R 2 . 5 . 9 .
EQUINE RHINOPNEUMONITIS
SUMMARY
Equine rhinopneumonitis (ER) is a collective term for any one of several highly contagious, clinical
disease entities of equids that may occur as a result of infection by either of two closely related
herpesviruses, equid herpesvirus-1 and -4 (EHV-1 and EHV-4).
Infection by either EHV-1 or EHV-4 is characterised by a primary respiratory tract disease of varying
severity that is related to the age and immunological status of the infected animal. Infections by
EHV-1 in particular are capable of progression beyond the respiratory mucosa to cause the more
serious disease manifestations of abortion, perinatal foal death, or neurological dysfunction.
Identification of the agent: The standard method of identification of the herpesviral agents of ER
continues to be laboratory isolation of the virus from appropriate clinical or necropsy material,
followed by seroconfirmation of its identity. The viruses can be isolated in equine cell culture from
nasopharyngeal samples taken from horses during the febrile stage of respiratory tract infection,
from liver, lung, spleen, or thymus of aborted fetuses and early foal deaths, and from the leukocyte
fraction of the blood of animals with acute EHV-1 disease. Positive identification of viral isolates as
EHV-1 or EHV-4 can be achieved by immunofluorescence with type-specific monoclonal antibodies.
A rapid presumptive diagnosis of rhinopneumonitis abortion can be achieved by direct
immunofluorescent detection of viral antigen in cryostat sections of tissues from aborted fetuses,
using conjugated polyclonal antiserum.
Sensitive and reliable methods for EHV-1/4 detection by polymerase chain reaction or
immunoperoxidase staining have been developed and are useful adjuncts to standard virus
cultivation techniques for diagnosis of ER.
Post-mortem demonstration of histopathological lesions of EHV-1 in tissues from aborted fetuses,
cases or perinatal foal death or in the central nervous system of neurologically affected animals
complements the laboratory diagnosis of ER.
Serological tests: Because most horses will possess some level of antibody to EHV-1/4, the
demonstration of specific antibody in the serum collected from a single blood sample is not
sufficient for a positive diagnosis of recent, active ER. Paired, acute and convalescent sera from
animals suspected of being infected with EHV-1 or EHV-4 can be tested for a four-fold or greater
rise in virus-specific antibody titre by either virus neutralisation, or enzyme-linked immunosorbent
assay, or complement fixation.
Requirements for vaccines and diagnostic biologicals: Both live attenuated and inactivated
viral vaccines of varying composition are commercially available for use in assisting in the control of
ER. While vaccination is helpful in reducing the incidence of abortion in mares, and in ameliorating
the severity of clinical signs of respiratory infection in young horses, it should not be considered to
be a substitute for strict adherence to the well established tenets of sound management practices
known to reduce the risk of rhinopneumonitis. Revaccination at frequent intervals is recommended
with each of the products, as the duration of vaccine-induced immunity is relatively short.
Standards for production and licensing of both attenuated and inactivated EHV vaccines are
established by appropriate veterinary regulatory agencies in the countries of vaccine manufacture
and use. A single set of internationally recognised standards for ER vaccines is not available. In
each case, however, vaccine production is based on the system of a detailed outline of production
employing a well characterised cell line and a master seed lot of vaccine virus that has been
validated with respect to virus identity, safety, virological purity, immunogenicity, and the absence of
extraneous microbial agents.
Chapter 2.5.9. - Equine rhinopneumonitis
A. INTRODUCTION
Equine rhinopneumonitis (ER) is an historically derived term that describes a constellation of several
disease
entities of horses that may include respiratory disease, abortion, neonatal foal pneumonitis, or
myeloencephalopathy (1, 2, 5, 7). The disease has been recognised for over 60 years as a threat to the
international horse industry, and is caused by either of two members of the Herpesviridae family, equid
herpesvirus-1 and -4 (EHV-1 and EHV-4). EHV-1 and EHV-4 are closely related alphaherpesviruses of
horses
with nucleotide sequence identity within individual homologous genes ranging from 55% to 84%, and amino
acid
sequence identity from 55% to 96% (13, 14). The two herpesviruses are enzootic in all countries in which
large
populations of horses are maintained as part of the cultural tradition or agricultural economy. There is no
recorded
evidence that the two herpesviruses of ER pose any health risks to humans working with the agents.
ER is highly contagious among susceptible horses, with viral transmission to cohort animals occurring by
inhalation of aerosols of virus-laden respiratory secretions. Extensive use of vaccines has not eliminated
EHV
infections, and the world-wide annual financial burden from these equine pathogens is immense.
In horses under 3 years of age, clinical ER usually takes the form of an acute, febrile respiratory illness that
spreads rapidly through the group of animals. The viruses infect and multiply in epithelial cells of the
respiratory
mucosa. Signs of infection become apparent 2–8 days after exposure to virus, and are characterised by
fever,
inappetence, depression, and nasal discharge. The severity of respiratory disease varies with the age of the
horse
and the level of immunity resulting from previous vaccination or natural exposure. Subclinical infections with
EHV-
1/4 are common, even in young animals. Although mortality from uncomplicated ER is rare and complete
recovery
within 1–2 weeks is the normal pattern, the respiratory infection is a frequent and significant cause of
interrupted
schedules among horses assembled for training, racing, or competitive equestrian events. Fully protective
immunity resulting from infection is of short duration, and convalescent animals are susceptible to reinfection
by
EHV-1/4 after several months. Although reinfections by the two herpesviruses cause less severe or clinically
inapparent respiratory disease, the risks of subsequent abortion and/or central nervous system (CNS)
disease are
not eliminated. The greatest clinical threats to individual breeding, racing, or pleasure horse operations
posed by
ER are the potential abortigenic and neurological sequelae of EHV-1 respiratory infection.
1. Identification of the agent (3)
Because ER is a highly contagious disease with the potential for occurring as explosive outbreaks with high
mortality from abortigenic or neurological sequelae, rapid diagnostic methods are important. Although
several
rapid and innovative diagnostic techniques based on enzyme-linked immunosorbent assay (ELISA),
polymerase
chain reaction (PCR), immunohistochemical staining with peroxidase, or nucleic acid hybridisation probes
have
been recently described, their use is often restricted to specialised reference laboratories, and thus the
method of
choice for diagnosis of ER by diagnostic virology laboratories handling many routine samples continues to
be the
traditional methodology of cell culture isolation followed by sero-identification of the isolated viruses.
Successful
laboratory isolation of EHV-1/4 depends on strict adherence to proper methods for both sample collection
and
laboratory processing.
a) Collection of samples
Samples of nasopharyngeal exudate for virus isolation are best obtained from horses during the very early,
febrile stages of the respiratory disease, and are collected via the nares by swabbing the nasopharyngeal
area with a 5 × 5 cm gauze sponge attached to the end of a 50 cm length of flexible, stainless steel wire
encased in latex rubber tubing. A guarded uterine swab devise can also be used. After collection, the swab
should be removed from the wire and transported immediately to the virology laboratory in 3 ml of cold (not
frozen) fluid transport medium (serum-free MEM [minimal essential medium] with antibiotics). Virus
infectivity
can be prolonged by the addition of bovine serum albumin or gelatine to 0.1% (w/v).
Virological examination of fetal tissues from suspect cases of EHV-1 abortion is most successful when
performed on aseptically collected samples of liver, lung, thymus, and spleen. The tissue samples should be
transported to the laboratory and held at 4°C until inoculated into tissue culture. Samples that cannot be
processed within a few hours should be stored at –70°C. In ante-mortem cases of EHV-1 neurological
disease, the virus can often be isolated from the leukocyte fraction of the blood of acutely infected horses or,
less often, from the nasopharynx of the affected animal or cohort animals. For attempts at virus isolation
from blood leukocytes, a 20 ml sample of sterile blood, collected in citrate, or heparin anticoagulant (EDTA
[ethylene diamine tetra-acetic acid] should not be used as it can destroy the cell cultures). The samples
should be transported without delay to the laboratory on ice, but not frozen. Although the virus has, on
occasion, been isolated from post-mortem cases of EHV-1 neurological disease by culture of samples of
brain and spinal cord, such attempts to isolate virus are often unsuccessful; however, they maybe useful for
PCR examination.
b) Virus isolation
For efficient primary isolation of EHV-4 from horses with respiratory disease, equine-derived cell cultures
must be used. Both EHV-1 and EHV-4 may be isolated from nasopharyngeal samples using primary equine
fetal kidney cells or cell strains of equine fibroblasts derived from dermal (E-Derm) or lung tissue. EHV-1 can
be isolated on other cell types, as will be discussed later. The nasopharyngeal swab and its accompanying 3
ml of transport medium are transferred into the barrel of a sterile 10 ml syringe. Using the syringe plunger,
the fluid is squeezed from the swab into a sterile tube. A portion of the expressed fluid is then filtered through
a sterile, 0.45 μm membrane syringe filter unit into a second sterile tube. Filtration will decrease bacterial
contamination, but may also lower virus titre. Recently prepared cell monolayers in 25 cm2 tissue culture
flasks are inoculated with 0.5 ml of the filtered, as well as the unfiltered, nasopharyngeal swab extract. Cell
monolayers in multiwell plates incubated in a 5% CO2 environment may also be used. Virus is allowed to
attach by incubating the inoculated monolayers at 37°C on a platform rocker for 1.5–2 hours. Monolayers of
uninoculated control cells should be incubated in parallel with sterile transport medium only.
At the end of the attachment period, the inocula are removed and the monolayers are rinsed twice with
phosphate buffered saline (PBS) to remove virus-neutralising antibody that may be present in the
nasopharyngeal secretions. After addition of 5 ml of supplemented maintenance medium (MEM containing
2% fetal calf serum [FCS] and twice the standard concentrations of antibiotics [penicillin, streptomycin,
gentamicin, and amphotericin B]), the flasks are incubated at 37°C. The use of positive control virus samples
to validate the isolation procedure carries the risk that this may lead to eventual contamination of diagnostic
specimens. This risk can be minimised by using routine precautions and good laboratory technique,
including the use of biosafety cabinets, inoculating positive controls after the diagnostic specimens,
decontaminating the surfaces in the hood while the inoculum is adsorbing and using a positive control of
relatively low titre. Inoculated flasks should be inspected daily by microscopy for the appearance of
characteristic herpesvirus cytopathic effect (CPE) (focal rounding, increase in refractility, and detachment of
cells). Cultures exhibiting no evidence of viral CPE after 1 week of incubation should be blind-passaged into
freshly prepared monolayers of cells, using small aliquots of both media and cells as the inoculum. Further
blind passage is usually not productive.
A number of cell types may be used for isolation of EHV-1 from the tissues of aborted fetuses or from
postmortem
cases of neurological disease (e.g. rabbit kidney [RK-13], baby hamster kidney [BHK-21], Madin–
Darby bovine kidney [MDBK], pig kidney [PK-15], etc.), but equine-derived cell cultures are most sensitive
and must be used if the infrequent cases of EHV-4 abortion are to be detected. Around 10% (w/v) pooled
tissue homogenates of liver, lung, thymus, and spleen (from aborted fetuses) or of CNS tissue (from cases
of neurological disease) are used for virus isolation. These are prepared by first mincing small samples of
tissue into 1 mm cubes in a sterile Petri dish with dissecting scissors, followed by macerating the tissue
cubes further in serum-free culture medium with antibiotics using a mechanical tissue grinder (e.g. Ten-
Broeck or Stomacher). After centrifugation at 1200 g for 10 minutes, the supernatant is removed and 0.5 ml
is inoculated into duplicate cell monolayers in 25 cm2 flasks. Following incubation of the inoculated cells at
37°C for 1.5–2 hours, the inocula are removed and the monolayers are rinsed twice with PBS. After addition
of 5 ml of supplemented maintenance medium, the flasks are incubated at 37°C for up to 1 week or until
viral
CPE is observed.
Culture of peripheral blood leukocytes for the presence of EHV-1 can be attempted in horses during the
early
stages of the myeloencephanlopathy. Buffy coats may be prepared from unclotted blood by centrifugation at
600 g for 15 minutes, and the buffy coat is taken after the plasma has been carefully removed. The buffy
coat is then layered onto Ficoll 1,090, centrifuged at 400 g for 20 minutes and the leukocyte-rich interface is
then layered onto Ficoll 1.077 and centrifuged in the same way. The PBMC interface (without most
granulocytes) is washed twice in PBS (300 g for 10 minutes) and resuspended in 1 ml of MEM containing
2% FCS. Then, 0.5 ml of the rinsed cell suspension is added to each of the duplicate monolayers of equine
fibroblast, equine fetal or RK-13 cell monolayers in 25 cm2 flasks containing 8–10 ml freshly added
maintenance medium. The flasks are incubated at 37°C for 7 days; the inoculum is not removed. Because
CPE may be difficult to detect in the presence of the massive inoculum of leukocytes, each flask of cells is
freeze–thawed after 7 days of incubation and the contents centrifuged at 300 g for 10 minutes. Finally, 0.5
ml
of the cell-free, culture medium supernatant is transferred to freshly made cell monolayers that are just
subconfluent. These are incubated and observed for viral CPE for at least 5–6 days before discarding as
negative.
c) Seroconfirmation of virus identity
The basis for identification of any herpesvirus isolate recovered from specimens submitted from suspected
cases of ER is its immunoreactivity with specific antisera. Specific identification of an isolate as EHV-1 or
EHV-4 can be quickly and simply accomplished by immunofluorescent (FA) detection of viral antigen in the
infected cell culture using type-specific monoclonal antibodies (MAbs), which are available from OIE
Reference Laboratories for equine rhinopneumonitis. The test, which is type-specific and accurate, can be
performed on a small aliquot of infected cells from the same container inoculated with clinical or post-
mortem
material. An isolate made in a laboratory that lacks MAbs or FA capability can be confirmed as EHV1/4 by
virus neutralization using a virus-specific polyclonal antiserum or by the PCR (see section B.1.f).
Cell monolayers infected with the isolate are removed by scraping from the flask when at least 75% CPE is
evident. The cells are pelleted from the culture medium and resuspended in 0.5 ml of PBS. 50 μl of the cell
suspension is placed into two wells of a multiwell microscope slide, air-dried, and fixed for 10 minutes with
100% acetone. Control cell suspensions (uninfected, EHV-1 infected, or EHV-4 infected) are also spotted
into each of two wells of the same slide. Control cells may be prepared in advance and stored frozen in
small
aliquots. A drop of an appropriate dilution of MAb specific for EHV-1 is added to one well of each cell pair,
and a drop of MAb specific for EHV-4 is added to each of the other wells. After 30 minutes’ incubation at
37°C in a humid chamber, unreacted antibody is removed by two 10-minute washes with PBS. MAbs bound
to viral antigen can be detected with goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC).
A drop of diluted conjugate is added to each well and, after 30 minutes at 37°C, the wells are again washed
twice with PBS. Cells are examined with a fluorescence microscope, and positive fluorescence with the
antibody of appropriate specificity indicates the virus type.
d) Virus detection by direct immunofluorescence
Direct immunofluorescent detection of EHV-1 antigens in samples of post-mortem tissues collected from
aborted equine fetuses provides an indispensable method to the veterinary diagnostic laboratory for making
a rapid preliminary diagnosis of herpesvirus abortion (9). Side-by-side comparisons of the
immunofluorescent and cell culture isolation techniques on more than 100 cases of equine abortion have
provided evidence that the diagnostic reliability of direct immunofluorescent staining of fetal tissues obtained
at necropsy approaches that of virus isolation attempts from the same tissues. In the United States of
America (USA), specific and potent polyclonal antiserum to EHV-1, prepared in swine and conjugated with
FITC, is provided to veterinary diagnostic laboratories for this purpose by the National Veterinary Services
Laboratories of the United States Department of Agriculture (USDA). The antiserum cross-reacts with EHV-4
and hence is not useful for serotyping. Freshly dissected samples (5 × 5 mm pieces) of fetal tissue (lung,
liver, thymus, and spleen) are frozen, sectioned on a cryostat at –20°C, mounted on to microscope slides,
and fixed with 100% acetone. After air-drying, the sections are incubated at 37°C in a humid atmosphere for
30 minutes with an appropriate dilution of the conjugated swine antibody to EHV-1. Unreacted antibody is
removed by two washes in PBS, and the tissue sections are then covered with aqueous mounting media
and
a cover-slip, and examined for fluorescent cells indicating the presence of EHV antigen. Each test should
include a positive and negative control consisting of sections from known EHV-1 infected and uninfected
fetal tissue.
e) Virus detection by immunoperoxidase staining
Enzyme immunohistochemical (IH) staining methods (e.g. immunoperoxidase) have been developed
recently as procedures for detecting EHV-1 antigen in paraffin-embedded tissues of aborted equine fetuses
or neurologically affected horses (12, 20). Such ancillary IH techniques for antigen detection may facilitate
identification of the virus in archival tissue samples or in clinical cases in which traditional laboratory
methods
for EHV-1 detection have been unsuccessful. Immunoenzymatic staining for EHV-1 is particularly useful for
the simultaneous evaluation of morphological lesions and the identification of the infectious agent.
Immunoperoxidase staining for EHV-1 or EHV-4 may also be done on infected cell monolayers (16).
Adequate controls must be included with each immunoperoxidase test run for evaluation of both the method
specificity and antibody specificity.
f) Virus detection by polymerase chain reaction
The PCR can be used for rapid amplification and diagnostic detection of nucleic acids of EHV-1 and -4 in
clinical specimens, paraffin-embedded archival tissue, or inoculated cell cultures (4, 10, 11, 17, 18). A variety
of type-specific PCR primers have been designed to distinguish between the presence of EHV-1 and EHV-4.
The correlation between PCR and virus isolation techniques for diagnosis of EHV-1 or EHV-4 is high (17).
Diagnosis of ER by PCR is rapid, sensitive, and does not depend on the presence of infectious virus in the
clinical sample. It now forms an integral part of a range of diagnostic tests currently available for ER, each
with its own advantages and limitations.
For diagnosis of active infection by EHV, PCR methods are most reliable with samples from aborted fetuses
or from nasopharyngeal swabs and peripheral blood leukocytes of foals and yearlings; they are most useful
in explosive epizootics of abortion or respiratory tract disease in which a rapid identification of the virus is
critical for guiding management strategies. PCR examinations of spinal cord and brain tissue, as well as
PBMC, are important in seeking a diagnosis on a horse with neurological signs. However, the interpretation
of the amplification by PCR of genomic fragments of EHV-1 or EHV-4 in lymph nodes or trigeminal ganglia
from adult horses is complicated by the high prevalence of latent EHV-1 and EHV-4 DNA in such tissues
(19).
A simple multiplex PCR assay for simultaneous detection of both EHV-1 and EHV-4 has been described
(18). A more sensitive protocol for nested PCR detection of EHV-1 or EHV-4 in clinical or pathological
specimens (nasal secretions, blood leukocytes, brain and spinal cord, fetal tissues, etc.) is described here
(4). This procedure has been used successfully; however, the technology in this area is changing rapidly and
other simpler more sensitive techniques are becoming available.
i) Prepare template DNA from test specimens: Following sample homogenisation and cell (and virion)
lysis in the presence of a chaotropic salt, nucleic acids bind selectively to silica or cationic resin
substrates. Substrate-bound nucleic acids are purified in a series of rapid wash steps followed by
recovery with low-salt elution. The reagents for performing such steps for rapid nucleic acid isolation
are available in kit format from a number of commercial sources (e.g. High Pure PCR Template
Preparation Kit, Roche Molecular Biochemicals, Indianapolis, USA; QIAamp DNA Kit, Qiagen,
Valencia, USA).
ii) Nested primer sequences specific for EHV-1 (Based on those described in reference 4):
BS-1-P1 = 5’-TCT-ACC-CCT-ACG-ACT-CCT-TC-3’ (917–936)
gB1-R-2 = 5’-ACG-CTG-TCG-ATG-TCG-TAA-AAC-CTG-AGA-G-3’ (2390–2363)
BS-1-P3 = 5’-CTT-TAG-CGG-TGA-TGT-GGA-AT-3’ (1377–1396)
gB1-R-a = 5’-AAG-TAG-CGC-TTC-TGA-TTG-AGG-3’ (2147–2127)
iii) Nested primer sequences specific for EHV-4 (4):
BS-4-P1 = 5’-TCT-ATT-GAG-TTT-GCT-ATG-CT-3’ (1705–1724)
BS-4-P2 = 5’-TCC-TGG TTG-TTA-TTG-GGT-AT-3’ (2656–2637)
BS-4-P3 = 5’-TGT-TTC-CGC-CAC-TCT-TGA-CG-3’ (1857–1876)
BS-4-P4 = 5’-ACT-GCC-TCT-CCC-ACC-TTA-CC-3’ (2456–2437)
iv) PCR conditions for first stage amplification: Specimen template DNA (1–2 ug in 2 μl) is added to a PCR
mixture (total volume of 50 μl) containing 1 × PCR buffer (50 mM KCl, 10 mM Tris/HCl, pH 9.0, 0.1%
Triton X-100), 200 μM of each deoxynucleotide triphosphate (dNTP), 2.5 mM MgCl2, 2.0 μM of each
outer primer (BS-1-P1 and gB1-R-2 for EHV-1 detection and, in a separate reaction mixture, BS-4-P1
and BS-4-P2 for EHV-4 detection) and 0.5 u Taq DNA polymerase. Cycling parameters are: initial
denaturation at 94°C for 4 minutes; 40 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C
for 90 seconds; with a final extension at 72°C for 10 minutes. Separate reaction mixtures containing
either known viral DNA or no DNA (water) should be prepared and amplified as positive and negative
controls.
v) PCR conditions for second stage (nested) amplification: Two μl of a 1/10 dilution of the first
amplification product is added to a fresh PCR mixture (total volume of 50 μl) containing 1 × PCR buffer,
200 μM of each dNTP, 2.5 mM MgCl2, 2.0 μM of each nested primer (BS-1-P3 and gB1-R-a for EHV-1
detection and, in a separate reaction mixture, BS-4-P3 and BS-4-P4 for EHV-4 detection) and 0.5 u
Taq DNA polymerase. Cycling parameters are: initial denaturation at 94°C for 4 minutes; 40 cycles of
94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute; with a final extension at 72°C for
10 minutes.
vi) Gel analysis of amplified products: 10 μl of each final amplified product, including controls, is mixed
with 2 μl of 6 × loading dye and electrophoresed on a 1.5% agarose gel in Tris/acetate or Tris-Borate
running buffer, along with a 100 base pairs (bp) DNA ladder. The gel is stained with ethidium bromide
and viewed by UV transillumination for amplified products of either 770 bp for EHV-1 or 580 bp for
EHV-4.
g) Histopathology
Histopathological examination of sections of formalin-fixed, paraffin-embedded tissues from aborted fetuses
or from neurologically affected horses is an essential part of the laboratory diagnosis of these two clinical
manifestations of ER. In aborted fetuses, typical herpetic intranuclear inclusion bodies present within
bronchiolar epithelium or in cells at the periphery of areas of hepatic necrosis are pathognomonic lesions for
EHV-1. The characteristic, but not pathognomonic, microscopic lesion associated with EHV-1 neuropathy is
a degenerative thrombotic vasculitis of small blood vessels in the brain or spinal cord (perivascular cuffing
and infiltration by inflammatory cells, endothelial proliferation and necrosis, and thrombus formation).
Because of the ubiquity of the viral agents of ER and the high seroprevalence among horses in most parts of
the
world, the demonstration of a negative antibody titre to EHV-1/4 by serological testing of horses designated
for
export is not part of present veterinary regulations that seek to prevent international spread of infectious
diseases
of horses. Serological testing can, however, be a useful adjunct procedure for assisting in the diagnosis of
ER in
horses. Serodiagnosis of ER is based on the demonstration of significant increases in antibody titres in
paired
sera taken during the acute and convalescent stages of the disease. The results of tests performed on sera
from
a single collection date are, in most cases, impossible to interpret with any degree of confidence. The initial
(acute
phase) serum sample should be taken as soon as possible after the onset of clinical signs, and the second
(convalescent phase) serum sample should be taken 2–4 weeks later. ‘Acute phase’ sera from mares after
abortion or from horses with EHV-1 neurological disease may already contain maximal titres of EHV-1
antibody,
with no increase in titres detectable in sera collected at later dates. In such cases, serological testing of
paired
serum samples from clinically unaffected cohort members of the herd for rising antibody titres against EHV-
1/4
may provide information useful for retrospective diagnosis of ER within the herd. Finally, the serological
detection
of antibodies to EHV-1 in heart or umbilical cord blood or other fluids of equine fetuses can be of diagnostic
value
in rare cases of virologically negative fetuses aborted as a result of EHV-1 infection; in some cases, the EHV
1/4
genome maybe identified from these tissues by PCR.
Serum antibody levels to EHV-1/4 may be determined by ELISA (8), virus neutralisation (VN) (15), or
complement
fixation (CF) tests (15). Because there are no internationally recognised reagents or standardised
techniques for
performing any of the serological tests for detection of EHV-1/4 antibody, antibody titre determinations on the
same serum may differ from one laboratory to another. Furthermore, all of the serological tests mentioned
detect
antibodies that are cross-reactive between EHV-1 and EHV-4. Nonetheless, the demonstration, by any of
the
tests, of a four-fold or greater rise in antibody titre to EHV-1 or EHV-4 during the course of a clinical illness
provides serological confirmation of recent infection with one of the viruses. The ELISA and CF test have the
advantage that they provide results faster and do not require cell culture facilities. Recently, a type-specific
ELISA
that can distinguish between antibodies to EHV-1 and EHV-4 was developed and made commercially
available
(6). The microneutralisation test is a widely used and sensitive serological assay for detecting EHV-1/4
antibody
and will thus be described here.
a) Virus neutralisation test
This serological test is most commonly performed in flat-bottom 96-well microtitre plates (tissue culture
grade) using a constant dose of virus and doubling dilutions of equine test sera. At least two replicate wells
for each serum dilution are required. Serum-free MEM is used throughout as a diluent. Virus stocks of
known
titre are diluted just before use to contain 100 TCID50 (50% tissue culture infective dose) in 25 μl.
Monolayers of E-Derm or RK-13 cells are monodispersed with EDTA/trypsin and resuspended at a
concentration of 5 × 105/ml. Note that RK-13 cells can be used with EHV-1 but do not give clear CPE with
EHV-4. Antibody positive and negative control equine sera and controls for cell viability, virus infectivity, and
test serum cytotoxicity, must be included in each assay. End-point VN titres of antibody are calculated by
determining the reciprocal of the highest serum dilution that protects 100% of the cell monolayer from virus
destruction in both of the replicate wells.
A suitable test procedure is as follows:
i) Inactivate test and control sera for 30 minutes in a water bath at 56°C.
ii) Add 25 μl of serum-free MEM to all wells of the microtitre assay plates.
iii) Pipette 25 μl of each test serum into duplicate wells of both rows A and B of the plate. The first row
serves as the serum toxicity control and the second row as the first dilution of the test. Make doubling
dilutions of each serum starting with row B and proceeding to the bottom of the plate by sequential
mixing and transfer of 25 μl to each subsequent row of wells. Six sera can be assayed in each plate.
iv) Add 25 μl of the appropriately diluted EHV-1 or EHV-4 virus stock to each well (100 TCID50/well) except
those of row A, which are the serum control wells for monitoring serum toxicity for the indicator cells.
Note that the final serum dilutions, after addition of virus, run from 1/4 to1/256.
v) A separate control plate should include titration of both a negative and positive horse serum of known
titre, cell control (no virus), virus control (no serum), and a virus titration to calculate the actual amount
of virus used in the test.
vi) Incubate the plates for 1 hour at 37°C in 5% CO2 atmosphere.
vii) Add 50 μl of the prepared E-Derm or RK-13 cell suspension (5 × 105 cells/ml) in MEM/10% FCS to
each well.
viii) Incubate the plates for 4–5 days at 37°C in an atmosphere of 5% CO2 in air.
ix) Examine the plates microscopically for CPE and record the results on a worksheet. Alternatively, the
cell monolayers can be scored for CPE after fixing and staining as follows: after removal of the culture
fluid, immerse the plates for 15 minutes in a solution containing 2 mg/ml crystal violet, 10% formalin,
45% methanol, and 45% water. Then, rinse the plates vigorously under a stream of running tap water.
x) Wells containing intact cell monolayers stain blue, while monolayers destroyed by virus do not stain.
Verify that the cell control, positive serum control, and serum cytotoxicity control wells stain blue, that
the virus control and negative serum control wells are not stained, and that the actual amount of virus
added to each well is between 101.5 and 102.5 TCID50. Wells are scored as positive for neutralisation of
virus if 100% of the cell monolayer remains intact. The highest dilution of serum resulting in complete
neutralisation of virus (no CPE) in both duplicate wells is the end-point titre for that serum.
xi) Calculate the neutralisation titre for each test serum, and compare acute and convalescent phase
serum titres from each animal for a four-fold or greater increase.
BIOLOGICALS
Both live attenuated and inactivated vaccines are available as licensed, commercially prepared products for
use
as prophylactic aids in reducing the burden of disease in horses caused by EHV-1/4 infection. Clinical
experience
has demonstrated that none of the vaccine preparations should be relied on to provide an absolute degree
of
protection from ER. Multiple doses, repeated annually, of each of the currently marketed ER vaccines are
recommended by their respective manufacturers, with vaccination schedules that vary with the particular
vaccine.
vaccine
At least sixteen vaccine products for ER, each containing different permutations of EHV-1, EHV-4, and the
two
subtypes of equine influenza virus, are currently marketed by five veterinary biologicals manufacturers.
The clinical indications stated on the product label for use of the several available vaccines for ER are either
herpesvirus-associated respiratory disease, abortion, or both. Only four vaccine products have met the
regulatory
requirements for claiming efficacy in providing protection from herpesvirus abortion as a result of successful
vaccination and challenge experiments in pregnant mares. None of the vaccine products has been
conclusively
demonstrated to prevent the occurrence of neurological disease sometimes associated with EHV-1 infection.
1. Seed management
a) Characteristics and culture
The master seed virus (MSV) for ER vaccines must be prepared from strains of EHV-1 and/or EHV-4 that
have been positively and unequivocally identified by both serological and genetic tests. Seed virus must be
propagated in a cell line approved for equine vaccine production by the appropriate regulatory agency. A
complete record of original source, passage history, medium used for propagation, etc., shall be kept for the
master seed preparations of both the virus(es) and cell stock(s) intended for use in vaccine production.
Permanently stored stocks of both MSV and master cell stock (MCS) used for vaccine production must be
demonstrated to be pure, safe and, in the case of MSV, also immunogenic. Generally, the fifth passage from
the MSV and the twentieth passage from the MCS are the highest allowed for vaccine production. Results of
all quality control tests on master seeds must be recorded and made a part of the licensee's permanent
records.
b) Validation as a vaccine
i) Purity
Tests for master seed purity include prescribed procedures that demonstrate the virus and cell seed
stocks to be free from bacteria, fungi, mycoplasmas, and extraneous viruses. Special tests must be
performed to confirm the absence of equine arteritis virus, equine infectious anaemia virus, equine
influenza virus, equine herpesvirus-2, -3, and -5, equine rhinovirus, the alphaviruses of equine
encephalomyelitis, bovine viral diarrhoea virus (BVDV – common contaminant of bovine serum), and
porcine parvovirus (PPV – potential contaminant of porcine trypsin). The purity check should also
include the exclusion of the presence of EHV-1 from EHV-4 MSV and vice versa.
ii) Safety
Samples of each lot of MSV to be used for preparation of live attenuated ER vaccines must be tested
for safety in horses determined to be susceptible to the virulent wild-type virus, including pregnant
mares in the last 4 months of gestation. Vaccine safety must be demonstrated in a ‘safety field trial’ in
horses of various ages from three different geographical areas. The safety trial should be conducted by
independent veterinarians using a prelicensing batch of vaccine. EHV-1 vaccines making a claim for
efficacy in controlling abortion must be tested for safety in a significant number of late gestation
pregnant mares, using the vaccination schedule that will be recommended by the manufacturer for the
final vaccine product.
iii) Immunogenicity
Tests for immunogenicity of the EHV-1/4 MSV stocks should be performed in horses on an
experimental test vaccine prepared from the highest passage level of the MSV allowed for use in
vaccine production. The test for MSV immunogenicity consists of vaccination of horses with low
antibody titres to EHV-1/4, with doses of the test vaccine that will be recommended on the final product
label. Second serum samples should be obtained and tested for significant increases in neutralising
antibody titre against the virus, 21 days after the final dose.
iv) Efficacy
An important part of the validation process is the capacity of a prelicensing lot of the ER vaccine to
provide a significant level of clinical protection in horses from the particular disease manifestation of
EHV-1/4 infection for which the vaccine is offered, when used under the conditions recommended by
the manufacturer's product label. Serological data are not acceptable for establishing the efficacy of
vaccines for ER. Efficacy studies must be designed to ensure appropriate randomisation of test animals
to treatment groups, blinding of the recording of clinical observations, and the use of sufficient numbers
of animals to permit statistical evaluation for effectiveness in prevention or reduction of the specified
clinical disease. The studies should be performed on fully formulated experimental vaccine products (a)
produced in accordance with, (b) at or below the minimum antigenic potency specified in, and, (c)
produced with the highest passage of MSV and MCS allowed by the approved ‘Outline of Production’
(see Section C.2). Vaccine efficacy is demonstrated by vaccinating a minimum of 20 EHV-1/4-
susceptible horses possessing serum neutralising antibody titres ≤32, followed by challenge of the
vaccinates and ten nonvaccinated control horses with virulent virus. A significant difference in the
clinical signs of ER must be demonstrated between vaccinates and nonvaccinated control horses. The
vaccination and challenge study must be performed on an identical number of pregnant mares and
scored for abortion if the vaccine product will make a label usage claim ‘for prevention of’ or ‘as an aid
in the prevention of’ abortion caused by EHV-1.
A detailed protocol of the methods of manufacture to be followed in the preparation of vaccines for ER must
be
compiled, approved, and filed as an Outline of Production with the appropriate licensing agency. Specifics of
the
methods of manufacture for ER vaccines will differ with the type (live or inactivated) and composition (EHV-1
only,
EHV-1 and EHV-4, EHV-4 and equine influenza viruses, etc.) of each individual product, and also with the
manufacturer.
Cells, virus, culture medium, and medium supplements of animal origin that are used for the preparation of
production lots of vaccine must be derived from bulk stocks that have passed the prescribed tests for
bacterial,
fungal, and mycoplasma sterility; nontumorgenicity; and absence of extraneous viral agents.
4. Batch control
Each bulk production lot of ER vaccine must pass tests for sterility, safety, and immunogenic potency.
a) Sterility
Samples taken from each batch of completed vaccine are tested for bacteria, fungi, and mycoplasma
contamination. Procedures to establish that the vaccine is free from extraneous viruses are also required;
such tests should include inoculation of cell cultures that allow detection of the common equine viruses, as
well as techniques for the detection of BVDV and PPV in ingredients of animal origin used in the production
of the batch of vaccine.
b) Safety
Tests to assure safety of each production batch of ER vaccine must demonstrate complete inactivation of
virus (for inactivated vaccines) as well as a level of residual virus-killing agent that does not exceed the
maximal allowable limit (e.g. 0.2% for formaldehyde). Safety testing in laboratory animals is also required.
c) Potency
Batch control of antigenic potency for EHV-1 vaccines only may be tested by measuring the ability of
dilutions of the vaccine to protect hamsters from challenge with a lethal dose of hamster-adapted EHV-1
virus. Although potency testing on production batches of ER vaccine may also be performed by vaccination
of susceptible horses followed by either viral challenge or assay for seroconversion, the recent availability of
virus type-specific MAbs has permitted development of less costly and more rapid in-vitro immunoassays for
antigenic potency. The basis for such in-vitro assays for ER vaccine potency is the determination, by use of
the specific MAb, of the presence of at least the minimal amount of viral antigen within each batch of vaccine
that correlates with the required level of protection (or seroconversion rate) in a standard animal test for
potency.
Tests to establish the duration of immunity to EHV-1/4 achieved by immunisation with each batch of vaccine
are not required. The results of many reported observations indicate that vaccination-induced immunity to
EHV-1/4 is not more than a few months in duration; these observations are reflected in the frequency of
revaccination recommended on ER vaccine product labels.
e) Stability
At least three production batches of vaccine should be tested for shelf life before reaching a conclusion on
the vaccine’s stability. When stored at 4°C, inactivated vaccine products generally maintain their original
antigenic potency for at least 1 year. Lyophilised preparations of the live virus vaccine are also stable during
storage for 1 year at 4°C. Following reconstitution, live virus vaccine is unstable and cannot be stored
without loss of potency.
Before release for labelling, packaging, and commercial distribution, randomly selected filled vials of the final
vaccine product must be tested by prescribed methods for freedom from contamination and safety in
laboratory
test animals.
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.
REFERENCES
1. ALLEN G.P. & BRYANS J.T. (1986). Molecular epidemiology, pathogenesis and prophylaxis of equine
herpesvirus-1 infections. In: Progress in Veterinary Microbiology and Immunology, Vol. 2, Pandey R., ed.
Karger, Basel, Switzerland & New York, USA, 78–144.
2. ALLEN G.P., KYDD J.H., SLATER J.D. & SMITH K.C. (1999). Recent advances in understanding the
pathogenesis, epidemiology, and immunological control of equid herpesvirus-1 (EHV-1) abortion. Equine
Infect. Dis., 8, 129–146.
3. ALLEN G.P., KYDD J.H., SLATER J.D. & SMITH K.C. (2004). Equid herpesvirus-1 (EHV-1) and -4 (EHV-4)
infections. In: Infectious Diseases of Livestock, Coetzer J.A.W., Thomson G.R. & Tustin R.C., eds. Oxford
University Press, Cape Town, South Africa.
4. BORCHERS K. & SLATER J. (1993). A nested PCR for the detection and differentiation of EHV-1 and EHV-4.
J. Virol. Methods, 45, 331–336.
5. BRYANS J.T. & ALLEN G.P. (1988). Herpesviral diseases of the horse. In: Herpesvirus Diseases of Animals,
Wittman G., ed. Kluwer, Boston, USA, 176–229.
6. CRABB B.S., MACPHERSON C.M., REUBEL G.H., BROWNING G.F., STUDDERT M.J. & DRUMMER H.E. (1995). A
type-specific serological test to distinguish antibodies to equine herpesviruses 4 and 1. Arch. Virol., 140,
245–258.
7. CRABB B.S. & STUDDERT M.J. (1995). Equine herpesviruses 4 (equine rhinopneumonitis virus) and 1
(equine
abortion virus). Adv. Virus Res., 45, 153–190.
8. DUTTA S.K., TALBOT N.C. & MYRUP A.C. (1983). Detection of equine herpesvirus-1 antigen and the specific
antibody by enzyme-linked immunosorbent assay. Am. J. Vet. Res., 44, 1930–1934.
9. GUNN H.M. (1992). A direct fluorescent antibody technique to diagnose abortion caused by equine
herpesvirus. Irish Vet. J., 44, 37–40.
10. LAWRENCE G.L., GILKERSON J., LOVE D.N., SABINE M. & WHALLEY J.M. (1994). Rapid, single-step
differentiation
of equid herpesvirus 1 and 4 from clinical material using the polymerase chain reaction and virus-specific
primers. J. Virol. Methods, 47, 59–72.
11. O’KEEFE J.S., JULIAN A., MORIARTY K., MURRAY A. & WILKS C.R. (1994). A comparison of the polymerase
chain
reaction with standard laboratory methods for the detection of EHV-1 and EHV-4 in archival tissue samples.
N.Z. Vet. J., 42, 93–96.
12. SCHULTHEISS P.C., COLLINS J.K. & CARMAN J. (1993). Use of an immunoperoxidase technique to detect
equine
herpesvirus-1 antigen in formalin-fixed paraffin-embedded equine fetal tissues. J. Vet. Diagn. Invest., 5, 12–
15.
13. TELFORD E.A.R., WATSON M.S., MCBRIDE K. & DAVISON A.J. (1992). The DNA sequence of equine
herpesvirus-1. Virology, 189, 304–316.
14. TELFORD E.A.R., WATSON M.S., PERRY J., CULLINANE A.A. & DAVISON A.J. (1998). The DNA sequence of
equine herpesvirus 4. J. Gen. Virol., 79, 1197–1203.
15. THOMSON G.R., MUMFORD J.A., CAMPBELL J., GRIFFITHS L. & CLAPHAM P. (1976). Serological detection of
equid
herpesvirus 1 infections of the respiratory tract. Equine Vet. J., 8, 58–65.
16. VAN MAANEN C., VREESWIJK J., MOONEN P., BRINKHOF J. DE BOER-LUIJTZE E. & TERPSTRA C. (2000).
Differentiation and genomic and antigenic variation among fetal, respiratory, and neurological isolates from
EHV1 and EHV4 infections in The Netherlands. Vet. Q., 22 (2): 88–93.
17. VARRASSO A., DYNON K., FICORILLI N., HARTLEY C.A., STUDDERT M.J. & DRUMMER H.E. (2001). Identification
of
equine herpesviruses 1 and 4 by polymerase chain reaction. Aust. Vet. J., 79, 563–569.
18. WAGNER W.N., BOGDAN J., HAINES D., TOWNSEND H.G.G. & MISRA V. (1992). Detection of equine
herpesvirus
and differentiation of equine herpesvirus type 1 from type 4 by the polymerase chain reaction. Can. J.
Microbiol., 38, 1193–1196.
19. WELCH H.M., BRIDGES C.G., LYON A.M., GRIFFITHS L. & EDINGTON N. (1992). Latent equid herpesviruses 1
and
4: detection and distinction using the polymerase chain reaction and cocultivation from lymphoid tissues. J.
Gen. Virol., 73, 261–268.
20. WHITWELL K.E., GOWER S.M. & SMITH K.C. (1992). An immunoperosidase method applied to the
diagnosis of
equine herpesvirus abortion, using conventional and rapid microwave techniques. Equine Vet. J., 24, 10–12.
*
**
NB: There are OIE Reference Laboratories for Equine rhinopneumonitis; see Table in Part 3 of this
Terrestrial
C H A P T E R 2 . 5 . 1 0 .
EQUINE VIRAL ARTERITIS
SUMMARY
Equine viral arteritis (EVA) is a contagious viral disease of equids caused by equine arteritis virus
(EAV), an RNA virus classified in the family Arteriviridae. Only one major serotype of the virus has
been identified so far. Equine arteritis virus is found in horse populations in many countries worldwide.
Although infrequently reported in the past, confirmed outbreaks of EVA appear to be on the
increase.
The majority of naturally acquired infections with EAV are subclinical. Where present, clinical signs
of EVA can vary in range and severity. The disease is characterised principally by fever,
depression, anorexia, dependent oedema, especially of the limbs, scrotum and prepuce in the
stallion, conjunctivitis, an urticarial-type skin reaction, abortion and, rarely, a fulminating pneumonia,
enteritis or pneumo-enteritis in young foals. Apart from mortality in young foals, the case-fatality rate
in outbreaks of EVA is very low. Affected horses almost invariably make complete clinical
recoveries. A long-term carrier state can occur in a variable percentage of infected stallions, but not
in mares, geldings or sexually immature colts.
Identification of the agent: EVA cannot be differentiated clinically from a number of other
respiratory and systemic equine diseases. Diagnosis of EAV infection is based on virus isolation,
detection of nucleic acid or viral antigen, or demonstration of a specific antibody response. Virus
isolation should be attempted from appropriate clinical or post-mortem specimens in rabbit, equine,
or monkey kidney cell culture. The identity of isolates of EAV should be confirmed by neutralisation
test, reverse-transcription polymerase chain reaction (RT-PCR) assay, or by immunocytochemical
methods, namely indirect immunofluorescence or avidin–biotin–peroxidase techniques.
Detection and identification of EAV nucleic acid in suspect cases of the disease can also be
attempted using the RT-PCR assay and appropriate viral-specific RNA primers.
Where mortality is associated with a suspected outbreak of EVA, a wide range of tissues should be
examined for histological evidence of panvasculitis that is especially pronounced in the small
arteries throughout the body. The characteristic vascular lesions present in the mature animal are
not a notable feature in EVA-related abortions. In such cases, equine arteritis viral antigens may be
visualised by immunohistochemical examination of placental and various fetal tissues.
Serological tests: A variety of serological tests, including virus neutralisation (VN), complement
fixation (CF), indirect fluorescent antibody, agar gel immunodiffusion, the enzyme-linked
immunosorbent assay (ELISA), and the fluorescent microsphere immunoassay assay (MIA) have
been used for the detection of antibody to EAV. The tests currently in widest use are the
complement-enhanced VN test and the ELISA. The VN test is a very sensitive and highly specific
assay of proven value in diagnosing acute infection and in seroprevalence studies. Several ELISAs
have been developed, none of which have been as extensively validated as the VN test though
some appear to offer comparable specificity and close to equivalent sensitivity. The CF test is less
sensitive than either procedure, but it can be used for diagnosing recent infection.
Requirements for vaccines and diagnostic biologicals: Two commercial tissue culture vaccines
are currently available against EVA. One is a modified live virus (MLV) vaccine prepared from virus
that has been attenuated for horses by multiple serial transfers in primary equine and rabbit cell
cultures. It has been shown to be safe and protective for stallions and nonpregnant mares.
Vaccination of foals under 6 weeks of age and of pregnant mares in the final 2 months of gestation
is contraindicated. There is no evidence of back reversion to virulence of the vaccine virus following
its use in the field over more than 20 years. The second vaccine is an inactivated, adjuvanted
product prepared from virus grown in equine cell culture that can be used in nonbreeding and
Chapter 2.5.10. - Equine viral arteritis
breeding horses. In the absence of appropriate safety data, the vaccine is not currently
recommended for use in pregnant mares.
A. INTRODUCTION
Equine viral arteritis (EVA) is a contagious viral disease of equids caused by equine arteritis virus (EAV), a
positive-sense, single-stranded RNA virus, and the prototype member of the genus Arterivirus, family
Arteriviridae,
order Nidovirales (10). Epizootic lymphangitis pinkeye, fièvre typhoide and rotlaufseuche are some of the
descriptive terms used in the past to refer to a disease that clinically very closely resembled EVA. The
natural host
range of EAV would appear to be restricted to equids, although very limited evidence would suggest it may
also
include new world camelids, viz. alpacas and llamas (63). The virus does not present a human health
hazard (59).
EAV is present in the horse population of many countries world-wide (59). There has been an increase in the
incidence of EVA in recent years that has been linked to the greater frequency of movement of horses and
use of
transported semen (2, 59).
While the majority of cases of acute infection with EAV are subclinical, certain strains of the virus can cause
disease of varying severity (59). Typical cases of EVA can present with all or any combination of the
following
clinical signs: fever, depression, anorexia, leukopenia, dependent oedema, especially of the limbs, scrotum
and
prepuce of the stallion, conjunctivitis, ocular discharge, supra or periorbital oedema, rhinitis, nasal discharge,
a
local or generalised urticarial skin reaction, abortion, stillbirths and, rarely, a fulminating pneumonia, enteritis
or
pneumo-enteritis in young foals. Regardless of the severity of clinical signs, affected horses almost
invariably
make complete recoveries. The case-fatality rate in outbreaks of EVA is very low; mortality is usually only
seen in
very young foals, particularly those congenitally infected with the virus (37, 59, 62), and very rarely in
otherwise
healthy adult horses.
EVA cannot be differentiated clinically from a number of other respiratory and systemic equine diseases, the
most
common of which are equine influenza, equine herpesvirus 1 and 4 infections, infection with equine rhinitis A
and
B viruses, equine adenoviruses and streptococcal infections, with particular reference to purpura
haemorrhagica.
The disease also has clinical similarities to equine infectious anaemia, African horse sickness fever, cases of
Hendra virus infection, Getah virus infection and toxicosis caused by hoary alyssum (Berteroa incana). After
infection, EAV replicates in macrophages and circulating monocytes (18) and is shed in various
secretions/excretions of acutely infected animals, in especially high concentration from the respiratory tract
(42).
A variable percentage of acutely infected stallions later become long-term carriers in the reproductive tract
and
constant semen shedders of the virus (59, 60). The carrier state, which has been shown to be androgen
dependant, has been found in the stallion, but not in the mare, gelding or sexually immature colt (59).
Unequivocal
evidence of the carrier state has only been found in stallions serologically positive for antibodies to the virus
(60).
While temporary down-regulation of circulating testosterone levels using a GnRH antagonist or by
immunisation
with GnRH would appear to have expedited clearance of the carrier state in some stallions, the efficacy of
either
treatment strategy has yet to be fully established. Concern has been expressed that such a therapeutic
approach
could be used to deliberately mask existence of the carrier state.
a) Virus isolation
Where an outbreak of EVA is suspected, or when attempting to confirm a case of subclinical EAV infection,
virus isolation should be attempted from nasopharyngeal and conjunctival swabs, unclotted blood samples,
and semen from stallions considered to be possible carriers of the virus (59). To optimise the chances of
virus isolation, the relevant specimens should be obtained as soon as possible after the onset of fever in
affected horses. There is evidence that heparin can inhibit the growth of EAV in rabbit kidney cells (RK-13
cell line) (1), and therefore, its use as an anticoagulant is contraindicated as it may interfere with isolation of
the virus from whole blood. Acid citrate dextrose or ethylenediaminetetraacetic acid (EDTA) are the
anticoagulants of choice to use in obtaining unclotted blood samples. Where EVA is suspected in cases of
mortality in young foals or older animals, isolation of EAV can be attempted from a variety of tissues,
especially the lymphatic glands associated with the alimentary tract and related organs, and also the lungs,
liver and spleen (42). In outbreaks of EVA-related abortion and/or cases of stillborn foals, placental and fetal
fluids and a wide range of placental, lymphoreticular and other fetal tissues (especially lung) can be
productive sources of virus (59).
Swabs for attempted isolation should be immersed in a suitable viral transport medium and these, together
with any fluids or tissues collected for virus isolation and/or reverse-transcription polymerase chain reaction
(RT-PCR) testing should be shipped either refrigerated or frozen in an insulated container to the laboratory,
preferably using an overnight delivery service. Unclotted blood samples must be transported refrigerated but
not frozen. Where possible, specimens should be submitted to a laboratory with established competency to
test for this infection.
Although reportedly not always successful in natural cases of EAV infection (43, 59), virus isolation should
be attempted from clinical specimens or necropsied tissues using rabbit, equine or monkey kidney cell
culture (43, 57, 59). Selected cell lines, e.g. RK-13 (ATCC CCL-37), LLC-MK2 (ATCC CCL-7), and primary
horse or rabbit kidney cell culture can be used, with RK-13 cells being the cell system of choice (57).
Experience over the years has shown that primary isolation of EAV from semen can present more difficulty
than from other clinical specimens or from infected tissues unless an appropriate cell culture system is used.
Several factors have been shown to influence primary isolation of EAV from semen in RK-13 cells (49).
Higher isolation rates have been obtained using 3–5-day-old monolayers, a large inoculum size in relation to
the cell surface area in the inoculated flasks or multiwell plates, and most importantly, the incorporation of
carboxymethyl cellulose (medium viscosity, 400–800 cps) in the overlay medium. It should be noted that
most RK-13 cells, including ATCC CCL-37, are contaminated with bovine viral diarrhoea virus, the presence
of which appears to enhance sensitivity of this cell system for the primary isolation of EAV, especially from
semen. There is considerable evidence to indicate that primary isolation rates of EAV may be increased by
using RK-13 cells of high passage history1 (57).
Inoculated cultures are examined daily for the appearance of viral cytopathic effect (CPE), which is usually
evident within 2–6 days. In the absence of visible CPE, culture supernatants should be subinoculated on to
confluent cell monolayers after 4–7 days. While the vast majority of isolations of EAV are made on the first
passage in cell culture, a small minority only become evident on the second or subsequent passages in vitro
(59, 60). The identity of isolates of EAV can be confirmed in a one-way neutralisation test, by standard
RTPCR
or real-time RT-PCR assay (2, 12, 51, 53) or by an immunocytochemical method (36), namely indirect
immunofluorescence (16) or the avidin–biotin–peroxidase (ABC) technique (36). A polyclonal rabbit
antiserum has been used to identify EAV in infected cell cultures. Mouse monoclonal antibodies (MAbs) to
the nucleocapsid protein (N) (2, 40) and major envelope glycoprotein (GP5) of EAV (2, 17) and a
monospecific rabbit antiserum to the unglycosylated envelope protein (M) (2, 39, 40) have also been
developed and these can detect various strains of the virus in RK-13 cells as early as 12–24 hours after
infection (2, 36).
• Virus isolation from semen (a prescribed test for international trade)
There is considerable evidence that short- and long-term carrier stallions shed EAV constantly in the semen,
but not in respiratory secretions or urine; nor has it been demonstrated in the buffy coat (peripheral blood
mononuclear cells) of such animals (59, 60). Stallions should first be blood tested using the virus
neutralisation (VN) test or an appropriately validated enzyme-linked immunosorbent assay (ELISA) or other
serological test procedure. Virus isolation should be attempted from the semen of stallions serologically
positive (titre ≥1/4) for antibodies to EAV that do not have a certified history of vaccination against EVA with
confirmation that they were serologically negative (titre <1/4) at time of initial vaccination. Virus isolation is
also indicated in the case of shipped semen where the serological status and possible vaccination history of
the donor stallion is not available. It is recommended that virus isolation from semen be attempted from two
samples, which can be collected on the same day, on consecutive days or after an interval of several days
or
weeks. There is no evidence that the outcome of attempted virus isolation from particular stallions is
influenced by the frequency of sampling, the interval between collections or time of the year. Isolation of EAV
should be carried out preferably on portion of an entire ejaculate collected using an artificial vagina or a
condom and a teaser or phantom mare. When it is not possible to obtain semen by this means, a less
preferable alternative is to collect a dismount sample at the time of breeding. Care should be taken to
ensure
that no antiseptics/disinfectants are used in the cleansing of the external genitalia of the stallion prior to
collection. Samples should contain the sperm-rich fraction of the ejaculate with which EAV is associated as
the virus is not present in the pre-sperm fraction of semen (59, 60). Immediately following collection, the
semen should be refrigerated on crushed ice or on freezer packs for transport to the laboratory with a
minimum of delay. Where there is likely to be a delay in submitting a specimen for testing, the semen can be
frozen at or below –20°C for a short period before being dispatched to the laboratory. Freezing a sample has
not been found to militate against isolation of EAV from the semen of a carrier stallion. In situations where it
is not feasible to determine the carrier status of a stallion by virus isolation or RT-PCR procedures, the
stallion can be test bred to two seronegative mares, which are checked for seroconversion to the virus
28 days after breeding (59).
i) On receipt in the laboratory, it should be noted whether each semen sample is frozen, chilled or at
ambient temperature. Every sample should be checked to ensure that it contains the sperm-rich
1 Such a line (RK-13-KY) is available from Dr P.J. Timoney, Maxwell H. Gluck Equine Research Center, University of
Kentucky, Lexington, Kentucky 40546-0099, United States of America (E-mail address: ptimoney@uky.edu).
fraction of the ejaculate. This can be established by microscopic examination of a wet-mount
preparation of a sample. Additionally, specimens of ejaculate should be visually inspected for colour
and presence of gross particulate contamination. If a semen specimen is contaminated with blood,
which can result from trauma to the external genitalia of the stallion at time of collection, a repeat
sample should be requested as testing such a specimen from a serologically positive stallion may
compromise the reliability of the virus isolation result.
ii) Although no longer considered an essential step, pretreatment of semen before inoculation into cell
culture by short-term sonication (for three 15-second cycles); facilitates effective mixing and dispersion
of a sample.
iii) After removal of culture medium, 3–5-day-old confluent monolayer cultures of RK-13 cells, either in
25 cm2 tissue culture flasks or multiwell plates, are inoculated with serial decimal dilutions (10–1–10–3) of
seminal plasma in tissue culture maintenance medium containing 2% fetal bovine serum and antibiotics.
An inoculum of 1 ml per 25 cm2 flask is used and no fewer than two flasks per dilution of seminal plasma
are inoculated. Inoculum size and number of wells inoculated per dilution of a specimen should be prorated
where multiwell plates are used. Appropriate dilutions of a virus positive control semen sample or
virus control of known titre diluted in culture medium should be included in each test.
iv) The flasks are closed, lids replaced on multiwell plates and inoculated cultures gently rotated to disperse
the inoculum over the cell monolayers.
v) Inoculated cultures are then incubated for 1 hour at 37°C either in an aerobic incubator or an incubator
containing a humidified atmosphere of 5% CO2 in air, depending on whether flasks or multiwell plates are
used.
vi) Without removing any of the inoculum or washing the cell monolayers, the latter are overlaid with 0.75%
carboxymethyl cellulose containing medium with antibiotics.
vii) The flasks or plates are reincubated at 37°C and checked microscopically for viral CPE, which is usually
evident within 2–6 days.
viii) In the absence of visible CPE, culture supernatants are subinoculated onto 3–5 day-old confluent cell
monolayer cultures of RK-13 cells after 5–7 days. After removal of the overlay medium, monolayers are
stained with 0.1% formalin-buffered crystal violet solution.
The identity of any virus isolates should be confirmed by VN, immunofluorescence (16) or ABC technique,
using a monospecific antiserum to EAV or MAbs to the structural proteins, N or GP5 of the virus (2, 18, 36,
37), or by standard RT-PCR (2, 6, 12, 27, 51, 53) or real-time RT-PCR assay (5, 38, 64).
In the one-way neutralisation test, serial decimal dilutions of the virus isolate are tested against a
neutralising
MAb or monospecific antiserum prepared against the prototype Bucyrus strain of EAV (ATCC VR 796) and
also a serum negative for neutralising antibodies to the virus. Corresponding titrations of the prototype
Bucyrus virus with the same reference antibody reagents are included as test controls. The test is performed
in either 25 cm2 tissue culture flasks or multiwell plates. Appropriate quantities of the known EAV positive
and negative antibody reagents are inactivated for 30 minutes in a water bath at 56°C and diluted 1/4 in
phosphate buffered saline, pH 7.2; then 0.3 ml of diluted antibody reagent is dispensed into five tubes for
each virus isolate to be tested. Serial decimal dilutions (10–1–10–5) of each virus are made in Eagles
Minimal
Essential Medium containing 10% fetal bovine serum, antibiotics and 10% freshly diluted guinea-pig
complement. Then, 0.3 ml of each virus dilution is added to the tubes containing positive and negative
antibody reagents. The tubes are shaken and the virus/antibody mixtures are incubated for 1 hour at 37°C.
The mixtures are then inoculated onto 3–5-day-old confluent monolayer cultures of RK-13 cells, either in
25 cm2 flasks or multiwell plates, using two flasks or wells per virus dilution. Each flask is inoculated with
0.25 ml of virus/antibody mixture; the inoculum size is pro-rated where multiwell plates are used. Inoculated
flasks or plates are incubated for 2 hours at 37°C, gently rocking after 1 hour to disperse the inoculum over
the cell monolayers. Without removing any of the inoculum or washing the cell monolayers, the latter are
overlaid with 0.75% carboxymethyl cellulose containing medium and incubated for 4–5 days at 37°C, either
in an aerobic incubator or an incubator containing a humidified atmosphere of 5% CO2 in air. After removal
of the medium, monolayers are stained with 0.1% formalin-buffered crystal violet solution. Plaques are
counted and the virus infectivity titre is determined both in the presence and absence of EAV antibodies
using the Spearman–Kärber method (34). Confirmation of the identity of an isolate is based on a reduction in
plaque count of at least 102 logs of virus in the presence of antibody positive serum against the Bucyrus
strain of EAV.
The vast majority of EAV isolates from carrier stallions are made in the first passage in cell culture using the
described test procedure (59, 60). The occurrence of nonviral cytotoxicity or bacterial contamination of
specimens is not considered significant problems when attempting isolation of this virus from stallion semen.
Nonviral cytotoxicity, if observed, usually affects monolayers inoculated with the 10–1 and, much less
frequently, the 10–2 dilution of seminal plasma. Treatment of seminal plasma with polyethylene glycol (Mol.
wt 6000) prior to inoculation has been used with some success in overcoming this problem (25). The method
described involves the addition of polyethylene glycol to the 10–1–10–3 dilutions of seminal plasma to give a
final concentration of 10% in each dilution. The mixtures are held overnight at 4°C with gentle stirring, after
which they are centrifuged at 2000 g for 30 minutes and the supernatants are discarded. The precipitates
are suspended in cell culture maintenance medium to one-tenth the volume of the original dilutions and the
mixtures are homogenised. They are then centrifuged at 2000 g for 30 minutes and the supernatants are
taken off and used for inoculation. There is no evidence to indicate that pretreatment of seminal plasma in
this manner reduces sensitivity of the virus isolation procedure (25). Where bacterial contamination of a
sample is a problem, it is preferable to request a repeat semen collection from the individual stallion. If this is
not possible, an attempt can be made to control the contamination by pre-treatment of the sample with
antibiotic containing viral transport medium, holding overnight at 4°C followed by ultracentrifugation and
resuspension of the pellet before diluting and inoculating the specimen into cell culture.
The presence of anti-EAV antibody activity in the seminal plasma of certain virus-shedding stallions has not
been found to prevent detection of the carrier state in these animals.
b) Nucleic acid recognition methods
The standard two-step RT-PCR, single-step RT-PCR and real-time RT-PCR (rRT-PCR) assays are gaining
greater acceptance and being more widely used as an alternative to virus isolation in cell culture for the
detection of EAV in diagnostic materials. The RT-PCR-based assays provide a means of identifying
virusspecific
RNA in clinical specimens, namely nasopharyngeal swab filtrates, buffy coats, raw and extended
semen and urine, and in post-mortem tissue samples (5, 6, 27, 55, 56, 64). Standard two-step RT-PCR,
single-step RT-PCR, RT-nested PCR (RT-nPCR), and one tube TaqMan® rRT-PCR assays have been
developed and evaluated for the detection of various strains of the virus in tissue culture fluid, semen and
nasal secretions (5, 6, 12, 27, 38, 50, 51, 53, 55, 56, 64, 66). These assays targeted six different open
reading frames (ORFs) in the EAV genome (ORFs 1b, 3–7). However, there is considerable variation in the
sensitivity and specificity among RT-PCR assays incorporating different primer pairs targeting various ORFs
(6). Results comparable to virus isolation have been obtained with some but not all standard single-step
RTPCR,
two-step RT-PCR, RT-nPCR or one tube TaqMan® rRT-PCR assays (5, 6, 9, 27, 38, 51, 53, 56, 66).
Compared with traditional virus isolation, these RT-PCR-based assays are frequently more sensitive, less
expensive and considerably more rapid to perform, the majority taking less than 24 hours to complete. In
addition, RT-PCR assays have the advantage of not requiring viable virus for performance of the test. The
one-tube rRT-PCR assay for EAV provides a simple, rapid and reliable method for the detection and
identification of viral nucleic acid in equine semen and tissue culture fluid (5, 38). The one tube rRT-PCR has
the following important advantages over the standard two-step RT-PCR: 1) eliminating the possibility of
cross contamination between samples with previously amplified products as the sample tube is never
opened; and 2) reducing the chance of false-positive reactions because the rRT-PCR product is detected
with a sequence-specific probe. Because of the high sensitivity of the RT-PCR assay, however, and in the
absence of appropriate safeguards in the laboratory, there is the potential for cross-contamination between
samples, giving rise to false-positive results. For example, the RT-nPCR assay, while it provides enhanced
sensitivity for the detection of EAV, it also increases the likelihood of false-positive results (6). The risk of
cross-contamination is greater using the RT-nPCR assay because of the second PCR amplification step
involving the product from the first RT-PCR reaction. To minimise the risk of cross-contamination,
considerable care needs to be taken, especially during the steps of RNA extraction and reaction setup.
Relevant EAV positive and negative template controls and, where appropriate, nucleic acid extracted from
the tissue culture fluid of uninfected cells, need to be included in each RT-PCR assay. Thus, in many
circumstances, use of the single-step RT-PCR or the one tube rRT-PCR assay would largely circumvent the
problems associated with cross contamination.
Primer selection is critical to the sensitivity of the RT-PCR assay with primers (and probe in the case of the
rRT-PCR assay) preferably designed from the most conserved region(s) of the EAV genome. Comparative
nucleotide sequence analysis has shown that ORF 1b (encodes the viral polymerase), ORF 6 (M protein)
and 7 (N protein) are more conserved than the other ORFs among EAV strains so far analysed from North
America and Europe (3, 4, 38, 64). The most conserved gene among different strains of EAV is ORF7 and
primers specific for ORF7 (and probe for rRT-PCR) have detected a diversity of strains of the virus of
European and North American origin (5, 6, 38, 53, 56). Furthermore, the use of multiple primer pairs specific
for different ORFs 1b ([forward: 5’-GAT-GTC-TAT-GCT-CCA-TCA-TT-3’ and reverse: 5’-GGC-GTA-
GGCTCC-
AAT-TGA-A-3’]) (12) and/or [forward: 5’-CCT-GAG-ACA-CTG-AGT-CGC-GT-3’ and reverse 5’-CCTGAT-
GCC-ACA-TGG-AAT-GA-3’]) (27), ORF 6 ([forward: 5’-CTG-AGG-TAT-GGG-AGC-CAT-AG-3’ and
reverse: 5’-GCA-GCC-AAA-AGC-ACA-AAA-GC-3’]) (51) and ORF 7 ([forward 5’-ATG-GCG-TCA-AGA-
CGATCA-
CG-3’ and reverse 5’-AGA-ATA-TCC-ACG-TCT-TAC-GGC-3’]) (53) markedly increases the likelihood
of detecting North American and European strains of EAV in the RT-PCR assay. The two primer pairs
specific for ORF 1b are suitable for use in the RT-nPCR assay (6, 12, 27). While the RT-PCR has been
found to be highly sensitive for viral nucleic acid detection in raw semen, there is evidence to that it is not of
equivalent reliability when testing extended or cryopreserved semen of very low virus infectivity (66).
In addition to the foregoing RT-PCR assays, 2 TaqMan® fluorogenic probe-based one-tube rRT-PCR
assays have been described for the detection of EAV nucleic acid (5); primers ([forward: 5’-GGC-GAC-
AGCCTA-
CAA-GCT-ACA-3’, reverse: 5’-CGG-CAT-CTG-CAG-TGA-GTG-A-3’] and probe [5’FAM-TTG-CGGACC-
CGC-ATC-TGA-CCA-A-TAMRA-3’] and (64); primers [forward: 5’-GTA-CAC-CGC-AGT-TGG-TAA-CA-
3’, reverse: 5’-ACT-TCA-ACA-TGA-CGC-CAC-AC-3’] and probe [5’FAM-TGG-TTC-ACT-CAC-TGC-
AGATGC-
CGG-TAMRA-3’]). It should be noted, however, that genomic variation among field isolates of EAV
could reduce the sensitivity of both RT-PCR and rRT-PCR assays, even when the primers and probe are
based on the most conserved region of the EAV genome (ORF 7 [38]).
In the absence of general agreement on a complete consensus or universal primer set for EAV, and since no
RT-PCR assay can determine the actual infectivity of a sample, there is a value to performing virus isolation
in conjunction with RT-PCR or rRT-PCR for the identification of virus in clinical or post-mortem specimens.
Strains of EAV isolated from different regions of the world have been classified into different phylogenetic
groups by sequence analysis of the GP3, GP5 and M envelope protein genes (ORFs 3, 5 & 6 respectively)
and the nucleocapsid (N) protein gene (ORF 7 [2, 8, 13, 54, 65]). The GP5 gene has been found to be most
useful and reliable for this purpose (8, 54, 65). The relationships between strains demonstrated by
nucleotide
sequencing are a useful molecular epidemiological tool for tracing the origin of outbreaks of EVA (2, 4, 65).
c) Histopathological and immunohistochemical methods
Where mortality is associated with a suspected outbreak of EVA, a wide range of tissues should be
examined for histological evidence of panvasculitis that is especially pronounced in the small arteries
throughout the body, particularly in the caecum, colon, spleen, associated lymphatic glands and adrenal
cortex (16, 18, 33). The presence of a disseminated necrotising arteritis involving endothelial and medial
cells of affected vessels is considered to be pathognomonic of EVA. The characteristic vascular lesions
present in the mature animal are not, however, a prominent a feature in many cases of EAV-related abortion
(32).
EAV antigen can be identified in various tissues of EVA-affected animals either in the presence or absence
of lesions (18). Antigen has been demonstrated in lung, heart, liver and spleen and the placenta of aborted
fetuses (18, 56). Immunohistochemical examination of biopsied skin specimens has also been investigated
as a means of confirming acute EAV infection. Though of some value, it is not entirely reliable for the
diagnosis of the disease. Viral antigen can be detected within the cytoplasm of infected cells by
immunofluorescence using conjugated equine polyclonal anti-EAV serum (16), or by the ABC technique
using mouse MAbs to the GP5 (28) or N proteins of the virus (18, 37, 40, 56).
A variety of serological tests including neutralisation (microneutralisation [52] and plaque reduction [41]
(VN)), the
complement fixation (CF) test (22), the indirect fluorescent antibody test (16), the agar gel immunodiffusion
(16),
the ELISA (11, 14, 30, 31, 35, 48) and the fluorescent microsphere immunoassay (MIA) (Go, pers. comm.)
have
been used to detect antibody to EAV.
The test currently in widest international use to diagnose infection, carry out seroprevalence studies, and
test
horses for export, is a microneutralisation test in the presence of complement. It has also been used to
screen
fetal heart blood for the retrospective diagnosis of cases of EVA-related abortion (56). Apart from the VN
test, the
CF test has been used for diagnosing recent EAV infection as complement-fixing antibodies are relatively
shortlived
in duration (22). In contrast, neutralising antibody titres to EAV frequently persist for several years after
natural infection (59). Although a number of ELISAs have been developed (11, 14, 30, 31, 35, 48), none has
as
yet been as extensively validated as the VN, though some appear to offer nearly comparable sensitivity and
specificity (11, 30, 31, 48). Unlike the VN test, a positive reaction in the ELISA is not necessarily reflective of
the
protective immune status of an individual horse to EAV as both non-neutralising and neutralising antibodies
are
involved.
Antiserum to unpurified EAV has been prepared in horses and in rabbits using conventional immunisation
protocols. Also, mouse monoclonal and monospecific rabbit antibodies have been developed to the
nucleocapsid
protein (N) major envelope glycoprotein (GP5), and unglycosylated envelope protein (M) of EAV (7, 17, 28,
39,
40).
OIE Standard Sera for EAV are available2 and these can facilitate international standardisation of the
microneutralisation test and ELISA.
2 Available from Dr P.J. Timoney, Maxwell H. Gluck Equine Research Center, Dept of Veterinary Science, University of
Kentucky, Lexington, Kentucky 40546-0099, United States of America; E-mail address: ptimoney@uky.edu.
Only one major serotype of EAV has been recognised so far (41, 59). This is represented by the prototype
Bucyrus strain (ATCC VR 796). The reference virus used in the EAV VN test is the CVL-Bucyrus
(Weybridge)
strain. Virus stock is grown in the RK-13 cell line, clarified of cellular debris by low-speed centrifugation and
stored
in aliquots at –70°C. Several frozen aliquots are thawed and the infectivity of the stock virus is determined
by
titration in RK-13 cells.
a) Virus neutralisation (a prescribed test for international trade)
The VN test is used to screen stallions for evidence of EAV infection and to determine whether there is a
need to attempt virus detection in semen using cell culture or RT-PCR assay. It is also used for diagnostic
purposes to confirm infection in suspect cases of EVA. The VN test procedure in current widest use is that
developed by the National Veterinary Service Laboratories of the United States Department of Agriculture
(52). It is important to obtain a sterile blood sample as bacterial contamination of serum can interfere with
the
test result. It is recommended that the test be carried out in RK-13 cells using the approved CVL-Bucyrus
(Weybridge) strain of EAV as reference virus3 (20). Although originally derived from the prototype Bucyrus
virus, the passage history of the CVL (Weybridge) strain is not fully documented. The sensitivity of the VN
test for detection of antibodies to EAV can be significantly influenced by several factors, especially the
source and passage history of the strain of virus used (20, 21). The CVL-Bucyrus (Weybridge) strain and the
highly attenuated MLV vaccine strain of EAV are of comparable sensitivity for detecting low-titred positive
sera, especially from EVA-vaccinated horses. Efforts are continuing to bring about greater uniformity in the
testing protocol and serological results among laboratories providing the VN or other comparable serological
assays for this infection.
i) Sera are inactivated for 30 minutes in a water bath at 56°C (control sera, only once).
ii) Serial twofold dilutions of the inactivated test sera in serum-free cell culture medium (25 μl volumes)
are made in a 96-well, flat-bottomed, cell-culture grade microtitre plate starting at a 1/2 serum dilution
and using duplicate rows of wells for each serum to be tested. Most sera are screened initially at a 1/4
and 1/8 serum dilution (i.e. final serum dilution after addition of an equal volume of the appropriate
dilution of stock virus to each well). Positive samples at the 1/8 dilution can, if desired, be retested and
titrated out for end-point determination. Individual serum controls, together with negative and known
low- and high-titred positive control sera must also be included in each test.
iii) A dilution of stock virus to contain from 100 to 300 TCID50 (50% tissue culture infective dose) per 25 μl
is prepared using as diluent, serum-free cell culture medium containing antibiotics and fresh guinea-pig
or rabbit complement at a final concentration of 10%.
iv) 25 μl of the appropriate dilution of stock virus is added to every well containing 25 μl of each serum
dilution, except the test serum control wells.
v) A virus back titration of the working dilution of stock virus is included, using four wells per tenfold
dilution, to confirm the validity of the test results.
vi) The plates are covered and shaken gently to facilitate mixing of the serum/virus mixtures.
vii) The plates are incubated for 1 hour at 37°C in a humid atmosphere of 5% CO2 in air.
viii) A suspension of cells from 3–5-day-old cultures of RK-13 cells are prepared using a concentration that
will ensure confluent monolayers in the microtitre plate wells within 18–24 hours after seeding.
ix) 100 μl of cell suspension is added to every well, the plates covered with plate lids or sealed with tape
and shaken gently.
x) The plates are incubated at 37°C in a humid atmosphere of 5% CO2 in air.
xi) The plates are read microscopically for nonviral CPE after 12–18 hours and again for viral CPE after
48–72 hours’ incubation. The validity of the test is confirmed by establishing that the working dilution of
stock virus contained 30–300 TCID50 virus and that the titres of the positive serum controls are within
0.3 log10 units of their predetermined titres.
A serum dilution is considered to be positive if there is an estimated 75% or preferably a 100% reduction in
the amount of viral CPE in the serum test wells compared with that present in the wells of the lowest virus
control dilution. End-points are then calculated using the Spearman–Kärber method (34). A titre of 1/4 or
greater is considered to be positive. A negative serum should only have a trace (less than 25%) or no virus
neutralisation at the lowest dilution tested. Antibody titres may, on occasion, be difficult to define as partial
neutralisation may be observed over a range of several serum dilutions. Infrequently, sera will be
3 Available from: Dr T.W. Drew, Mammalian Virology Department, Veterinary Laboratories Agency, Weybridge, New Haw,
Addlestone KT15 3NB, United Kingdom; E-mail address: tdrew.vla@gfnet.gov.uk.
encountered that cause toxic changes in the lower dilutions tested. In such cases it may not be possible to
establish whether the sample is negative or a low-titred positive. The problem may be overcome by testing
another serum sample from the animal in question or by retesting the toxic sample using microtitre plates
with confluent monolayers of RK-13 cells that had been seeded the previous day. It has been reported that
the toxicity of the serum can, in some cases, be reduced or eliminated if the sample is adsorbed with packed
RK-13 cells prior to testing or by substituting rabbit in place of guinea-pig complement in the virus diluent.
Vaccination status for equine herpesviruses should be considered when evaluating sera causing non-viral
cytotoxicity. One of the equine herpesvirus vaccines currently available in Europe has been shown to
stimulate antibodies to rabbit kidney cells used in the vaccine production. These, in turn, can give rise to
cytotoxicity, usually in the 1/4 and/or 1/8 serum dilutions and cause difficulties in interpretation of the test
results (26, 47).
b) Enzyme-linked immunosorbent assay
A number of direct or indirect ELISAs have been developed for the detection of antibodies to EAV (11, 14,
30, 31, 35, 48). These have been based on the use of purified virus or recombinant-derived viral antigens.
The usefulness of earlier assays was compromised by the frequency of false-positive reactions (15). The
latter were associated with the presence of antibodies to various tissue culture antigens in the sera of horses
that had been vaccinated with tissue-culture-derived antigens (15). Identification of the importance of the
viral GP5 protein in stimulation of the humoral antibody response to EAV led to the development of several
ELISAs that employ a portion of, or the entire recombinant protein produced in a bacterial or baculovirus
expression system (14, 17, 30). Most recently, an ovalbumin-conjugated synthetic peptide representing
amino acids 81–106 of the GP5 protein has been used (48). Some of these assays appear to offer nearly
comparable sensitivity and specificity to the VN test and may detect EAV-specific antibodies prior to a
positive reaction being obtainable in the VN test (11). False-negative reactions can occur, however, with
some of these assays. Screening a random peptide-phage library with polyclonal sera from EAV-infected
horses led to the identification of ligands, which were purified and used as antigen in an ELISA for EAV (31).
No correlation was found, however, between absorbency values obtained with this assay and neutralising
antibody titres, indicating that the antibodies being detected were largely against nonsurface epitopes of the
virus. An ELISA based on the use of a combination of the GP5, M or N structural proteins of EAV expressed
from recombinant baculoviruses successfully detected viral antibody in naturally or experimentally infected
horses but not in EVA-vaccinated animals (30). Of major importance with respect to any GP5 protein-based
ELISA for EAV is the fact that test sensitivity will vary depending on the ectodomain sequence(s) of this viral
protein used in the assay. Considerable amino acid sequence variation within this domain has been found
between isolates of EAV (7). To maximise sensitivity of a GP5-based ELISA, it may be necessary to include
multiple ectodomain sequences representative of known phenotypically different isolates of EAV rather than
depend on a single ectodomain sequence. Two more recently described ELISAs appear to offer most
promise as reliable serodiagnostic tests for EAV infection (14, 48). A blocking ELISA involving MAbs
produced against the GP5 protein was reported to have a sensitivity of 99.4% and a specificity of 97.7%
compared with the VN test (14). Another assay, a GP5 ovalbumin-conjugated synthetic peptide ELISA was
shown to have a sensitivity and specificity of 96.75% and 95.6%, respectively, using a panel of 400 VN
positive sera and 400 VN negative samples (48). It is expected that an ELISA will soon be available, having
very similar if not equivalent sensitivity and specificity to the VN test.
BIOLOGICALS
A number of experimental and commercial vaccines have been developed against EVA. Currently, there are
two
commercially available vaccines, both tissue-culture derived. The first is a modified live virus (MLV) vaccine
prepared from virus that has been attenuated for horses by multiple serial transfers in equine and rabbit cell
cultures (19, 41). This vaccine is licensed for use in stallions, nonpregnant mares and in nonbreeding
horses.
Whereas nonbreeding horses can be vaccinated at any time, stallions and mares should be vaccinated not
less
than 3 weeks prior to breeding. The vaccine is not recommended for use in pregnant mares, especially in
the last
2 months of gestation, nor in foals under 6 weeks of age unless in the face of significant risk of exposure to
natural infection. The vaccine is commercially available in the USA and Canada. It has also been used in
New
Zealand, subject to ministerial controls, to aid in that country’s EVA eradication programme.
The second commercially available vaccine against EVA is an inactivated product prepared from virus grown
in
equine cell culture, which is filtered, chemically inactivated and then combined with a metabolisable
adjuvant. This
vaccine is licensed for use in nonbreeding and breeding horses. In the absence of appropriate safety data,
the
vaccine is currently not recommended for use in pregnant mares. The initial vaccination regimen involves
two
doses of vaccine administered intramuscularly 3–6 weeks apart. Booster vaccination at 6-month intervals is
recommended by the manufacturer. The inactivated vaccine is licensed for commercial use in certain
European
countries, including Denmark, France, Germany, Hungary, Ireland, Sweden and the United Kingdom.
An additional inactivated vaccine against EVA has been developed in Japan for use should an outbreak of
EVA
occur in that country (24). It is an aqueous formalin-inactivated vaccine that has been shown to be safe and
effective for use in nonbreeding and breeding horses. For optimal immunisation with this vaccine, horses
require a
primary course of two injections given at an interval of 4 weeks, with a booster dose administered every 6–
12 months. As the vaccine is currently not commercially available, no details can be provided on its
production.
vaccine
1. Seed management
Both MLV and inactivated commercial vaccines are derived from the prototype Bucyrus strain of EAV (ATCC
VR 796). Available evidence points to the existence of only one major serotype of the virus, and strain
variation is not considered to be of significance in relation to vaccine efficacy (41, 59).
In the case of the MLV vaccine, the prototype virus was attenuated by serial passage in primary cultures of
horse kidney (HK-131), rabbit kidney (RK-111), and a diploid equine dermal cell line, ATCC CCL57 (ECID-
24) (19, 29, 41). The indications from the use of this vaccine are that the virus is safe and immunogenic
between its 80th and 111th passage in primary rabbit kidney (19, 29, 41, 44, 45, 61).
The inactivated adjuvanted vaccine is prepared from the unattenuated prototype Bucyrus strain of EAV
(ATCC VR 796) that has been plaque purified and in its fourth serial passage in the diploid equine dermal
cell line (ECID-4). After growth in cell culture, the virus is then purified by filtration before being chemically
inactivated and adjuvanted.
Suitable lots of master seed virus for each vaccine should be maintained in liquid nitrogen or its equivalent.
The virus for both MLV and inactivated vaccines should be grown in a stable cell culture system, such as
equine dermal cells, using an appropriate medium supplemented with sterile bovine serum or bovine serum
albumin as replacement for bovine serum in the growth medium. Cell monolayers should be washed prior to
virus inoculation to remove traces of bovine serum. Extensive virus growth as evidenced by the appearance
of cytopathic changes in 80–100% of the cells should be obtained within 2–3 days.
In the case of both MLV and inactivated vaccines, the respective virus strains should be grown in an
appropriate cell culture system that has been officially approved for vaccine production and confirmed to be
free from extraneous bacteria, fungi, mycoplasmas and viruses (46). The identity of the vaccine virus in the
master seed should be confirmed by neutralisation with homologous anti-EAV serum. Incomplete
neutralisation of EAV by homologous horse or rabbit antisera has been scientifically documented (46, 52)
and is a problem when screening master seed virus for extraneous viruses and when attempting to confirm
the identity of the vaccine virus. The problem has been circumvented by reducing the infectivity titre of the
master seed virus below that required for seed virus production before conducting a neutralisation test on
the
diluted virus. Virus/serum mixtures are tested for residual live virus by serial passage in cell culture. No
evidence of cytopathic viruses, haemadsorbing viruses, or noncytopathic strains of bovine virus diarrhoea
virus should be found, based on attempted virus isolation in cell culture. If cells of equine origin are used,
they should be confirmed to be free from equine infectious anaemia virus. The newer technologies of PCR
and antigen-capture ELISA may be used as adjuncts to virus isolation in screening for adventitious agents.
The MLV vaccine has been shown to be both safe and effective for use in stallions and nonpregnant mares
(44, 45, 61). Although not recommended by the manufacturer for pregnant mares, especially in the last
2 months of gestation, the vaccine has been used to immunise pregnant mares in the face of high risk of
natural exposure to EAV, with minimal, if any, reported adverse effects. Vaccination confers a high level of
protective immunity that persists for at least several years (29, 41, 59). Based on experimental studies and
extensive field use of the vaccine since 1985, there is no evidence of back reversion to virulence of the
vaccine virus, nor of recombination of the vaccine virus with naturally occurring strains of EAV. Furthermore,
there is no confirmed evidence that the attenuated strain of EAV in the current vaccine localises and sets up
the carrier state in the reproductive tract of the vaccinated stallion (45, 59, 60, 61).
The commercial inactivated vaccine has been shown to be nonreactive and safe for use in healthy
nonbreeding and breeding horses. Transient local reactions may be observed in less than 10% of horses
vaccinated with the inactivated vaccine. Limited field studies of this vaccine indicate that it is immunogenic,
stimulating a satisfactory degree of immunity, the duration of which has yet to be reported.
Although there are no published reports on the efficacy of either commercial vaccine in preventing
establishment of the carrier state in the stallion, an experimental aqueous formalin inactivated vaccine
against EVA has been shown to prevent virus persistence in the reproductive tract of vaccinated stallions
following subsequent challenge with EAV (23).
Both the MLV and inactivated vaccines are produced by cultivation of the respective seed viruses in an
equine
dermal cell system. Cell monolayers should be washed prior to inoculation with seed virus to remove traces
of
bovine serum in the growth medium. Inoculated cultures should be maintained on an appropriate
maintenance
medium. Harvesting of infected cultures should take place when almost the entire cell sheet shows the
characteristic CPE. Supernatant fluid and cells are harvested and clarified of cellular debris and unwanted
material by filtration. In the case of the inactivated vaccine, the purified virus is then chemically inactivated
and
adjuvanted with a metabolisable adjuvant.
The MLV and inactivated vaccines should be produced in a stable cell line that has been tested for identity
and
confirmed to be free from contamination by bacteria, fungi, mycoplasmas or other adventitious agents. In
addition
to the preproduction testing of the master seed virus for each vaccine and the cell line for adventitious
contaminants, the cell cultures infected with the respective vaccine viruses should be examined
macroscopically
for evidence of microbial growth or other extraneous contamination during the incubation period. If growth in
a
culture vessel cannot be reliably determined by visual examination, subculture, microscopic examination, or
both
should be carried out.
4. Batch control
a) Sterility
b) Safety
In the case of both MLV and inactivated vaccines, each production lot of vaccine should be checked for
extraneous bacterial, fungal and mycoplasmal contaminants. The vaccine should be safety tested by the
intramuscular inoculation of at least two horses seronegative for neutralising antibodies to EAV with one
vaccine dose of lyophilised virus each (46). None of the inoculated horses should develop any clinical signs
of disease other than mild pyrexia during the ensuing 2-week observation period. In addition,
nasopharyngeal swabs should be collected daily from each horse for attempted virus isolation; white blood
cell counts and body temperatures should also be determined on a daily basis. No significant febrile or
haematological changes should supervene following vaccination (45, 59, 61). Limited shedding of vaccine
virus by the respiratory route for at most 7 days may be demonstrated in the occasional vaccinated horse
(61). There is no evidence of persistence of the vaccine virus in the reproductive tract after vaccination of
stallions (45, 60, 61).
To ensure complete inactivation of the vaccine virus, each serial lot of the inactivated vaccine should be
checked for viable virus by three serial passages in equine dermal cells and by direct fluorescent antibody
staining with specific EAV conjugate before being combined with adjuvant. This should be followed by a
safety test in guinea-pigs and mice.
c) Potency
Potency of the vaccine in the final containers is determined by plaque infectivity assay in monolayer cultures
of equine dermal cells and by a vaccination challenge test in horses (46). The vaccine must be tested in
triplicate in cell culture, the mean infectivity titre calculated and the dose rate determined on the basis that
each dose of vaccine shall contain not less than 3 × 104 plaque-forming units of attenuated EAV. The in-vivo
potency of the MLV and inactivated vaccines is evaluated in a single vaccination challenge test using 17–
20 vaccinated and 5–7 control horses or in two tests each comprising ten vaccinates and five controls.
The viral antigen concentration in the inactivated vaccine is over one-thousand times the concentration of
viral antigen present in the MLV vaccine.
Detectable neutralising antibody titres to EAV should develop in the majority of horses within 1-2 weeks of
vaccination with the MLV vaccine (44, 45, 58, 59, 61). Reported responses to primary vaccination have been
variable in a couple of studies. In one stallion vaccination study, there was a rapid fall in antibody titres with
a
significant number of animals reverting to seronegativity 1–3 months after vaccination (61). On the other
hand, other studies have been characterised by an excellent durable response, with persistence of high VN
levels for at least 1–2 years (58). Revaccination with this vaccine results in an excellent anamnestic
response, with the development of high antibody titres that remain relatively undiminished for several years
(59).
Experimental studies have shown that most horses vaccinated with the inactivated vaccine develop low to
moderate neutralising antibody titres to EAV by day 14 after the second vaccination. There is no published
information on the duration of immunity conferred by this vaccine.
e) Stability
The lyophilised MLV vaccine can be stored for at least 3–4 years at 2–7°C without loss in infectivity,
provided
it is kept in the dark (29). Infectivity is preserved for much longer periods if vaccine is frozen at
–20°C or below. Once rehydrated, however, the vaccine should be used within 1 hour or else destroyed. The
inactivated vaccine is stored as a liquid suspension at 2–8°C, with no loss of potency for at least 1 year,
provided it is protected from light.
f) Preservatives
The preservatives added to the MLV and inactivated vaccines are neomycin, polymyxin B and amphotericin
B.
Pregnant mares should not be vaccinated with the MLV vaccine during the last 2 months of gestation, as
there is a risk, albeit minimal, of fetal invasion by the vaccine virus. The possibility of a vaccinally induced
anaphylactic reaction, though very rare, could result from the administration of either the MLV or inactivated
vaccine. In the absence of appropriate safety data, the inactivated vaccine is currently not recommended for
use in pregnant mares.
a) Safety
With the exception of the inactivated vaccine, which needs to be sterility tested a second time to ensure
freedom from contamination, no further safety tests are required on the inactivated or MLV vaccines.
b) Potency
No potency tests additional to those conducted on each production lot of the MLV or inactivated vaccines
are
required on either final product.
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*
**
NB: There are OIE Reference Laboratories for Equine viral arteritis Table in Part 3 of this Terrestrial Manual
or
consult the OIE Web site for the most up-to-date list: www.oie.int).
CHAPTER2.1.13.
RABIES
SUMMARY
Rabies is a major zoonosis for which diagnostic techniques have been standardised inter-nationally.
As there is no gross pathognomonic lesion for rabies, diagnosis can only be made in the laboratory.
Laboratory techniques are preferably conducted on central nervous system (CNS) tissue removed
from the cranium. A composite of CNS samples should be tested and the brain stem is the most
important component of the sample.
Identification of the agent: Agent identification is preferably done using the fluorescent antibody
test (FAT). A drop of purified immunoglobulin previously conjugated with fluorescein isothiocyanate
is added to an acetone-fixed brain tissue smear, preferably made from several parts of the brain
stem. FAT provides a reliable diagnosis in 98–100% of cases for all serotypes if a potent conjugate
is use. For a large number of samples, as in an epidemiological survey, polymerase chain reaction
(PCR) or immunoenzyme techniques can provide rapid results.
Infected neuronal cells have been demonstrated by histological tests and these procedures will
reveal aggregates of viral material (the Negri bodies) in the cytoplasm of neurones. However, the
sensitivity of histological techniques is much less than that of immunological methods, especially if
there has been some autolysis of the specimen. Consequently, histological techniques can no
longer be recommended.
As a single negative test on fresh material does not rule out the possibility of infection, cell culture
or mouse inoculation tests should be carried out simultaneously. A monolayer culture of susceptible
cells is inoculated with a pool of several CNS tissues, including the brain stem. FAT carried out after
appropriate incubation will demonstrate the presence or absence of viral antigen. Alternatively,
newborn or 3–4-week-old mice may be inoculated intracerebrally with a similar pool tissues and
then kept under observation for 28 days. For any mouse that dies between 5 and 28 days postinoculation,
the cause of death should be confirmed by FAT. Wherever possible, virus isolation in
cell culture should replace mouse inoculation tests.
The identification of the agent can be supplemented in specialised laboratories by identifying any
variant virus strains through the use of monoclonal antibodies, specific nucleic acid probes, or the
polymerase chain reaction followed by DNA sequencing of genomic areas. Such techniques can
distinguish between field and vaccine strains, and possibly identify the geographical origin of the
field strains. These very sensitive tests should be used by well trained personnel in specialised
laboratories.
Serological tests: Virus neutralisation (VN) assays in cell cultures are the prescribed tests for
international trade. Alternatively, use may be made of a test that is known to correlate with these,
notably an enzyme-linked immunosorbent assay using antibody to the G protein or the
neutralisation test in mice. Results are expressed in International Units or equivalent units relative to
an international standard antiserum.
Requirements for vaccines: Rabies vaccines for use in animals contain either live virus
attenuated for the target species (such as Flury low egg passage, Flury high egg passage, Street-
Alabama-Dufferin or Kelev), or virus inactivated by chemical or physical means, or recombinant
vaccines. The virus is cultivated in embryonated egg, or in cell cultures.
Rabies vaccines are usually lyophilised, but inactivated virus vaccines, preferably with an adjuvant,
may be stored in liquid form.
Before newly developed vaccines can be licensed, the duration of immunity resulting from their use
should be determined in vaccinated animals of the target species. Vaccines should confer
protective immunity for at least 1 year.
Chapter 2.1.13. — Rabies
For live virus vaccines, the minimum virus content that will elicit an adequate immune response
must be established.
The potency of inactivated virus vaccines is established and controlled by mouse vaccination
followed by intracerebral challenge using tests formulated by the United States Department of
Agriculture in the United States of America or the European Pharmacopoeia elsewhere. The final
products of both types of vaccine are subjected to tests for innocuity and absence of toxicity.
For live vaccines that are prepared for oral vaccination of wild (or domestic) animals, safety and
efficacy in target animals and safety in nontarget species must be demonstrated.
A. INTRODUCTION
Rabies is caused by a neurotropic virus of the genus Lyssavirus of the family Rhabdoviridae, and is
transmissible
to all mammals. As it is transmissible to humans by inoculation or inhalation of infectious virus, all suspected
infected material must be handled under the appropriate safety conditions specified by the World Health
Organisation (WHO) (45).
Seven distinct genetic lineages can be distinguished within the genus Lyssavirus by cross-protection tests
and
molecular biological analysis (6, 16, 26), namely the classical rabies virus itself (RABV, genotype 1, serotype
1),
Lagos bat virus (LBV, genotype 2, serotype 2), Mokola virus (MOKV, genotype 3, serotype 3), and
Duvenhage
virus (DUVV, genotype 4, serotype 4). The European bat lyssaviruses (EBLV), subdivided into two biotypes
(EBLV1, genotype 5 and EBLV2, genotype 6) and the Australian bat lyssavirus (ABLV, genotype 7), isolated
in
Australia (30), are also members of the Lyssavirus genus, but are not yet classified into serotypes. Viruses
of
serotypes 2–4, EBLV and ABLV are known as rabies-related viruses. The use of monoclonal antibodies
(MAbs)
directed against viral nucleocapsid or glycoprotein antigens, and the sequencing of defined genomic areas
has
made possible the definition of numerous subtypes within each serotype. Lyssaviruses cause a clinical
disease
indistinguishable from classical rabies. Conserved antigenic sites on the nucleocapsid proteins permit
recognition
of all lyssaviruses with modern commercial preparations of anti-rabies antibody conjugates used for
diagnostic
tests on brain tissue. There exist two lyssavirus phylogroups with distinct pathogenicity and immunogenicity
(5).
For RABV, DUVV, EBLV and ABLV, conserved antigenic sites on the surface glycoproteins allow
crossneutralisation
and cross-protective immunity to be elicited by rabies vaccination. Little or no cross-protection
against infection with MOKV or LBV is elicited by rabies vaccination and most anti-rabies virus antisera do
not
neutralise these lyssaviruses. Four new rabies-related viruses (Aravan, Khujand, Irkut, and West Caucasian
bat
viruses) have been isolated recently from Eurasian bats, and are described as new putative lyssavirus
species.
There is a reduced protection with pre-exposure vaccination and with conventional rabies post-exposure
prophylaxis against all four new bat variants of rabies virus (29).
Humans working with suspect material must be vaccinated against lyssaviruses or other pathogens that may
be
present in diagnostic samples. The laboratory must comply with national biocontainment and biosafety
regulations
to protect staff from contact with pathogens; it should also comply with the guidelines in Chapter 1.1.2
Biosafety
and biosecurity in the veterinary microbiological laboratory and animal facilities.
WHO recommends the preventive immunisation of exposed staff. The immunisation protocol includes three
injections, e.g. at days 0, 7, and 28. The serological evaluation of immunisation is made 1–3 weeks after the
last
injection, and checked every 6 months in the case of laboratory workers or every 2 years for other
diagnosticians.
Booster vaccination must be given when the titre falls below 0.5 International Units (IU) per ml. In the
absence of
serological monitoring, the vaccination regimen should consist of a booster vaccination at 1 year and
thereafter
every 1–3 years.
As no clinical sign or gross post-mortem lesion can be considered pathognomonic in domestic or wild
animals, the
diagnosis of rabies has to rely on laboratory testing. Serological evidence of infection is rarely useful
because of
late seroconversion and the high mortality rate of host species, although such data may be used in some
epidemiological surveys.
Clinical observation may only lead to a suspicion of rabies because signs of the disease are not
characteristic and
may vary greatly from one animal to another (43). The only way to perform a reliable diagnosis of rabies is to
identify the virus or some of its specific components using laboratory tests.
As rabies virus is rapidly inactivated, refrigerated diagnostic specimens should be sent to the laboratory by
the
fastest means available. Shipment conditions must be considered to be part of the ‘rabies diagnostic chain’.
Several laboratory techniques may be used, and have been detailed and standardised in the fourth edition
of the
WHO’s Laboratory Techniques in Rabies (45). The methods vary in their efficiency, specificity and reliability.
They
are classically applied to brain tissue, but they can also be applied, though less effectively, to other organs
(e.g.
salivary glands). In the brain, rabies virus is particularly abundant in the thalamus, pons and medulla. The
hippocampus (Ammon’s horn), cerebellum and different parts of the cerebrum have been reported to be
negative
in 3.9–11.1% of the positive brains. The structure of choice is the thalamus as it was positive in all cases. It
is
recommended that a pool of brain tissues that includes the brain stem should be collected and tested (13).
To
reach these parts of the brain, it is necessary to remove the entire organ after having opened the skull in a
necropsy room. Under some conditions (e.g. in the field or when sampling for large epidemiological studies),
a
simplified method of sampling through the occipital foramen (11), or through the orbital cavity (32), can be
used.
Precautions should be taken when handling central nervous system tissues from suspected rabies cases.
Gloves
should always be worn and precautions must be taken to prevent aerosols. The use of cutting tools, scissors
and
scalpels, should be used with care to prevent injury and contamination.
a) Shipment of samples
During the shipment of suspect material for diagnosis (animal heads, brain or other tissue samples), no risk
of human contamination should arise: brains must be placed in a leak-proof rigid container (animal heads
will
be wrapped in absorbent material) as prescribed in the International Air Transport Association (IATA)
Dangerous Goods Regulations must be followed. These regulations are summarised in Chapter 1.1.1.
Collection and shipment of diagnostic specimens.
When it is not possible to send refrigerated samples, other preservation techniques may be used. The
choice of the preservative is closely linked to the tests to be used for diagnosis:
• Formalin inactivates the virus, thus the isolation tests cannot be used and diagnosis depends on using
a modified and less sensitive direct fluorescent antibody test (FAT), immunohistochemistry or histology
(39, 45);
• Infectivity at room temperature may be extended for several days if brain material is kept in a mixture of
50% glycerol in phosphate buffered saline (PBS). Glycerol/PBS slows bacterial action and therefore
protects against the chemical and biological effects of putrefaction. It does not protect against titre
decline due to thermal conditions and therefore, because rabies is thermo-labile, the virus titre will
decline during glycerol/PBS storage. Under normal transport conditions in the tropics, this protection
may only be effective for a matter of several days. Therefore, whenever possible samples in
glycerol/saline should be kept refrigerated. As the virus is not inactivated by glycerol/PBS, all laboratory
tests can be used on these samples.
b) Collection of samples
Usually the brain is collected following the opening of the skull in a necropsy room, and the appropriate
samples are collected. This step may be hazardous if laboratory technicians are not fully trained, or under
field conditions. In such cases, there are two possible methods of collecting some brain samples without
opening the skull:
• Occipital foramen route for brain sampling
A 5 mm drinking straw (11) or a 2 ml disposable plastic pipette (17) is introduced into the occipital foramen in
the direction of an eye. Samples can be collected from the rachidian bulb, the base of the cerebellum,
hippocampus, cortex, and medulla oblongata. Bovine spongiform encephalopathy (BSE) should be
considered in the differential diagnosis of most cattle that are considered to be ‘rabies suspect’. Sampling of
brain specimens for both diseases can be done using the ‘brain scoop or tool’ developed for BSE tissue
sampling rather than a straw or pipette. The resulting samples are relatively easily recognised as to the area
of brain sampled.
• Retro-orbital route for brain sampling
In this technique (32), a trocar is used to make a hole in the posterior wall of the eye socket, and a plastic
pipette is then introduced through this hole. The sampled parts of the brain are the same as in the former
technique, but they are taken in the opposite direction.
c) Routine laboratory tests
Laboratory diagnosis can be performed by using three kinds of procedure.
i) Immunochemical identification of rabies virus antigen
• Fluorescent antibody test
The most widely used test for rabies diagnosis is the FAT, which is recommended by both WHO and
OIE. This test may be used directly on a smear, and can also be used to confirm the presence of rabies
antigen in cell culture or in brain tissue of mice that have been inoculated for diagnosis. The FAT gives
reliable results on fresh specimens within a few hours in more than 95–99% of cases. The sensitivity of
the FAT depends on the specimen (the degree of autolysis and how comprehensively the brain is
sampled, see Section B.1) (1, 9), on the type of lyssavirus and on the proficiency of the diagnostic staff.
Sensitivity may be lower in samples from vaccinated animals due to localisation of antigen, which is
confined to the brainstem. For direct rabies diagnosis, smears prepared from a composite sample of
brain tissue, that includes the brain stem, are fixed in high-grade cold acetone and then stained with a
drop of specific conjugate. Anti-rabies fluorescent conjugates may be prepared in the laboratory. Those
available commercially are either polyclonal conjugates specific to the entire virus or specific to the
rabies nucleocapisid protein, or they may be prepared from a mix of different MAbs. In the FAT, the
specific aggregates of nucleocapsid protein are identified by their fluorescence. The specificity and
sensitivity of these anti-rabies fluorescent conjugates for locally predominant virus variants should be
checked before use.
The FAT may be applied to glycerol-preserved specimens. If the specimen has been preserved in a
formalin solution, the FAT may be used only after the specimen has been treated with a proteolytic
enzyme (7, 8, 38, 39). However, the FAT on formalin-fixed and digested samples is always less reliable
and more cumbersome than when performed on fresh tissue.
• Immunochemical tests
(FITC). This conjugate may be used for direct diagnosis with the same sensitivity as FAT (27), but
attention should be paid to the risk of nonspecific false-positive results. This risk is considerably
reduced by the thorough training of the technicians. It must also be emphasised that this technique
needs one incubation step more than the FAT.
Peroxidase conjugate may be used on sections of formalin-fixed tissue for immunohistochemical tests.
ii) Detection of the replication of rabies virus after inoculation
These tests detect the infectivity of a tissue suspension in cell cultures or in laboratory animals. They should
be used if the FAT gives an uncertain result or when the FAT is negative in the case of known human
exposure.
• Cell culture test
Neuroblastoma cell lines, e.g. CCL-131 in the American Type Culture Collection (ATCC)1, is used for
routine diagnosis of rabies. The cells are grown in Dulbecco’s modified Eagle’s medium (DMEM) with
5% foetal calf serum (FCS), incubated at 36°C with 5% CO2. Its sensitivity has been compared with
that of baby hamster kidney (BHK-21) cells (34). This cell line is sensitive to street isolates without any
adaptation step, but should be checked for susceptibility to locally predominant virus variants before
use. Presence of rabies virus in the cells is revealed by the FAT. The result of the test is obtained after
at least 18 hours (one replication cycle of virus in the cells); generally incubation continues for 48 hours
(10) or in some laboratories up to 4 days.
This test is as sensitive as the mouse inoculation test. Once a cell culture unit exists in the laboratory,
this test should replace the mouse inoculation test as it avoids the use of live animals, is less expensive
and gives more rapid results.
It is often advisable to carry out more than one type of test on each sample, particularly when there has
been human exposure.
• Mouse inoculation test
Five-to-ten mice, 3–4 weeks old (12–14 g), or a litter of 2-day-old newborn mice, are inoculated
intracerebrally. The inoculum is the clarified supernatant of a 20% (w/v) homogenate of brain material
(cortex, Ammon’s horn, cerebellum, medulla oblongata) in an isotonic buffered solution containing
antibiotics. To reduce animal pain, mice should be anaesthetised when inoculated. The young adult
mice are observed daily for 28 days, and every dead mouse is examined for rabies using the FAT. For
faster results in newborn mice, it is possible to check one baby mouse by FAT on days 5, 7, 9 and 11
post-inoculation. Any deaths occurring during the first 4 days are regarded as nonspecific (due to
stress/bacterial infection etc.).
This in-vivo test should be avoided when possible on animal welfare grounds. It is also expensive,
particularly if SPF mice are used, and does not give rapid results (compared with in-vitro inoculation
tests), but when the test is positive, a large amount of virus can be isolated from a single mouse brain
1 American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Virginia 20108, United States of America . (USA)
for strain identification purposes. Another advantage of this low-tech test is that it can be easily and
practicably applied in situations where skills and facilities for other tests (e.g. cell culture) are not
available
iii) Histological identification of characteristic cell lesions
Negri bodies correspond to the aggregation of viral proteins, but the classical staining techniques detect only
an affinity of these structures for acidophilic stains. Immunohistochemical tests are the only histological test
specific to rabies.
An unfixed tissue smear may be stained by the Seller’s method, diagnosis is then obtained in under 1 hour.
Generally, histological tests, such as Mann’s test, are performed on fixed material after a paraffin-embedding
step, and the result of the test is obtained within 3 days. These techniques have the advantage that the
laboratory equipment needed to perform them is inexpensive and any need to keep specimens cold after
fixation is avoided. Whichever staining method is used, the evidence of infection is provided by
intracytoplasmic acidophilic bodies. These histological methods, especially the Seller’s method, can no
longer be recommended because they have very low sensitivity and should be abandoned.
d) Other identification tests
An enzyme-linked immunosorbent assay (ELISA) that detects rabies antigen is a variation of the
immunochemical test. It is useful for large epidemiological surveys (46). It should only be used after
validation against numerous samples in different laboratories. The specificity and sensitivity of these
antirabies
enzyme conjugates for locally predominant virus variants should be checked before use. This test
should be used in combination with confirmatory tests by FAT or virus isolation.
The tests above describe methods to accurately diagnose rabies and to isolate and identify the virus. Typing
of the virus can provide useful epidemiological information and should be carried out in specialised
laboratories (such as OIE or WHO Reference Laboratories). These techniques would include the use of
MAbs, nucleic acid probes, or the polymerase chain reaction (PCR), followed by DNA sequencing of
genomic areas for typing the virus (17). This characterisation enables a distinction to be made between
vaccine virus and a field strain of virus, and possibly the geographical origin of the latter.
Serological tests are rarely used in epidemiological surveys, due to late seroconversion and the low
percentage of
animals surviving the disease and therefore having post-infection antibodies. The main application of
serology is
to determine responses to vaccination, either in domestic animals prior to international travel, or in wildlife
populations following oral immunisation of rabies reservoirs. For follow-up investigations in oral vaccination
campaigns, virus neutralisation (VN) tests in cell culture are preferred. However, if poor quality sera are
submitted,
the VN tests in cell culture are sensitive to cytotoxicity, which could lead to false-positive results. For such
samples, the use of an indirect ELISA with rabies glycoprotein-coated plates has been shown to be as
sensitive
and specific as the VN test on cells (22).
a) Virus neutralisation test in cell culture: fluorescent antibody virus
neutralisation test (a prescribed
test for international trade)
The principle of the fluorescent antibody virus neutralisation (FAVN) test (21) is the neutralisation in vitro of a
constant amount of rabies virus (‘challenge virus standard’ [CVS] strain adapted to cell culture) before
inoculating cells susceptible to rabies virus: BHK-21 C13 cells (ATCC number: CCL-10).
The serum titre is the dilution at which 100% of the virus is neutralised in 50% of the wells. This titre is
expressed in IU/ml by comparing it with the neutralising dilution of the OIE serum of dog origin under the
same experimental conditions. The WHO standard for rabies immunoglobulin [human]2 No. 2, or an internal
control calibrated against the international control may be used. The WHO standard or internal control
should only be used as a control in the test and should not be used to calculate the IU/ml titre of the sera.
This microplate method uses 96-well plates, and is an adaptation of the technique of Smith et al. (36),
modified by Zalan et al. (47) and by Perrin et al. (33). Several publications (18, 21) have shown that the
FAVN test and the rapid fluorescent focus inhibition test (RFFIT) give equivalent results.
• Essential equipment
Humidified incubator at 35°C/37°C with 5% CO2; dry incubator at 37°C; biocontainment cabinet;
fluorescence microscope suitable for FITC fluorescence equipped with ×10 eye-piece and ×10 objective.
The
global magnification of the microscope ranges between ×100 and ×125 due to the extra magnification of
some epi-fluorescence systems.
2 National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire
EN6 3QG, United Kingdom (UK).
• Reagents and biologicals
PBS buffer, pH 7.2, without Ca2+ and Mg2+, stored at 4°C;
Trypsin ethylene diamine tetra-acetic acid (EDTA);
High-grade acetone 80% (diluted with deionised water), stored at 4°C;
Dulbecco’s modified Eagle’s medium (DMEM) + 10% heat-inactivated FCS;
FITC anti-rabies conjugate;
Cells: BHK-21 C13 (ATCC CCL-10);
Virus: CVS-11 (ATCC VR 959) strain, which is available from the ATCC or the OIE Reference Laboratory for
Rabies, Nancy, France (see Table given in Part 3 of this Terrestrial Manual). Vials are stored at –80°C;
OIE Standard Serum of dog origin (OIE Reference Laboratory for Rabies, Nancy, France [see Table given in
Part 3 of this Terrestrial Manual] stored at +4°C and diluted to 0.5 IU/ml with sterile deionised or distilled
water according to the titre of the batch);
Naive serum: The pool of negative dog sera is stored at –20°C.
� CVS production
i) Cell growth: the BHK-21 C13 cells (ATCC CCL-10) used to produce the CVS virus (ATCC VR 959
CVS-11) are trypsinised during the rapid growth phase, i.e. cells are in the exponential phase of their
kinetic growth. If the confluence of the layer is complete, a new passage should be made. The cells in
the cell suspension should not be aggregated; 2 × 107 cells are used for a 75 cm2 cell culture flask.
Cells are collected within a volume of 20–30 ml in cell culture medium with 10% heat-inactivated FCS.
ii) Infection of cells: the multiplicity of infection (number of infective particles per cell) is adjusted to
between 0.1 and 0.5. The glass bottle containing the virus/cell suspension is incubated for 60 minutes
at 35.5–37°C. The contents of the bottle are gently stirred every 10–15 minutes.
iii) Virus growth: the virus/cell suspension is then centrifuged at 800–1000 g for 15 minutes and the cell
pellet is resuspended in cell culture medium mixed with 10% heat-inactivated FCS. Virus is harvested
2 days later.
iv) Harvest and storage: the supernatant is centrifuged at 800–1000 g for 15 minutes at 4°C. If several
flasks have been used, the different centrifuged supernatants are mixed and then aliquoted and frozen
at –80°C. The infective titre of the harvest is established at least 3 days after freezing.
� Titration of virus in TCID50 (50% tissue culture infective dose)
This titration method uses BHK-21 C13 cells (ATCC CCL-10) in microtitre plates.
Different steps in this procedure may be adapted according to the safety requirements and to the working
practices of the laboratory, but the following should not be changed:
� inoculation of a 24-hour cell layer,
� tenfold dilutions prepared using 0.9 ml of diluent and 0.1 ml of virus suspension,
� six 50 μl replicates per dilution,
� incubation for 72 hours,
� qualitative reading (i.e. the well is positive or negative),
� in every titration session, a vial of a control batch of virus is titrated and this titre is integrated in a
control card to validate the titration process,
� calculation according to neoprobit graphic or Spearman–Kärber methods.
i) Cell suspension: the day before titration, a cell suspension containing 105 cells/ml is prepared in cell
culture medium containing 10% heat-inactivated FCS, and is distributed, 200 μl per well, into 96-well
microtitre plates. The plates are then incubated for 24 hours at 35.5°C–37°C with 5% CO2.
ii) Dilution of the virus: the serial dilutions are performed in 5 ml tubes using a cell culture medium without
FCS as diluent. Tenfold dilutions from 10–1 to 10–12 are prepared (0.9 ml of diluent with 0.1 ml of the
previous dilution).
iii) Infection of the cells: the medium in the microtitre plates is discarded using an aspiration system. Fifty
μl of each virus dilution is distributed per well. Six replicates are used per dilution. The microtitre plate is
then incubated for 1 hour at 35.5–37°C with 5% CO2. Then 200 μl of cell culture medium, containing
5% FCS, is added.
iv) Incubation: incubate for 3 days at 35.5–37°C in 5% CO2.
v) Staining and calculation of titre: The cells are stained using the FAT, as detailed below. Reading is
qualitative, every well that shows specific fluorescence is considered to be positive. The titre calculation
is made using either the neoprobit graphic method (2) or the Spearman–Kärber formula.
vi) The CVS titration must be performed by FAVN test to establish the infective dose in TCID50.
Fig. 1. Proposed use of microplates for the fluorescent antibody virus neutralisation test. Wells
to which undiluted
sera must be added are filled with the indicated ‘50 μl’. Wells to which 50 μl of diluted
challenge virus standard
must be added are shaded. Dilutions are given in log10.
Plate 1: Controls
HGFEDCBA
Challenge virus 1
standard 2
titration 3
4
OIE 50 μl 50 μl 5 Naive dog
standard serum 50 μl 50 μl 6 serum
(0.5 IU/ml) 50 μl 50 μl 7 (negative)
50 μl 50 μl 8
Serum or internal 50 μl 9
positive control 50 μl 10
or WHO Standard 50 μl 11
serum 50 μl 12
log (dilution) 0.48 0.95 1.43 1.91 2.39 2.87 CVS
virus
control
Cells
control
Plate 2: Sera to be tested
log dilution 0.48 0.95 1.43 1.91 2.39 4.23 0.48 0.95 1.43 1.91 2.39 4.23
1 2 3 4 5 6 7 8 9 10 11 12
A 50 μl 50 μl
Serum 1 B 50 μl 50 μl Serum 3
C 50 μl 50 μl
D 50 μl 50 μl
E 50 μl 50 μl
Serum 2 F 50 μl 50 μl Serum 4
G 50 μl 50 μl
H 50 μl 50 μl
• Test procedure
i) The microplates are used according to the pattern shown in Figure 1. Plate No. 1 is used for the
titration of CVS (rows 1 to 4), and for the controls, standard sera and naive dog serum are used. All
other plates are used for the sera to be tested.
ii) Medium is added to the wells as follows: plate 1, rows 1 to 4 and cells A9 to A12: add 150 μl per well; in
the other plates, rows 6 and 12: add 200 μl per well; all other wells: add 100 μl.
iii) Sera to be tested are heat inactivated for 30 minutes at 56°C. As indicated in Figure 1, 50 μl of each
undiluted serum to be tested is added to four adjacent wells.
iv) Dilutions of sera are conducted in the microplates as follows:
OIE serum, the WHO serum, the internal control and the naive dog serum: with a 50–200 μl
multichannel pipette, mix the first dilution wells by sucking in and out at least eight times, transfer 50 μl
from one row to the next one, until the last one is reached. Discard 50 μl from the last row.
If there is a serum to be tested on the control plate, see below for the dilution step.
A minimum of four three-fold dilutions is required.
Sera being tested (all plates): as above, transfer successively 50 μl from one row to the following one
until rows 5 and 11 (dil. 10–2.39). With a 5–50 μl multichannel pipette, transfer 10 μl from rows 5 and 11
to rows 6 and 12, respectively (from dil. 10–2.39 to dil. 10–4.23). Using a multichannel pipette adjusted to
90 μl, mix rows 6 and 12 and discard 180 μl. Then add 70 μl of medium to these rows. This final step
does not lend itself to high throughput testing. To attain or exceed the recommended final dilution
alternative procedures may be used. These may require modifications to the plate layout.
• Addition of challenge virus standard
i) Stock CVS is stored in 1 ml microtubes at –80°C. One tube is thawed rapidly under cold running water,
and placed in melting ice.
ii) One dilution from this tube is prepared in order to obtain 100 TCID50 in 50 μl. Of this dilution, 50 μl is
added to each serum-filled well (see Figure 1). For virus titration, 50 μl is added to wells H1 to H4 (plate
1). Next, transfer 50 μl from row to row (plate 1, lines 1–4). Discard 50 μl from the last row (plate 1,
wells A1 to A4). No virus is added to wells A9 to A12 of plate 1 (controls). The range allowed for the
virus dose titre must be between 30 and 300 TCID50/50 μl.
iii) Incubate the microplates at 35–37°C in a humid incubator with 5% CO2 for 1 hour.
iv) Addition of cells: trypsinise a subconfluent culture of 3-day-old BHK-21 cells. Resuspend the cells to
obtain a 4 × 105 cells/ml suspension in DMEM supplemented with 10% heat-inactivated FCS. Add 50 μl
of the cell suspension to each well.
v) Incubate the microplates for 48 hours at 35–37°C in a humid incubator with 5% CO2.
• Fixation and staining
i) After the 48-hour incubation period, the medium is discarded, and the microplates are rinsed once in
PBS, pH 7.2, and once in 80% acetone. The microplates are then fixed in 80% acetone at room
temperature for 30 minutes, and are dried at room temperature for at least 30 minutes.
ii) Add 50 μl of the FITC anti-rabies conjugate, at the working dilution, to each well, gently rock the
microplates and incubate at 35–37°C for 30 minutes. Discard the fluorescent conjugate and rinse the
microplates twice with PBS. Excess PBS is removed by briefly inverting the microplates on absorbent
paper.
• Reading and interpreting the results
i) The total surface of each well is observed. The reading evaluation is qualitative (plus or minus): no
fluorescent cell – a minus score is recorded for the well; fluorescent cells (one cell or more) – a plus
score is recorded for the well.
ii) Cell and virus controls are read first. For titration of CVS, naïve serum, and OIE standard serum, titres
are calculated according to the Spearman–Kärber method or the neoprobit graphic method (2).
iii) Results of titration of CVS (TCID50), naive serum (D50 [median dose]) and positive standard (D50) are
reported on a control card for each of these three controls. The control results of the current test are
compared with the accumulated control test results from previous tests using the same batch of control.
The test is validated if the values obtained for the three controls in the current test are not statistically
different from the mean (± 2 SD) of all the values obtained in the tests conducted previously according
to this technique.
iv) The result of the test corresponds to the non-neutralised virus after incubation with the reference serum
or with the serum to be tested. These titres are calculated with the neo-probit graphic method (2) or
with the Spearman–Kärber formula (45).The comparison of the measured titre of the tested sera with
that of the OIE positive standard serum of a known neutralising titre allows determination of the
neutralising titre of the tested sera in IU/ml. The conversion to IU/ml can be made by using either the
log D50 value of the day or the mean value of the OIE standard serum.
� Formula to convert the log D50 value in IU/ml titre:
Serum titre (IU/ml) = [(10 (serum log D
50
value)) × theoretical titre of OIE serum 0.5 IU/ml]
(10 (log D
50
of OIE serum 0.5 IU/ml))
Example of conversion:
� log D50 of the serum = 2.27
� theoretical titre of OIE serum 0.5 IU/ml = 0.5 IU/ml
� log D50 of OIE serum = 1.43
(for the log D50 of OIE, the value of the day or the mean value can be considered)
Serum titre (IU/ml) = [(102.27 × 0.5 = 3.46 IU/ml]
(101.43)
The following parameters have to be strictly respected:
� Rabies virus: only the CVS-11 strain (ATCC number = VR 959 should be used.
� Cells culture: only BHK-21 cells (ATCC number – CCL 10) should be used.
� The FAVN test must be performed only in 96 wells microplate.
� Control charts should be used for rabies virus, naïve serum and OIE standard serum.
� The back titration of the CVS virus, as well as naïve serum and OIE serum, must be present on control
plate.
� A minimum of four three-fold dilutions of sera are required. The reading method is ‘all or nothing’ only.
� Four replicates of each serum should be diluted.
� For the conversion of log D50 in IU/ml, the laboratories should use only the log D50 value of the OIE
standard serum of dog origin.
b) The rapid fluorescent focus inhibition test (RFFIT) for determining rabies
virus-neutralising
antibody (a prescribed test for international trade)
� Standard procedure (from WHO Laboratory Techniques in Rabies, 1996;
[ref. 45])
� Preparation of seed virus suspension
i) Trypsinise one 3-day-old 150 ml flask culture of mouse neuroblastoma (MNA) cells. These cells prefer
an acidic medium, supplemented with vitamins (31). A similar cell line (CCL-131) may be obtained on
request from the ATCC (see footnote 1).
ii) Resuspend 3 × 107 cells in a 50 ml conical centrifuge tube in 2.7 ml of Eagle’s minimal essential
medium supplemented with 10% fetal bovine serum (EMEM-10).
iii) Using standard rabies safety procedures, add 1 × 107 infectious units of CVS-11 rabies virus (ATCC,
VR959) and vortex/mix once. Incubate the cells and virus for 15 minutes at 37°C; vortex/mix the cells
once during this time.
iv) Add 10 ml EMEM-10, vortex/mix, and centrifuge the cells at 500 g for 10 minutes.
v) Discard the supernatant. Resuspend the cells in 30 ml of growth medium and transfer to a 150 ml flask.
vi) Gently rock the flask to mix the cell suspension, and then prepare three eight-well tissue-culture
chamber slides by pipetting 0.2 ml of the cell suspension into one well of each slide.
vii) Incubate the flask and slides at 37°C in a humidified incubator with 0.5% carbon dioxide (CO2). The
flask should be incubated as a closed culture (tighten the cap).
viii) At 20, 40 and 64 hours after infection, acetone fix and stain one slide using an immunofluorescence
technique (21) to determine the virus infectivity. The supernatant should be harvested 24 hours after
the cells reach 100% infectivity (typically 40 hours after infection).
ix) Transfer the supernatant to a 50 ml centrifuge tube and centrifuge at 4000 g for 10 minutes.
x) Distribute the supernatant into 0.5 ml aliquots and store at –70°C.
� Titration of seed virus suspension
i) Thaw one aliquot of the seed virus and prepare serial tenfold dilutions (from 10–1 to 10–8) in EMEM-10.
ii) Distribute 0.1 ml of each virus dilution into one well of an eight-well tissue-culture chamber slide. Add
0.2 ml of MNA cells suspended in EMEM-10 (concentration 5 × 104 cells per 0.2 ml) to each well.
iii) Mix the cells and virus by gently rocking the slide, then incubate at 37°C in a humidified incubator with
0.5% CO2 for 40 hours.
iv) Acetone fix and stain the slide using an immunofluorescence technique. Evidence of virus infection
should be observed at the 10–6 dilution of virus, indicating a virus stock suspension containing at least
1 × 106 infectious units per 0.1 ml. Prepare sufficient seed virus so that frequent serial passage of the
virus is unnecessary.
� Preparation of stock virus suspension
i) Infect 3 × 107 MNA cells with 1 × 107 infectious units of the seed virus preparation (see above).
ii) Harvest the supernatant 24 hours after the cells reach 100% infectivity (typically 40 hours after
infection).
iii) Distribute the supernatant into 0.5 ml aliquots and store at –70°C.
� Titration of stock virus suspension
i) Thaw one aliquot of the stock virus and use this to prepare serial tenfold dilutions (from 10�1 to 10�6) in
EMEM-10.
ii) Distribute 0.1 ml of each virus dilution into one well of an eight-well tissue-culture chamber slide. Add
0.2 ml of MNA cells suspended in EMEM-10 (concentration 1 × 105 cells per 0.2 ml) to each well.
iii) Mix the cells and virus suspension by gently rocking the slide, then incubate at 37°C in a humidified
incubator with 0.5% CO2 for 20 hours.
iv) Acetone fix and stain the slide using an immunofluorescence technique.
Each well of an eight-well tissue-culture chamber slide contains 25–50 distinct microscopic fields when
observed at ×160–200 magnification. One unit of virus for the RFFIT is determined as the dilution at which
50% of the observed microscopic fields contain one or more foci of infected cells (the focus-forming dose,
FFD50). The stock virus suspension should contain at least 1 × 104 FFD50 per 0.1 ml (i.e. the well with cells
infected with the 10–4 dilution of the virus should contain at least one focus of infected cells in 50% of the
observed microscopic fields). A stock virus suspension of this titre can then be diluted to 10–2.3 to obtain a
challenge virus containing 50 FFD50.
� Reference sera
A national or international reference serum standard diluted to a potency of 2.0 IU/ml should be included in
each test. The reference serum used at the Centres for Disease Control and Prevention is the first
international standard for rabies immunoglobulin (32), which may be obtained from the NIBSC (see footnote
2). The reference serum should be maintained as frozen aliquots in amounts sufficient for 1 week of tests. A
positive serum control standard diluted to a potency of 0.5 IU/ml and a negative serum control standard with
a potency of <0.1 IU/ml should also be prepared by the laboratory and included in each test.
� Test sera
Serum samples should be heated at 56°C for 30 minutes before testing in order to inactive complement. If
sera are frozen, they should be reheated after thawing. Serial dilutions of test sera may be prepared in an
eight-well tissue-culture chamber slide. Screening dilutions of 1/5 and 1/50 are sufficient for routine
evaluation of vaccination efficacy and may be made as follows:
i) Prepare a 1/2.5 dilution by adding 0.1 ml of inactivated serum and 0.15 ml of EMEM-10 to one of the
slides. Mix by gently rocking the slide.
ii) Transfer 0.05 ml of the 1/2.5 dilution to a second well containing 0.45 ml of EMEM-10. Discard all but
0.1 ml from the well containing the 1/2.5 dilution.
iii) Mix the second well and discard all but 0.1 ml.
iv) Add 0.1 ml of the challenge virus preparation (containing 32–100 FFD50) to all serum dilutions.
v) Mix and incubate at 35°C in a humidified incubator with 0.5% CO2 for 90 minutes.
� Addition of cells
i) During the incubation period, trypsinise a stock culture of 3–5-day-old MNA cells.
ii) Resuspend the cells in EMEM-10 to give a final concentration of 1 × 105 cells per 0.2 ml.
iii) Distribute 0.2 ml of the cell suspension into each well of the slide and incubate at 35°C in a humidified
incubator with 0.5% CO2 for a further 20 hours.
� Acetone fixation and staining by immunofluorescence
i) After 20 hours, remove the slides from the incubator and pour off the medium into a virucidal solution.
ii) Rinse the slides once in PBS and then fix for 10 minutes at room temperature in cold acetone (–20°C).
iii) Leave the slides to dry for 10 minutes before adding FITC-conjugated anti-rabies serum. The conjugate
may be prepared in EMEM-10 or PBS; there is no need to adsorb the conjugate with tissue or cells.
The working dilution of the conjugate should be determined by titration. The slides should be stained for
20–30 minutes at 37°C and then rinsed in PBS and distilled water, respectively.
iv) Observe the slides under a fluorescence microscope.
� Calculation of virus-neutralising antibody titres
Residual virus is detected using a standard fluorescence microscope. The serum neutralisation end-point
titre is defined as the dilution factor of the highest serum dilution at which 50% of the observed microscopic
fields contain one or more infected cells (i.e. a 97% reduction in the virus inoculum). This value may be
obtained by mathematical interpolation. Alternatively, a 100% neutralisation titre may be determined by
recording the highest serum dilution at which 100% of the challenge inoculum is neutralised and there are
no
infected cells in any of the observed fields. For both titration methods, the titre of antibody in the test serum
(in IU/ml) can be obtained by comparison with the titre of the national reference standard included in each
test. It should be noted that it is also valid to perform the RFFIT using BHK-21 cells instead of
neuroblastoma cells. A modified protocol for this has been published (45).
The following parameters have to be strictly adhered to:
� Rabies virus; only the CVS-11 strain (ATCC number VR 959) should be used.
� Cells cultures: only BHK-21 cells (ATCC number CCL10) or MNA cells (ATCC number CCL131) should
be used.
� The test should be performed only on Labteck chamber slides.
� Control charts should be used for rabies virus, naïve serum and OIE standard serum.
� The back titration of the CVS virus, as well as the naïve serum and OIE serum, must be present on
control plate.
� Reading method for the test: each chamber slide should contain 25–50 fields and be observed at
×160–200 magnification.
� A minimum of three-to-five-fold dilutions of sera is required.
� For the conversion of log D50 to IU/ml, only the log D50 value of the OIE standard serum of dog origin
should be used.
c) Virus neutralisation in mice
This method is no longer recommended by either OIE or WHO and should be discontinued.
d) Enzyme-linked immunosorbent assay (a prescribed test for international
trade)
Commercial kits are available for indirect ELISA that allow a qualitative detection of rabies antibodies in
individual dog and cat serum samples following vaccination. In accordance with the WHO recommendations
(41), 0.5 IU per ml rabies antibodies is the minimum measurable antibody titre considered to represent a
level of immunity that correlates with the ability to protect against rabies
infection. The ELISA provides a rapid (~ 4 hours) test that does not require handling of live rabies virus, to
determine if vaccinated dogs and cats have sero-converted. The sensitivity and specificity of any kit
used should be determined by comparison with virus neutralisation methods. The ELISA is acceptable
as a Prescribed Test for international movement of dogs or cats provided that a kit is used that
has been validated and adopted on the OIE Register as fit for such purposes (see
http://www.oie.int/vcda/eng/en_VCDA_registre.htm?e1d9)]. Virus neutralisation methods may be used as
confirmatory tests if desired.
ELISA methods are also useful for monitoring of vaccination campaigns in wildlife populations, provided the
kit used has been validated for the wildlife species under study.
BIOLOGICALS
Rabies vaccines prepared from Pasteur’s original 1885 strain and its derivative strains (Pasteur Virus,
Challenge
Virus Standard, Pitman-Moore, etc.), and strains isolated more recently (Flury, Street-Alabama-Dufferin
[SAD],
Vnukovo and Kelev), protect against all strains of genotype 1 isolated so far. Conventional rabies virus
vaccines
may not provide adequate cross-protection against other lyssaviruses, especially in phylogroup II; there is
no
protection provided against Mokola virus (37) and the recently identified West Caucasian Bat Virus (29).
Cross
neutralisation using conventional rabies virus vaccines has been demonstrated against two phylogroup I
viruses:
EBLV type-1 and EVLV type-2 (20). The principles governing the preparation of inactivated rabies vaccines
are
identical whether they are to be used in humans or animals, although an adjuvant may be added to vaccines
for
animal use.
Recombinant vaccine (e.g. vaccinia rabies-glycoprotein recombinant) has also proved to be effective (19,
31). The
rabies-glycoprotein recombinant vaccines are not live rabies vaccines. They are prepared by inserting
noninfectious
rabies nucleic acid into a vector such as vaccinia or canary pox. Since these do not contain live rabies
virus, animals vaccinated with rabies-glycoprotein recombinant vaccines should not be restricted from entry
into
countries that have restrictions on entry of animals vaccinated with live rabies vaccines.
For animals, live and recombinant vaccines are effective by the oral route and can be distributed in baits in
order
to immunise wild (or domestic) animals.
vaccine
Different standards apply to vaccines containing live virus modified by passage in eggs or cell cultures to
reduce
its virulence for the target animal, and to vaccines prepared from inactivated virus. Both types of vaccine
have
their advantages and disadvantages (6), but they can both be used to immunise animals for periods of
between 1
and 3 years. Live attenuated rabies vaccines are not accepted in some countries. They are not to be relied
on to
protect previously unvaccinated animals that have been exposed to infection (15). Only in humans has the
efficiency of post-exposure prophylaxis with vaccine alone been proven and even in these cases there is an
additional strong recommendation to administer anti-rabies immunoglobulin.
All handling of the virus during manufacture and testing of vaccines must conform to the strict safety
precautions
specified by WHO (43, 45), the OIE (Chapter 1.1.2) and to national guidelines and regulations.
1. Seed management
Any strain belonging to serotype 1, which has been proved to protect against field rabies viruses (currently
found in the country where the vaccine is to be used), is suitable. The strain of virus used should have
wellknown
biological (e.g. pathogenicity) and antigenic properties (typing by MAbs). If it is to be used as a live
vaccine, the master seed virus must be shown not to cause clinical rabies. At least two animals (preferably
five to six per group) of each of the species for which the vaccine is intended and, so far as possible, any
species that might be in contact with vaccine or vaccinated animals, should be tested. This can be done by
inoculating in or adjacent to a major nerve, a dose equivalent to ten times the intended viral titre in one dose
of the proposed final product. Animals should be observed for at least 90 days for any adverse effect
attributable to the master seed.
A master cell stock of the seed virus should be prepared and kept at or below –70°C. Subculture from this
stock will be used for vaccine production. Virus multiplication is verified by titration during growth of the seed
virus.
Before a vaccine is licensed, evidence of efficacy should be established by the challenge of vaccinated and
control animals of each target species. The challenge should be performed at the end of the period after
vaccination for which the manufacturer claims maintenance of immunity. Antibody kinetics should also be
determined in order to establish the correlation between antibody titre and resistance to challenge.
The efficacy of the produced vaccine is assessed by studies on every target species previously vaccinated
as recommended. Protection at the end of the period of immunity is monitored by a measurement of specific
neutralising antibodies and by challenge with rabies virus. The experimental conditions of this challenge
should mimic the natural conditions of infection. The challenge virus should preferably be prepared from
locally isolated strains. In animals vaccinated with inactivated vaccines, the percentage of seroconversion
and the mean level of antibody allow a good prognosis for survival to challenge (3).
The correlation between potency in the target species and antigenic value as estimated in mice should be
established (see Section C.4.c below).
For the purposes of licensing a vaccine, safety tests should be conducted in the target species. In the case
of live virus vaccines used in oral vaccination campaigns (including recombinant vaccines), safety tests
should also be carried out on those other species that live in the area of vaccination and could become
exposed to the vaccine (6).
Vaccine stability is ascertained by testing batches after prolonged storage, usually 1–2 years. A process of
accelerated ageing, by storage at 37°C for 1 week, is sometimes used. The storage life claimed by the
manufacturer is checked by the national licensing authority. In general, it is 12–18 months for fluid vaccines,
and possibly 24 months for lyophilised vaccines.
Whatever method is adopted, close attention should be paid to the quality of the substrate. Eggs should be
of SPF
origin, and the cell cultures, such as BHK cell lines, should conform to international standards of sterility and
innocuity.
During manufacture, the multiplication of the virus in one of the substrates mentioned above is monitored,
followed by harvesting at the most appropriate time, usually 4–6 days after inoculation of eggs or cell
cultures. The
virus harvest is suspended in a buffer solution at a dilution that will provide an optimum antigenicity of the
endproduct.
If required, the suspension is either inactivated or lyophilised. An adjuvant is recommended for vaccines
prepared from inactivated virus, as well as for other vaccine antigens that may be incorporated in polyvalent
vaccines.
a) In cell cultures
Cultures are infected with cell-culture-adapted strains of rabies virus and incubated at 35–36°C. These may
then be used as live virus vaccines (as in Flury and SAD vaccines), or as inactivated vaccines after the
addition of phenol (Semple vaccine) or some other chemical, such as beta-propiolactone.
Cell culture can also be used to grow the vector viruses (e.g. vaccinia virus) harbouring the gene coding for
the expression of rabies virus glycoprotein (31).
b) In eggs
A modified egg-adapted strain of virus is inoculated into SPF-embryonated chicken eggs, which are then
incubated at 38°C for 5–6 days. The virus is harvested in the form of infective embryo tissues, and is usually
lyophilised and used as a live vaccine. Examples of such vaccines include those that contain the Flury low
egg passage (LEP), or the more desirable high egg passage (HEP) variant strain, which is safer for some
animal species such as the cat.
c) In animals
Nervous tissue vaccines prepared in animals are no longer considered safe or effective, and their use
should
be discontinued.
This consists of monitoring virus growth to provide an optimum titre and ensure the absence of undesirable
microbial contamination.
In live virus vaccines, kinetics of virus growth should be established in order to ensure a final titre of virus
correlated to the desired protection in target species.
In inactivated virus vaccines, immunogenic properties of the final product may be evaluated by in-vitro
techniques
(e.g. ELISA, agar gel immunodiffusion, antibody-binding tests or infected cell staining). These evaluations
will
indicate the best time for harvesting the virus in cell cultures.
4. Batch control
a) Sterility
b) Safety
Safety tests for batches of inactivated virus vaccines are carried out by inoculation of cell culture or
intracerebrally into mice to detect viable virus. A suitable safety test for live rabies vaccines should be carried
out on each lot of vaccine, in the intended host species. At least three, preferably five to six animals of the
intended host species should be given a dose equivalent to ten times the recommended field dose, by the
recommended route of administration. The animals should be observed for 180 days for adverse reactions
attributable to the vaccine (23).
c) Potency/biological activity
The amount of virus present in live attenuated and recombinant vaccines is determined by titration. Once a
correlation has been established between the activity of the vaccine in the target species and virus titres,
virus titrations become reliable indicators of vaccine efficacy. This is carried out using cell cultures or by the
intracerebral inoculation of unweaned mice (in mice it is only possible with a few attenuated viruses).
Recombinant vaccines should be monitored for the expressed rabies protein until assured that expression
stablilty is maintained in the manufacturing process. Titre of the vector can then be used as a reliable
indicator of vaccine efficacy.
For inactivated virus vaccines, correlation between potency in the target species and antigenic value as
estimated in mice provides a reliable indicator of vaccine activity. The potency of the vaccine is established
in the USA by the National Institutes of Health (NIH) test. Elsewhere, the European Pharmacopoeia test is
widely adopted.
Groups of at least ten mice, aged 3–4 weeks, are inoculated with single, decreasing doses of vaccine in
accordance with the European Pharmacopoeia (23), or with two doses, 1-week apart, according to the NIH
test (45). A sufficient number of dilutions of vaccine are compared to estimate the dilution at which 50% of
the mice are protected against intracerebral challenge 14 days later (23, 45).
A WHO international standard vaccine is available (see footnote 2) for calibration of national standards, so
that the results of testing for antigenicity can be expressed in IUs. The test is not valid unless:
i) For both the vaccine to be examined and the standard preparation, the PD50 (50% protective dose) lies
between the largest and smallest doses given to the mice.
ii) The titration of the challenge virus suspension shows that 0.03 ml of the suspension contained
25 mouse intra-cranial LD50 (MIC LD50). The challenge dose should be in the range 12–50 LD50 for a
valid test.
iii) The confidence interval (p = 0.95) for the test should not be less than 25% and not more than 400% of
the estimated potency: statistical analysis should show a significant slope and no significant deviations
from linearity or parallelism of the dose–response lines.
The vaccine passes the test if the estimated potency is not less than 1 IU per dose, in the smallest
prescribed dose.
A simplified test can also be used for the purpose of anticipating which vaccines are likely to be of an
antigenic value ≥1 IU per dose (4). This test used as a screening test is a good way to reduce the number of
mice used in vaccine potency control tests.
Duration of immunity must be established for the product licence in the target species with a defined
vaccination protocol.
e) Stability
The proposed shelf life must be verified by appropriate tests. These experiments include biological and
physico–chemical stability tests, and should be performed on a sufficient number of batches of vaccine
stored under recommended conditions.
The thermostability of live virus vaccines in liquid form is generally poor. For freeze-dried inactivated virus
vaccines, stability is generally granted for 2 years at 4°C.
f) Preservatives
Inactivated virus vaccines may contain preservatives (formalin, merthiolate). The nature and quantity of
these preservatives should comply with national control regulations.
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.
6. Oral vaccination
The concept of oral vaccination is unique: as stray or wild animals are out of physical reach, dropping
vaccine
baits into their environment is the only way to immunise them. In the 1980s and 1990s, the Veterinary Public
Health Department of WHO organised several meetings of rabies experts to define the requirements for
guaranteeing the safety and efficacy of vaccines both for the target species (red fox, raccoon dog, skunk,
dog,
etc.) and nontarget species, namely wild rodents and any other wild and domestic species that might be in
contact
with baits or a recently vaccinated animal (42, 44).
Several guidelines have been established for the quality criteria that vaccines have to satisfy before
marketing;
the most precise documents are those produced by WHO, the European Pharmacopoeia and the European
Commission (24, 25, 44). Available oral vaccines have been extensively tested by different routes (cerebral,
muscular and oral) in a variety of species: puppies and adults of carnivores, avian species, nonhuman
primates,
rodents and immunocompromised mice. Nonhuman primates have been added to this list since the
discovery in
1992 that the original SAD Bern strain is highly pathogenic for baboons by the oral route (12).
All vaccines currently used for oral vaccination programmes are either modified live-virus vaccines or live
recombinant vaccines. At the present time, two oral vaccines are recommended by WHO (44): a
recombinant
vaccine – VRG vaccine, and a highly attenuated vaccine – SAG2.
The production controls are closely related to the ones used for parenteral vaccines. The major differences
concern three points:
i) Safety of the vaccine for man, target and non target species.
ii) Efficacy of the protection induced by the vaccine.
iii) Monitoring of the impact of oral vaccination campaigns in the field.
a) Safety considerations
For oral vaccination, either attenuated rabies strains or live-recombinant vaccines may be used. The vaccine
should not induce any adverse signs in target and nontarget species. For vaccines used for dog
immunisation, saliva should be checked for the absence of vaccinal virus because of possible contact with
humans.
The attenuated rabies virus-based vaccines must achieve the lowest residual pathogenicity for target and
nontarget species (24); this is of utmost importance in the case of oral vaccination of dogs as dogs are often
in close contact with humans (44).
The recombinant vaccines cannot induce any risk of rabies; the safety controls concern only the possible
residual pathogenicity of the recombined parental virus.
b) Protection induced by the vaccine
The protection induced by the vaccine must be tested not only with the virus itself (to determine the minimal
vaccinating dose) but also with manufactured baits ready to be used in the field. For foxes for instance, the
vaccine should have a minimal titre corresponding to at least ten times the 100% protective dose (obtained
with the same vaccine experimentally by direct oral instillation) (14).
The protection status cannot be then checked by serology only; a virulent challenge with the homologous
street rabies virus is necessary because of the important implication of cell-mediated immunity in response
to oral vaccination (25).
c) Monitoring the impact of oral vaccination
The stability of the vaccine in the field is important. The European Commission stresses the importance of
checking the 100% protective dose after 7 days of exposure at 25°C (24). Each vaccine bait should be
tested for stability with a melting point above 40°C, and the blister or sachet containing the vaccine should
still be covered by the bait casing after 7 days exposure at 40°C (24).
Aerial distribution of baits is the only way to perform an homogenous, rapid and sufficient distribution for
wildlife vaccination. Quality control measures should be used to monitor different key points of baiting;
control of vaccine titre, control of area coverage by air and of baiting density should at least be constantly
monitored. The cross border cooperation between neighbouring countries is also needed to avoid any
unvaccinated area along the border.
For wildlife in Europe, two campaigns are performed yearly: the spring one aims at vaccinating the young
population of the target species, its period should then be fixed according to the biology of the target
species.
The autumn campaign concerns both adult and young animals. It is generally admitted that four campaigns
(i.e. 2 years) should be conducted after the last rabies diagnosis.
The impact of vaccination on the host/vector population is monitored in two different ways:
• Directly by measuring the bait uptake by the wild target species. This supposes that a biomarker
(generally tetracycline) is included in the bait casing. The same examination allows the age of animals
to be determined.
• Directly by measuring the serological response of target animals. This serological control is better done
using validated ELISA techniques (22, 35) as they are more robust than seroneutralisation tests when
testing poor quality field specimens.
• Indirectly by measuring the incidence of rabies in the vaccinated area. Typing of field isolates should be
performed (44) either by using MAbs or by sequencing positive samples from areas where the target
species have been vaccinated with attenuated vaccines to possibly distinguish vaccine and field virus
strains.
The first two controls should be performed on specially killed animals to collect good quality samples.
Rabies
monitoring is more sensitive when performed in found dead or ill animals.
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40. WIKTOR T.J., DOHERTY P.C. & KOPROWSKI H. (1977). In vitro evidence of cell-mediated immunity after
exposure of mice to both live and inactivated rabies virus. Proc. Natl Acad. Sci. USA, 74, 334–338.
41. WORLD HEALTH ORGANIZATION EXPERT COMMITTEE ON BIOLOGICAL STANDARDS. THIRTY-FIFTH REPORT (1985).
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on oral rabies vaccination of dogs and wild carnivores, Geneva, 1–2 March 1989, Doc.
WHO/Rab.Res./89.32.
43. WORLD HEALTH ORGANIZATION EXPERT COMMITTEE ON RABIES. EIGHTH REPORT (1992). World Health
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44. WORLD HEALTH ORGANIZATION EXPERT COMMITTEE ON RABIES, FIRST REPORT (2005). World Health
Organisation, WHO Technical Report Series, 931, WHO, Geneva, Switzerland, 1–87.
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M.M. & Koprowski H., eds. WHO, Geneva, Switzerland.
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*
**
NB: There are OIE Reference Laboratories for Rabies (see Table in Part 3 of this Terrestrial Manual or
consult
the OIE Web site for the most up-to-date list: www.oie.int).
CHAPTER2.1.17.
TRYPANOSOMA EVANSI INFECTIONS
(INCLUDING SURRA)
SUMMARY
Definition of the disease: Trypanosoma evansi causes a disease known as Trypanosomosis1
(‘surra’). It affects a number of species of domesticated animals in Asia, Africa and Central and
South America. The principal host species affected varies geographically, but buffalo, cattle, camels
and horses are particularly affected, although other animals, including wildlife, are also susceptible.
It is an arthropod-borne disease. Several species of haematophagous flies, including Tabanus spp.
and Musca spp., are implicated in transferring infection from host to host mechanically vectors. In
Brazil, vampire bats have been implicated in transmission.
Description of the disease: The disease in susceptible animals is manifested by pyrexia, directly
associated with parasitaemia, together with a progressive anaemia, loss of condition and lassitude.
Recurrent episodes of fever and parasitaemia occur during the course of the disease. Oedema,
particularly of the lower parts of the body, urticarial plaques and petechial haemorrhages of the
serous membranes are often observed. Abortions have been reported in buffaloes and camels.
There are indications that the disease causes immunodeficiencies.
Identification of the agent: The general clinical signs of T. evansi infection are not sufficiently
pathognomonic for diagnosis. Laboratory methods for detecting the parasite are required.
Examination of the host blood is problematic as trypanosomes can be detected only when there is a
high parasitaemia. Under these circumstances examination of wet blood films, stained blood
smears or lymph node material might reveal the trypanosomes. In other, more chronic cases, such
as the carrier state, the examination of thick blood smears, as well as methods of parasite
concentration and the inoculation of laboratory animals are required.
Serological tests: Infection gives rise to specific antibody responses and a variety of antibody
detection tests have been introduced for laboratory and field use. Some have been partially
validated, but await large-scale evaluation and standardisation. Among those that are used
regularly in the laboratory are immunoenzyme assays, card agglutination tests and latex
agglutination tests. For field use both card agglutination tests (CATT) for T. evansi and latex can be
applied, yet an individual test format (pen side test) is currently unavailable. Assays for detection of
circulating antibodies have high measures of validity. Estimates of predictive values of different
serological tests indicate that enzyme linked immunosorbent assays (ELISA) for detecting IgG
antibodies are more likely to classify correctly uninfected animals, and CATT are more likely to
classify correctly truly infected animals. An IgG ELISA would thus be suitable for verifying that
animals are free from infection, prior to movement or during quarantine. In situations where there is
overt disease, CATTs can be used to target individual animals for treatment with trypanocidal
drugs. For declaring a disease-free status, serial testing – ELISA followed by re-testing of suspect
samples by CATT – is recommended. It must be stressed however, that there are considerable
antigenic similarities among the different species of pathogenic trypanosomes, hence in areas
where tsetse-transmitted trypanosomoses occur cross-reactions may occur with any serological test
employed.
Requirements for vaccines and diagnostic biologicals: No vaccines are available for the
disease.
1 Nomenclature of parasitic diseases: see the note in Chapter. 2.4.18 Trypanosomosis (Tsetse-borne).
Chapter 2.1.17. — Trypanosoma evansi infections (including surra)
A. INTRODUCTION
The clinical signs of surra, the disease caused by Trypansoma evansi, are indicative but are not sufficiently
pathognomonic and diagnosis must be confirmed by laboratory methods. The disease in susceptible
animals,
including cattle, buffalo, camels (dromedary and bactrian), horses, pigs, sheep and goats, is manifested by
pyrexia, directly associated with parasitaemia, together with a progressive anaemia, loss of condition and
lassitude. Recurrent episodes of fever and parasitaemia occur during the course of the disease. Oedema,
particularly of the lower parts of the body, urticarial plaques and petechial haemorrhages of the serous
membranes are often observed. Abortions have been reported in buffaloes and camels (8, 17) and there are
indications that the disease causes immunodeficiency (5, 21).
There is considerable variation in the pathogenicity of different strains and the susceptibility of different host
species to disease. Disease may manifest as an acute or chronic condition, and in the latter case may
persist for
many months. The disease is often rapidly fatal in camels, buffaloes, horses, cattle, llama and dogs, but mild
and
subclinical infections can also occur in these species. Wild animals such as deer and capybara can become
infected. Animals subjected to stress – malnutrition, pregnancy, work – are more susceptible to disease.
Biologically T. evansi is very similar to T. equiperdum, the causative agent of dourine (2), and
morphologically
resembles the slender forms of the tsetse-transmitted trypanosomes, T. brucei, T. gambiense and T.
rhodesiense.
Molecular characterisation indicates that various strains of T. evansi isolated from Asia, Africa and South
America
have a single origin. Molecular characterisation using random amplified polymorphic DNA techniques and
endonuclease fingerprinting showed that isolates of T. evansi and T. equiperdum formed a closely
homogeneous
group. One possibility of this finding is that T. equiperdum is not a genuine species per se and that the
clinical
outcome of disease is related primarily to the hosts’ immune response. Like all pathogenic trypanosomes,
T. evansi is covered by a dense protein layer consisting of a single protein called the variable surface
glycoprotein. This acts as a major immunogen and elicits the formation of specific antibodies. The parasites
are
able to evade the consequences of these immune reactions by switching the variant surface glycoprotein,
the
phenomenon known as antigenic variation.
The classical direct parasitological methods for the diagnosis of trypanosomosis, namely examining blood or
lymph node material, are not highly sensitive. In regions where other Trypanozoon spp. occur in addition to
T. evansi, specific identification by microscopy is not possible. Specific DNA probes (19, 24) may enable
identification of trypanosome species by nonradioactive DNA hybridisation. A species-specific polymerase
chain
reaction (PCR) based on T. evansi specific antigen (RoTat 1.2 VSG) has been developed, but has not been
validated in the field (3).
• Direct methods
a) Usual field methods
i) Blood sampling
Trypansoma evansi is a parasite of the blood and tissues often inhabiting the deep blood vessels in
cases of low parasitaemia. For this reason, it is recommended that blood for diagnosis be obtained
from both the peripheral and deep blood vessels. However it should be realised that less than 50% of
infected animals may be identified by examination of peripheral blood.
Peripheral blood is obtained by puncturing a small vein in the ear or tail. Deeper samples are taken
from a larger vein by syringe. Cleanse an area of the ear margin or tip of the tail with alcohol and, when
dry, puncture a vein with a suitable instrument. Ensure that instruments are sterilised or disposable
instruments are used between individual animals, so that infection cannot be transmitted by residual
blood.
ii) Wet blood films
Place a small drop of blood on to a clean glass slide and cover with a cover-slip to spread the blood as
a monolayer of cells. Examine by light microscopy (×200) to detect any motile trypanosomes.
iii) Stained thick smears
Place a large drop of blood on the centre of a microscope slide and spread with a toothpick or the
corner of another slide so that an area of approximately 1.0–1.25 cm in diameter is covered. Air-dry for
1 hour or longer, while protecting the slide from insects. Stain the unfixed smear with Giemsa’s Stain
(one drop of commercial Giemsa + 1 ml of phosphate buffered saline [PBS, 2.4 g Na2HPO4.2H2O,
0.54 g NaH2PO4.2H2O, 0.34 g NaCl], pH 7.2), for 25 minutes. After washing, examine the smears by
light microscopy at high magnification (×500–1000). The advantage of the thick smear technique is that
it concentrates the drop of blood into a small area, and thus less time is required to detect the
parasites. The disadvantage is that the trypanosomes may be damaged in the process, and the method
is therefore not suited for species identification in case of mixed infections.
iv) Stained thin smears
Place a drop of blood 20 mm from one end of a clean microscope slide and draw out a thin film in the
usual way. Air-dry briefly and fix in methyl alcohol for 2 minutes and allow to dry. Stain the smears in
Giemsa (one drop Giemsa + 1 ml PBS, pH 7.2) for 25 minutes. Pour off, stain and wash the slide in tap
water and dry. Unfixed smears can be stained by covering them with May–Grünwald stain for
2 minutes, then adding an equal volume of PBS, pH 7.2, and leaving the slides for a further 3 minutes.
Pour off and add diluted Giemsa for 25 minutes. Pour off, wash the slides with tap water, and dry.
Examine at high magnification (×400–1000). This technique permits detailed morphological studies and
identification of the trypanosome species. Rapid staining techniques also exist (Field’s stain, Diff
Quick®).
v) Lymph node biopsies
Samples are usually obtained from the prescapular or precrural (subiliac) lymph nodes. Select a
suitable node by palpation and cleanse the site with alcohol. Puncture the node with a suitable gauge
needle, and draw lymph node material into a syringe attached to the needle. Expel lymph on to a slide,
cover with a cover-slip and examine as for the fresh blood preparations. Fixed thin or thick smears can
also be stored for later examination.
b) Concentration methods
In most hosts T. evansi can induce mild clinical or subclinical carrier state infections with low parasitaemia in
which it is difficult to demonstrate the parasites. In these circumstances, concentration methods become
necessary.
i) Haematocrit centrifugation
Collect blood (70 μl) into at least two heparinised capillary tubes (75 × 1.5 mm). Seal at the dry end by
heat and centrifuge, sealed end down, at 3000 g for 10 minutes. Place the capillary tube between two
pieces of glass (25 × 10 × 1.2 mm) glued to a slide. Place a cover-slip on top at the level of the buffy
coat junction where the trypanosomes will be concentrated. Flood the space around this part of the
tube with water or immersion oil, and examine the buffy coat area under the microscope (×100–200).
This technique can detect around 400 trypanosomes/ml. A simpler alternative is to examine the
centrifuged capillary tube by placing a drop of immersion oil on the tube and ensuring that there is
contact between the objective lens and the immersion oil.
ii) Dark-ground/phase-contrast buffy coat technique
Collect blood into heparinised capillary tubes and centrifuge as above. Scratch the tube with a glasscutting
diamond and break it 1 mm below the buffy coat layer – the upper part thus contains the top
layer of red blood cells (RBCs), the buffy coat (white blood cells) and some plasma.
Partially expel the contents of this piece on to a slide, cover with a cover-slip and examine under
darkground,
phase-contrast or ordinary illumination.
As an alternative to the electrically powered haematocrit centrifuge, hand-powered micro-centrifuges
have been used successfully for detection of trypanosomosis in cattle and camels (9, 14).
iii) Haemolysis techniques
Sodium dodecyl sulphate (SDS) can be used as a reagent to haemolyse RBCs to facilitate detection of
motile trypanosomes in parasitised blood samples. As SDS is toxic, contact with skin, inhalation and
ingestion should be avoided. SDS solution can be stored for several months at ambient temperature.
Both the SDS solution and the blood samples should be used at a temperature above 15°C. At lower
temperatures the trypanosomes may be destroyed.
Two general procedures, namely wet blood film clarification and haemolysis centrifugation, can be used2.
2 All necessary materials and instructions can be obtained from the Institute of Tropical Medicine, Laboratory of Serology,
Nationalestraat 155, B-2000 Antwerp, Belgium.
� Wet blood film clarification method
This method uses the partial lysis of RBCs to facilitate the detection of motile trypanosomes. The
method requires an SDS solution: 1% SDS dissolved in Tris/glucose/saline, pH 7.5, inoculating loops
(10 μl), slides and cover-slips (24 × 24 mm), and a drop of fresh meat or heparinised blood. Dissolve
100 mg of SDS in 100 ml of isotonic Tris/NaCl/glucose buffer, pH 7.5 (Trizma base 14.0 g, NaCl 3.8 g,
glucose 10.8 g; dissolve chemicals in 750 ml distilled H2O, add 90–100 ml 1 N HCl and adjust pH to 7.5
then make up to final volume of 1000 ml with distilled H2O). This buffer can be stored in small vials at
ambient temperature for several months. The test does not give a significantly higher sensitivity than
wet film technique. In addition, the SDS can cause problems when focusing the microscope and the
movements of trypanosomes can be severely curtailed owing to the high viscosity of the SDS.
Put approximately 10 μl of blood on a slide. Add 10 μl of SDS solution using a dip inoculation loop and
mix gently. Apply a cover-slip and examine the preparation without delay at low magnification (×100 or
×200).
� Haemolysis centrifugation technique
Nearly complete lysis of RBCs is required for this procedure. The materials needed include: SDS
solution (0.1% SDS dissolved in Tris/glucose/saline, pH 7.5), conical centrifuge tubes, ordinary test
tubes, large and fine plastic tapering pipettes with attached bulb, slides, cover-slips (24 × 24 mm or
24 × 32 mm), and heparinised blood.
Using a pipette or syringe, transfer nine volumes (maximum 6.3 ml) of SDS solution into an ordinary
test tube. Aspirate one volume (maximum 0.7 ml) of heparinised blood into a bulb pipette and expel it
just above the surface of the SDS solution; mix quickly and thoroughly. Avoid foam formation, which
may result in destruction of the trypanosomes. Wait for 10 minutes so as to achieve complete
haemolysis.
Pour the haemolysed suspension into a conical centrifuge tube and spin at approximately 500 g for
10 minutes. With a clean bulb pipette, remove as much supernatant as possible without disturbing the
sediment. Using a fine tapering bulb pipette, remove more supernatant, leaving 10–20 μl of undisturbed
sediment at the bottom.
Very carefully collect the entire sediment and put on to a microscope slide. Apply a cover-slip and
examine the preparation without delay at low magnification (×100 or ×200).
iv) Mini-anion exchange centrifugation technique
When a blood sample from animals infected with salivarian trypanosomes is passed through an
appropriate anion-exchange column, the host blood cells, being more negatively charged than
trypanosomes, are adsorbed onto the anion-exchanger, while the trypanosomes are eluted, retaining
viability and infectivity (15). A simplified field method for detection of low parasitaemia has been
developed (26). The sensitivity of this technique can be increased by approximately tenfold by the use
of buffy coat preparations rather than whole blood (25).
� Preparation of phosphate buffered saline glucose, pH 8
Na2HPO4 (anhydrous) (13.48 g); NaH2PO4.2H2O (0.78 g); NaCl (4.25 g); distilled water (1 litre).
Solutions of different ionic strength are made by diluting the stock PBS, pH 8, and adding glucose to
maintain a suitable concentration. For blood of mice, domestic and wild ruminants and dog, add four
parts PBS to six parts distilled water and adjust the final glucose concentration to 1%. For blood of pigs
and rabbits, add three parts PBS to seven parts distilled water and adjust the final glucose
concentration to 1.5%. The PBS/glucose solution (PSG) must be sterile.
� Equilibration of DEAE-cellulose
Suspend 500 g of DEAE-cellulose (diethylamino-ethylcellulose) in 2 litres of distilled water. Adjust the
pH to 8 with phosphoric acid. Allow to settle for 30 minutes. Discard the supernatant fluid containing the
fine granules. Repeat the procedure three times. Store the equilibrated concentrated suspension of
DEAE-cellulose (slurry) at 4°C or in small aliquots at –20°C.
� Packing of equilibrated DEAE-cellulose
Place a 2 ml syringe without the plunger on a test-tube rack complete with needle (20 G × 1.5 inch). Put
a disc of Whatman No. 41 filter paper at the bottom of the syringe and moisten by adding a few drops
of PSG. Pour 2–2.5 ml of the slurry of equilibrated cellulose into the syringe and allow to pack by
elution of the buffer. The height of the sediment should be approximately 3 cm. Wash and equilibrate
the column with 2 ml of PSG without disturbing the surface.
� Adsorption of blood eluate of the trypanosomes
Gently place 100–300 μl of heparinised blood above the surface of the cellulose column. Add ten drops
of PSG and discard the first ten drops of the eluate. Progressively add 1.5 ml of PSG and start
collecting the eluate into a finely tapered Pasteur pipette with a sealed end. Put the filled pipette,
protected by a conical plastic pipette tip, in a tube and centrifuge at 525 g (or up to 1000 g) for
10 minutes. Examine the bottom of the pipette under the microscope (×100 or ×200) using a special
mounting device. Alternatively, the eluate could be collected into 50 ml plastic tubes, with conical
bottoms, centrifuged at 1000 g and the sediment examined by dark-ground microscopy.
The cellulose column should remain wet throughout the procedure.
c) Animal inoculation
Laboratory animals may be used to reveal subclinical (nonpatent) infections in domesticated animals.
Trypanosoma evansi has a broad spectrum of infectivity for small rodents, and so rats and mice are often
used. Rodent inoculation is not 100% sensitive (18) but further improvement in its efficacy can be obtained
by the use of buffy coat material. Such a procedure was able to detect as few as 1.25 T. evansi/ ml blood
(25).
Inoculate heparinised blood intraperitoneally into rats (1–2 ml) or mice (0.25–0.5 ml). Inoculate a minimum of
two animals. Bleed animals from the tail three times a week to detect parasitaemia. The incubation period
before appearance of the parasites and their virulence depends on the strain of trypanosomes, their
concentration in the inoculum, and the strain of laboratory animal used. Sensitivity of this in-vivo culture
system may perhaps be increased by use of immunosuppressed laboratory animals. Drugs such as
cyclophosphamide or hydrocortisone acetate, or X-ray irradiation or splenectomy are used for this purpose.
d) Recombinant DNA probes
Specific DNA probes have been used to detect trypanosomes in infected blood or tissue but are not routinely
applied (28, 31). Although molecular methods have a potentially high analytical sensitivity there have been
few convincing studies to critically evaluate the diagnostic sensitivity of these tests as compared with other
techniques, such as serology.
e) Detection of trypanosomal DNA
Detection of minute amounts of trypanosomal DNA using a PCR procedure is a possible means of
identifying
animals with active infections, and could have the sensitivity and specificity required (3, 20, 32).
Experimental studies in buffalo (10) showed the diagnostic sensitivity of a PCR was only 78%, which is
similar to mouse inoculation.
• Indirect methods
These methods involve tests that demonstrate the effects of the parasite on its host rather than directly
detecting
the parasite itself.
a) Haematology
Anaemia is usually a reliable indicator of trypanosome infection, although it is not in itself pathognomonic.
However, animals with a mild subclinical infection can have parasitaemia without evidence of anaemia (4).
Anaemia can be estimated by measuring the packed cell volume and may be used in surveys of herds at
risk. The technique is identical to that of haematocrit centrifugation. The capillary tube is examined and the
results are expressed as a percentage of packed RBCs to total blood volume.
Historically, many different methods have been used to detect specific humoral antibodies to trypanosomal
antigens but, more recently, there has been a tendency to concentrate on more easily standardised
techniques
such as ELISA (6, 7, 12, 22, 23, 24, 27) card agglutination tests (CATT) (1, 20, 24) and latex agglutination
tests
(LAT) (11, 16). Extensive evaluation of ELISA and CATT has been carried out in buffaloes in Indonesia and
Vietnam (6, 11, 29). The collection of samples can be simplified by using filter paper blood spots for later use
in
the ELISA, while for the CATT whole blood can be substituted for serum (11). Other innovative modifications
that
might be developed in the future are the use of a colloidal-dye dipstick test (13) that could enable the tests to
be
carried out under field conditions. It is vitally important that serological tests are validated and standardised if
they
are to be suitable for correctly identifying infected animals. This means that standard criteria for interpreting
the
tests might have to be developed for each animal species as well as each laboratory operating the
procedure.
a) Enzyme-linked immunosorbent assay
The principle of this technique is that specific antibodies to trypanosomes can be detected by enzyme-linked
anti-immunoglobulins using solid-phase polystyrene plates coated with soluble antigen. The enzyme may be
peroxidase, alkaline phosphatase or any other suitable enzyme. The enzyme conjugate binds to the
antigen/antibody complex and then reacts with a suitable substrate to yield a characteristic colour change
either of the substrate itself or of an added indicator (the chromogen).
The antigen for coating the plates is derived from the blood of a heavily parasitaemic rat. The trypanosomes
are separated on a DEAE-cellulose column and washed three times by centrifugation in cold PSG, pH 8
(PBS with 1% glucose). The final pellet is suspended in cold PSG to a concentration of 3–5%, and briefly
ultrasonicated on ice for 30–120 seconds until disintegration of the organisms is complete. This preparation
is centrifuged at 4°C and 40,000 g for 60 minutes. The supernatant is diluted in water so as to obtain a
protein concentration of 1 mg/ml. The reagent thus obtained can be stored in small aliquots at –70°C for
several months. It can also be freeze-dried and stored at –20°C. Various treatments of the antigen
preparations have been applied to improve the accuracy of antibody detection (32).
� Test procedure
i) Dilute or reconstitute the frozen or freeze-dried antigen with freshly prepared 0.01 M
carbonate/bicarbonate buffer, pH 9.6. Add 100 μl to each well of a 96-well microtitre plate and incubate
overnight at 4°C or for 1 hour at 37°C.
ii) Remove excess antigen by washing plate with 0.01 M PBS containing 0.05% Tween 20 (PBST). Add
test serum dilutions in PBST (100 μl). Include control negative and positive sera. Dilutions must be
determined empirically, but are usually between 1/100 and 1/1000.
iii) Incubate plates at 37°C for 30 minutes. Eject contents and wash three times with PBST.
iv) Add a specific peroxidase conjugated species-specific anti-globulin (100 μl) appropriately diluted in
PBST (usually between 1/1000 and 1/50,000).
v) Incubate the plates at 37°C for 30 minutes, eject contents and wash three times with PBST.
vi) For peroxidase conjugates a number of substrate/chromogen solutions can be used, consisting of
hydrogen peroxide with a chromogen, such as tetramethylbenzidene (TMB), 2,2’-azino-bis-(3-
ethylbenzothiazoline-6-sulphonic acid) (ABTS) and ortho-diphenylenediamine (OPD). A suitable
substrate/chromogen solution for peroxidase conjugates is 30% hydrogen peroxide (0.167 ml and
35 mg) in citrate buffer (100 ml), pH 6.0. The citrate buffer is made up as follows: Solution A (36.85 ml):
(0.1 M citric acid [21.01 g/litre]); Solution B (65.15 ml): (0.2 M, Na2HPO4 [35.59 g/litre]); and distilled
water (100 ml). Dissolve 10 mg TMB in 1 ml dimethyl sulphoxide and add to 99 ml of the citrate buffer.
A number of these combinations are available commercially in ready-to-use formulations that remain
stable at 4°C for up to 1 year.
vii) Add the substrate chromogen (100 μl) to the plates and incubate at room temperature for 15–
20 minutes.
viii) Stop the reaction by adding 50 μl 1 M sulphuric acid. Read the absorbance of each well at 450 nm for
TMB chromogen. Other chromogens may require the use of a different wavelength. All tests should
include known high and medium positive control sera, a negative control serum, and a buffer control.
A large variety of other test procedures exists. For closely related animal species, cross-reacting reagents
may often be used (e.g. anti-bovine immunoglobulin for buffaloes) and the use of monospecific anti-IgG
conjugates is generally recommended. There are a number of methods that can be used to determine a
cutoff
point to discriminate between positive and negative results. The simplest method is to base the cut-off on
visual inspection of the test results from known positive and negative populations. These results are likely to
show some overlap. The operator can choose the most appropriate point to modify the false negative or
false positive results depending on the required application of the assay. An alternative is to base the cut-off
on the mean +2 standard deviations (SD) or +3 SD values from a large sample of negative animals. Finally,
if no suitable negative/positive samples are available a cut-off can be based on the analysis of the data from
animals in an endemic situation. If a bimodal distribution separates infected from uninfected animals, then
an
appropriate value can be selected. The ELISA is likely to correctly identify uninfected animals. Equivocal
results can be re-tested using CATT. The Institute of Tropical Medicine in Antwerp developed an ELISA
using the VSG from a T. evansi RoTat 1.2 clone. It was shown (30) that this antigen is a predominant
antigen in T. evansi and absent in T.b brucel. This ELISA/RoTat 1.2 was successfully used in the field in
Vietnam (11, 29). Protocols are available at ITM Antwerp for use in equines, camelidae and water buffaloes.
b) Card agglutination tests
It is well known that certain predominant variable antigen types (VATs) are expressed in common in different
strains of salivarian trypanosomes from different areas. This finding was used as a basis for a test for the
diagnosis of T. evansi, the card agglutination test – CATT/T.evansi – was developed at the Laboratory of
Serology, Institute of Tropical Medicine, Antwerp3. The test makes use of fixed and stained trypanosomes of
a defined VAT known as RoTat 1.2. Both variable and invariable surface antigens take part in the
agglutination reaction. The CATT is available in kit form from ITM, Antwerp. It consists of lyophilised antigen,
PBS, pH 7.4, plastic-coated cards, spatulas, positive and negative control sera and a rotator. The lyophilised
antigen can be stored at 2–8°C for up to 1 year. Reconstituted antigen can be stored at 2–8°C for 2 days,
but preferably should be used within 8 hours.
For screening, dilute test sera 1/4 or 1/8 in PBS on to circles inscribed on the plastic cards. Add 45 μl of the
prepared antigen suspension onto circles inscribed on the plastic cards. Add 25 μl of each test serum. Mix
and spread the reagents with a spatula and rotate the card for 5 minutes using the rotator provided in the kit.
Compare the pattern of agglutination with the illustrations of different reactions provided in the kit. Blue
granular deposits reveal a positive reaction visible to the naked eye.
c) Latex agglutination tests
A kit is available from ITM, Antwerp. It comprises a lyophilised latex suspension coated with T. evansi RoTat
1.2 variable antigens, PBS, positive and negative controls, test cards, plastic spatulas and a rotator.
Reconstitute the antigen-coated latex particles using distilled, deionised water. Mix gently. Add 20 μl of latex
suspension onto each black spot on the test cards.
Dilute test sera with PBS (1/2 to 1/4) and add 20 μl to the latex suspension. Include appropriate controls.
Mix
the reagents carefully with a plastic spatula.
Rotate test cards at 70 rotations/minutes for 5 minutes and view cards under a good light source at the end
of the incubation. Positive sera will cause agglutination of the latex particles.
BIOLOGICALS
No vaccines are available for this disease.
REFERENCES
1. BAJYANA SONGA E. & HAMERS R. (1988). A card agglutination test (CATT) for veterinary use based on an
early
VAT RoTat 1–2 of Trypanosoma evansi. Ann. Soc. Belg. Med. Trop., 68, 233–240.
2. CLAES F., AGBO E.C., RADWANSKA M., TE PAS M.F.W., BALTZ T., DE WAAL D.T., GODDEERIS B.M., CLASSEN E.
&
BUSCHER P. (2003). How does Trypanosoma equiperdum fit into the Trypanozoon group? A cluster analysis
by RAPD and multiplex-endonuclease genotyping approach. Parasitology, 26, 425–431
3. CLAES F., RADWANSKA M., URAKAWA T., MAJIWA P.A., GODDEERIS B. & BUSCHER P. (2004) Variable surface
glycoprotein RoTat 1.2 PCR as a specific diagnostic tool for the detection of Trypanosoma evansi infections.
Kinetoplastid Biol. Dis., 3, 3.
4. DARGANTES A.P., REID S.A. & COPEMAN D B. (2005). Experimental Trypanosoma evansi infection in the
goat. I
Clinical signs and pathology. J. Comp. Pathol., 133, 261–266.
5. DARGANTES A.P., REID S.A. & COPEMAN D.B. (2005). Experimental Trypanosoma evansi infection in the
goat.
II Pathology. J. Comp. Pathol., 133, 267–276.
6. DAVISON H.C., THRUSFIELD M.V., MUHARSINI S., HUSEIN A., PARTOUTOMO S., MASAKE R. & LUCKINS A.G.
(1999).
Evaluation of antigen- and antibody-detection tests for Trypanosoma evansi infections of buffaloes in
Indonesia. Epidemiol. Infect., 123, 149–155.
7. FRANKE C.R., GREINER M. & MEHLITZ D. (1994). Investigations on naturally occurring Trypanosoma evansi
infections in horses, cattle, dogs and capybaras (Hydrochaeris hydrochaeris) in Pantanal de Pocone (Mato
Grosso, Brazil). Acta Trop., 58, 159–169.
3 CATT/T. evansi kits are available at the Laboratory of Parasite Diagnostic, Institute of Tropical Medicine, Nationalestraat
155, B-2000 Antwerp, Belgium. (pbuscher@itg.be ; fclaes@itg.be)
8. GUTIERREZ C., CORBERA J.A., JUSTE M.C., DORESTE F. & MORALES I. (2005). An outbreak of abortions and
high
neonatal mortality associated with Trypanosoma evansi infection in dromedary camels in the Canary
Islands.
Vet. Parasit., 130, 163–168.
9. HOLMSTEDT H. (1965). Simple microcentrifuge for use in the field. Science, 149, 977–978.
10. HOLLAND W.G., CLAES F., MY L.N., THANH N.G., TAM P.T., VERLOO D., BUSCHER P., GODDEERIS B. &
VERCRUYSSE J. (2001). A comparative evaluation of parasitological tests and a PCR for Trypanosoma evansi
diagnosis in experimentally infected water buffaloes. Vet. Parasit., 97, 23–33.
11. HOLLAND W.G., THANH N.G., MY L.N., MAGNUS E., VERLOO D., BUSCHER P., GODDEERIS B. & VERCRUYSSE J.
(2002). Evaluation of whole fresh blood and dried blood on filter paper discs in serological tests for
Trypanosoma evansi in experimentally infected water buffaloes. Acta Trop., 81, 159–165.
12. INTERNATIONAL ATOMIC ENERGY AGENCY (IAEA) (1993). Improving the diagnosis and control of
trypanosomiasis and other vector-borne diseases of African livestock using immunoassay methods.
International Atomic Energy Agency, TECDOC, 707, 171 pp.
13. KASHIWAZAKI Y., KANITPUN R., SUTEERAPARP P. & BOONCHIT S. (2000). A preliminary comparative study of a
dipstick colloidal dye immunoassay and two antigen-detection ELISAs for diagnosis of Trypanosoma evansi
infection in cattle. Vet. Res. Commun., 92, 533–544.
14. KELLY S. & SCHILLINGER D. (1983). Improved field diagnostic technique for trypanosomiasis by use of a
microcentrifuge. Vet. Rec., 113, 219.
15. LANHAM S.M. & GODFREY D.G. (1970). Isolation of salivarian Trypanosomes from man and other
mammals
using DEAE – Cellulose. Exp. Parasitol., 28, 521–528.
16. LEJON V., CLAES F., VERLOO D., MAINA M., URAKAWA T., MAJIWA P.A. & BUSCHER P. (2005). Recombinant
RoTat 1.2 variable surface glycoprotein as antigen for diagnosis of Trypanosoma evansi in dromedary
camels. Int. J. Parasitol., 35, 455–460.
17. LOHR K.F., PHOLPARK S., SIRIWAN P., LEESIRIKUL N., SRIKITJAKARN & STAAK C. (1986). Trypanosoma evansi
infection in buffaloes in North-East Thailand. II. Abortions. Trop. Anim. Health Prod., 18, 103–108.
18. MONZON C.M., MANCEBO O.A. & ROUX J.P. (1990). Comparison between 6 parasitological methods for
diagnosis of Trypanosoma evansi in the subtropical area of Argentina. Vet. Parasitol., 36, 141–146.
19. MASIGA D.K. & GIBSON W.C. (1990). Specific probes for Trypanosoma (Trypanozoon) evansi based on
kinetoplast DNA minicircles. Mol. Biochem. Parasitol., 40, 279–284.
20. NJIRU Z.K., CONSTANTINE C.C., NDUNG’U J.M., ROBERTSON I., OKAYE S., THOMPSON R.C. & REID S.M.
(2004).
Detection of Trypanosoma evansi in camels using PCR and CATT/T. evansi tests in Kenya. Vet. Parasitol.,
124, 187–199.
21. ONAH D.N., HOPKINS J.A. & LUCKINS 5A.G. (1998). Increase in CD5 B cells and depression of immune
responses in sheep infected with Trypanosoma evansi. Vet. Immunol. Immunopathol., 63, 209–222.
22. RAE P.F., THRUSFIELD M.V., HIGGINS A., AITKEN C.G.G., JONES T.W. & LUCKINS A.G. (1989). Evaluation of
enzyme immunoassays in the diagnosis of camel (Camelus dromedarius) trypanosomiasis: a preliminary
investigation. Epidemiol. Infect., 102, 297–307.
23. REID S.A. & COPEMAN D.B. (2002). Evaluation of an antibody-ELISA using five crude antigen
preparations for
the diagnosis of Trypansoma evansi infection in cattle. Vet. Parasitol., 104, 79–84.
24. REID S.A. & COPEMAN D.B. (2003). The development and validation of an antibody-ELISA to detect
Trypanosoma evansi infection in cattle in Australia and Papua New Guinea. Prev. Vet. Med., 61, 195–208.
25. REID S.A., HUSEIN A. & COPEMAN D.B. (2001). Evaluation and improvement of parasitological tests for
Trypanosoma evansi infection. Vet. Parasitol., 102, 291–297.
26. SACHS R. (1984). Improvements in the miniature anion exchange centrifugation technique for detecting
trypanosomes in domestic pigs. Trans. R. Soc. Trop. Med. Hyg., 78, 561.
27. TUNTASUVAN D., CHOMPOOCHAN T., VONGPAKORN M. & MOHKAEW K. (1996). Detection of Trypanosoma
evansi
antibodies in pigs using an enzyme linked immunosorbent assay. J. Thai Vet. Med. Assoc., 47, 45–53.
28. VENTURA R.M., TAKEDA G.F., SILVA R.A., NUNES V.L., BUCK G.A. & TEIXEIRA M.M. (2002). Genetic
relatedness
among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a
synapomorphic DNA fragment for species-specific diagnosis. Int. J. Parasitol., 32, 53–63.
29. VERLOO D., HOLLAND W., MY L.N., THANH N.G., TAM P.T., GODDEERIS B., VERCRUYSSE J. & BUSCHER P.
(2000).
Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from north
Vietnam. Vet. Parasitol., 92, 87–96.
30. VERLOO D., MAGNUS E. & BUSCHER P. (2001). General expression of RoTat 1.2 variable antigen type in
Trypanosoma evansi isolates from different origin. Vet. Parasitol., 97, 183–189.
31. VISESHAKUL N. & PANYIM S. (1990). Specific DNA probe for the sensitive detection of Trypanosoma
evansi.
Southeast Asian J. Trop. Med. Public Health, 21, 21–27.
32. WUYTS N., CHODESAJJAWATEE N. & PANYIM S. (1994). A simplified and highly sensitive detection of
Trypanosoma evansi by DNA amplification. Southeast Asian J. Trop. Med. Public Health, 25, 266–271.
*
**
NB: There are OIE Reference Laboratories for Trypanosoma evansi infections (including surra) (see Table in
Part
3 of this Terrestrial Manual or consult the OIE Web site for the most up-to-date list: www.oie.int).
C H A P T E R 2 . 1 . 9 .
LEPTOSPIROSIS
SUMMARY
Leptospirosis is a transmissible disease of animals and humans caused by infection with any of the
pathogenic members of the genus Leptospira. Laboratory diagnosis of leptospirosis can be complex
and involves tests which fall into two groups. One group of tests is designed to detect antileptospiral
antibodies and the other group of tests is designed to detect leptospires, leptospiral
antigens, or leptospiral nucleic acid in animal tissues or body fluids. The particular testing regimen
selected depends on the purpose of testing (e.g. herd surveys or individual animal testing) and on
the tests or expertise available in the area.
Identification of the agent: The isolation or demonstration of leptospires in:
a) the internal organs (such as liver, lung, brain, and kidney) and body fluids (blood, milk,
cerebrospinal, thoracic and peritoneal fluids) of clinically infected animals gives a definitive
diagnosis of acute clinical disease or, in the case of a fetus, chronic infection of its mother.
b) the kidney, urine, or genital tract of animals without clinical signs is diagnostic only of a chronic
carrier state.
Isolation of leptospires from clinical material and identification of isolates is time-consuming and is a
task for specialised reference laboratories. Isolation followed by typing from renal carriers is
important and very useful in epidemiological studies to determine which serovars are present within
a particular group of animals, an animal species, or a geographical region.
The demonstration of leptospires by immunochemical tests (immunofluorescence and
immunohistochemistry) is more suited to most laboratory situations. However, the efficacy of these
tests is dependent on the number of organisms present within the tissue, and these tests lack the
sensitivity of culture. Unless specially prepared reagents are used, immunochemical tests do not
identify the infecting serovar and results must be interpreted in conjunction with serological results.
Reagents for immunofluorescence are best prepared with high IgG titre anti-leptospire sera, which
are not available commercially. Rabbit leptospiral-typing serum or monoclonal antibodies can be
used for immunohistochemistry and are available from leptospiral reference laboratories.
Genetic material of leptospires can be demonstrated in tissues or body fluids using a variety of
assays based on the polymerase chain reaction (PCR), either in real-time or traditional formats.
PCR assays are sensitive, but quality control procedures and sample processing for PCR are
critical and must be adjusted to the tissue, fluid and species being tested. Like immunochemical
tests, PCR assays do not identify the infecting serovar.
Serological tests: Serological testing is the most widely used means for diagnosing leptospirosis,
and the microscopic agglutination test (MAT) is the standard serological test. Antigens selected for
use in the MAT should include representative strains of the serogroups known to exist in the
particular region plus those known to be maintained elsewhere by the host species under test.
The MAT is used to test individual animals and herds. As an individual animal test, the MAT is very
useful for diagnosing acute infection: a four-fold rise in antibody titres in paired acute and
convalescent serum samples is diagnostic. To obtain useful information from a herd of animals, at
least ten animals, or 10% of the herd, whichever is greater, should be tested and the vaccination
history of the animals documented.
The MAT has limitations in the diagnosis of chronic infection in individual animals and in the
diagnosis of endemic infections in herds. Infected animals may abort or be renal/genital carriers
with MAT titres below the widely accepted minimum significant titre of 1/100 (final dilution).
Chapter 2.1.9. - Leptospirosis
Enzyme-linked immunosorbent assays (ELISAs) can also be useful for detection of antibodies
against leptospires. Numerous assays have been developed and are primarily used for the
detection of recent infections and the screening of experimental animals for use in challenge
studies. Animals that have been vaccinated against the serovar of interest may be positive in some
ELISAs, thus complicating interpretation of the results.
Requirements for vaccines and diagnostic biologicals: Vaccines for veterinary use are most
often suspensions of one or more serovars of Leptospira spp. inactivated in such a manner that
immunogenic activity is retained. While a range of experimental vaccines based on cellular extracts
have been tested, commercial vaccines are, with few exceptions, whole cell products. The
leptospires are grown in suitable culture media, which often contain serum or serum proteins. If
used, serum or serum proteins should be removed from the final products. Vaccines may contain
suitable adjuvants.
A. INTRODUCTION
Leptospirosis is a transmissible disease of animals and humans caused by infection with the spirochete
Leptospira. All the pathogenic leptospires were formerly classified as members of the species Leptospira
interrogans, however the genus has recently been reorganised and pathogenic leptospires are now
identified in 17
named species and four genomospecies of Leptospira (14, 61, 71, 105). There are more than 200 distinct
leptospiral serovars recognised and these are arranged in 23 serogroups (52, 98).
The use, interpretation, and value of laboratory diagnostic procedures for leptospirosis vary with the clinical
history
of the animal or herd, the duration of infection, and the infecting serovar. Acute leptospirosis should be
suspected
in the following cases: sudden onset of agalactia (in adult milking cattle and sheep); icterus and
haemoglobinuria,
especially in young animals; meningitis; and acute renal failure or jaundice in dogs. Chronic leptospirosis
should
be considered in the following cases: abortion, stillbirth, birth of weak offspring (may be premature);
infertility;
chronic renal failure or chronic active hepatitis in dogs; and cases of periodic ophthalmia in horses. Two
major
chronic microbiological sequelae of leptospiral infection present particular diagnostic problems: the
localisation
and persistence of leptospires in the kidney and in the male and female genital tract. Chronically infected
animals
may remain carriers for years to life and serve as reservoirs of the infection for other animals and humans.
The demonstration of leptospires in blood and milk of animals showing clinical signs suggestive of acute
leptospirosis is considered to be diagnostic. However, isolation from blood is not often successful because
bacteraemia is transient and not always accompanied by clinical signs. Dogs are often treated with
antibiotics
before samples are collected for testing for Leptospira, which further decreases the likelihood of identifying
the
agent in blood. The demonstration of generalised leptospiral infection in a range of organs taken at necropsy
is
also considered to be diagnostic. However, if the animal lives long enough or has been treated with
antibiotics, it
may be difficult to detect intact organisms systemically; immunohistochemistry can be particularly helpful in
identifying residual leptospiral antigen in these cases. Demonstration of leptospires in the genital tract,
kidneys, or
urine only must be interpreted with full consideration of the clinical signs and serological results as these
findings
may merely indicate that the animal was a carrier.
Failure to demonstrate leptospires in the urine of an animal does not eliminate the possibility that the animal
is a
chronic renal carrier, it merely indicates that the animal was not excreting detectable numbers of leptospires
at the
time of testing. Collection of urine following treatment of the animals with a diuretic enhances the chances of
detecting the organism (63). In important cases involving individual animals (e.g. clearing an infected stallion
to
return to breeding), negative tests on three consecutive weekly urine samples has been considered to be
good
evidence that an animal is not shedding leptospires in the urine.
The demonstration of leptospires in body fluids or internal organs (usually kidney, liver, lung, brain, or
adrenal
gland) of aborted or stillborn fetuses is considered to be diagnostic of chronic leptospirosis of the mother,
and is
evidence of active infection of the fetus.
In experienced hands, the isolation of leptospires is the most sensitive method of demonstrating their
presence,
provided that antibiotic residues are absent, that tissue autolysis is not advanced, that tissues are processed
for
culture rapidly after collection, and – in the case of urine – at a suitable pH. If tissues or fluids cannot be
transported promptly to the laboratory for leptospiral culture, the sample should be kept at 2–5°C to prevent
overgrowth with other bacteria and autolysis of tissue samples. Liquid culture medium or 1% bovine serum
albumin (BSA) solution containing 5-fluorouracil at 100–200 μg/ml should be used as transport medium for
the
submission of samples.
Culture should be carried out in a semisolid (0.1–0.2% agar) medium containing BSA and either Tween 80
(e.g.
Tween 80/BSA medium or EMJH) (44) or containing BSA and a combination of Tween 80 and Tween 40
(30).
Contamination may be controlled by the addition of a variety of selective agents, e.g. 5-fluorouracil (48),
nalidixic
acid (49), fosfomycin (64), and a mixture of rifamycin, polymyxin, neomycin, 5-fluorouracil, bacitracin, and
actidione (1). However, use of selective agents may reduce the chances of isolation when there are only
small
numbers of viable leptospires, and some strains of leptospires will not grow in selective media containing
multiple
antibiotics. Addition of 0.4–1% rabbit serum to semisolid culture medium enhances the chances of isolating
fastidious leptospiral serovars.
Cultures should be incubated at 29 ± 1°C for at least 16 weeks, and preferably for 26 weeks (30). The time
required for detection of a positive culture varies with the leptospiral serovar and the numbers of organisms
present in the sample. Less fastidious serovars (e.g. Pomona and Grippotyphosa) may result in positive
cultures
as soon as 7–10 days after inoculation; other serovars (e.g. Hardjo and Bratislava) may take much longer.
Cultures should be examined by dark-field microscopy every 1–2 weeks. It is important to use a 100 watt
light
source and a good quality dark-field microscope.
Leptospires may also be demonstrated by a variety of immunochemical staining techniques, e.g.
immunofluorescence (10, 31), and various immunohistochemical techniques (5, 33, 79, 81, 99, 106). These
are
useful in diagnosing infection in pathological material that is unsuitable for culture or where a rapid diagnosis
is
required. As the success of these techniques is dependant on the number of organisms present, they are
less
suitable for diagnosing the chronic carrier state, where the numbers of organisms may be very low or
localised.
Leptospires do not stain satisfactorily with aniline dyes, and silver-staining techniques lack sensitivity and
specificity, although they are a useful adjunct for histopathological diagnosis (7).
Polymerase chain reaction (PCR)-based assays are now used in some diagnostic and many reference
laboratories for the detection of leptospires in tissues and body fluids of animals. A variety of primer sets for
the
conduct of PCR assays have been described (3, 6, 13, 37, 39, 44, 50, 51, 54, 59, 60, 66, 84, 93, 97, 103)
with
some primers only specific for the genus Leptospira and others designed to identify only pathogenic species.
These assays do not identify the infecting serovar, although some primer sets may permit further
identification to
the species or serovar level if the PCR amplicons are sequenced. This further analysis is not a routine
diagnostic
method. Many of the PCR primer sets have been designed and evaluated for use in human rather than
animal
specimens and general agreement about the PCR primers to be used for testing of animal samples is
lacking.
Therefore, the individual laboratory is generally responsible for the validation of the particular assay they use
for
the tissue, fluid, and species being tested. PCR assays can be quite sensitive, but lack of specificity (i.e.
falsepositive
results) can be a problem. Presence of amplification inhibitors in clinical samples can cause falsenegative
results, particularly in animal specimens that may be compromised by contamination with faeces or
autolysis. Quality control of PCR assays used for diagnosis of leptospirosis requires careful attention to
laboratory
design and workflow to prevent contamination of reagents, and use of appropriate control samples (29, 57).
In
addition, sample processing for PCR is critical and must be suited to the tissue, fluid, and species being
tested. A
procedure for the preparation of urine samples for PCR using magnetic beads coated with anti-leptospiral
antibody shows promise in enhancing the detection of pathogenic leptospires in urine (88).
The identification of leptospiral isolates is a task for specialised reference laboratories. For complete
identification,
a combination of procedures is used to determine: 1) if the isolate is a pathogen or a saprophyte; 2) the
species of
Leptospira to which the isolate belongs; and 3) the serogroup and serovar of the isolate. A pure leptospiral
culture
may be identified as belonging to a pathogenic or saprophytic species by a variety of tests: the ability to
infect
animals; the relative resistance to 8-azaguanine; lipase activity; salt and temperature tolerance (46, 47);
PCRassay-
based amplification of 23S rDNA (102); and G+C content of DNA (46).
New leptospiral species have been identified based on DNA–DNA hybridisation analysis (14, 71, 105). In
most
cases, the type strain for each serovar was used in these analyses; for a few serovars, clinical isolates have
also
been tested to determine the new species designations. Different isolates belonging to a single serovar
usually
belong to the same species, but this is not always the case. Species identification of field isolates is still
cumbersome but can be done by sequence analysis of the 16S rDNA, by genetic analysis of the 16S or 23S
ribosomal RNA genes (17, 53, 61, 68, 70, 100, 101), by multilocus sequence typing (4, 77), by sequencing
the
DNA gyrase subunit B encoding gene (82), or by PCR using species-specific ompL1 primer sets (73).
Strains belonging to Leptospira can be differentiated to the serogroup level by cross-agglutination reactions
(28).
Further differentiation to the serovar level was traditionally by cross-agglutination absorption, although for
most
isolates this is now being done using less time-consuming methods: factor analysis (28), monoclonal
antibodies
(MAbs) (89, 90), restriction endonuclease analysis (43, 56, 91, 92), and various other molecular strategies
(17, 21,
26, 38, 61, 69, 70, 72, 75, 77, 78, 82, 83, 107, 108). However, genetic-based tests may not always give the
same
results as the cross-agglutination absorption test.
Serological testing is the laboratory procedure most frequently used to confirm the clinical diagnosis, to
determine
herd prevalence, and to conduct epidemiological studies. Leptospiral antibodies appear within a few days of
onset
of illness and persist for weeks or months and, in some cases, years. Unfortunately, antibody titres may fall
to
undetectable levels while animals remain chronically infected. To overcome this problem, sensitive methods
are
needed to detect the organism in urine or the genital tract of chronic carriers.
A wide variety of serological tests, which show varying degrees of serogroup and serovar specificity, have
been
described. Two tests have a role in veterinary diagnosis: the microscopic agglutination test (MAT) and the
enzyme-linked immunosorbent assay (ELISA).
a) Microscopic agglutination test
The MAT using live antigens is the most widely used serological test. It is the reference test against which all
other serological tests are evaluated and is used for import/export testing. For optimum sensitivity, it should
use antigens representative of all the serogroups known to exist in the region in which the animals are found
and, preferably, strains representing all the known serogroups. The presence of a serogroup is usually
indicated by frequent reaction in serological screening but can only be definitively identified by isolation of a
serovar from clinically affected animals. The sensitivity of the test can be improved by using local isolates
rather than reference strains, but reference strains assist in the interpretation of results between
laboratories.
The specificity of the MAT is good; antibodies against other bacteria usually do not cross-react with
Leptospira to a significant extent. However, there is significant serological cross-reactivity between serovars
and serogorups of Leptospira and an animal infected with one serovar is likely to have antibodies against
the
infecting serovar that cross-react with other serovars (usually at a lower level) in the MAT. Therefore,
serology cannot be used to definitively identify the infecting serovar in an individual infection or outbreak –
this requires isolation of the agent. However, in areas where the serovars of Leptospira present have been
well described by isolation studies, serological examination of the infected animal(s) may suggest, but not
definitively identify, the infecting serovar. In addition, animals that have been vaccinated against
leptospirosis
may have antibodies against the serovars present in the vaccine used. Therefore, it is particularly important
to consider the vaccination history of the animals under test. The two methods for carrying out the test have
been described in detail (36, 62).
The strains selected should be grown in liquid leptospiral culture medium (e.g., EMJH, Tween 80 BSA, or
other suitable medium) at 29 ± 1°C and the culture should be at least 4 days old, but no more than 8 days.
Live cultures with densities of approximately 2 × 108 leptospires per ml are to be used as the antigens. The
culture density can be determined by counting the cells directly using a bacterial counting chamber and
darkfield
microscopy. Alternatively, cell counts can be estimated by measuring transmittance in a
spectrophotometer with a 400 nm filter or by nephelometry. If indirect methods are used, direct bacterial cell
counts should be correlated with the readings on the specific instrument being used. The number of
antigens
to be used is determined and a screening test may be performed with a 1/50 serum dilution (or a different
starting dilution based on the purpose of the test). A volume of each antigen, equal to the diluted serum
volume, is added to each well, making the final serum dilution 1/100 in the screening test. The microtitration
plates are incubated at 29 ± 1°C for 2–4 hours. The plates are examined by dark-field microscopy.
The endpoint is defined as that dilution of serum that shows 50% agglutination, leaving 50% free cells
compared with a control culture diluted 1/2 in phosphate buffered saline. The result of the test may be
reported as the endpoint dilution of serum (e.g. 1/100 or 1/400) or as a titre that is the reciprocal of the
endpoint serum dilution (e.g. 100 or 400). Many laboratories perform a screening test at a final serum
dilution
of 1/100 and then retest sera with titres of ≥100 to determine an endpoint using doubling dilutions of sera
beginning at 1/100 through to 1/12,800 or higher.
Identity of antigens is a crucial factor in conducting the MAT. Antigens should be evaluated for identity, using
hyperimmune rabbit sera, MAbs, or a molecular method that confirms passages over time, preferably each
time the test is run, but at least twice a year. Hyperimmune rabbit serum for this purpose can be obtained
from a reference laboratory or prepared using a protocol such as that given by the Subcommittee on the
Taxonomy of Leptospira (45). Briefly, healthy rabbits weighing 3–4 kg that lack detectable anti-leptospiral
antibodies are selected. Each rabbit is given an intravenous injection in a marginal vein of the ear with a
well-growing live or formalin-treated cloned culture with a density of approximately 2 × 108 leptospires/ml.
The culture should be grown in Tween 80 BSA medium or another appropriate medium. Five injections of 1
ml, 2 ml, 4ml, 6 ml, and 6 ml each are given at 7-day intervals. One week following the final injection, the
homologous antibody titre is determined by MAT. If the titre is ≥1/12,800, the rabbit is anaesthetised and
bled by cardiac puncture 7 days later (i.e. 14 days after the final injection). If the titre is <1/12,800, a further
injection of 6 ml of culture can be given; 7 days after this injection the homologous titre is again determined.
Unless the titre is ≥1/12,800 the procedure should be repeated with another rabbit. Two rabbits are used to
prepare each antiserum. If the titres are satisfactory in both rabbits, the sera may be pooled. To preserve
potency, it is preferable to freeze-dry the antiserum in 2 ml volumes and store it at 2–5°C. Alternatively, the
serum can be stored in 2 ml volumes at –15 to –20°C. All animal inoculations should be approved and
conducted according to the relevant standards for animal care and use. Other immunisation protocols may
be considered based on the intended use of the antiserum.
Purity of antigens used in the MAT should be checked regularly by culture on blood agar and in thioglycolate
broth. Stock cultures of antigens may be stored at –70 to –80°C or in liquid nitrogen. There may be a low
survival rate of leptospires after lyophilisation. Repeated passage of antigens in liquid medium results in a
loss of antigenicity. In this case, a new liquid culture should be derived from the stock culture.
As an individual animal test, the MAT is very useful in diagnosing acute infection; the demonstration of a
four-fold change in antibody titres in paired acute and convalescent serum samples is diagnostic. In addition,
a diagnosis of leptospirosis is likely based on the finding of very high titres in an animal with a consistent
clinical picture. The test has limitations in diagnosis of chronic infection in individual animals, both in the
diagnosis of abortion (32) and in the identification of renal or genital carriers (30). This is particularly true
with
the host-adapted leptospiral infections, e.g. serovar Hardjo infection in cattle: when a titre of 1/100 or greater
is taken as significant, the sensitivity of the test is only 41%, and even when the minimum significant titre is
reduced to 1/10, the sensitivity of the test is only 67% (30). The demonstration of antibodies in fetal blood is
diagnostic, but the titres are often very low, i.e. 1/10, requiring a modified testing procedure for most
laboratories.
As leptospirosis is a herd problem, the MAT has much greater use as a herd test. To obtain useful
information, Cole et al. (20) suggested that samples be taken from at least ten animals, or 10% of the herd,
whichever is the greater. In a study of Hardjo infection in cattle, Hathaway et al. (42) found that a 10-cow
sample usually indicated the presence or absence of infection in a herd. Increasing the sample size
markedly improved epidemiological information, investigations of clinical disease, and public health
tracebacks.
In making a serological diagnosis of leptospirosis, the infecting serovar and the clinical condition involved
must be fully considered. In the case of serovar Pomona-induced abortion in cattle, a high titre is commonly
found at the time of abortion because the clinical incident occurs relatively soon after infection. Abortion in
cattle due to serovar Hardjo is a chronic event; in this case, the serological response at the time of abortion
is more variable, with some animals seronegative and others showing high titres. Cattle may experience a
drop in milk production during the acute phase of Hardjo infection and this clinical sign is associated with
high titres. Vaccination history must also be considered in the interpretation of MAT results as widespread
vaccination contributes significantly to the number of seropositive animals and may mask the presence of
chronic infections in the herd – particularly with serovar Hardjo.
b) Enzyme-linked immunosorbent assays
ELISAs for detection of anti-leptospiral antibodies have been developed using a number of different antigen
preparations, assay protocols and assay platforms, including plate tests and dipstick tests. Information
regarding the surface antigens of Leptospira has been reviewed (25). In general, ELISAs are quite sensitive,
but lack the serovar specificity of the MAT. An ELISA that measures canine IgG and IgM against various
leptospiral serovars has been developed and evaluated in Europe (40, 41). Anti-leptospiral IgM is detectable
in this assay as early as 1 week after infection, before agglutinating antibodies are present. IgG antibodies
are detectable in infected dogs beginning 2 weeks after infection and persist for long periods of time.
Therefore, dogs with acute leptospirosis have high IgM titres and relatively low IgG titres; dogs that are
vaccinated or have had previous leptospiral infections have high IgG titres but low IgM titres. Similar assays
to detect anti-leptospiral bovine, porcine, and ovine antibodies have also been developed (2, 19, 24, 58, 74,
85–87, 94, 104). The major identified role of ELISA in livestock species is the use of an IgM ELISA for
identification of recent infections (24) and for screening herds in regions where vaccination for leptospirosis
is not practiced. A total-Ig ELISA is useful in identification of fully susceptible animals suitable for
experimental challenge work (34). ELISAs have also been developed for use in milk from individual cows or
in bulk tank milk for the detection of serovar Hardjo antibodies. These tests have been helpful in identifying
Hardjo-infected herds. However, herds that are vaccinated against serovar Hardjo will also be positive in
these various ELISAs decreasing their usefulness in regions where vaccination is a routine practice. New
ELISAs have been developed based on detection of antibodies against surface proteins or lipoproteins of
Leptospira (12, 27, 55, 65, 67) but these tests are not yet widely available.
BIOLOGICALS
Leptospiral vaccines for veterinary use are suspensions of one or more strains of pathogenic Leptospira
inactivated in such a manner that immunogenic activity is retained. While experimental vaccines based on
cellular
extracts have been tested (9), commercial vaccines are, with few exceptions, whole-cell products. The
leptospires
are grown in suitable culture media that may contain serum or serum proteins. If used, serum or serum
proteins
should be removed from the final product. Vaccines may contain suitable adjuvants.
vaccine
1. Seed management
Proper selection of vaccine production strains is of utmost importance. Immunity induced by vaccination is
largely serovar specific (18). A vaccine should be formulated for use in a particular animal species in a
particular geographical region. It should contain only those serovars – and preferably those genotypes – that
cause problems in the animal species, or that are transmitted by the animal species to other species in the
region. Strains selected for use as master seed culture should be cloned on solid medium to ensure the
absence of saprophytic Leptospira contaminants and uniformity of the culture.
Suitable strains should be further selected by their ability to grow to high yields under batch culture
conditions.
b) Methods of culture
Each component strain to be included in the final vaccine should be grown separately in liquid medium;
preferably in a protein-free (8, 80) or low-protein medium (8).
The volume of each master seed culture should be amplified by growth for 2–10 days at 29°C ±1°C in a
series of subcultures until a volume sufficient for use as a production seed culture is achieved. Cultures
should be aerated and agitated as required.
Each subculture of the master seed culture should be checked for purity and for satisfactory growth. Purity
can be checked by inoculating a loopful of culture into blood agar plates or into thioglycolate broth for
incubation at 35–37°C for 2–5 days, and by examining a Gram-stained smear of culture sediment. Growth
can be checked by dark-field microscopy. Each production seed culture should also be checked against its’
homologous rabbit antiserum (28) to ensure purity and homology. MAbs may also be used for this purpose.
There is a large volume of literature describing the efficacy of leptospiral vaccines. In most cases, vaccines
provide significant protection against disease produced by homologous challenge under field conditions.
Vaccines are less efficacious at preventing infection in animals and a percentage of vaccinated animals will
become infected with the relevant serovar and may shed the organism in their urine despite a lack of clinical
signs of disease.
Efficacy trials and vaccine validation must be conducted in the target species for the vaccine. The vaccine
should be administered as recommended on the label, and immunity should be tested by challenge with
virulent field strains of each serovar by natural routes of infection, i.e. by conjunctival and/or vaginal
challenge. Validation studies have often been conducted with challenge of immunity by intravenous or
intramuscular injections of leptospires. Vaccines validated in this way have not always been shown to be
protective against field challenge, which occurs by exposure of mucous membranes of the eye, mouth, and
genital tract to leptospires. Most notably, commercial leptospiral vaccines containing serovar Hardjo have
not
always protected cattle from conjunctival or field challenge with serovar Hardjo (11). A draft monograph for
the efficacy testing of serovar Hardjo vaccines has been prepared and specifies the use of more natural
routes of challenge (35).
Manufacture is carried out by batch culture in appropriately sized fermentor vessels. These should be
equipped
with ports for the sterile addition of seed culture, air, and additional medium. They should also have
sampling
ports so that the purity and growth of the production culture can be monitored.
Ideally, low-protein or protein-free media are used for production. However, some strains require the
presence of
animal protein to achieve suitable yields; this is usually supplied as BSA. All media components that are not
degraded by heat should be heat sterilised. This reduces the risk of contamination by water-borne
saprophytic
leptospires that are not removed by filter sterilisation.
After addition of the seed culture, the growth of the production culture is monitored at frequent intervals for
the
start of log-phase growth. Once this is observed, the vessel is then agitated and aerated. The final yield can
often
be improved by the addition of more Tween 80 to the culture when log-growth is first observed to be slowing
down. Adequate growth may require up to 10 days of incubation at 29 ± 1°C.
Inactivation is usually by the addition of formalin, but phenol, merthiolate, and heat inactivation have also
been
used.
After the appropriate inactivation period, the culture may be concentrated and extraneous protein material
may be
removed by ultrafiltration. Suitable volumes of the various strains to be included in the final vaccine can then
be
blended, and adjuvant and preservative added, if appropriate.
During production, daily or twice daily subsamples should be taken and monitored for growth of leptospires
and
absence of contaminants. Growth is monitored either by counting leptospires in a counting chamber under
darkfield
microscopy or by a nephelometer. The absence of contamination can be monitored by the microscopic
examination of Gram-stained preparations of centrifuged culture.
Immediately prior to inactivation, a sample should be taken for checking against its homologous antibody in
a
MAT. The inactivated culture must be checked for freedom from viable leptospires. This is done by
inoculating
aliquots of inactivated culture into an appropriate growth medium, such as the medium of Johnson & Harris
(47),
incubating at 29 ± 1°C for at least 4 weeks, and examining weekly by dark-field microscopy for the presence
of
viable leptospires.
After blending, the levels of free inactivating agents, minerals present in adjuvants (such as aluminum), and
preservative (such as thiomersal) must be within prescribed limits.
4. Batch control
a) Sterility
Selected samples of the completed vaccine should be tested for the absence of viable bacteria and fungi
(16, 22, 23, 95). Tests for sterility and freedom from contamination of biological materials may be found in
Chapter 1.1.9.
b) Safety
Samples of completed product should be tested for safety. Methods for this have been described elsewhere
(15, 22, 96). The test should be carried out for each route of inoculation indicated on the label and in two
healthy animals of each category (e.g. pregnant animals, young stock) for which the vaccine is intended.
The
animals must be susceptible to the serovars used in the vaccine and their sera must be free from
agglutinating antibodies to those serovars. Each animal is given an injection of the vaccine by the
recommended route with twice the recommended dose, as stated on the label. The animals are observed for
14 days and should show no adverse local or systemic effects attributable to the vaccine.
c) Potency
Samples of completed vaccine should be tested for potency in hamsters or guinea-pigs. Potency is usually
measured by the vaccine’s ability to prevent the death of the animal when challenged with between 10 and
10,000 LD50 (50% lethal dose). With some serovars that are not hamster or guinea-pig lethal, such as
serovar Hardjo, potency is measured against prevention of renal infection when the animals are challenged
with between 10 and 10,000 ID50 (50% infectious dose) or by induction of a suitable antibody titre in rabbits.
An example protocol is to inject 1/40 dog dose of the vaccine into each of ten healthy hamsters no more
than
3 months old. After 15–30 days, each vaccinated hamster, and each of ten unvaccinated hamsters of the
same age, is injected intraperitoneally with a suitable quantity of a virulent culture of leptospires of the
serovar used to make the vaccine (or a suspension of liver or kidney tissue collected from an experimentally
infected animal). In the case of bivalent vaccines, each serovar is tested separately. For the vaccine to pass
the test, at least 8/10 of the vaccinated animals should remain in good health for 14 days after the death of
the controls. Other protocols may apply to cattle and pig vaccines, which contain as many as five or six
components.
In-vitro potency tests for leptospiral vaccines are being developed based on quantifying the protective
antigen in the vaccine using MAbs in a capture ELISA (76). These assays are being standardised using
reference vaccines and correlation with existing hamster or antibody-based potency assays and target–host
efficacy data.
Duration of immunity should be determined in the animal species for which the vaccine is intended using
natural routes of challenge (11). Duration of immunity should not be estimated based on the duration of MAT
titres in vaccinated animals as protection against clinical disease may be present with very low titres.
Vaccinal immunity should persist for at least 6 months or longer depending on the label claim.
e) Stability
When stored under the prescribed conditions, the vaccines may be expected to retain their potency for 1–
2 years. Stability should be assessed by determining potency after storage at 2–5°C, room temperature, and
35–37°C.
5. Tests on the final project
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.
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*
**
NB: There are OIE Reference Laboratories for Leptospirosis (see Table in Part 3 of this Terrestrial Manual or
consult the OIE Web site for the most up-to-date list: www.oie.int).
PART 2
OIE LISTED DISEASES AND OTHER DISEASES
OF IMPORTANCE TO INTERNATIONAL TRADE

S E C T I O N 2 . 1 .
MULTIPLE SPECIES
C H A P T E R 2 . 1 . 1 .
ANTHRAX
SUMMARY
Definition of the disease: Anthrax is primarily a disease of herbivorous animals, although all
mammals, including humans, and some avian species can contract it. Mortality can be very high,
especially in herbivores. The aetiological agent is the spore-forming, Gram-positive rod-shaped
Bacillus anthracis. The disease has world-wide distribution and is a zoonosis.
Description of the disease: The disease is mediated by exotoxins. Peracute, acute, subacute and,
rarely, chronic forms of the disease are reported. Ante-mortem clinical signs may be virtually absent
in peracute and acute forms of the disease. Subacute disease may be accompanied by progressive
fever, depression, inappetence, weakness, prostration and death. Acute, subacute, and chronic
disease may show localised swelling, fever; and in chronic disease the only sign may be enlarged
lymph glands.
Identification of the agent: Bacillus anthracis is readily isolated in relatively high numbers from
blood or tissues of a recently dead animal that died of anthrax, and colony morphology of
B. anthracis is quite characteristic after overnight incubation on blood agar. The colony is relatively
large, measuring approximately 0.3–0.5 cm in diameter. It is grey-white to grey, non-haemolytic with
a rough, ground-glass appearance and has a very tacky, butyrous consistency. The vegetative cells
of B. anthracis are large, measuring 3–5 μm in length and approximately 1 μm in width. Ellipsoidal
central spores, which do not swell the sporangium, are formed at the end of the exponential cell
growth phase. The cells stain strongly Gram positive, and long chains are often seen in vitro, while
paired or short chains are seen in vivo. Visualisation of the encapsulated bacilli, usually in large
numbers, in a blood smear stained with polychrome methylene blue (M’Fadyean reaction) is fully
diagnostic.
Serological tests: Antibody detection in serum from infected animals is rarely used for diagnostic
purposes and is essentially a research tool. The predominant procedure today is the enzyme-linked
immunosorbent assay (ELISA).
Requirements for vaccines and diagnostic biologicals: The most widely used livestock anthrax
vaccine developed by Max Sterne in 1937, is a live, non-encapsulated, spore former held in
suspension. In Russia and Eastern Bloc countries, an equivalent type of vaccine is used (strain 55).
The Pasteur vaccine is no longer used in Italy. A new vaccine, Carbosap, has been developed that
retains both plasmids and exhibits very low virulence. A list of producers is given in the World
Health Organization anthrax guidelines.
A. INTRODUCTION
Anthrax, an acute bacterial disease of primarily herbivores, is transmissible to humans. The aetiological
agent,
Bacillus anthracis, is a Gram-positive spore-forming rod-shaped bacterium. Anthrax is known by many
names
around the world including charbon, woolsorters disease, ragpickers disease, malignant carbuncle, and
malignant
pustule.
Chapter 2.1.1. - Anthrax
Animals become infected by ingesting spores or possibly by being bitten by flies that have fed on an infected
animal or carcass. Infected animals are usually found dead as death can occur within 24 hours. A careful
postmortem
examination of recently dead animals may show any number of lesions, none of which is pathognomonic
or entirely consistent. To avoid environmental contamination, post-mortem examinations of carcasses of
animals
suspected to have died of anthrax is discouraged. Lesions most commonly seen are those of a generalised
septicaemia often accompanied by an enlarged spleen having a ‘blackberry jam’ consistency and poorly
clotted
blood. Haemorrhage from the nose, mouth, vagina and/or anus at death is not a common sign.
Gram-positive rod-shaped Bacillus anthracis is the only obligate pathogen within the genus Bacillus. Most of
the
other species of Bacillus are common ubiquitous environmental saprophytes, although a number, notably
B. cereus, B. licheniformis and B. subtilis, are occasionally associated with food poisoning in humans and
with
other clinical manifestations in both humans and animals.
More than 95% of human anthrax cases take the cutaneous form and result from handling infected
carcasses or
hides, hair, meat or bones from such carcasses. Bacillus anthracis is not invasive and requires a lesion to
infect.
Protection for veterinarians and other animal handlers involves wearing gloves, and other protective clothing
when
handling specimens from suspected anthrax carcasses and never rubbing the face or eyes. The risk of
gastrointestinal anthrax may arise if individuals eat meat from animals infected with anthrax.
The risk of inhaling infectious doses becomes significant in occupations involving the processing of animal
byproducts
for manufacturing goods (industrial anthrax). These include the tanning, woollen, carpet, bone
processing, and other such industries, where the potential for aerosolisation of substantial numbers of
spores
increases the risk of exposure to infectious doses.
Demonstration of encapsulated B. anthracis in smears of blood or tissues from fresh anthrax-infected
carcasses
and growth of the organism on blood agar plates is relatively uncomplicated and within the capability of most
bacteriology laboratories. Difficulty may be encountered in the case of pigs and carnivores in which the
terminal
bacteraemia is frequently not marked, or in animals that received antibiotics before death.
Recovery of B. anthracis from old decomposed carcasses, processed specimens (bone meal, hides), or
environmental samples (contaminated soil) is also often difficult, requiring demanding and labour-intensive
procedures.
a) Culture and identification of Bacillus anthracis
i) Fresh specimens
Bacillus anthracis grows readily on most types of nutrient agar, however, 5–7% horse or sheep blood agar is
the diagnostic medium of choice. Blood is the primary clinical material to examine. Swabs of blood, other
body fluids or swabs taken from incisions in tissues or organs can be spread over blood agar plates. After
overnight incubation at 37°C, B. anthracis colonies are grey-white to grey, 0.3–0.5 mm in diameter,
nonhaemolytic,
with a ground-glass moist surface, and very tacky when teased with an inoculating loop. Tailing
and prominent wisps of growth trailing back toward the parent colony, all in the same direction, are
sometimes seen. This characteristic has been described as a ‘medusa head’ appearance. Confirmation of
B. anthracis should be accomplished by the demonstration of a capsulated, spore-forming, Gram-positive
rod in blood culture. Absence of motility is an additional test that can be done.
The susceptibility of B. anthracis to the gamma bacteriophage was first described by Brown & Cherry in
1955
(3). The phage is available from various national central veterinary laboratories and anthrax reference
laboratories. The procedure for the test is simply to streak a lawn on a blood or nutrient agar plate, or portion
of a plate (several tests can be done on one plate) with the suspect organism and place a 10–15 μl drop of
the phage suspension on one side of the streaked area and place a 10-unit penicillin disk to the other side.
Allow the drop of phage suspension to soak in and incubate the plate at 37°C. A control culture should be
included; the Sterne vaccine or the NCTC strain 10340 can be used for this. If the culture is B. anthracis, the
area under the phage will be devoid of bacterial growth, due to lysis, and a clear zone will be seen around
the penicillin disk after overnight incubation. (Note: phage-resistant B. anthracis isolates are encountered
occasionally; similarly, there are a few reports in the literature of penicillin-resistance.)
ii) Capsule visualisation
Virulent encapsulated B. anthracis is present in tissues and blood and other body fluids from animals that
have died from anthrax. The bacteria should be looked for in smears of these specimens that have been
dried, fixed and stained with polychrome methylene blue (M’Fadyean’s reaction). The capsule stains pink,
whereas the bacillus cells stain dark blue. The cells are found in pairs or short chains and are often
squareChapter
2.1.1. - Anthrax
ended (the chains are sometimes likened to a set of railway carriages – so-called ‘box-car’ appearance).
Gram and Giemsa stains do not reveal the capsule. The capsule is not present on B. anthracis grown
aerobically on nutrient agar or in nutrient broths, but can be seen when the virulent bacterium is cultured for
a few hours in a few millilitres of blood (defribrinated horse or sheep blood seems to work best).
Alternatively,
the capsule is produced when the virulent B. anthracis is cultured on nutrient agar containing 0.7% sodium
bicarbonate and incubated in the presence of CO2 (20% is optimal, but a candle jar works well). The agar is
prepared by reconstituting enough nutrient agar base powder for 100 ml of agar in 90 ml of water. Autoclave
and cool to 50°C in a water bath. Add 10 ml of a filter-sterilised (0.22–0.45 μm filter) 7% solution of sodium
bicarbonate. Mix and pour into Petri dishes. The encapsulated B. anthracis will form mucoid colonies and
the
capsule can be visualised by making thin smears on microscope slides, fixing, and staining with polychrome
methylene blue.
iii) Other specimens
Identification of B. anthracis from old, decomposed specimens, processed materials, and environmental
samples, including soil, is possible but these samples often have saprophytic contaminants that outgrow and
obscure B. anthracis on nonselective agars. The following procedure is suggested:
a) The sample is blended in two volumes of sterile distilled or deionised water and placed in a water bath
at 62.5 ± 0.5°C for 30–60 minutes.
b) Tenfold dilutions to 10–2 or 10–3 are then prepared. From each dilution, 10–100 μl are plated on to
blood agar and optionally 250–300 μl on to PLET agar (polymyxin, lysozyme, EDTA [ethylene diamine
tetra-acetic acid], thallous acetate) (7, 11). All plates are incubated at 37°C.
c) Blood agar plates are examined for typical colonies as previously described after overnight incubation,
and the PLET plates are examined after 40–48 hours. Confirmation of the identity of suspect colonies
as B. anthracis is done as described above.
PLET medium (7, 11) is prepared by using heart-infusion agar base (DIFCO) made up to the manufacturer’s
instructions with the addition of 0.25–0.3 g/litre EDTA and 0.04 g/litre thallous acetate. (NOTE: thallous
acetate is poisonous and should be handled with care.) The mixture is autoclaved and uniformly cooled to
50°C before adding the polymyxin at 30,000 units/litre and lysozyme at 300,000 units/litre. After mixing
thoroughly, the agar is dispensed into Petri dishes.
Reports of procedures for direct detection of B. anthracis in soils and other environmental specimens using
the polymerase chain reaction (PCR) are emerging. None of these has become routinely applicable at the
present time.
Animal inoculation may be considered for recovery of B. anthracis if all other methods fail. Examples of
when
this might occur are specimens from animals that received antibiotic therapy before death or environmental
samples containing sporostatic chemicals. Due to the increasing concern to eliminate the use of animals for
biological testing, this approach should be used as a last resort and only if justified. Adult mice or guineapigs
are the animals of choice. If the samples involved are soils, the animals should be pretreated, the day
before testing, with both tetanus and gas gangrene antiserum. The samples are prepared as described for
culturing, including heat-shocking at 62.5°C for 15 minutes. Mice are injected subcutaneously with 0.05–
0.1 ml; guinea pigs are inoculated intramuscularly with up to 0.4 ml (0.2 ml in each thigh muscle). Any
B. anthracis present will result in death in 48–72 hours and the organism can be cultured from the blood as
described above.
b) Immunological detection and diagnosis
It needs to be borne in mind that B. anthracis is antigenically very closely related to B. cereus, which is
considered a ubiquitous component of the environmental microflora. The only unshared antigens that lend
themselves to differentiating these two species by immunological approaches are the anthrax toxin antigens,
produced during the exponential phase of growth, and the capsule of B. anthracis. This places considerable
constraints on the extent to which immunological methods can be used in routine detection methodology.
i) Ascoli test
In 1911, Ascoli (1) published a procedure for the detection of thermostable anthrax antigen in animal tissue
being used for by-products. This uses antiserum raised in rabbits to produce a precipitin reaction. The test
lacks high specificity, in that the thermostable antigens of B. anthracis are shared by other Bacillus spp., and
is dependent on the probability that only B. anthracis would proliferate throughout the animal and deposit
sufficient antigen to give a positive reaction. Nowadays, it appears to be used in Eastern Europe only.
To perform the Ascoli test, put approximately 2 g of sample in 5 ml of saline containing 1/100 final
concentration of acetic acid and boil for 5 minutes. The resultant solution is cooled and filtered through filter
paper. A few drops of rabbit antiserum (see preparation below) are placed in a small test tube. The filtrate
from the previous step is gently layered over the top of the antiserum. A positive test is the formation of a
visible precipitin band in under 15 minutes. Positive and negative control specimen suspensions should be
included.
Antiserum is prepared in rabbits by the subcutaneous inoculation of Sterne anthrax vaccine on days 1 and
14. On days 28 and 35, the rabbits receive 0.5 ml of a mixture of several strains of virulent B. anthracis not
exceeding 105 colony-forming units (CFU)/ml suspended in saline. Alternatively, the live virulent bacteria can
be inactivated by prolonged suspension in 0.2% formalised saline, but the antigen mass needs to be
increased to 108–109 CFU/ml. The suspension should be checked for inactivation of the B. anthracis before
animal inoculation by culture of 0.1 ml into 100 ml of nutrient broth containing 0.1% histidine and, after
incubation at 37°C for 7 days, subculture on to blood or nutrient agar. The dose regimen for the formalised
suspension after initial vaccination on days 1 and 14 is increasing doses of 0.1, 0.5, 1, and 2 ml given
intravenously at intervals of 4–5 days. Following either procedure, a test bleed at 10 days after the last
injection should determine whether additional 2 ml doses should be administered to boost the precipitin titre.
ii) Immunofluorescence
While some success has been achieved with immunofluorescence for capsule observation in the research
situation (4), it does not lend itself to routine diagnosis.
c) Confirmation of virulence with the polymerase chain reaction
Full confirmation of virulence can be carried out using the PCR. The following instructions are taken from ref.
11. Template DNA for PCR can be prepared from a fresh colony of B. anthracis on nutrient agar by
suspension of a loop of growth in 25 μl sterile deionised (or distilled) water and heating to 95°C for
20 minutes. Following cooling to approximately 4°C, and brief centrifugation, the supernatant can be used
for
the PCR reaction.
Suitable primers (2, 5) for confirming the presence of the pX01 and pX02 plasmids are given in the table
below.
Target Primer ID Sequence 5’–3’ Product size Concentration
Protective
antigen (PA)
PA 5
3048–3029
TCC-TAA-CAC-TAA-CGA-AGT-CG 596 bp 1 mM
PA 8
2452–2471
GAG-GTA-GAA-GGA-TAT-ACG-GT
Capsule 1234
1411–1430
CTG-AGC-CAT-TAA-TCG-ATA-TG 846 bp 0.2 mM
1301
2257–2238
TCC-CAC-TTA-CGT-AAT-CTG-AG
PCR can be carried out in 50 μl volumes using the above primers, 200 μM each of dATP, dCTP, dTTP and
dGTP, 1.5 mM MgCl2 and 2.5 units of ampli Taq DNA polymerase™1, all in NH4 buffer, followed by the
addition of 5 μl of template DNA. A 2% agarose gel has been found to work best with these small fragments.
Alternatively, ‘Ready-To-Go™’ beads are available from Pharmacia Biotech2. These are premixed,
predispensed, dried beads, stable at room temperature, containing all the necessary reagents, except
primer
and template, for performing 25 μl PCR reactions. The template can be added in a 2.5 μl volume.
The following PCR cycle can be used: 1 × 95°C for 5 minutes; 30 × 95°C for 0.5 minute followed by 55°C for
0.5 minute followed by 72°C for 0.5 minute; 1 × 72°C for 5 minutes; cool to 4°C.
It should be noted that, in use for some years now in an anthrax reference facility, the primers given in the
table above have proved successful for confirming the presence or absence of pXO1 and/or pXO2 in pure
cultures of isolates from animal (including human) or environmental specimens or samples. They are
unsuitable, however, for direct detection of B. anthracis in such specimens or samples. A choice of
alternatives can be found in reference 6 and 9. For the rare possibility that an isolate may lack both pXO1
and pXO2, a chromosomal marker should also be run; primers for these are also supplied in references 6
and 9.
1 This product is available from Applied Biosystems;
(https://products.applied biosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catlD=601622
2 Uppsala, Sweden, product number 27-9555-01
BIOLOGICALS
The most widely used vaccine for prevention of anthrax in animals was developed by Sterne in 1937 (10).
He
derived a rough variant of virulent B. anthracis from culture on serum agar in an elevated CO2 atmosphere.
This
variant, named 34F2, was incapable of forming a capsule and was subsequently found to have lost the pX02
plasmid, which codes for capsule formation. It has become the most widely used strain world-wide for
animal
anthrax vaccine production. In Central and Eastern Europe, an equivalent pX02– derivative, Strain 55, is the
active ingredient of the current livestock vaccine. A list of manufacturers of anthrax vaccine for use in
animals is
given in Appendix V of reference 11.
The following information concerning preparation of the anthrax vaccine for use in animals is based on
references
8 and 12. Generalised procedures are given; national regulatory authorities should be consulted in relation
to
Standard Operating Procedures that may pertain locally.
1. Seed management
Anthrax vaccine production is based on the seed-lot system. A seed lot is a quantity of spores of uniform
composition processed at one time and maintained for the purpose of vaccine preparation. Each seed lot is
no more than three passages from the parent culture and must produce a vaccine that is efficacious and
safe for use in animals. It is recommended that a large seed lot be prepared from the parent strain and
preserved by lyophilisation for future production lots. The parent culture can be purchased3. The seed lot is
acceptable for anthrax vaccine if a vaccine prepared from the seed lot or a suspension harvested from a
culture derived from a seed lot meets the requirements for control of final bulk with respect to freedom from
bacterial contamination, safety and efficacy (immunogenicity).
b) Preparation of the master seed
Seed lots are cultured on solid media formulated to promote sporulation of the organism (see Section C.2
below). The solid medium formula given in reference 8 is: 50 g tryptic digest of casein; 10 g yeast extract;
0.1 g CaCl2.6H2O; 0.01 g FeSO4.7H2O; 0.05 g MgSO4.7H2O; 0.03 g MnSO4.4H2O; 5.0 g K2HPO4; 1.0 g
KH2PO4; 22 g agar; 1000 ml deionised or distilled water. The ingredients are dissolved in the water with the
appropriate amount of heating; the solution is adjusted to pH 7.4, distributed into Roux bottles (120 ml per
bottle) or other appropriate container, sterilised by autoclaving and cooled in the horizontal position. After the
agar has solidified, excess liquid should be removed aseptically and the bottles left in an incubator (37°C) for
at least 2 days to dry and to check the sterility.
Volumes of 2 ml of vaccine seed from a reference laboratory should be spread across the agar in Roux
bottles, which should be incubated at 37°C until at least 80% sporulation is apparent by microscopic
examination of aseptically extracted loopfuls (at least 72 hours). The growth is harvested with 10 ml per
bottle of sterile deionised or distilled water and checked for purity. After washing three times in sterile
deionised or distilled water with final suspension, also in sterile deionised or distilled water, sterilised
lyophilisation stabiliser is added and the suspension is dispensed into lyophilisation vials and freeze-dried.
c) Preparation and testing of the working seed
Reconstitute a vial of seed stock and inoculate several slants (approximately 10 ml) of sporulation (casein
digest) agar. Incubate at 37°C for 72 hours and store in a refrigerator. Test the slants for purity by culture on
to nutrient agar plates and in nutrient broth (0.1 ml in 100 ml of nutrient broth). The latter should be
subcultured on to nutrient agar after incubation at 37°C for 7 days and should be a pure culture of
B. anthracis. A sample of the broth culture should also be checked for lack of motility.
Volumes of seed needed for a production run should be calculated on the basis of harvesting the spores
from each slant with 10 ml of sterile deionised or distilled water and using this to inoculate five Roux bottles.
3 National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6
3QG,
United Kingdom.
d) Safety of the seed lot
Not less than 5 × 109 culturable spores should be injected subcutaneously into each of three healthy, 1–2-
year-old, unvaccinated sheep, which must survive an observation period of at least 10 days.
e) Immunogenicity of the seed lot
At least 10 healthy guinea-pigs, 300–500 g in weight should be inoculated with 5 × 106 viable spores and
observed for 21 days. At least 80% of the animals should survive. The immunised animals, together with
three unimmunised controls, should then be challenged with 10 median lethal doses (LD50) of the strain
17 JB of B. anthracis. During a 10-day observation period, none of the immunised animals should succumb
to the challenge while all the controls should die from anthrax. The test should be repeated if one of the
immunised animals dies.
2. Method of production
a) Preparation of vaccine concentrate
Roux bottles with casein digest agar are prepared as for the master seed in Section C.1.b above. One Roux
bottle can be expected to yield about 2000 doses of vaccine. Each Roux bottle is inoculated with 2 ml of
working seed suspension and incubated at 37°C with porous plugs for several days until small loopfuls of
culture from randomly selected bottles show at least 90% of the organisms to be in sporulated forms when
examined in wet mounts by phase contrast (phase bright spores) or following staining for spores. The
growth
from each bottle is then harvested with 20 ml of physiological saline. Tests for contaminants should be
carried out by subculture to nutrient agar plates and inoculation of 100 ml nutrient broth with 0.1 ml of
harvested spores followed by subculture to nutrient agar after 7 days at 37°C and by tests for motility.
Acceptable harvests (i.e. those showing no evidence of contaminants) are pooled.
b) Glycerination
Twice the volume of sterile, pure, neutral glycerol should be added to the bulk pool. Saponin (0.1% final
concentration) may also be added at this point if it is to be included as an adjuvant. Mix thoroughly (the
inclusion of sterilised glass beads may be helpful). Carry out a purity test as before and hold for 3 weeks at
ambient temperature to allow lysis of any vegetative bacteria, determine the viable spore count and store
under refrigeration thereafter.
c) Determining titre and dilution for use
The number of culturable spores in the product is then calculated by spreading tenfold dilutions on nutrient
agar plates. The suspension is diluted so that the final bulk contains the number of culturable spores
desired. The diluent should contain the same proportions of saline, glycerol and (if being included) saponin
as present in the vaccine concentrate. The vaccine should contain not less than 1 × 107 viable spores per
dose for cattle, buffaloes and horses, and not less than 1–5 × 106 viable spores per dose for sheep, goats
and pigs.
d) Safety
Safety testing is performed on two healthy sheep or goats and consists of inoculating subcutaneously twice
the recommended vaccination dose. The animals are observed for 10 days. The final bulk passes the test if
no systemic reactions develop and if not more than a transient oedema is observed at the injection site. If
the test is carried out in sheep only, a progressive oedema indicates that the vaccine may be unsuitable for
goats.
e) Filling the containers
Distribution of aliquots of vaccine into single and multidose containers is performed as outlined in World
Health Organization Technical Report No. 363 series entitled General Requirements for Manufacturing
Establishments and Control Laboratories (Requirements for Biological Substances No. 1), 1965, 16–17.
Basically, the final bulk is distributed to containers in an aseptic manner in an area not used for production,
and any contamination or alteration of the product must be avoided. The vaccine may be lyophilised after
distribution into appropriate dosage containers. Containers are sealed as soon as possible with a material
that is not detrimental to the product and that is capable of maintaining a hermetic seal for the life of the
vaccine.
Purity tests consist of microscopic examination of stained smears with culture and motility tests as described
in
Section C.2.a.
4. Batch control and tests on the final product
a) Sterility
The vaccine is a live culture of B. anthracis spores; sterility does not apply, but the batches must be tested
for freedom from contamination (see Chapter 1.1.9 Tests for sterility and freedom from contamination of
biological materials).
b) Efficacy
Efficacy or immunogenicity is tested on the final bulk as follows: at least ten healthy 300–500 g guinea-pigs
are inoculated with a sheep dose of the vaccine. The guinea-pigs are observed for 21 days, and at least
80%
of the animals must survive the observation period. Surviving immunised guinea-pigs and three
nonvaccinated
controls are challenged with an appropriate dose of virulent B. anthracis. A recommended
challenge is 200 LD50 of the Pasteur II strain (17JB), which is available from the same source as the Sterne
34F2 vaccine strain. If, by 10 days after challenge, all vaccinated guinea-pigs survive and control animals
die, the final bulk is deemed to be satisfactory. If any vaccinated animals die during the post-challenge
observation period from a cause other than anthrax, and death is not associated with the vaccine, the test
may be repeated.
c) Dose
The recommended dose for cattle and horses is a minimum of 1 × 107 culturable spores; for sheep, goats
and pigs, it is 1–5 × 106 culturable spores. The vaccine should contain these spores in an appropriate
volume, e.g. 1 × 107/ml.
Most experts agree that immunity is good for at least 1 year and it is recommended that an annual booster
be given. Horses may be slow to develop immunity following initial vaccination; some manufacturers
therefore recommend a two-dose initial vaccination, administered 1 month apart, followed by a single annual
booster.
e) Stability
As there is no generally acceptable test for stability of anthrax vaccines, it is recommended that, in each
filling lot, the number of culturable spores be determined before and after holding at an appropriate
temperature for an appropriate period. There should be no evidence of a fall in the number of culturable
spores.
f) Preservatives and storage
Bacillus anthracis spores are stable in unlyophilised or lyophilised vaccine and preservatives are not
required. Storage under refrigeration is recommended (4°C).
The vaccine has been shown to cause disease in some goats and llamas; this may be related to the saponin
adjuvant. The vaccine is not recommended for use in pregnant animals, nor in animals destined for
slaughter
within 2–3 weeks of vaccination. Local regulations may specify other time periods in some countries or
regions, but there is no scientific reason for regarding meat from clinically healthy animals as unfit for human
handling or consumption after a holding period of 2 weeks following vaccination. Concurrent administration
of
antibiotics to vaccinated animals is contraindicated as the antibiotic will interfere with the vaccine. Antibiotics
should not be given for several days before and after vaccination. Leftover vaccine, empty vials, and
equipment used for vaccinating are contaminated with the live spores and should be autoclaved, disinfected,
or incinerated. Accidental human inoculation is treated by expressing as much of the inoculum as possible
from the injection site and washing the wound thoroughly with soap and water. Medical attention should be
sought if infection develops.
a) Safety
Every batch of vaccine will be tested for safety as described in Section C.2.d.
b) Potency
Every batch of vaccine will be tested for potency, as described in Section C.4.b.
6. Propagation of the diagnostic ‘gamma’ bacteriophage
Anthrax-specific phages were first isolated in the 1950s, and the specifically named gamma phage was first
reported in 1955 and quickly became the standard diagnostic phage for anthrax. Gamma phage belongs to
a
family of closely related anthrax phages (11). On occasion a phage-negative B. anthracis or phage-positive
B. cereus is encountered. The phage must be used in conjunction with other tests for confirmation, yet it is a
useful and reliable test.
Phage suspensions may be obtained from central veterinary laboratories or central public health
laboratories.
The phage can be propagated and concentrated by the following protocol. Store phage at 2–4°C and do not
freeze phage as it will quickly become non-viable.
Stage one
i) Spread a blood agar (BA) plate of the Sterne vaccine strain of B. anthracis. Incubate overnight at 37°C.
ii) Inoculate approximately 10 ml of nutrient broth (NB) with growth from the BA plate and incubate at 37°C
for
approximately 4 hours or until just cloudy, then refrigerate.
iii) Spread 100 μl of the culture from step ii on three pre-dried BA plates and incubate at 37°C for 30–
60 minutes.
iv) Spread 100 μl of the phage suspension to be amplified over the same plates. Incubate at 37°C overnight.
v) Harvest the phage-lysed growth on the BA plate in 5 ml of NB followed by a second ‘wash’ of 5 ml NB.
Incubate at 37°C overnight.
vi) Filter (0.45 μm) and count by dropping 20 μl drops (three drops per dilution) of tenfold dilutions of the
filtrate
in saline onto lawns of the B. anthracis culture prepared as in step iii.
Stage two
This is essentially the same procedure as Stage one, only using the filtrate from step vi to harvest the phage
from
the plates.
vii) Prepare three Sterne strain lawns on BA, as in step iii. Incubate at 37°C for 30–60 minutes.
viii) Spread 100 μl phage from step vi. Incubate at 37°C overnight.
ix) To 9 ml of filtrate from step vi, add 1 ml of 10× concentrated NB.
x) Harvest the phage from step viii with 5 ml of the solution from step ix, followed by a second 5 ml wash
with
the rest of the solution from step ix.
xi) Add 10 ml of 1× NB.
xii) Incubate at 37°C overnight, filter and count.
Stage three
xiii) Inoculate 100 ml of brain–heart infusion broth with approximately 2.5 ml of the culture from step ii.
Incubate
on a rotary shaker at 37°C until just turbid.
xiv) Add the 20 ml of filtrate from step xii and continue incubation overnight.
xv) The resultant filtrate is checked for sterility and titrated in ten-fold dilutions on lawns of the vaccine strain
as
in step vi to determine the concentration of the phage. This should be of the order of 108–109 plaque forming
units per ml.
7. Polychrome methylene blue (M’Fadyean’s stain)
Polychrome methylene blue is prepared as follows: 0.3 g of methylene blue is dissolved in 30 ml of 95%
ethanol;
100 ml of 0.01% potassium hydroxide (KOH) is mixed with the methylene blue solution. Ideally, this should
be
allowed to stand exposed to the air, with occasional shaking, for at least 1 year to oxidise and mature.
Addition of
K2CO3 (to a final concentration of 1%) hastens the ‘ripening’ of the stain, but before it is regarded as
diagnostically reliable, its efficacy should be established by testing it in parallel with an earlier, functional
batch of
stain on bona fide samples. It has now been found that stains that give positive reactions with cultures of
B. anthracis cultured artificially in horse blood sometimes do not give positive results in the field.
In making smears for staining, only small drops of blood or tissue fluid are needed and a thin, small smear is
best.
After fixing and drying, a small (approximately 20 μl) drop of stain is placed on the smear and spread over it
with
an inoculating loop. After 1 minute, the stain is washed with water, blotted, air-dried and observed initially
using
the ×10 objective lens under which the short chains appear like short hairs; once found, these can be
observed
under oil immersion (×1000) for the presence of the pink capsule surrounding the blue/black-staining bacilli.
To
avoid laboratory contamination, the slide and blotting paper should be autoclaved or left for some hours in a
10%
sodium hypochlorite solution.
REFERENCES
1. ASCOLI A. (1911). Die Präzipitindiagnose bei Milzbrand. Centralbl. Bakt. Parasit. Infeckt., 58, 63–70.
2. BEYER W., GLOCKNER P., OTTO J. & BOHM R. (1996). A nested PCR and DNA-amplification-fingerprinting
method for detection and identification of Bacillus anthracis in soil samples from former tanneries. Salisbury
Med. Bull., No. 87, Special Suppl., 47–49.
3. BROWN E.R. & CHERRY W.B. (1955). Specific identification of Bacillus anthracis by means of a variant
bacteriophage. J. Infect. Dis., 96, 34–39.
4. EZZELL J.W. & ABSHIRE T.G. (1996). Encapsulation of Bacillus anthracis spores and spore identification.
Salisbury Med. Bull., No 87, Special Suppl., 42.
5. HUTSON R.A., DUGGLEBY C.J., LOWE J.R., MANCHEE R.J. & TURNBULL P.C.B. (1993). The development and
assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis. J. Appl.
Bacteriol., 75, 463–472.
6. JACKSON P.J., HUGH-JONES M.E., ADAIR D.M., GREEN G., HILL K.K., KUSKE C.R., GRINBERG L.M., ABRAMOVA,
F.A. & KEIM P. (1998). PCR analysis of tissue samples from the 1979 Sverdlovsk anthrax victims: The
presence of multiple Bacillus anthracis strains in different victims. Proc. Natl Acad. Sci. USA, 95, 1224–
1229.
7 KNISELY R.F. (1966). Selective medium for Bacillus anthracis. J. Bacteriol., 92, 784–786.
8. MISRA R.P. (1991). Manual for the Production of Anthrax and Blackleg Vaccines. Food and Agriculture
Organisation of the United Nations (FAO) Animal Production and Health Paper 87, FAO, Rome, Italy.
9. RAMISSE V., PATRA G., GARRIGUE H., GUESDON J.L. & MOCK M. (1996). Identification and characterization of
Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosal
DNA. FEMS Microbiol. Lett., 145, 9–16.
10. STERNE M. (1937). The effect of different carbon dioxide concentrations on the growth of virulent anthrax
strains. Onderstepoort J. Vet. Sci. Anim. Ind., 9, 49–67.
11. TURNBULL P.C.B., BOEHM R., COSIVI O., DOGANAY M., HUGH-JONES M.E., LALITHA M.K. & DE VOS V. (1998).
Guidelines for the Surveillance and Control of Anthrax in Humans and Animals. WHO/EMC/ZDI/98.6. World
Health Organization, Geneva, Switzerland.
12. WORLD HEALTH ORGANIZATION EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION (1967). Requirements
for
Anthrax Spore Vaccine (Live – for Veterinary Use) (Requirements for Bioiogical Substances No. 13). World
Health Organization (WHO) Technical Report Series No. 361. WHO, Geneva, Switzerland.
FURTHER READING
A. LOGAN N.A. & TURNBULL P.C.B. (1998). Bacillus and recently derived genera. In: Manual of Clinical
Microbiology, Seventh Edition, Murray P.R., Baron E.J., Pfaller M.A., Tenover F.C. & Yolken R.H., eds.
American Society for Microbiology, Washington DC, USA, 357–369.
B. QUINN C.P. & TURNBULL P.C.B. (1998). Anthrax. In: Topley & Wilson’s Microbiology and Microbial
Infections,
Vol. 3, Ninth Edition, Collier L., Balows A. & Sussman M., eds. Arnold, London, UK, 799–818.
C. TURNBULL P.C.B., (ED.) (1996). Proceedings of the International Workshop on Anthrax. Salisbury Med.
Bull.,
Special Supple. No. 87, 139 pages.
D. TURNBULL P.C.B. (1998). Anthrax. In: Zoonoses. Biology, Clinical Practice, and Public Health Control.
Palmer
S.R., Soulsby E.J.L. & Simpson D.I.H., eds. Oxford University Press, Oxford, UK, 3–16.
E. WHITFORD H.W. & HUGH-JONES M.E. (1994). Anthrax. In: Handbook Series in Zoonoses, Second Edition,
Beran G.W., editor-in-chief. CRC Press, Boca Raton, Florida, USA, 61–82.
*
**
NB: There are OIE Reference Laboratories for Anthrax (see Table in Part 3 of this Terrestrial Manual or
consult
the OIE Web site for the most up-to-date list: www.oie.int).
OIE Terrestrial Manual 2009 xi

LIST OF TESTS FOR INTERNATIONAL TRADE
The table below lists diagnostic tests in two categories: ‘prescribed’ and ‘alternative’. Prescribed tests
are required by the OIE Terrestrial Animal Health Code for the international movement of animals
and animal products and are considered optimal for determining the health status of animals. In the
Terrestrial Manual these tests are printed in blue. At present it is not possible to have prescribed
tests for every listed disease. Alternative tests are those that are suitable for the diagnosis of disease
within a local setting, and can also be used in the import/export of animals after bilateral agreement.
There are often other tests described in the chapters that may also be of some practical value in local
situations or that may still be under development.
Chapter No. Disease name Prescribed tests Alternative tests

2.1.1. Anthrax – –
2.1.2. Aujeszky’s disease ELISA, VN –
2.1.3. Bluetongue Agent id., ELISA,
PCR
AGID, VN
2.1.4. Echinococcosis/Hydatidosis – –
2.1.5. Foot and mouth disease ELISA*, VN CF
2.1.6. Heartwater – ELISA, IFA
2.1.7. Japanese encephalitis – –
2.1.8. Leishmaniosis – Agent id.
2.1.9. Leptospirosis – MAT
2.1.10. New World screwworm (Cochliomyia
hominivorax) and Old World screwworm
(Chrysomya bezziana)
– Agent id.
2.1.11. Paratuberculosis (Johne’s disease) – DTH, ELISA
2.1.12. Q fever – CF
2.1.13. Rabies ELISA, VN –
2.1.14. Rift Valley fever VN ELISA, HI
2.1.15. Rinderpest ELISA VN
2.1.16. Trichinellosis Agent id. ELISA
2.1.17. Trypanosoma evansi infections (including
surra)
––
2.1.18. Tularemia – Agent id.
2.1.19. Vesicular stomatitis CF, ELISA, VN –
2.1.20. West Nile fever – –
2.2.1. Acarapisosis of honey bees – –
2.2.2. American foulbrood of honey bees – –
* Please refer to Terrestrial Manual chapters to verify which method is prescribed.
List of tests for international trade
xii OIE Terrestrial Manual 2009
2.2.3. European foulbrood of honey bees – –
2.2.4. Nosemosis of bees – –
2.2.5. Small hive beetle infestation
(Aethina tumida)
––
2.2.6. Tropilaelaps infestation of honey bees
(Tropilaelaps spp.)
––
2.2.7 Varroosis of honey bees – –
2.3.1. Avian chlamydiosis – –
2.3.2. Avian infectious bronchitis – ELISA, HI, VN
2.3.3. Avian infectious laryngotracheitis – AGID, ELISA, VN
2.3.4. Avian influenza Virus isolation with
pathogenicity
testing
AGID, HI
2.3.5. Avian mycoplasmosis
(Mycoplasma gallisepticum, M. synoviae)
– Agg., HI
2.3.6. Avian tuberculosis – Agent id.,
Tuberculin test
2.3.7. Duck virus enteritis – –
2.3.8. Duck virus hepatitis – –
2.3.9. Fowl cholera – –
2.3.10. Fowl pox – –
2.3.11. Fowl typhoid and Pullorum disease – Agent id., Agg.
2.3.12. Infectious bursal disease
(Gumboro disease)
– AGID, ELISA
2.3.13. Marek’s disease – AGID
2.3.14. Newcastle disease Virus isolation HI
2.3.15. Turkey rhinotracheitis (avian
metapneumovirus)
––
2.4.1. Bovine anaplasmosis – CAT, CF
2.4.2. Bovine babesiosis – CF, ELISA, IFA
2.4.3. Bovine brucellosis BBAT, CF,
ELISA, FPA
–
2.4.4. Bovine cysticercosis – Agent id.
2.4.5. Bovine genital campylobacteriosis Agent id. –
2.4.6. Bovine spongiform encephalopathy – –
2.4.7. Bovine tuberculosis Tuberculin test Gamma interferon test
2.4.8. Bovine viral diarrhoea Agent id. –
2.4.9. Contagious bovine pleuropneumonia CF, ELISA –
2.4.10. Dermatophilosis – –
2.4.11. Enzootic bovine leukosis AGID, ELISA PCR
2.4.12. Haemorrhagic septicaemia – Agent id.
2.4.13. Infectious bovine rhinotracheitis/
infectious pustular vulvovaginitis
Agent id. (semen
only), ELISA, PCR,
VN
–
OIE Terrestrial Manual 2009 xiii
2.4.14. Lumpy skin disease – VN
2.4.15. Malignant catarrhal fever – IFA, PCR, VN
2.4.16. Theileriosis Agent id., IFA –
2.4.17. Trichomonosis Agent id. Mucus agg.
2.4.18. Trypanosomosis (Tsetse-transmitted) – IFA
2.5.1. African horse sickness CF, ELISA Agent id. (real-time
PCR), VN
2.5.2. Contagious equine metritis Agent id. –
2.5.3. Dourine CF ELISA, IFA
2.5.4. Epizootic lymphangitis – –
2.5.5. Equine encephalomyelitis
(Eastern and Western)
– CF, HI, PRN
2.5.6. Equine infectious anaemia AGID ELISA
2.5.7. Equine influenza – HI
2.5.8. Equine piroplasmosis ELISA, IFA CF
2.5.9. Equine rhinopneumonitis – VN
2.5.10. Equine viral arteritis Agent id. (semen
only), VN
–
2.5.11. Glanders CF, Mallein test –
2.5.12. Horse mange – Agent id.
2.5.13. Horse pox – –
2.5.14. Venezuelan equine encephalomyelitis – CF, HI, PRN
2.6.1. Myxomatosis – AGID, CF, IFA
2.6.2. Rabbit haemorrhagic disease – ELISA, HI
2.7.1. Border disease Agent id. –
2.7.2. Caprine and ovine brucellosis
(excluding Brucella ovis)
BBAT, CF,
ELISA, FPA
Brucellin test
2.7.3/4. Caprine arthritis/encephalitis & Maedi-visna AGID, ELISA –
2.7.5. Contagious agalactia – –
2.7.6. Contagious caprine pleuropneumonia CF –
2.7.7. Enzootic abortion of ewes
(ovine chlamydiosis)
– CF
2.7.8. Nairobi sheep disease – –
2.7.9. Ovine epididymitis (Brucella ovis) CF ELISA
2.7.10. Ovine pulmonary adenocarcinoma
(adenomatosis)
––
2.7.11. Peste des petits ruminants VN ELISA
2.7.12. Salmonellosis (S. abortusovis) – –
2.7.13. Scrapie – –
2.7.14. Sheep pox and goat pox – VN
2.8.1. African swine fever ELISA IFA
2.8.2. Atrophic rhinitis of swine – –
xiv OIE Terrestrial Manual 2009
2.8.3. Classical swine fever (hog cholera) ELISA, FAVN,
NPLA
–
2.8.4. Nipah virus encephalitis – –
2.8.5. Porcine brucellosis BBAT, CFT,
ELISA, FPA
–
2.8.6. Porcine cysticercosis – –
2.8.7. Porcine reproductive and
respiratory syndrome
– ELISA, IFA, IPMA
2.8.8. Swine influenza – –
2.8.9. Swine vesicular disease VN ELISA
2.8.10. Teschovirus encephalomyelitis (previously
enterovirus encephalomyelitis or
Teschen/Talfan disease)
– VN
2.8.11. Transmissible gastroenteritis – VN, ELISA
2.9.1. Bunyaviral diseases of animals (excluding
Rift Valley fever)
––
2.9.2. Camelpox – –
2.9.3. Campylobacter jejuni and C. coli – –
2.9.4. Cryptosporidiosis – –
2.9.5. Cysticercosis – Agent id.
2.9.6. Hendra and Nipah virus diseases – –
2.9.8. Listeria monocytogenes – –
2.9.8. Mange – Agent id.
2.9.9. Salmonellosis – Agent id.
2.9.10. Toxoplasmosis – –
2.9.11. Verocytotoxigenic Escherichia coli – –
2.9.12. Zoonoses transmissible from non-human
primates
––
Note: The tests prescribed by the Terrestrial Animal Health Code for the purposes of international trade are
printed
in blue in this Terrestrial Manual.
Abbreviations
Agent id. Agent identification HI Haemagglutination inhibition
Agg. Agglutination test IFA Indirect fluorescent antibody
AGID Agar gel immunodiffusion IPMA Immunoperoxidase monolayer assay
BBAT Buffered Brucella antigen test MAT Microscopic agglutination test
CAT Card agglutination test NPLA Neutralising peroxidase-linked assay
CF Complement fixation PCR Polymerase chain reaction
DTH Delayed-type hypersensitivity PRN Plaque reduction neutralisation
ELISA Enzyme-linked immunosorbent assay VN Virus neutralisation
FAVN Fluorescent antibody virus neutralisation – No test designated yet
FPA Fluorescence polarisation assay

Equine Influenza

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Equine Influenza

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EQUINE INFLUENZA

OIE Terrestrial Manual 2008 135

OIE Terrestrial Manual 2009 xi

Chapter No. Disease name Prescribed tests Alternative tests

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