Vol. 22, No.
4 April 2000
CE Refereed Peer Review
FOCAL POINT
★Serology combined with
polymerase chain reaction–based
assays can be used to rapidly
and accurately document
pasteurellosis in rabbits.
KEY FACTS
■ Pasteurella multocida is one of
the most common disease-
causing agents in rabbits.
■ Culture accuracy of P. multocida
depends on the transport
medium, storage temperature
of the swab, previous antibiotic
treatment, and laboratory culture
conditions.
■ A polymerase chain reaction–based
assay is a more sensitive test than
is culture, partially because the
former detects the presence of
both viable and nonviable bacteria.
■ Serology can be used to detect P.
multocida antibodies, particularly in
chronically infected rabbits.
■ Nasopharyngeal swabs are
recommended for obtaining culture
samples to detect the presence of
P. multocida; however, sample
collection from conscious rabbits
is difficult.
Pasteurellosis in Rabbits
University of Georgia
Susan Sanchez, PhD Shaikh Mizan, PhD
Branson W. Ritchie, DVM, PhD Margie D. Lee, DVM, PhD
ABSTRACT: Pasteurella multocida infections in rabbits may remain subclinical or can cause a
variety of acute or chronic diseases in most organs, particularly tissue in the repiratory and
genital tracts. Although clinical signs are frequently used to diagnose pasteurellosis, other or-
ganisms can cause similar clinical signs. The status of a rabbit with respect to P. multocida
can be best determined using a polymerase chain reaction–based assay in conjunction with a
sensitive and specific ELISA.
P
asteurella multocida can cause various clinical changes in rabbits. As one
of the most common disease-causing agents in this genus, P. multocida
has been associated with rhinitis (snuffles), abscessation, pneumonia, oti-
tis, conjunctivitis, genital tract infections, abortions, neonatal mortality, and sep-
ticemia.1,2 Although this organism is highly infectious, many rabbits are subclini-
cally infected until stress or concomitant problems precipitate recognizable
disease. The reported prevalence of P. multocida in clinically stable rabbits ranges
from 20% to 90%, depending on the animal’s age, sex, and health status and the
chosen testing method.3,4
Based on studies by Webster and Smith, it is generally believed that rabbits in-
fected with P. multocida can resist or clear infection, become chronically but sub-
clinically infected, or develop acute or chronic disease. The type of infection is
believed to be influenced by the strain of infecting Pasteurella and the specific
host resistant to that strain.5 Generally, Pasteurellaceae should be considered
commensal organisms that may be pathogenic in immunosuppressed rabbits.
Peracute pasteurellosis usually causes septicemia and death in seemingly
healthy rabbits. Upper respiratory tract disease (e.g., snuffles) associated with P.
multocida infection starts as a serous nasal discharge that usually turns mucopu-
rulent (Figure 1). Hair on the animal’s forelimbs is usually moist from repeated
attempts to clean the muzzle. Epiphora, sneezing, and coughing may accompany
the upper respiratory tract form of pasteurellosis. Anorexia, weight loss, dyspnea,
pleuropneumonia, and, in the most severe cases, death may occur if untreated
rhinitis progresses to more generalized disease.
P. multocida is a common cause of abscessation in rabbits, irrespective of the
presence or absence of respiratory tract disease. These abscesses are filled with
thick, yellow–gray mucopurulent exudate and can occur in any location on or
within the body (Figures 2 and 3). Microscopic examination of material collected
from these abscesses usually reveals small gram-negative coccobacilli suggestive of
P. multocida infection (Figure 4). P. multocida can also cause otitis externa and oti-
tis media. If the vestibular system is involved, torticollis with the head tilted to
Compendium April 2000 Small Animal/Exotics

Figure 1—Mucopurulent rhinitis suggestive of progressive Figure 2—Pasteurella multocida infection can cause abscessa-
pasteurellosis. tion in any organ. In this rabbit, the scrotum is ulcerated and
its necrotic abscessed testicle was surgically removed.

the affected side and head shaking are common (Figure

5). Pasteurella-associated metritis, which also occasional-
ly affects rabbits, typically follows breeding or pseudo-
pregnancy and is manifested by chronic purulent dis-
charge from the vulva. Surviving rabbits are frequently
sterile.3,6

DIAGNOSIS
Suggestive clinical signs are frequently used to diag-
nose pasteurellosis. However, other organisms alone or
in combination with viruses and parasites can cause
clinical signs that are characteristic of pasteurellosis.5
Accurate diagnosis and treatment of pasteurellosis are
important to reduce the spread of P. multocida infec-
tion, particularly in multiple-rabbit households or rab-
bitries. Isolating the organism from the rabbit’s nostrils,
conjunctiva, ears, or abscessed tissue has been the most
common method for confirming pasteurellosis. Figure 3—Mucopurulent, necrotizing pneumonia in a rabbit
In a classic study5 involving a rabbit colony with en- with chronic pasteurellosis. The affected lung was surgically
demic pasteurellosis, 58 of 100 rabbits had gross upper removed.
respiratory changes at necropsy. P. multocida was recovered
from the nares of 55 of these rabbits and from 8 rabbits
with no signs of upper respiratory disease.5 However, cul- ture process can produce false-negative results. Immediate
ture of P. multocida in living rabbits can be problematic transfer of samples to culture medium is not always possi-
because even deep nasal cultures frequently fail to confirm ble, and transport medium must preserve the organism
upper respiratory tract infection when an organism in sub- until samples reach the laboratory. Compounding this
clinically infected rabbits is present in low numbers. Local- problem is the fact that P. multocida is extremely delicate
ized infection in the tympanic bullae, caudal oropharynx, and does not survive well in most common transport me-
genital tract, and other internal organs renders ante- dia (e.g. Stuart’s, Amies), in some instances surviving less
mortem sampling for culture difficult.7,8 In other cases, P. than 24 hours at room temperature.9 The organism may
multocida cannot be cultured from obviously diseased or- survive for several days in Cary-Blair medium. Because re-
gans because the rabbit has received antibiotics. frigeration can reduce survival of P. multocida,9 samples for
In addition to problems with sample collection, the cul- culture should be maintained at room temperature or

MUCOPURULENT RHINITIS ■ TORTICOLLIS ■ TRANSPORT MEDIUM

Small Animal/Exotics
Figure 4—Pasteurella species are gram-negative coccobacilli
that can frequently be detected in cytologic preparations of
secretions or exudates.
frozen. Use of fresh blood agar plates containing 5% rab-
bit blood can increase the sensitivity of P. multocida isola-
tion, but this modification of routine cultures is not com-
mon in commercial bacteriology laboratories usually used
by exotic animal clinicians.5 In addition, some strains of P.
multocida grow best at 34˚C to 35˚C,5 which approxi-
mates the temperature of rabbit nares but is lower than
that used for routine cultures (37˚C).
As a rule, polymerase chain reaction (PCR)–based as-
says are more sensitive than are those for cultures, be-
cause PCR assays detect target nucleic acid sequences
even from nonviable organisms. Cultures and the most
sensitive PCR assays can produce false–negative results
if the sample does not contain viable organism (cul-
tures) or sufficient quantities of nucleic acid (PCR as-
says).
Serology can be a useful adjunct to cultures or PCR as-
says to establish whether a rabbit has active infection (ris-
ing antibody titer in paired serum samples tested in the
same laboratory at the same time) and is the only method
currently available for demonstrating previous infection.
ELISAs are considered reliable for epidemiology studies in
rabbit colonies.10–12 Rabbits with detectable antibodies
may have cleared an infection in the immunologically de-
tectable past or may be seropositive yet remain subclinical-
ly infected. In general, high levels of anti-Pasteurella im-
munoglobulin (Ig) G correlate with chronic infection.13
Routinely used ELISAs may not detect acute infection be-
cause it generally takes rabbits 2 to 3 weeks to develop de-
tectable IgG antibody titers.10 In rabbits younger than 8
weeks of age, detectable antibodies are probably derived
from the female. Antibodies from normal bacteria found
POLYMERASE CHAIN REACTION ■ ANTIBODY TITER ■ OTITIS MEDIA/INTERNA
Compendium April 2000
Figure 5—Torticollis secondary to Pasteurella-associated otitis
media/interna.
in rabbits cross-react with the ELISA at low levels. Thus it
has been suggested that the ideal antibody assay would de-
tect antibody to an epitope unique to P. multocida.5
CLINICAL TRIALS
A clinical study14 compared routine culture, a recent-
ly developed PCR assay,a and a recently developed sero-
logic testb for confirming P. multocida infection in 58
rabbits with clinical signs suggestive of pasteurellosis.
Clinical signs in tested rabbits included sneezing, con-
gestion, chronic nasal discharge, chronic ocular dis-
charge, otitis, ataxia, head tilt, anorexia, recumbency, or
the presence of abscesses in various locations. All sam-
ples were submitted to the laboratory by regular mail,
which is typical for routine culture.c
A summary of results for each test as an independent
assay is shown in Table I. Although samples from a to-
tal of 58 rabbits were used in the study, not all samples
were submitted for each rabbit. For testing purposes,
rabbits were placed in subgroups based on the samples
a
P. multocida PCR, Infectious Diseases Laboratory, College of
Veterinary Medicine, The University of Georgia, Athens,
Georgia.
b
P. multocida ELISA, Infectious Diseases Laboratory, College
of Veterinary Medicine, The University of Georgia, Athens,
Georgia.
c
For each hospital, swabs for culture and PCR evaluation were
taken from the nostrils, ocular discharge, inner ear, or abscess-
es; placed in transport medium for storage; and shipped. Sam-
ples took an average of 3 days to arrive at the laboratory. For
P. multocida isolation, swabs were plated onto primary (blood
and chocolate agar) and selective (modified Knight’s and
Avril’s)13,14 media maintained in aerobic and microaerophilic
conditions at 37˚C for 48 hours.
Compendium April 2000 Small Animal/Exotics

TABLE I TABLE II
Summary of the Test Results Results of Serology and Culture
to Diagnosis Pasteurellosis Testing in 52 Rabbits with Clinical
Total Signs Suggestive of Pasteurellosis
Tested Positive Negative No. of Rabbits Serology Culture

Culture 55 2 (3.6%) 53 (96.4%) 27 (52%) – –

Polymerase chain 38 11 (29%) 27 (71%) 23 (44%) + –

reaction assay
2 (4%) + +
Serology 55 25 (45%) 30 (55%)

TABLE III TABLE IV

Results of Serology and Polymerase Results of Polymerase Chain Reaction
Chain Reaction Testing in 35 Rabbits with and Culture Testing in 38 Rabbits with
Clinical Signs Suggestive of Pasteurellosis Clinical Signs Suggestive of Pasteurellosis
Polymerase Polymerase
No. of Rabbits Chain Reaction Serology No. of Rabbits Culture Chain Reaction

16 (46%) – – 27 (71%) – –

10 (29%) + + 9 (24%) – +

8 (23%) – + 2 (5%) + +

1 (3%) + –

PCR assay and serology. Samples from eight rabbits

available for each animal. In total, swabs from 55 rab-
bits were cultured. P. multocida was recovered from
only two (3.6%) of the swabs (Table I). The serum
from these two rabbits contained anti-Pasteurella anti-
bodies that could be detected using an ELISA with re-
combinant neuraminidase as an antigen.14 These results
suggest that the selective media (Avril and modified
Knight’s media containing antibiotics that inhibit fungi
and other bacteria) were effective in preventing the
growth of other microorganisms in the swabs, thereby
reducing the possibility of other organisms overgrowing
P. multocida.16,17
Samples from 52 rabbits were used to compare serol-
ogy and culture results (Table II). Serum and swabs
from 27 rabbits (52%) had negative results on both
culture and serology. Samples from 23 of 52 rabbits
(44%) tested serologically positive but had negative
culture results, and two of the 52 rabbits (4%) tested
positive on both culture and serology.
Samples from 35 rabbits were used to compare serol-
ogy and PCR assay results (Table III). Serum and swab
results for 16 rabbits (46%) were negative for both
(23%) were serologically positive but negative on the
PCR assay. Ten rabbits (29%) were positive on both
PCR assay and serology. One rabbit (3%) tested posi-
tive on PCR assay and negative on serology.
Samples from 38 rabbits were available for testing us-
ing culture and PCR assay (Table IV). Twenty-seven
swabs (71%) had negative results by both PCR assay
and culture; nine swabs (24%) tested positive on PCR
assay but negative on culture. Results of two swabs
(5%) from which P. multocida was cultured were also
positive on PCR assay.
Samples from 35 rabbits were available for testing us-
ing serology, culture, and PCR assay (Table V). Sixteen
rabbits (46%) tested negative and only two rabbits
(6%) tested positive on all three tests. The results of
eight of 35 rabbits (23%) were positive on both PCR
assay and serology. The same number of rabbits also
had negative results on PCR assay but had detectable
levels of serum antibodies. P. multocida nucleic acid was
detected on a swab from one rabbit (3%) that tested
serologically negative; the remaining 16 rabbits also
tested negative on PCR assay.
The results of this study showed that rabbits in which

ANTI-PASTEURELLA ANTIBODIES ■ RECOMBINANT NEURAMINIDASE ■ SEROLOGY

Small Animal/Exotics
TABLE V
Results of Culture, Serology, and
Polymerase Chain Reaction Testing in 35 Rabbits with
Clinical Signs Suggestive of Pasteurellosis
No. of Polymerase
Rabbits Culture Serology Chain Reaction
16 (46%) – – – la bronchiseptica, Pseudomonas species, Staphylococcus
2 (6%) + + + multocida is a ubiquitous bacteria, it may be an over-
8 (23%) – + + stated cause of respiratory disease in rabbits when clini-
8 (23%) – + –
1 (3%) – – + in combination with serology using a neuraminidase–
P. multocida was detected by culture and/or PCR assay
also tested positive on a recently developed ELISA.14 Fur-
thermore, positive serology results indicated that eight
rabbits (23%) had been infected with P. multocida in
their immunologically detectable past even though the
organism was not demonstrated on are nasal swabs for
PCR assay or culture. Most of the submitted swabs were
obtained from the nasal passages, which may have re-
duced the number of bacteria available for detection on
culture or PCR assay. P. multocida has an affinity for the
mucosal epithelium of the nasopharynx.8,15 Nasopharyn-
geal swabs are reportedly 36% more sensitive than are
nasal swabs in detecting P. multocida; however, collecting
a sample from the nasopharynx is difficult in a conscious
rabbit.8 The use of nasal swabs in this study, instability of
P. multocida during shipment, and ability of PCR assay
to detect nucleic acid from inactive organisms likely con-
tributed to the higher sensitivity of PCR assay compared
with that of culture. To screen for bacteria in rabbits
with rhinitis, a No. 4 calcium alginate swab can be in-
serted 1 to 4 cm into the nares along the nasal septum
on both sides.5
The ELISA used in this study detected anti-Pasteurella
IgG in 25 of 55 or 45.5% of the sera samples tested. By
comparison, only 11 of 38 rabbits (29%) had positive
results on PCR assay, and only two of 55 rabbits
(3.64%) had positive culture results. All except one of
the rabbits with positive results on PCR assay and/or
culture also had positive results on serology. Because no
additional sample was available from the one rabbit
with positive PCR results and negative serology results,
whether the serology was a false-negative reaction, the
swab was contaminated with target nucleic acid, or the
rabbit had an early infection to which the immune sys-
tem had not mounted a detectable immune-response
cannot be determined. Serology detected more positive
results than did any of the other methods, but more
NASOPHARYNGEAL SWABS ■ MUCOSAL EPITHELIUM ■ NASAL SEPTUM
Compendium April 2000
than half of the sera submitted (30 of 55) tested nega-
tive for anti-Pasteurella IgG antibodies by ELISA.
Although samples were submitted for rabbits with
clinical signs suggestive of pasteurellosis, the results of
this study suggest that many cases of suspected pas-
teurellosis may be caused by other organisms alone or
in combination. Candidate organisms include Bordetel-
species or other Pasteurella species.5 Thus although P.
cal signs alone are used for diagnosis. The clinical trial
reviewed in this manuscript suggests that PCR testing
based ELISA can help confirm pasteurellosis as the
cause of disease in rabbits with suspicious clinical signs.
ACKNOWLEDGMENTS
This research was supported by the University of
Georgia Research Foundation Animal Health Fund and
the Veterinary Medical Experiment Station. The au-
thors thank Drs. William Chavez, Joe Deck, Cheryl
Greenacre, Heather McClure, Masako Mori, Robert Ness,
Richard Nye, Robert Owen, Pamela Slack, Samuel
Vaughn, and Heather Wilson for submitting samples
used in this project.
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Small Animal/Exotics
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About the Authors
Dr. Sanchez is affiliated with the Athens Diagnostic Labo-
ratory, Drs. Mizan and Lee with the Department of Medi-
cal Microbiology and Parasitology, and Dr. Ritchie with
the Department of Small Animal Medicine and Surgery,
College of Veterinary Medicine, The University of Geor-
gia, Athens, Georgia.