Effects of Temperature and Stabilizer on the Viability of a Live Attenuated Avian

Metapneumovirus Vaccine
Author(s): M. A. Ramakrishnan , Binu T. Velayudhan , Senthilvelan Anantharaman , Sally L. Noll ,
David A. Halvorson , Kakambi V. Nagaraja , and Sagar M. Goyal
Source: Avian Diseases, 51(4):979-981. 2007.
Published By: American Association of Avian Pathologists
DOI: http://dx.doi.org/10.1637/7962-030107.1
URL: http://www.bioone.org/doi/full/10.1637/7962-030107.1

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AVIAN DISEASES 51:979–981, 2007

Research Note—
Effects of Temperature and Stabilizer on the Viability of a Live Attenuated Avian
Metapneumovirus Vaccine
M. A. Ramakrishnan,A Binu T. Velayudhan,B Senthilvelan Anantharaman,A Sally L. Noll,C David A. Halvorson,B
Kakambi V. Nagaraja,B and Sagar M. GoyalAD
A
Department of Veterinary Population Medicine
B
Department of Veterinary and Biomedical Sciences
C
Department of Animal Science
University of Minnesota, St. Paul, MN 55108
Received 1 March 2007; Accepted and published ahead of print 3 July 2007

SUMMARY. A commercial live attenuated, freeze-dried avian metapneumovirus vaccine, PneumomuneTM, was assessed for its
viability at three different temperatures (5.6 C, 21 C, and 37 C). No significant reduction in virus titer was observed when the
vaccine was stored at 5.6 C for a period of 24 hr. However, reductions in virus titer of 1 log10 and 2 log10 were observed after 24 hr
at 21 C and 37 C, respectively. Batch-to-batch variation in virus reduction was also observed. The addition of a dye or a vaccine
stabilizer to the vaccine preparation did not have any deleterious effect on the survival of vaccine virus.
RESUMEN. Nota de Investigación—Efecto de la temperatura y del estabilizador sobre la viabilidad de una vacuna viva atenuada
de Metapneumovirus aviar.
Una vacuna comercial liofilizada, viva y atenuada contra metapneumovirus aviar (PneumomuneH), fue evaluada para su
viabilidad a tres temperaturas diferentes (5.6 C, 21 C y 37 C). No se observó una reducción significante en el tı́tulo del virus
cuando la vacuna fue almacenada a 5.6 C durante un periodo de 24 horas. Sin embargo, se observaron reducciones en el tı́tulo del
virus de uno y dos logaritmos10 después de 24 horas a 21 C y 37 C, respectivamente. También se observó variación en la reducción
del virus entre lote y lote de vacuna. La adición de un colorante o de un estabilizador a la vacuna preparada no tuvo ningún efecto
perjudicial sobre la supervivencia del virus vacunal.
Key words: avian metapneumovirus, vaccine, dye, stabilizer, temperature, viability
Abbreviations: aMPV 5 avian metapneumovirus; TCID50 5 50% tissue culture infective dose

Avian metapneumovirus (aMPV) causes serious respiratory disease
in turkeys (8) and belongs to the genus Metapneumovirus within the
subfamily Pneumovirinae, family Paramyxoviridae (16). Four sub-
groups of aMPV, designated as A, B, C, and D, have been defined.
European isolates of aMPV belong to subgroups A, B, and D and
the U.S. isolates belong to subgroup C (7,14). Currently, only one
commercial, live attenuated vaccine is available against aMPV-C
(PneumomuneTM; Biomune, Lenexa, KS) in the United States.
Because the titer of live virus is likely to decrease over time, the
live virus vaccine should be administered as soon as possible after
reconstitution so that the birds may receive the required dosage of
the vaccine. The survival of viruses is affected by many physical and
chemical variables, including temperature, humidity, pH, presence
of organic matter, and exposure to various chemicals (4,15,17).
Many techniques are used in the poultry industry for vaccine
administration including drinking water, spray/nebulization, in-
traocular administration, scarification, parenteral injection, and in-
ovo vaccination. Live vaccines are usually administered to birds by
the drinking water route or by aerosol spray. To differentiate
between vaccinated and nonvaccinated birds, a dye is often added to
the water or the reconstituted vaccine so that it can stain the mouth
(tongue/beak), choanal cleft, or feathers of birds vaccinated via
drinking water, intraocular route, or spray application, respectively
(9,10). Water quality factors such as chlorine, detergents, and
organic matter often affect the viability of live vaccines in water (5).
D
Corresponding author. E-mail: goyal001@umn.edu
To neutralize such elements and maintain vaccine viability,
stabilizers such as skimmed milk powder are commonly added to
water (2,5,18).
The preparation of PneumomuneTM for use in vaccination is
a two-step process involving initial reconstitution of freeze-dried
vaccine in distilled water followed by additional dilution for
administration via spray or drinking water. Prior to use, final
vaccine dilution may include dye or stabilizer. In the present study,
we evaluated the effect of temperature, dye,and stabilizer on the
viability of the PneumomuneTM vaccine.
MATERIALS AND METHODS
Cells. Vero cells were grown and maintained in Eagle minimum
essential medium with Earle salts (Media Tech, Herndon, VA). The
additives were antibiotics (150 IU/ml penicillin, 150 mg/ml streptomy-
cin, 50 mg/ml neomycin, 2.5 mg/ml amphotericin B), 0.5% lactalbumin
hydrolysate (Edamin S), 1 mM sodium pyruvate, and 8% fetal bovine
serum. Maintenance medium was the same as above except that no fetal
bovine serum was added. The cells were grown at 37 C in the presence
of 5% CO2.
Sources of vaccine, dye, and stabilizer. PneumomuneTM vaccine is
commercially available (Biomune). Two serials of the vaccine (serials
010 and 011) were used. The expiration date for serial 010 was May 30,
2006 and that for serial 011 was July 7, 2006. All experiments were
conducted in April and May of 2006. A water-soluble blue dye (Hi-
Light tablets; Becker Underwood, Ames, IA) and a vaccine stabilizer
(Vac Pro Tec; International Nutrition, Omaha, NE) were also used in
this study.

979
980 M. A. Ramakrishnan et al.

Table 1. The effect of time and temperature on titers of PneumomuneTM vaccine.A

Virus titer (log10 TCID50/ml) at indicated temperature
5.6 C 21 C 37 C
Time/vial 1 2 3 Mean (6 SD)B 1 2 3 Mean (6 SD) 1 2 3 Mean (6 SD)
0 min 5.75 5.50 5.75 5.67 (0.14) a 5.75 5.50 5.75 5.67 (0.14) a 5.75 5.50 5.75 5.67 (0.14) a
30 min 5.50 5.50 5.75 5.58 (0.14) ab 5.75 5.50 5.25 5.50 (0.25) abc 5.75 5.75 5.25 5.58 (0.29) ab
1 hr 5.50 5.25 5.50 5.42 (0.14) abc 5.25 5.50 5.50 5.42 (0.14) abc 5.50 5.75 5.50 5.58 (0.14) ab
2 hr 5.50 5.25 5.50 5.42 (0.14) abc 5.50 5.50 5.25 5.42 (0.14) abc 5.50 5.50 5.50 5.50 (0.00) abc
4 hr 5.50 5.50 5.50 5.50 (0.00) abc 5.25 5.50 5.50 5.42 (0.14) abc 5.25 5.25 5.00 5.17 (0.14) bcd
8 hr 5.50 5.50 5.50 5.50 (0.00) abc 5.00 5.50 4.75 5.08 (0.38) cde 4.75 5.25 4.75 4.92 (0.29) de
24 hr 5.25 5.75 5.50 5.50 (0.25) abc 4.50 4.75 4.75 4.67 (0.14) e 3.50 3.75 3.75 3.67 (0.14) f
A
SEM for vial, temperature, and time were 0.0318, 0.0318, and 0.0486, respectively.
B
Means followed by different lowercase letters are significantly different (P , 0.05).

Effect of storage temperature on virus viability. In Experiment 1,
three vials of PneumomuneTM (serial number 011; 5000 doses/vial)
were used. As per the manufacturer’s recommendation, the contents of
each vial were reconstituted with 150 ml of sterile distilled water (30 ml
per 1000 doses). The reconstituted suspension from each vial was
divided into three parts and stored at 5.6 C, 21 C, or 37 C. Samples
taken from each vaccine vial immediately after reconstitution were
labeled as ‘zero’ time samples. Additionally, samples were removed from
reconstituted vaccine stored at three different temperatures at 30 min
and 1, 2, 4, 8, and 24 hr. After indicated incubation time, serial 10-fold
dilutions of all samples were made immediately in maintenance medium
and titrated in monolayers of Vero cells grown in 96-well microtiter
plates using four wells per dilution (100 ml/well). The inoculated plates
were incubated at 37 C in the presence of 5% CO2 for 5 days and
aMPV antigen was detected by indirect immunofluorescence assay (11).
The 50% tissue culture infective dose (TCID50) of the vaccine virus was
calculated using the Spearman–Karber method as modified by Finney
(6,12,13). The titers were expressed as log10 TCID50 per milliliter.
The effect of dye and stabilizer on viability of vaccine virus. In
Experiment 2, two vials of PneumomuneTM vaccine (one each of serials
010 and 011) were reconstituted in sterile, cold distilled water at 23
concentration. One day before the start of the experiment, the Hi-Light
dye tablet was dissolved in sterile distilled water at 23 concentration and
stored at 37 C (recommended dilution is 1 tablet in 1.9 liter of water). A
23 concentration of the stabilizer (recommended quantity is 12 g/liter
for water vaccination) was prepared in warm water (42 C) and then
cooled prior to use. Equal volumes of 23 vaccine suspensions were
mixed with distilled water, 23 dye, or 23 stabilizer solutions and stored
at room temperature (21 C). Duplicate samples were taken at 0 min and
after 15 min, 30 min, and 1, 2, 4, 6, and 24 hr and titrated on Vero cell
monolayers. Duplicate samples were prepared in two separate
laboratories to assess lab-to-lab variation. To ensure that the dye and
the stabilizer were not toxic to the cells, 13 solutions of dye and
stabilizer were prepared in the maintenance medium and inoculated on
to Vero cells, which were then incubated at 37 C in the presence of 5%
CO2 for 5 days and observed for morphological changes.
Statistical analysis. Virus titers as affected by the above experimental
factors were determined in both experiments by analysis of variance using
statistical software Statistix 8 (Analytical Software, Tallahassee, FL) for
a factorial design. Mean differences were determined using the Tukey
honestly significant difference procedure at 5% significance level. In
Experiment 1, the model included the main effects of incubation time,
vaccine vial, and temperature as well as the two-way interactions. In
Experiment 2, the model included the main effects of vaccine serial, diluent
(distilled water, dye, or stabilizer), testing laboratory, and incubation time as
well as the interactions. The effects were considered significant at P , 0.05.
RESULTS
In Experiment 1, both temperature and time had significant (P ,
0.0001) effect on viral titers (Table 1). Increasing incubation times
decreased virus titer. A significant two-way interaction existed for
time by temperature (P , 0.0001) and time by vial (P , 0.02). The
time-by-temperature interaction occurred because there was no
reduction in virus titer after 24 hr of storage at refrigeration
temperature (5.6 C), whereas at 21 C and 37 C, there was
a reduction of 1 log10 and 2 log10, respectively (Table 1).
In Experiment 2, the virus titer was affected only by time (P ,
0.0001) and vaccine serial (P , 0.0001) but not for diluent or lab
(data not shown). Titer means for the significant main effects of
serial and time are presented in Table 2. Titer was reduced
significantly from the initial value after 30 min of incubation time
(Table 2) and titer was less in serial 010 than in serial 011 (Table 2).
A significant interaction was observed for time by serial (P ,
0.0001) indicating that the amount of virus reduction over time was
dependent on the vaccine serial tested. Virus titer declined more
rapidly in serial 010 as compared to serial 011 as incubation time
increased. After 24 hr storage, 1 log10 and 2 log10 virus reduction
were observed in serials 011 and 010, respectively (Table 2). The
viability of the virus was similar in the presence or absence of the dye
or the stabilizer. The dye and the stabilizer were not toxic to Vero
cells (data not shown).
DISCUSSION
The virus in freeze-dried, live attenuated vaccines is usually stable
for several months if stored properly at refrigeration temperature.
Once reconstituted in the diluent, however, the virus titers begin to
decrease. Therefore, reconstituted vaccine should be used within
a short period of time. PneumomuneTM is recommended for
vaccination of healthy turkeys at 1 wk of age or older using the
drinking water, intraocular, or spray route of administration. The
manufacturer recommends that the vaccine should be administered
to the birds within 1 hr of reconstitution. In practice, however, the
turkey producers may not adhere strictly to these guidelines.
Temperature was selected as one of the study variables because
high temperature in turkey barns may be deleterious to virus
survival. The temperature in turkey barns depends on the type of
housing and on the age of the turkeys housed. In general, brooding
temperature (in the first week of life) could range from 32 C to
35 C (3). For birds at 6 wk of age, an average temperature of 21 C is
desired and for older turkeys the temperature should be maintained
within a range of 10–24 C (1,3). Because initial vaccination may
occur at warm barn temperatures, we evaluated the viability of
PneumomuneTM at three different temperatures. The temperature
was found to significantly affect the viability of the vaccine virus,
which is in agreement with the observation of Townsend et al. (15).
The titer of reconstituted PneumomuneTM was stable for at least
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