J. Appl. Poult. Res.
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J APPL POULT RES 2008. 17:278-282. doi:10.3382/japr.2007-00110
© 2008 Poultry Science Association
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Research Reports

Stabilization of Live Mycoplasma gallisepticum Vaccines During Vaccination with Second-Generation Spray-Vac Vaccine Stabilizer1

S. A. Leigh2, S. L. Branton and S. D. Collier

USDA, Agricultural Research Service, Mid-South Area Poultry Research Unit, Mississippi State, MS 39762

2 Corresponding author: spencer.leigh{at}ars.usda.gov


   SUMMARY
TOP
 SUMMARY
DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Dilution and application of live Mycoplasma gallisepticum vaccines without the use of vaccine-stabilizing compounds may lead to significant loss of vaccine viability and loss of vaccine efficacy. Vaccine viability may decrease because of osmotic lysis of the mycoplasma as well as the presence of free chlorine or other detrimental chemicals in the water. Second-generation Spray-Vac vaccine stabilizer was developed and shown to maintain live Mycoplasma gallisepticum vaccine viability during exposure to free chlorine while protecting from the other factors that appear to decrease vaccine survival in solution. Increased vaccine survival in solution should lead to increased survival of the vaccine during vaccination. Field trial results demonstrate that second-generation Spray-Vac vaccine stabilizer yields excellent results without the need for distilled water or other vaccine-stabilizing compounds.

Key Words: Mycoplasma gallisepticum • vaccine • stabilizer • Spray-Vac


   DESCRIPTION OF PROBLEM
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Mycoplasma gallisepticum (MG) causes chronic respiratory disease in chickens and infectious sinusitis in turkeys [1]. Three live MG vaccines are currently commercially available for use in layer chickens. One vaccine, containing strain ts-11, is supplied as a frozen, ready-to-use product [2]. The other 2 vaccines, Mycovac-L and FVAX-MG, are supplied as lyophilized pellets, and the manufacturers recommend rehydration in distilled or chlorine-free water before application [2, 3]. The manufacturers of FVAX-MG also recommend the addition of evaporated milk, an inexpensive stabilizer used for removal of free chlorine. However, the absence of free chlorine alone is insufficient to ensure mycoplasma survival. The use of distilled or nonchlorinated water for rehydrating and diluting MG vaccines is a concern because, unlike most bacteria, mycoplasmas lack a cell wall [4]. This leaves them susceptible to osmotic lysis when the vaccine is diluted in distilled water [5, 6]. Although MG is known to be more resistant to osmotic lysis than other mycoplasma species [4, 7], it is not as resistant as bacteria with cell walls. Previous work has demonstrated that the F-strain of MG is stable in distilled water for at least 2 h [8]. However, this previous work was performed before commercial live vaccines were available, and the results were accurate only to 1 log10 difference at best.

Application of MG vaccines is generally performed by spraying because of ease and cost factors [3, 9], and a stabilizer is commonly added during dilution to maintain vaccine viability. Phosphate-buffered saline has been shown to stabilize MG vaccines in water [10], but commercial PBS concentrates are not marketed for use in field conditions. Spray-Vac concentrate is marketed to stabilize live vaccines during spray administration. The objectives of this research were to evaluate whether first-generation Spray-Vac vaccine stabilizer could stabilize live attenuated MG vaccine viability at a level comparable to 1x PBS and to determine whether a reformulated second-generation product would provide effective stabilization when the first-generation product did not.


   MATERIALS AND METHODS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Vaccine Strains
Mycoplasma gallisepticum vaccines FVAX-MG [11], in 1,000-dose vials, and Mycovac-L [12], in 2,000-dose vials, were obtained from their respective commercial sources. The vaccines were stored at 4°C. The same respective vaccine serial lot for each vaccine was used for all experiments.

Conditions for Testing Salt Concentrations
All solutions were made with distilled, deionized water. Phosphate-buffered saline was made from a commercial powdered concentrate [13] at the manufacturer’s recommended concentration. First- and second-generation Spray-Vac concentrates [14] were diluted as recommended by the manufacturer [4 oz/gal (125 mL/4 L)]. Tap water was simulated by adding sodium hypochlorite to distilled, deionized water to a final concentration of 4 ppm of chlorine. The following solutions were tested in this work: PBS at the manufacturer’s recommended concentration (1x PBS), simulated tap water (Tap), first-generation Spray-Vac in simulated tap water (Tap + SV), first-generation Spray-Vac and 1x PBS in simulated tap water (Tap + SV + 1x PBS), second-generation Spray-Vac in simulated tap water (Tap + 2SV).

Vaccines were rehydrated by using 10 mL of distilled, deionized water per vial of vaccine. The rehydrated vaccine was then diluted into the individual solutions to be tested at a rate of 1 dose of vaccine per 800 µL. Experiments were performed at room temperature by using 3 independent replicates of each condition. At 15, 30, and 60 min postrehydration, 100-µL samples of each condition or vaccine tested were removed for enumeration of surviving mycoplasma.

Color Change Units and Statistics
Mycoplasma gallisepticum survival was measured by determining the number of color change units (CCU50) per milliliter. At each time point, 100 µL was removed from each sample and immediately diluted in Frey’s medium [15]. The CCU50 was determined as described previously [10]. Briefly, serial 10-fold dilutions were incubated at 37°C in a sealed microtiter plate. The CCU50 for each sample was calculated by the method of Reed and Muench [16]. The results are expressed as percent survival, determined by dividing the CCU50 obtained for each sample per time point by the average starting (t=0) CCU50 of the diluted vaccine. All data were analyzed with PROC ANOVA in SAS [17], and differences in the treatment means were separated by using Tukey’s honestly significant difference [17]. Differences were considered to be significant at P ≤ 0.05.

Field Testing
Half of the 10-wk-old Hy-Line W-36 pullets in a commercial pullet house containing 75,000 birds were vaccinated with FVAX-MG as described previously [3], with vaccine diluted in distilled water containing 0.1x PBS (as is standard practice at that location) and previously shown to yield results equivalent to 1x PBS up to 30 min of postrehydration [10]. The remaining half of the 75,000 pullets were vaccinated with FVAX-MG diluted in well water with the manufacturer-recommended 4 oz/gal of second-generation Spray-Vac concentrate added. The time required for vaccination was less than 20 min from the time that vaccine vials were opened until spraying was completed. Six weeks after vaccination, blood was collected from either 10 or 20 randomly selected pullets from each group. All serum samples were analyzed by serum plate agglutination (SPA) analysis as described previously [3], and results were reported on a scale of 0 to 4, where 0 is no clumps formed and 4 is very large clumps formed and most were in the periphery [18]. Serum plate agglutination scores were reported by the number of birds that were positive and the averaged SPA score for all the birds in a treatment group. Significance of differences was determined by using an unpaired 2-tailed t-test. Differences were considered to be significant if P ≤ 0.05.


   RESULTS AND DISCUSSION
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Spray-Vac vaccine stabilizer concentrate is recommended for stabilization of vaccines during spray application. However, the first-generation Spray-Vac product was only intended to neutralize free chlorine and other oxidizing compounds in the water. This product was tested to determine whether it could stabilize the viability of MG vaccines diluted in distilled water. Figure 1Go shows that both FVAX-MG and Mycovac-L rapidly lost viability in the presence of 4 ppm of chlorine (simulated tap water). The addition of first-generation Spray-Vac to simulated tap water protected MG vaccines from the effects of free chlorine. However, it did not provide the same level of stabilization as provided by 1x PBS and instead gave results similar to rehydration of the vaccines in distilled water [10]. Addition of 1x PBS to the first-generation Spray-Vac product provided the same level of vaccine survival as obtained by using 1x PBS in distilled water. We hypothesize that first-generation Spray-Vac neutralizes the effects of free chlorine but fails to prevent osmotic lysis. Addition of 1x PBS reduces the effects of osmotic stress, as evidenced by increased vaccine survival.


Figure 1
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  		Figure 1. Survival of Mycoplasma gallisepticum in vaccines FVAX-MG (A) and Mycovac-L (B) in PBS (1x PBS), simulated tap water (Tap), simulated tap water containing first-generation Spray-Vac vaccine stabilizer (Tap + SV), or simulated tap water containing PBS and first-generation Spray-Vac vaccine stabilizer (Tap + SV + 1x PBS). Bars with the same letter do not have significantly different means. Average SD for all means not approaching zero is 21.1 for FVAX-MG and 32.6 for Mycovac-L.

 
In working with Animal Science Products Inc., a new formulation, referred to as second-generation Spray-Vac, was developed. The second-generation stabilizer includes pH buffering and protection from osmotic stresses in addition to removal of free chlorine and other oxidizing compounds. A comparison of the protective effects of second-generation Spray-Vac in simulated tap water with PBS in distilled water showed that for both FVAX-MG and Mycovac-L, second-generation Spray-Vac provided protection from loss of vaccine viability equal to that obtained by using PBS in addition to providing protection from the free chlorine in solution (Figure 2Go).


Figure 2
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  		Figure 2. Survival of Mycoplasma gallisepticum in vaccines FVAX-MG (A) and Mycovac-L (B) in PBS (1x PBS) or simulated tap water containing second-generation Spray-Vac vaccine stabilizer (Tap + 2SV). Average SD for all means not approaching zero is 9.0 for FVAX-MG and 48.6 for Mycovac-L.

 
The results of the previous tests demonstrated that second-generation Spray-Vac concentrate provides protection for live MG vaccines rehydrated with tap water equivalent to that obtained by using PBS concentrate in distilled water, without the need for purchasing distilled water. However, those experiments addressed the survival of MG vaccines only in solution and not during spray application. Two live field trials were conducted to compare the 2 methods of vaccine stabilization on subsequent seroconversion. Results from both trials showed that all birds were 100% positive for MG by SPA analysis (Table 1Go). Averaged SPA scores demonstrated no gross difference in the immune response, with PBS-stabilized vaccine-treated birds having scores equivalent to those receiving the second-generation Spray-Vac stabilized vaccine (Table 1Go).


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  		Table 1. Field trial comparison of second-generation Spray-Vac vaccine stabilizer (2SV) and PBS
 
The results of this work demonstrate that removal of free chlorine from water is insufficient for maintaining live MG vaccine viability in solution. Because of the absence of a cell wall in mycoplasma, they are susceptible to osmotic lysis [6, 7]. Phosphate-buffered saline has been used to maintain the viability of MG vaccines in solution [10]. However, PBS alone requires the use of distilled water to ensure that no oxidizing compounds are present, which would lead to decreased viability. The results using second-generation Spray-Vac vaccine stabilizer demonstrate that the product is able to remove the deleterious effects of free chlorine and also stabilize the vaccine from the osmotic stresses of a hypotonic solution, providing results equivalent to PBS-stabilized vaccines during field application and removing the requirement for distilled water.


   CONCLUSIONS AND APPLICATIONS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES
 

  1. Second-generation Spray-Vac vaccine stabilizer increases the survival of live MG in vaccine solutions and provides protection from the harmful effects of oxidizing compounds, including free chlorine.
  2. Application of live MG vaccines stabilized with second-generation Spray-Vac vaccine stabilizer results in the same level of successful vaccination as obtained when using PBS as a stabilizer.


   ACKNOWLEDGMENTS
 
The authors thank David Ley and Todd Pharr for reviewing this manuscript, Clint Benoit for technical assistance, and William Dozier for assistance with statistical analysis.


   FOOTNOTES
 
1 Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. Back


   REFERENCES AND NOTES
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 

  1. Ley, D. H. 2003. Mycoplasma gallisepticum infection. Pages 722–744 in Diseases of Poultry. 11th ed. Y. M. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, L. R. McDou-gald, and D. E. Swayne. Iowa State Press, Ames.
  2. Bermudez, A. J., and B. Stewart-Brown. 2003. Disease prevention and diagnosis. Pages 17–55 in Diseases of Poultry. 11th ed. Y. M. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald, and D. E. Swayne. Iowa State Press, Ames.
  3. Branton, S. L., W. B. Roush, B. D. Lott, J. D. Evans, W. A. Dozier III, S. D. Collier, S. M. D. Bearson, B. L. Bearson, and G. T. Pharr. 2005. A self-propelled, constant-speed spray vaccinator for commercial layer chickens. Avian Dis. 49:147–151.[CrossRef][Web of Science][Medline]
  4. Razin, S. 1978. The mycoplasmas. Microbiol. Rev. 42:414–470.[Free Full Text]
  5. Razin, S. 1992. Mycoplasma taxonomy and ecology. Pages 3–22 in Mycoplasmas: Molecular Biology, and Pathogenesis. J. Maniloff, R. N. McElhaney, L. R. Finch, and J. B. Baseman. Am. Soc. Microbiol., Washington, DC.
  6. Wieslander, Å., M. J. Boyer, and H. Wróblewski. 1992. Membrane protein structure. Pages 93–112 in Myco-plasmas: Molecular Biology and Pathogenesis. J. Maniloff, R. N. McElhaney, L. R. Finch, and J. B. Baseman. Am. Soc. Microbiol., Washington, DC.
  7. Lim, P., B. B. Sears, and K. L. Klomparens. 1992. Membrane properties of a plant-pathogen mycoplasmalike organism. J. Bacteriol. 174:682–686.[Abstract/Free Full Text]
  8. Kleven, S. H. 1985. Stability of the F strain of Myco-plasma gallisepticum in various diluents at 4, 22, and 37°C. Avian Dis. 29:1266–1268.[CrossRef][Web of Science][Medline]
  9. Marangon, S., and L. Busani. 2006. The use of vaccination in poultry production. Rev. Sci. Technol. Off. Int. Epiz. 26:265–274.
  10. Leigh, S. A., J. D. Evans, S. L. Branton, and S. D. Collier. 2008. The effects of increasing sodium chloride concentration on Mycoplasma gallisepticum vaccine survival in solution. Avian Dis. 52:136–138.[CrossRef][Medline]
  11. Schering-Plough Animal Health, Omaha, NE.
  12. Intervet Inc., Millsboro, DE.
  13. Thermo Fisher Scientific, Waltham, MA.
  14. Animal Science Products Inc., Nacogdoches, TX.
  15. Frey, M. C., R. P. Hanson, and D. P. Anderson. 1968. A medium for the isolation of avian mycoplasma. Am. J. Vet. Res. 29:2164–2171.
  16. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27:493–497.
  17. SAS Institute. 2004. SAS User’s Guide. Statistics. Version 9.1 Edition. SAS Inst. Inc., Cary, NC.
  18. Intervet International. 2001. The serum plate agglutination test (SPA). Intervet International BV, Boxmeer, the Netherlands.




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