The Role Of Vaccine Inoculation Routes On Protective Immunity Against Avian Pneumovirus

Devi P. Patnayak, BVSc, MVSc

Khalid Munir, DVM, MSc

Sagar M Goyal, BVSc, MVSc, PhD

 

Department of Veterinary Diagnostic Medicine

College of Veterinary Medicine

University of Minnesota

St. Paul, Minnesota

 

This study was supported in part by grants from Minnesota Turkey Research and Promotion Council and the Rapid Agricultural Response Fund.

 

KEY WORDS: Avian pneumovirus,
vaccine, immunity

Abstract: To determine the optimum route of vaccination for protection against avian pneumovirus, 1-week-old turkeys were inoculated with an experimental live vaccine via 3 different routes: aerosol, oculonasal, and oral. They were then challenged with virulent avian pneumovirus (APV) 2 weeks after vaccination. The birds vaccinated by any of the three routes did not show clinical signs after vaccination. The birds vaccinated via the oculonasal route did not develop clinical signs after challenge and did not shed the challenge virus. The birds vaccinated via the aerosol and the oral routes also did not develop clinical signs after the challenge. However, they did shed virus 5 days after challenge (53% birds in aerosol route and 13% in oral route). Low levels of antibodies were detected in birds inoculated with any of the 3 routes. These results indicate that this vaccine provides protection against APV when given by any of the three routes.

Introduction

Avian pneumovirus (APV) causes one of the major economically important diseases of the turkeys in the United States.1 The disease is caused by an RNA virus, which has recently been classified under genus matapneumovirus in the family Paramyxoviridae.2 The infection is characterized by couging, ocular and nasal discharge and swelling of infraorbital sinuses.3 The disease is similar to the one found in Europe, which was initially named as turkey rhinotracheitis4 and subsequently has been referred to as avian pneumovirus infection. In the United States, the disease was first reported in Colorado in late 19965 and subsequently in Minnesota.6 The disease continues to be a major problem in areas where it has been reported and causes high mortality in the presence of secondary bacterial infections.7

The control of APV in Europe has mainly been accomplished with the use of live and killed vaccines.8 The European APV isolates are divided into subgroups A and B9 while the US isolates belongs to subgroup C.10 We have developed a live vaccine against subgroup C by serially passing the virus cell cultures. The vaccine has been found to be protective under experimental and field conditions by the oculonasal route.11 In the present study, we compared 3 routes of vaccination (aerosol spray, oculonasal, and oral) in 1-week-old turkeys to determine the optimum route of vaccination.

Material and Methods

Turkeys

Seventy-five tom turkeys (BUT strain) were procured from a commercial APV-free hatchery on day of hatch. The birds were reared in the animal housing facility at the University of Minnesota throughout the course of the study.

Vaccine and Vaccination

The vaccine was prepared by serial propagation of a Minnesota isolate of APV (APV/MN/turkey/1-a/97) in Vero cells for 63 passages (designated as P63).11 The titer of the vaccine virus in Vero cells was 3.16 ¥ 106 TCID₅₀/mL.

At 1 week of age, the birds were divided into 5 groups (1 through 5) of 15 each. In the first 3 groups, the vaccine was administered by aerosol (spray), oculonasal, and oral routes, respectively. The remaining 2 groups were inoculated with mock-infected Vero cell culture fluid by the oculonasal route and served as nonvaccinated-challenged and nonvaccinated-nonchallenged groups, respectively. For aerosol administration, 2 mL of vaccine was mixed with 500 mL of dechlorinated water containing 1.25 g of nonfat dairy milk powder as a stabilizer. This vaccine was sprayed well over birds in group 1 using a hand-held garden sprayer. Birds in the second group were inoculated with 50 µL of vaccine in each eye and nostril (200 µL/bird). In the third group, 200 µL of the vaccine was deposited as a single dose in the mouth of each bird.

Challenge

At 2 weeks after vaccination, birds in groups 1 though 4 were challenged with virulent APV (thirteenth passage of APV/MN-2a). This virus had a titer of 1.9 x 104 TCID₅₀/mL. Each bird was challenged by inoculating 50 µl of this virus into each eye and nostril. Birds in group 5 were challenged with mock-infected Vero cell culture fluid.

Clinical Signs

The birds were monitored daily for the appearance of respiratory clinical signs after vaccination and after challenge. Clinical scores were calculated as reported previously.11 Briefly, scores were given for unilateral or bilateral nasal discharge, watery eyes, and sinus swelling. Severity of sinus swelling (moderate vs severe) was also noted.

Serology

All birds were bled before vaccination and 1 and 2 weeks after vaccination and 10 days after challenge. All sera were tested for APV antibodies using an ELISA test.12

Virus shedding

Five days after vaccination, choanal clefts of all birds were swabbed.11 The choanal swabs from each group of birds were pooled into 3 pools each and tested by reverse transcriptase polymerase chain reaction (RT-PCR)13 for the detection of viral RNA. Swabs collected at 5 days after challenge were tested individually for the detection of viral RNA by RT-PCR.

Results

Clinical Signs

Birds in all groups were free of clinical signs for up to 2 weeks after vaccination (Table 1). After challenge, none of the vaccinated birds showed clinical signs irrespective of route of vaccination. Sixty percent of birds in group 4 (nonvaccinated-challenged) showed clinical signs, with a mean clinical score of 6.5/bird (Table 1). All birds in the nonvaccinated-nonchallenged group remained free of clinical signs throughout the study.

Serology

The data on seroconversion are shown in Table 2. None of the birds, except one bird in the oculonasal group, showed evidence of APV antibody 1 week after vaccination. At 2 weeks after vaccination, some of the birds in the oculonasal and oral groups showed seroconversion, but none of the birds in the aerosol group was positive for antibodies. Ten days after challenge, birds in groups 1 through 4 showed evidence of seroconversion. All birds in the nonvaccinated-nonchallenged group remained negative for APV antibodies throughout the study.

RT-PCR

After vaccination, pools of swabs from birds in all 3 vaccinated groups were positive for viral RNA (Table 3), and pools from birds in groups 4 and 5 were negative. Five days after challenge, 15 of 15 birds in group 4 (nonvaccinated-challenged) and 0 of 15 in group 5 (nonvaccinated-nonchallenged) were positive for viral RNA (Table 3). Fifty-three and 13% of birds were positive for viral nucleic acid in the aerosol and oral groups, respectively.

Discussion

For a successful vaccination program, an effective vaccine administered by an appropriate route is necessary. It has been well documented that vaccination can protect birds against APV infection.11,14–18  For mass vaccination, it is important that vaccine administration is easy and that all birds receive an adequate dose of vaccine.8 In this respect, the aerosol and drinking water routes are methods of choice.

Many studies are available that have determined the effect of different routes of administration on immunity against avian diseases such as avian encephalomyelitis,19 infectious bursal disease,20 and infectious laryngotracheitis.21 Because such a study for APV vaccination is not available, the purpose of this study was to compare 3 different routes of vaccination against APV in 1-week-old turkeys. Because the oculonasal route has previously been shown to be an effective method for APV vaccination,11 we compared oculonasal, aerosol, and oral routes in the present study. The amount of vaccine administered by oculonasal and oral routes was exactly the same, and it was a little low in the aerosol route. It should be realized, however, that determining the exact amount of vaccine inhaled by each bird via aerosol route is difficult.

As was the case in the previous study,11 no clinical signs were seen for up to 2 weeks after vaccination in birds vaccinated via any of the 3 routes, indicating safety of the vaccine. Ten days after challenge, none of the birds in 3 vaccinated groups developed clinical signs, but 60% of the birds in the nonvaccinated-challenged group did. These results clearly indicate that all 3 routes of vaccination afforded protection against clinical APV. This is not surprising because in our earlier study with experimental evaluation of this vaccine, no clinical signs were produced in the vaccinated birds after challenge.11 The birds were not monitored for more than 10 days after challenge, and hence no conclusion can be drawn on the duration of immunity afforded by this vaccine.

The results of virus shedding indicated that 53% and 13% birds in the aerosol and oral groups, respectively, were shedding viral RNA at 5 days after challenge. At this point, it is not known if the virus being shed is the vaccine virus or the challenge virus. However, based on our earlier studies,11,22 it would seem that the challenge virus is being shed. Stoppage of virus shedding after challenge is one measure of protection. Although the birds were clinically protected using all 3 routes, virus shedding by a few birds in the oral and aerosol routes is of some concern. This would seem to indicate that although birds vaccinated with any of the 3 routes would be protected against clinical disease, they would shed virulent virus if they encounter a field challenge with virulent virus.

However, this may not be of major concern, however, because hopefully all birds in that flock would have been protected by vaccination. Additionally, after the marketing of these birds, the premises would be thoroughly cleaned and disinfected to avoid posing a danger to incoming birds. In any event, it seems that only 13% of the birds in the oral group shed virus. It is possible that these birds did not shed virus at 6 to 7 days after challenge, but we did not test at these time points.

Poor seroconversion seen in this study is not surprising. European studies have also indicated poor seroconversion following the use of this vaccine.11 It seems, therefore, that protection against APV is provided by low levels of hormonal antibodies, local or cellular immunity, or a combination thereof.11,15,23 That the live vaccine does prime the birds for antibody protection is shown by post challenge data on seroconversion, since average antibody titers were higher in the vaccinated-challenged group than in the nonvaccinated-challenged group.

References

1. Senne DA, Edson RK, Pederson JC, Panigrahy B: Avian pneumovirus update. Proceedings of the 134th Annual Meeting of the American Veterinary Medicine Association, Reno, NV, p. 190, 1997.

2. Anonymous: Turkey rhinotracheitis of unknown aetiology in England and Wales. Vet.Rec 117:653-654, 1985.

3. Kleven SH: Report of the committee on transmissible diseases of poultry and other avian species. In Proceeding of the US Animal Health Association, 101st Annual Meeting, Louisville, KY, pp. 473-491, 1997.

4. Anonymous: Turkey rhinotracheitis of unknown aetiology in England and Wales. Vet.Rec 117:653-654, 1985.

5. Kleven SH: Report of the committee on transmissible diseases of poultry and other avian species. In Proceeding of the US Animal Health Association, 101st Annual Meeting, Louisville, KY, pp. 473-491, 1997.

6. Goyal SM, Chiang S, Dar A, et al: Isolation of avian pneumovirus from an outbreak of respiratory illness in Minnesota turkeys. J Vet Diag Invest 12:116-168, 2001.

7. Kumar M: Avian pneumovirus infection Minnesota field perspective. In the Proceedings of the 50th Annual Meeting of the North Central Avian Disease Conference, Minneapolis, MN, pp. 18-20, 1999.

8. Cook JKA: Avian rhinotracheitis. Rev Sci Tech Off Int Epiz 19:602-613, 2000.

9. Patnayak DP, Sheikh AM, Gulati BR, Goyal SM: Experimental and field evaluation of a live vaccine against avian pneumovirus. Avian Pathol 31:377-382, 2002.

10. Chiang SJ, Dar AM, Goyal SM, et al: A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies. J Vet Diag Invest 12:381-384, 2000.

11. Patnayak DP, Sheikh AM, Gulati BR, Goyal SM: Experimental and field evaluation of a live vaccine against avian pneumovirus. Avian Pathol 31:377-382, 2002.

12. Chiang SJ, Dar AM, Goyal SM, et al: A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies. J Vet Diag Invest 12:381-384, 2000.

13. Shin HJ, Rajashekara G, Jirjis FF, et al: Specific detection of avian pneumovirus (APV) isolates by RT-PCR. Arch Virol 145:1239-1246, 2000.

14. Cook JKA, Ellis MM, Dolby CA, et al: A live attenuated turkey rhinotracheitis virus vaccine: 1. Stability of the attenuated strain. Avian Pathol18:511-522, 1989.

15. Cook JKA, Holmes HC, Finney PM, et al: A live attenuated turkey rhinotracheitis virus vaccine: 2. The use of the attenuated strain as an experimental vaccine. Avian Pathol 18:523-534, 1989.

16. Gulati BR, Patnayak DP, Sheikh AM, et al: Protective efficacy of high passage avian pneumovirus (APV/MN/turkey/1-a/97) in turkeys. Avian Dis 45:593-597, 2001.

17. Williams RA, Savage CE, Jones RC: Development of a live attenuated vaccine against turkey rhinotracheitis. Avian Pathol 20:45-55, 1991.

18. Williams RA, Savage CE, Worthington KJ, Jones RC: Further studies on the development of a live, attenuated vaccine against turkey rhinotracheitis. Avian Pathol 20:585-596, 1991.

19. Shafren DR, Tannock GA, Groves PJ: Antibody responses to avian encephalomyelitis virus vaccines when administered by different routes. Aust Vet J 69:272-275, 1992.

20. Kembi FA, Delano OO, Oyekunle MA: Effect of three different routes of administration on the immunogenicity of infectious bursal disease vaccine. Rev Elev Med Vet Pays Trop 48:33-35, 1995.

21. Fulton RM, Schrader DL, Will M: Effect of route of vaccination on the prevention of infectious laryngotracheitis in commercial egg-laying chickens. Avian Dis 44:8-16, 2000.

22.  Patnayak DP, Gulati BR, Sheikh MA, Goyal SM: Cold adapted avian pneumovirus for use as live, attenuated vaccine in turkeys. Accepted for publication.

23.  23.   Jones RC, Naylor CH, Al-Afaleq A, et al: Effect of cyclophosphamide immunosuppression on the immunity of turkeys to viral rhinotracheitis. Res Vet Sci 53:38-41, 1992.

 

Table 1. Clinical Signs In Turkeys Following Vaccination And Challenge*

 

                                   Birds showing clinical signs/Total no of birds

 

 

        1                              Aerosol                                               0/15 (0)                           0/15 (0)

        2                            Oculonasal                                             0/15 (0)                           0/15 (0)

        3                                 Oral                                                  0/15 (0)                           0/15 (0)

        4                              NV-Ch†                                               0/15 (0)                          9/15 (6.5)

        5                             NV-NCh‡                                              0/15 (0)                           0/15 (0)

 

  *Groups 1 through 3 were vaccinated by the indicated routes. Group 4 and 5 were inoculated with mock-infected cell culture fluid by the oculonasal route. Birds in groups 1 through 4 were challenged with virulent virus 2 weeks after vaccination. Birds in group 5 were challenged with mock-infected Vero cell culture fluid.

  †Nonvaccinated-challenged.

  ‡Nonvaccinated-nonchallenged.   

 

 

Table 2. Seroconversion After Vaccination and Challenge*

 

 

                                                                               1-week                     2-weeks                    10-days

 

        1                             Aerosol                            0/15 (10)                0/15 (10)                  6/15 (19)

        2                          Oculonasal                         1/15 (11)                6/15 (17)                 11/15 (35)

        3                                Oral                               0/15 (10)                3/15 (14)                  8/15 (23)

        4                             NV-Ch†                            0/15 (10)                0/15 (10)                  3/15 (24)

        5                            NV-NCh‡                           0/15 (10)                0/15 (10)                  0/15 (10)

                                              

  *Groups 1 through 3 were vaccinated by the indicated routes. Group 4 and 5 were inoculated with mock-infected cell culture fluid by the oculonasal route. Birds in groups 1 through 4 were challenged with virulent virus 2 weeks after vaccination. Birds in group 5 were challenged with mock-infected Vero cell culture fluid.

  †Nonvaccinated-challenged.

  ‡Nonvaccinated-nonchallenged.

  A geometric mean titer (GMT) of 10 or less was considered to be negative for APV antibodies.

 

 

Table 3. Detection of Viral RNA After Vaccination and After Challenge by RT-PCR

 

                  Pools of choanal swabs positive Choanal swabs positive

                     5 days after vaccination  5 days after challenge

 

  1                        Aerosol                                  3/3                                                   8/15

  2                      Oculonasal                               3/3                                                   0/15

  3                           Oral                                     3/3                                                   2/15

  4                        NV-Ch†                                  0/3                                                  15/15

  5                       NV-NCh‡                                 0/3                                                   0/15

 

  †Nonvaccinated-challenged.

  ‡Nonvaccinated-nonchallenged.