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Logo of cjvetresCVMACanadian Journal of Veterinary ResearchSee also Canadian Journal of Comparative MedicineJournal Web siteHow to Submit
Can J Vet Res. Jan 2006; 70(1): 65–67.
PMCID: PMC1325097

Language: English | French

Duration of immunity engendered by a single dose of a cold-adapted strain of Avian pneumovirus

Abstract

The duration of immunity after a single dose of a cold-adapted strain of Avian pneumovirus (APV) was studied. Turkeys were vaccinated at 1 wk of age and challenged with virulent virus 3, 7, 10, and 14 wk later. Nonvaccinated groups were also challenged at the same times. No clinical signs were observed in the vaccinated birds after vaccination or after any challenge. No viral RNA was shed by the vaccinated birds after any challenge. The nonvaccinated birds shed viral RNA after all challenges. Avian pneumovirus-specific humoral antibodies were detected in the vaccinated birds until 14 wk after vaccination. The results of this preliminary study indicate that inoculation with a single dose of a cold-adapted strain of APV at 1 wk of age provides protection until 15 wk of age.

Résumé

La durée de l’immunité suite à l’administration d’une dose unique d’une souche adaptée au froid du pneumovirus aviaire (APV) a été étudiée. Des dindes ont été vaccinées à 1 semaine d’âge et une infection défi avec une souche virulente de virus effectuée 3, 7, 10 et 14 semaines plus tard. Des groupes non-vaccinés ont également été infectés au même moment. Chez les oiseaux vaccinés, aucun signe clinique n’a été observé après la vaccination ou après les infections. Aucun ARN viral n’a été excrété par les oiseaux vaccinés après les infections. Par contre, les oiseaux non-vaccinés ont excrétés de l’ARN viral après les infections. Des anticorps spécifiques à APV ont été détectés chez les oiseaux vaccinés jusqu’à 14 semaines après la vaccination. Les résultats de cette étude préliminaire indiquent que l’inoculation à une semaine d’âge avec une dose unique d’une souche adaptée au froid conférait une protection jusqu’à l’âge de 15 semaines.

(Traduit par Docteur Serge Messier)

Avian pneumovirus (APV) causes a serious respiratory disease in turkeys and heavy economic losses to the turkey industry (1). This enveloped, nonsegmented, negative-sense RNA virus belongs to the genus Metapneumovirus in the family Paramyxoviridae (2,3). The APV-associated disease in the United States (US) is similar to the turkey rhinotracheitis (TRT) observed in Europe since the mid-1980s (4,5). Secondary bacterial infections may complicate the disease and result in a mortality rate of up to 30% in turkey poults (6). It is now known that the European isolates of APV belong to subgroups A, B, and D and that the US isolates belong to subgroup C (79).

Since killed vaccines against APV were found not to be effective (10), the main focus in the US has been on the development of live, attenuated vaccines. Our laboratory has reported on the development of live, attenuated APV vaccines with 2 different approaches: serial propagation of APV in cell culture (11) and cold adaptation of APV (12). Experimentally, the cold-adapted strain protected 2-wk-old turkeys against virulent APV challenge (12). In this paper we report on the duration of immunity afforded by a single dose of coldadapted APV vaccine.

The APV strain APV/MN/turkey/1-a/97 (also called APV/MN-1a) was isolated from a respiratory disease outbreak in Minnesota (1) and used to develop the cold-adapted strain, as described earlier (12). Briefly, APV/MN-1a was blind-passaged in chicken embryo fibroblasts (7 passages) and Vero cells (34 passages). The virus, designated P41 (13), was developed as a cold-adapted strain through growth at successively lower temperatures (35°C, 33°C, and 31°C) for 8 passages each, until it was finally able to grow at 31°C (12). The cold-adapted virus was plaque-purified (to a titre of 106.3 plaque-forming units [pfu]/mL) before use in this study. A virulent strain of APV (APV/Mn-2a) at 13th cell culture passage in Vero cells (titre 104.3 tissue culture infective dose [TCID]50/mL) was used as the challenge virus.

We obtained 140 tom turkeys from a commercial APV-free hatchery. The birds were reared in animal housing facilities at the College of Veterinary Medicine, University of Minnesota, St. Paul, throughout the study. At 1 wk of age, 120 birds were randomly divided into 2 groups of 60 birds each (groups 1 and 2). To determine APV serostatus before the experiment, we took blood from the remaining 20 birds, which were then killed. The birds in group 1 were inoculated with 50 μL of vaccine in each eye and each nostril (200 μL/bird); thus, each bird received 3.9 × 105 pfu. The birds in group 2 were inoculated with mock-infected Vero cell culture fluid. At 3, 7, 10, and 14 wk after inoculation, 15 birds from each group were transferred to another 2 rooms and challenged by inoculation with the virulent virus (50 μL in each eye and nostril). Ten days after each challenge, blood was drawn and the birds were then killed.

The 3 main criteria for protective efficacy of the vaccine after challenge were absence of clinical signs in the vaccinated birds, lack of demonstrable APV-RNA in the vaccinated birds 5 d after challenge, and development of an anamnestic antibody response in the vaccinated birds 10 d after challenge.

After vaccination and after each challenge, the birds were monitored for the appearance of clinical signs. Clinical scores were assigned as described previously (11). At 5 d after vaccination, choanal swabs from all 120 birds in groups 1 and 2 were collected in Eagle’s minimum essential medium containing 1% fetal bovine serum and antibiotics (150 IU/mL of penicillin, 150 μg/mL of streptomycin, 50 μg/mL of neomycin, and 1 μg/mL of amphotericin B). The samples were pooled (15 samples in each pool) and examined by reverse transcription polymerase chain reaction (RT-PCR) for APV-RNA (14). In addition, 5 d after each challenge, choanal swabs were collected from the vaccinated and challenged birds (n = 15 at each time) and from the nonvaccinated and challenged birds (n = 15 at each time) and tested individually by RT-PCR for APV-RNA to determine if vaccination prevented the shedding of viral RNA. The APV-free status of the birds before the experiment was confirmed by testing serum samples from the 20 birds killed before the experiment. To determine seroconversion, we collected blood from the birds in groups 1 and 2 immediately before and 10 d after each challenge. All samples were tested by enzyme-linked immunosorbent assay (ELISA) (15); statistical analysis of the titres was performed using computer software (Systat, version 6.0; SAS Institute, Cary, North Carolina, USA).

No clinical signs were observed (clinical score = 0) after vaccination or after any challenge in any of the vaccinated birds. The nonvaccinated, nonchallenged birds were also free of clinical signs throughout the study (45, 30, and 15 birds at 4, 8, and 11 wk of age). After the challenge at 4 wk of age, 12 of the 15 nonvaccinated birds showed clinical signs (mean clinical score 14.8/bird within the 10-d observation period). Initially the birds appeared depressed, then unilateral or bilateral watery nasal discharge appeared; the discharge became thick and mucoid in some birds. Unilateral or bilateral swelling of infraorbital sinuses was also observed in some birds. The maximum number of sick birds was observed 5 d after challenge. After challenge at 8, 11, and 15 wk of age (7, 10, and 14 wk after mock vaccination), no clinical signs were observed in the nonvaccinated birds.

At 5 d after vaccination, all pools of choanal swabs from the vaccinated group were positive for viral RNA, whereas those from the mock-vaccinated group were negative. At 5 d after challenge at 4 wk of age (3 wk after vaccination), only 1 of the 15 birds in the vaccinated group was positive for APV-RNA, whereas all 15 birds in the nonvaccinated group were positive. After challenge at 8, 11, and 15 wk of age (7, 10, and 14 wk after vaccination), all birds in the vaccinated group were negative for APV-RNA, whereas all birds in the mock-vaccinated group were positive.

None of the 20 birds tested at 1 wk of age had APV antibodies. The antibody findings after vaccination and 10 d after each challenge are shown in Table I. At 3 wk after vaccination, 12 of the 15 vaccinated and challenged birds were positive for APV antibodies (geometric mean titre [GMT] = 61). At 7 wk and until 14 wk after vaccination, all birds in the vaccinated and challenged groups were positive, the GMT rising, declining at 10 wk, and then rising again. The difference between the GMTs at 10 and 14 wk after vaccination was statistically significant (P < 0.002), as was the difference in pre- and post-challenge titres between the vaccinated and nonvaccinated groups (P < 0.05). After challenge, the GMTs rose in all the groups, but they were lower in the nonvaccinated birds than in the vaccinated birds.

Table I
Seroconversion in turkeys after vaccination with a cold-adapted strain of Avian pneumovirus (APV) and after challenge with virulent APV

In the initial report on experimental evaluation of a cold-adapted strain of APV, no clinical signs were observed in turkey poults after vaccination (12). The same was true in this study. When the birds were challenged at 4 wk of age (3 wk after vaccination), the complete absence of clinical signs in the vaccinated birds as compared with signs in 12 of the 15 nonvaccinated birds further confirmed that the vaccine was protective at this age. Birds in the nonvaccinated group served as nonvaccinated, nonchallenged controls (45, 30, and 15 birds at 4, 8, and 11 wk of age). No clinical signs were observed in this group at any time. In addition, this group was free of humoral APV antibodies when tested immediately before challenge (Table I).

Since no clinical signs were observed in either vaccinated and challenged or nonvaccinated and nonchallenged birds at 8, 11, and 15 wk of age (7, 10, and 14 wk after vaccination), it was difficult to make a conclusion about the protective efficacy of the vaccine at these ages. The absence of clinical signs in older birds is not surprising, because age is a limiting factor in the production of consistent clinical signs of APV infection (12). Furthermore, the disease has been reported to be more severe under field conditions than under experimental conditions, probably because of secondary bacterial infections and close proximity of birds under field conditions (16).

In all pools of choanal swabs collected after vaccination but before challenge, RT-PCR detected APV-RNA. Although pools may test positive if even 1 swab is positive, individual swabs were not tested before challenge because the main focus of this study was not to determine if viral RNA is shed by vaccinated birds (11,12). After all challenges, the swabs from all the birds were tested individually. Further, we tested the pools at only a single point, 5 d after vaccination, because the largest amounts of both attenuated and virulent APV are recovered about 5 d after inoculation; subsequently, virus excretion is much reduced, and virus is completely cleared in about 10 d (17,18).

Because of inconsistent reproduction of clinical signs under experimental conditions, we used the lack of viral RNA shedding as 1 of the 3 indications of protection. The RT-PCR results suggest that the vaccinated birds were protected up to 15 wk of age (11,12). After challenge at 4, 8, 11, and 15 wk of age, all vaccinated birds were free of viral RNA 5 d after challenge (except for 1 bird at 4 wk of age), whereas all nonvaccinated birds were positive. These results are similar to those obtained with another live, attenuated vaccine strain (P63), which was developed by 63 passages of an APV isolate in cell culture and was found to be protective until 14 wk after vaccination (19). In the present study, we did not attempt to isolate and titrate live virus from birds after challenge because we have found the procedure to have low sensitivity (personal observation). Also, the virus is present in birds for only a short time after inoculation; hence, virus isolation is often difficult unless performed early (20).

Antibodies against APV were detected in 12 of 15 birds 3 wk after vaccination and lasted until 15 wk of age (Table I). However, the GMT was significantly lower at 11 wk of age than at 15 wk of age. The reason for this discrepancy is not known. All vaccinated birds showed a 2- to 3-fold increase in titre after challenge, which indicated an anamnestic response.

The results of this preliminary study indicate that this cold-adapted strain of APV provided protection until 14 wk after vaccination, as determined by the lack of viral RNA shedding in the vaccinated and challenged birds as compared with the nonvaccinated and challenged birds. However, these results are based on a single experimental trial and should be confirmed with larger samples to determine the application of these results in the field.

Acknowledgments

This study was supported in part by grants from the Rapid Agricultural Response Fund and the Minnesota Turkey Research and Promotion Council. The authors thank Claudia Munoz-Zanzi for her helpful suggestions.

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