J.R. ROBERTS,
W. BALL
University of New England, Armidale NSW
Australia
In the trial reported here, egg production and egg quality response to challenge at 62 weeks with T-strain IBV were evaluated in 320 vaccinated birds. The birds were vaccinated at 4, 28 and 198 days with one of A3 (A) or VicS (V) vaccine strains to form four vaccination treatment groups AAA, AVA, VVV and VAV. Egg production declined significantly for three weeks post-exposure to T-strain IBV. Moderate negative effects of IBV challenge were found on shell quality measurements, but no deleterious effects were found on internal quality measurements. All birds were still partially immune to some of the egg quality effects of T-strain IBV when challenged late in lay.
Introduction
Since the earliest reports in laying flocks, infectious bronchitis virus (IBV) has been connected with declines in egg production and quality (Sevoian and Levine, 1957). The negative impacts on quality most commonly associated with IBV infection are a thinning of the albumen component, lightening of shell colour, reduced shell thickness, decrease in egg weight and increased incidence of abnormal shell formation (Sevoian and Levine, 1957; McDougall, 1968; Box et al., 1980; Wernery and Daivi, 1984). The importance of IBV to the commercial egg industry is clear, it has the potential to directly impact both egg production and quality.
Currently all pullets destined for commercial egg production in Australia are vaccinated against IBV. Only live attenuated vaccines are presently available for use in this country and the two most widely used vaccines are VicS and A3, both derived from local field strains (Ratanasethakul and Cumming, 1983). The administration protocol for IBV vaccines most commonly involves three vaccinations during the rearing phase, before point of lay. Some producers practice revaccination during the lay cycle. ELISA assessments of circulating IBV antibody titres are used in industry to varying degrees as indicators of immune status and to confirm field infections. Work both within Australia and in other countries has raised questions about the potential negative effects of administering a live attenuated vaccine to a flock in lay (Box, 1988; Sulaiman, et al., 2004). The duration of immunity after vaccination with live vaccines is not clearly understood, but is believed to decline over time from last vaccination (Alexander and Gough, 1978). Work evaluating the impact of vaccine protocols and challenge with IB during lay on a whole flock basis is very limited due to the high costs associated with such trials. Obtaining information about the impacts of vaccination protocol on subsequent egg quality and production, following IB challenge during lay, is fundamental to any future discussion about vaccination best practice.
The availability of only live attenuated vaccines and the potential for a decline in immunity with time, results in a conflict between providing the best level of protection in a way that is cost effective, both in terms of revaccination costs, and potential losses in production and product quality.
The work presented in this paper investigated whether vaccination during the rearing phase was sufficient to prevent decline in egg production and quality when birds were challenged with IBV late in the lay cycle and whether different rearing period vaccination protocols had a lasting impact on such a response.
Materials and methods
Three hundred and twenty pullets of four different commercial strains of birds were obtained at day old and reared together on litter in isolation sheds at the University of New England. All birds were vaccinated for IBV by coarse spray at day old and at 4 and 14 weeks of age, with one of two commercial live attenuated vaccines, VicS (serotype B, Ignatovic and McWaters, 1991) and A3 (serotype C, Ignatovic and McWaters, 1991). Four vaccination treatment groups were formed by the combination of the vaccine strains given at each age as shown in Table 1. The flock was also vaccinated for Marek's disease at day old and Avian Encephalomyelitis at 12 weeks of age (all vaccines, Fort Dodge Australia).
At 14 weeks of age, the hens were moved to conventional layer cages in two large isolated sheds, at a stocking rate of two birds per cage. At this time the birds were also lightly beak trimmed and divided into groups. Birds from each vaccination treatment were divided into groups of ten with four replicates of each treatment being housed in each shed. The flock was then transferred to a pre-layer diet before being maintained on a commercially formulated layer diet, with regular monitoring of egg production and egg quality.
At 62 weeks of age, the birds in one of the sheds (160 hens) were exposed to T-strain IBV (serotype C, Ignatovic and McWaters 1991) by eye drop of virus suspension 107.4EID50/mL) to one in every two birds (IB Challenge group), while the other shed was left unchallenged (Control group). This resulted in each bird within the IB Challenge group receiving an estimated dose of 2 x 105 EID50.
From three weeks before challenge, egg production was recorded daily for each replicate group and averaged over weekly intervals. For statistical analysis of the production data, the challenge period was divided into 3 time intervals, A: three weeks before exposure, B: three weeks immediately after exposure and C: three weeks following interval B.
To compare any inherent differences between the challenge and control groups, egg production was measured and eggs collected in the two groups four weeks prior to challenge. During the challenge trial, eggs were collected for analysis at weekly intervals for 6 weeks after challenge. At each collection, 10 eggs per replicate group (320 eggs per collection) were analysed. Shell quality parameters were assessed including deformation, breaking strength, reflectivity, egg weight, shell weight, percentage shell and shell thickness. Albumen height, Haugh units and yolk colour were assayed as indicators of internal egg quality (Technical Services and Supply, U.K.).
Heparinised blood samples were taken from 5 individually identified birds per treatment group immediately prior to challenge and at 3 weeks after challenge. Plasma samples were analysed using the TropBio® ELISA kit for IBV antibody titres.
Statistical analysis was carried out using ANOVA and Fisher's PLSD to establish significant differences between group means. (Stat View 5.0.1, SAS Institute Inc).
Results
a) Egg Production
The three weeks prior to challenge, interval A, was analysed separately for differences between vaccination groups unrelated to challenge. The results indicated that the VAV (81.1 ± 2.43), VVV (80.1 ± 1.75) and AVA (75.8 ± 2.35) groups were not different and the AAA (72.3 ± 2.27) and AVA groups were not significantly different (P < 0.05). The two groups which received VicS at day old had significantly higher production, immediately prior to challenge, than the AAA group but not the AVA group.
Over the six weeks following challenge with IBV (intervals B and C), comparison of the IB Challenge and Control birds was analysed. The birds infected with IBV had a significantly lower production (66.7 ± 1.34 eggs/hen/100 days) than the Control group (72.15 ± 1.15 eggs/hen/100 days) (P<0.01). For the same 6-week post-challenge collection period, the VAV vaccination group (75.6 ± 1.78) had significantly higher production than the VVV (69.5 ± 1.52), AVA (67.2 ± 2.08) and AAA (66.5 ± 1.57) groups, which were not different from one another (P<0.01).
Among the three time intervals, for the control and challenge groups combined, A (77.3 ± 1.15 eggs/hen/100 days) was significantly higher than the other two intervals B (70.3 ± 1.31 eggs/hen/100 days) and C (71.6 ± 1.15 eggs/hen/100 days), which were not different (P < 0.01).
The overall significantly lower production in the IB Challenge group was due almost entirely to the interaction between challenge and time interval (P <0.05). For the three weeks prior to challenge (interval A), production was not different between the Control and IB Challenge treatment groups (Figure 1). Over the three weeks following challenge (interval B), production declined slightly in the control group but dropped significantly in the IB Challenge group. During the subsequent three weeks (interval C), production was not significantly different between the Control and IB Challenge groups, although it was lower in both groups than during interval A.
Across the six weeks after challenge there was a significant interaction between the challenge and vaccination group treatments (P <0.05). In the VAV and AVA groups there was significantly higher production in the Control than in the IB Challenge groups in this post challenge period (Table 2).
b) Egg Quality
The baseline collection 4 weeks prior to challenge was to investigate inherent differences between the Control and IB Challenge groups prior to the challenge experiment. There were significant differences between the control and IB challenged groups for both breaking strength and percentage shell, prior and subsequent to challenge, as shown in Figures 2 and 3.
Four weeks before challenge there were significant differences between vaccination groups in breaking strength, albumen height and Haugh units (Table 3). Vaccination group AVA performed best of all groups for all three parameters, although group differences were not large.
The IB Challenge group had significantly lower egg weight, shell weight and yolk colour compared to the Control group for six weekly egg collections following challenge (Table 4). Further, the IB Challenge group had significantly higher shell reflectivity (Table 4), breaking strength and percentage shell (Figures 2 and 3).
Egg weight was significantly lower for the IB Challenged group in all vaccination treatment groups (Table 5). Shell colour was significantly darker (lower reflectivity) for the Control group only within vaccination group VAV (Table 5). Yolk colour was significantly paler for the IB Challenged group within vaccination groups AAA and VVV only (Table 5).
c) IBV Antibody titres
There was a significant interaction between time of blood collection and challenge group (P <0.01) for IBV antibody titres. The challenge treatment groups were not significantly different prior to challenge, while at 3 weeks post-challenge, the titres of IB challenged birds were significantly higher than the Control birds (P <0.01), which had not varied significantly between the two collections (Figure 4).
The increase in antibody titre of the IB Challenge group was due largely to the response in the vaccination groups that had received VicS at day old. These vaccination groups had lower titres prior to challenge and greater rises in antibody titre after challenge than the two groups that had received A3 at their first IBV vaccination (Figure 5). Only the VVV and VAV vaccination treatment groups showed significant increase in antibody titres after exposure.
Discussion
This experiment involved birds that had not been revaccinated beyond the rearing phase of the production cycle. The success of the experimental challenge at 62 weeks is shown by the rise in the IB Challenge group antibody titres after exposure. When these birds were exposed to T-strain infectious bronchitis virus, some deleterious effects on egg production and egg quality were recorded.
There was an obvious and significant decline in production over the challenge period, which was unrelated to the effects of the virus. These birds were reaching the end of their first lay period at this time and therefore a decline in the numbers of eggs produced would be expected (Nys, 1986). However, as well as the decline in production with age, there was a significant drop in the IB challenged birds during the three weeks immediately after challenge. The depression in production associated with IBV infection is consistent with the work of others (Sevoian and Levine, 1957; Jones and Jordan, 1972). The significant difference between the IB Challenge and Control groups for the VAV vaccination group across the whole trial period was due to the Control group from this vaccination treatment having a very high level of production for this stage of lay, 80.3%, and as such is perhaps a case of the control group being high rather than, or as well as, the T-strain group having declined in production. All the vaccination treatment groups had numerically lower production in the T-strain infected birds, but between-replicate variation was such, in the VVV and AAA groups, as to prevent statistically significant differences.
The baseline egg collection was carried out four weeks prior to challenge, rather than immediately before. The flock was in the later stages of lay when this trial was carried out, so at this point there are age related changes in egg and eggshell quality. This base-line collection was not included in the statistical analysis of the post-challenge egg collections as variations related to age, rather than response to challenge, would have influenced the findings. It can be seen from Figures 2 and 3 that the control and IB challenged groups followed very similar patterns over the progressive collections after one group was exposed to virus. Consequently any differences seen after challenge in shell breaking strength and percentage shell are due to inherent differences between the two groups of birds, which were present before challenge occurred.
Based on the one egg collection prior to challenge there appear to be some lasting effects of rearing phase vaccination protocol on egg quality measurements (Table 3). The vaccination treatment groups initiated with the A3 attenuated virus had better albumen quality and shell breaking strength. At this point in the lay cycle, 58 weeks of age, the egg quality would already be on the decline. Hence it may be that the treatment groups, which received A3 at day old, have persisted at higher quality for longer than the VicS at day old groups. These results suggest that use of A3 for vaccination at day old has potential benefits late in lay.
The significant effects of challenge on egg quality measures were on egg weight, shell reflectivity, shell weight and yolk colour. The egg weight of the IB Challenge group following challenge was significantly lower than that of the control group. A decrease in egg weight with IBV infection, along with a decline in production, was one of the first noted signs of the disease in naive birds in the field (Hill and Lorenz, 1956) and it appears to still occur in these vaccinated birds. There was a slight but significant decrease in the mean shell weight of the IB challenged group compared to the control group. This is probably in line with the decline in egg weight, but is too small to account for all of the variation seen in egg weight. The smaller egg weight in the challenged birds is possibly also contributed to by changes in the amount of fluid added to the egg during the formation of the albumen components. This would appear to agree with the work of others, which suggests that some IBV strains target the cells of the upper reproductive tract, and impair albumen formation (Butler et al., 1972; Davidson, 1986). However, this has yet to be established for T-strain IBV, traditionally viewed as a nephropathogenic strain (Ignatovic and McWaters, 1991). The shell colour of the IB challenged group was significantly paler than the control group over the six weekly collections following exposure of half the flock to the T-strain virus. A lightening of 2-3% reflectivity is unlikely to be detected by the naked eye or commercial grading equipment and consequently may not be of great practical importance to industry. Despite the very small magnitude of the shell lightening as measured by objective equipment in this case, it coincides with the widely reported subjective assessment of pale shells in association with IBV in fully susceptible birds (McDougall, 1968).
Yolk colour was highly variable between treatment and vaccination groups, with no clear pattern, although it would appear that the yolk colour of the IBV challenged birds became lighter suggesting that these birds were eating less of the pigment-containing feed. The reduction in feed intake with IB infection has been reported by others (Sevoian and Levine, 1957; Heath, 1970) but has not been linked directly to IBV effects, rather a depression in general health. Feed intake measurements were not carried out on the birds in this experiment.
The significant differences between vaccination treatment groups that were noted before challenge in shell breaking strength and albumen quality persisted throughout the post-challenge collections. There were some differences in the response to IB Challenge when compared to the Control group within the same vaccination group. However, there was no clear pattern emerging from the post-challenge results of any of the vaccination protocols, or vaccine strains, being superior in the face of T-strain challenge.
It was expected that a measurable difference would have been detected between the AAA and VVV groups related to the level of homologous protection, since A3 and T-strain are of the same serotype (Ignatovic and McWaters, 1991). However, S1 glycoprotein sequence analysis has been utilised to divide all Australian strains into two groups with VicS, A3 and T-strain all belonging to Group I (Sapats et al., 1996). Whatever the relationship between the two vaccine strains and the challenge virus used in the study, no vaccination protocol performed consistently better across all the parameters analysed.
The vaccination groups that received VicS at day old had a larger increase in antibody titre and lower titres prior to challenge. The antibody titre reached during the growth phase was not substantially different among vaccination groups (data not shown). This suggests that the circulating antibody titres of these initial VicS groups declined more between the last vaccination at 14 weeks and the time of challenge at 62 weeks of age. These differences in circulating antibody titre prior to challenge did not translate into clear differences in egg quality responses to T-strain infection.
When birds were challenged with T-strain at 62 weeks of age, there was a significant drop in egg production. There were also some modest negative impacts on egg size and shell colour. The internal quality deterioration routinely associated with IBV (Sevoian and Levine, 1957; Box et al., 1980; Wernery and Daivi, 1984) was not observed in this trial. Consequently it can be assumed that the birds retained some level of immunity between 14 and 62 weeks of age, an interval of 48 weeks. While being adequate to prevent major direct effects of IBV, this immunity was unable to prevent more generalised effects on production and shell quality. The magnitude of these production and quality effects must be considered in the context of commercial production realities and the cost of ongoing regular revaccination.
Acknowledgments
The Authors would like to acknowledge Australian Egg Corporation Ltd for financially supporting this work.
References
Alexander, D.J. and Gough, R.E. (1978). Research in Veterinary Science, 24: 228-233.
Box, P.G. (1988). Proceedings of the International Symposium on Infectious Bronchitis, Rauischholzausen, pp.289-297.
Box, P.G., Beresford, A.V. and Roberts, B. (1980). The Veterinary Record, 106: 264-268.
Butler, E.J., Curtis, M.J., Pearson, A.W. and McDougall, J.S. (1972). Journal of the Science of Food and Agriculture, 23:359-369.
Davidson, M.F. (1986). British Poultry Science, 27: 353-354.
Heath, B.H. (1970). Avian Diseases, 14: 95-106.
Hill, R.W. and Lorenz, F.W. (1956). Poultry Science, 35: 409-417.
Ignatovic, J. and McWaters, P.G. (1991). Journal of General Virology, 72: 2915-2922.
Jones, R.C. and Jordan, F.T.W. (1972). Research in Veterinary Science, 13: 52-60.
McDougall, J.S. (1968). The Veterinary Record, 83: 84-86.
Nys, Y. (1986). British Poultry Science, 27: 253-259.
Ratanasethakul, C. and Cumming, R.B. (1983). Australian Veterinary Journal, 60: 214-216.
Sapats, S., Wright, P.J. and Ignatovic, J. (1996). Proceedings of the Australian Poultry Science Symposium, Sydney, pp. 33-38.
Sevoian, M. and Levine, P.P. (1957). Avian Diseases, 1: 136-164.
Sulaiman, A., Roberts, J.R. and Ball, W. (2004). Proceedings of the Australian Poultry Science Symposium, Sydney, pp. 89-92.
Wernery, U. and Daivi, K. (1984). Deutsche Tierarztliche Wochenschrift
From Proceedings of the "17th Australian Poultry Science Symposium", New South Wales, Australia.
