Effect Of Pullet Vaccination On Development And Longevity Of Immunity Part 2

4. Discussion

In the present study, we demonstrated that pullets serially administered live attenuated vaccines against IBV, NDV, and ILTV were protected against homologous challenge with IBV, NDV, or ILTV for at least 36 weeks, as determined by challenge virus detection, clinical signs, histopathology, and cryostasis at 5 days after challenge.

Additionally, our study showed that the age at vaccination and intervals between each vaccination did not interfere with the development of immunity to each virus and consequently protection against homologous challenges. We designed our vaccination protocol to represent a typical vaccination program for IBV, NDV, and ILTV in commercial pullets. With the knowledge that lives vaccine viruses can persist in flocks, it has been unclear, until now whether the immunity induced by live vaccines could be compromised because of viral interference, a phenomenon in which one replicating virus blocks the infection and/or replication of another virus [9,10]. 

Although the vaccines in the present study were administered at intervals of 2 or 4 weeks, it is feasible that the virus from a previous immunization was still present at the time of the subsequent vaccination. IBV vaccines have been detected in the respiratory tract up to 28 days post-vaccination [27], and IBV was isolated from tracheal and cloacal swabs collected at the point of lay and 19 weeks of age in hens that had been virus-negative for several weeks following recovery from inoculation at one day of age [4]. Fentie, et al. [28] reported that chickens vaccinated with NDV B1 shed the vaccine virus 14 days post-inoculation. In addition, a study by Hughes, et al. [29] demonstrated intermittent shedding in the trachea from ILTV-immunized chickens between 7 and 14 weeks post-vaccination.

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Few experimental studies of sequential virus infections have been published, and fewer yet have been considered in the context of poultry viral respiratory pathogens. Costa-Hurtado, et al. [30]demonstrated that chickens and turkeys serially infected with a mesogenic strain of NDV and highly pathogenic avian influenza virus (HPAIV) 3 days apart resulted in an initial decrease followed by a subsequent increase in replication of the second virus. In a subsequent study [11], the same group found that low pathogenic avian influenza virus given 3 days after a lentogenic strain of NDV did show viral interference whereas viral interference was not observed when the viruses were given simultaneously. We did not measure vaccine virus replication in the present study and, therefore, could not determine whether one vaccine virus compromised the infection and replication of a subsequent vaccine virus. 

However, our goal was to determine if chickens sequentially vaccinated with all three viruses were protected from viral replication and clinical signs following homologous challenge. Thus, regardless of viral interference, our data shows that immunity to individual vaccine viruses was not compromised following sequential administration of multiple live attenuated vaccines targeting different viral respiratory tract pathogens.

The detection of IBV RNA in the cecal tonsils of vaccinated/non-challenged birds at 20 and 24 WOA but not at subsequent times indicates that residual vaccine virus RNA remained in the cecal tonsils until at least 24 WOA, following the second IBV vaccination. IBV has been isolated from the cecal tonsils at 14 weeks post-infection and is known to persist for several months in various internal organs [4,31]. IBV RNA was also detected in non-challenged negative control HG at 24 WOA but was absent from all other tissues collected from negative controls, which displayed no clinical signs of IBV infection. It is not clear why we detected IBV RNA in samples from the HG in negative control birds but likely represents cross-contamination during the processing of the samples since IBV was not detected in any other tissue.

The tracheal histopathological lesions of deciliation in positive controls were consistent with previous reports of IBV-induced histopathology [32,33] and were further confirmed by the presence of cryostasis among positive controls. As expected, IBV-vaccinated/challenged birds were protected from cryostasis. The European Pharmacopoeia states that cryostasis can be used to evaluate IBV vaccine efficacy, in which a lack of cryostasis would indicate that the vaccine was efficacious [34]. Therefore, these observations further confirm that IBV-vaccinated birds were protected from homologous challenges.

The observation of robust IBV-specific serum IgG titers in vaccinated birds is consistent with previous studies showing that IBV infection stimulates a humoral response in chickens [35], but that circulating antibody titers do not correlate with resistance to infection [36]. Therefore, the presence of IBV-specific serum IgG titers indicates only that the bird has been exposed to a vaccine or challenge virus and should not be correlated with other measures of protection. The lack of significant titers in non-vaccinated/challenged birds can be explained by the early time of collection post-challenge. Orr-Burks, et al. [37] found that significant changes in IgG serum titer were not detected until 10 days post-inoculation.

Orr-Burks, et al. [37] also demonstrated a lack of significance in IBV-specific IgA titer in tears 5 days after both primary and secondary exposure to IBV, but IgA titer was significantly higher between 6 and 16 days after the primary exposure to IBV. In this study, IBV-specific IgA titers measured in tears at 5 dpc did not reveal consistent trends and may be explained by the early time of collection post-infection. It was beyond the scope of this study to collect samples after 5 dpc, but the production of IBV-specific IgA in tears at later time points was demonstrated in a different experiment in which tears were collected between 10 and 14 dpc. In that experiment, naïve chickens challenged with IBV showed a higher trend of specific IgA titer compared to non-challenged controls [38].

The decision to use the B1 vaccine for the NDV challenge was based on biosecurity regulations and the lack of appropriate biosafety level 3 facilities needed for a challenge with mesogenic or velogenic strains of NDV. Since we used a lentogenic strain of NDV as a challenge virus in our experimental design, protection was primarily based on significantly reduced or no virus detection at 5 days after the challenge, which is a measure of local immunity preventing virus infection and/or replication. Presumably, a bird protected from infection with a lentogenic strain of NDV would also show some level of protection from exposure to mesogenic or velogenic strains. Vaccinated birds at each sampling time had significantly lower or no challenge virus RNA compared to positive control groups, indicating that the vaccinated birds developed a local immune response and were indeed protected whereas the non-vaccinated positive controls were not. Because lentogenic NDV only causes a mild respiratory or enteric infection [3], it was not surprising that respiratory signs and histological changes were mild or absent despite the presence of viral RNA. We observed clinical signs of NDV infection only at 20 WOA in positive controls, while only a few birds showed mild clinical signs at 24 and 28 WOA, and no clinical signs of disease were observed at later challenge times. This observation is consistent with existing knowledge that ND tends to be more severe in younger birds [39].

The lack of cryostasis observed in NDV B1-infected positive controls contrasts with previous reports demonstrating that NDV caused cryostasis in tracheal explants. Butler, et al. [40] demonstrated that NDV caused ciliostasis within 2 to 6 days after infection of tracheal explants, and Malo, et al. [41] reported that following vaccination of one-day-old chicks with lentogenic NDV, the peak of cryostasis occurred at 5 and 7 dpi and waned by 13 dpi. The discrepancy between our results and the previous studies may be explained by the use of a different lentogenic NDV strain; B1 in our studies and LaSota in previous reports [41]. It is well established that the LaSota vaccine strain is more virulent than B1 [42], which may explain the iconostasis observed in LaSota-vaccinated chicks or LaSota-infected tracheal explants, whereas iconostasis was not observed in our study using B1 vaccine.

Robust NDV-specific circulating IgG antibody responses developed following NDV vaccination and stayed elevated, which was consistent with prior research indicating that antibodies may be detected for up to one year in birds immunized multiple times against NDV [39]. Except for 28 WOA, the IgG titers in non-vaccinated/challenged birds were not significantly increased compared to titers in negative controls. This observation is not surprising given that NDV-specific antibodies are not detected in the serum until 6–10 days after exposure [39].

For the ILTV-challenged birds, vaccination prevented challenge virus replication in the trachea and HG but did not completely block virus replication in the conjunctiva, especially at 32 and 36 WOA. This may suggest a waning of the local immunity against ILTV after the second vaccination at 16 WOA. In addition, the proximity of the conjunctiva to the inoculation site (eyedrop and intranasal) might explain why the local immune response was not able to completely block viral replication in the conjunctiva but successfully cleared the virus before it could replicate in HG and trachea.

Very few positive controls (non-vaccinated/challenged) demonstrated histological evidence of ILT infection in the trachea, though all positive controls tested positive for ILT DNA by PCR. This finding is not surprising in light of a report by Guy, et al. [43], which illustrated that histological detection of ILT is highly specific (98%) but poorly sensitive (42%). Notably, intranuclear inclusion bodies are present only during the initial infection (1–5 days) and disappear following epithelial cell necrosis and desquamation [44], which may explain the observation that by 5 dpc only 3/9 and 1/10 positive controls at 20 and 28 WOA, respectively, had histological evidence of ILT infection despite the presence of ILT DNA in the trachea.

The absence of cryostasis observed in both vaccinated and non-vaccinated, ILTV-challenged birds at 5 dpc was not surprising given that Butler, et al. [40] found that only some strains of ILTV caused cryostasis and did not correlate with virulence. Moreover, the authors showed that cryostasis rarely occurred before 6 days, and sometimes even 9 days, after inoculation. In addition, Gerganov and Surtmadzhiev [45] also demonstrated cryostasis in ILTV-infected tracheal organ cultures at 7–8 days post-infection. Therefore, our results combined with previous studies indicate that measuring iconostasis may not be a reliable marker of protection from ILTV infection and that cryostasis in ILTV infection studies may need to be evaluated at later times post-inoculation.

The lack of significant ILTV-specific serum IgG titers in non-vaccinated positive controls may be explained by the early time of collection post-challenge, as ILTV-specific antibodies do not become detectable until 5–7 dpi and peak at 21 dpi [46]. However, it is worth noting that antibody titers are not correlated to protection from ILTV infection [44]. IgG titers were robust in vaccinated birds throughout the study and support previous data that antibodies may be detectable for at least a year [44].

Taken together, our data indicate that a typical pullet vaccination program consisting of serially administered live attenuated vaccines against IBV, NDV, and ILTV does not interfere with immune responses to the individual vaccines, and the birds are adequately protected against homologous challenge until at least 36 WOA. This information is important because it shows that consecutively administered live attenuated vaccines against multiple respiratory pathogens can be an effective vaccination strategy for the development of protective immunity against each disease agent in long-lived birds.

Author Contributions:

Conceptualization, M.W.J.; Methodology, E.J.A., B.J.J., S.W.M., M.G., and M.W.J.; Formal analysis, E.J.A., B.J.J., S.W.M., and M.G.; Investigation, E.J.A., B.J.J., S.W.M., M.G. and M.W.J.; Resources, B.J.J. and M.W.J.; Data curation, E.J.A., B.J.J., S.W.M., M.G. and M.W.J.; Writing-original draft, E.J.A. and M.W.J.; Writing-review and editing, B.J.J., S.W.M., M.G. and M.W.J.; Supervision, B.J.J. and M.W.J.; Project administration, E.J.A.; Funding acquisition, M.W.J.

Funding:

This work was funded by the USDA, NIFA, and Poultry Respiratory Disease Coordinated Agricultural Project.

Conflicts of Interest:

The authors declare no conflict of interest.

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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.

Emily J. Aston 1, Brian J. Jordan 1,2, Susan M. Williams 1, Maricarmen García 1 and Mark W. Jackwood 1,*

1 Poultry Diagnostic and Research Center, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA; ejaston@ucdavis.edu (E.J.A.); brian89@uga.edu (B.J.J.); smwillia@uga.edu (S.M.W.); mcgarcia@uga.edu (M.G.) 2 Department of Poultry Science, College of Agricultural and Environmental Sciences, University of Georgia, Athens, GA 30602, USA.

Received: 9 January 2019; Accepted: 30 January 2019; Published: 2 February 2019

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