Optimizing Propagation of Putative Vaccine Strain of Haemorrhagic Enteritis Virus

Abstract

<p>The disease turkey haemorrhagic enteritis caused by the adenovirus, haemorrhagic enteritis virus (HEV), is controlled worldwide by either vaccination with commercial vaccines or crude homogenates of splenic material from turkeys infected with avirulent HEV. The virus is fastidious and the only cell line known to support efficient growth is a virus transformed lymphoblastoid line (MDTC-RP 19) which is not allowed into Australia due to quarantine restrictions. In addition, no specific pathogen free (SPF) turkeys are available in Australia for propagation in low disease risk turkeys. Australia does have an avirulent HEV vaccine candidate (HEV086), used in the past for controlled infection to limit the impact of HEV. The major goal of this thesis is to develop methods to propagate and titrate the infectivity of this vaccine candidate in Australia and to carry out a preliminary investigation into its safety and efficacy in turkey poults. </p> <p>Chapter 1 presents the review of literature of the thesis. Chapter 2 presents the results of three experiments investigating the ability of HEV to infect and replicate in SPF chickens, given previous findings that chickens can be successfully infected with HEV. These experiments showed that the virus replicates effectively in chickens with an optimum infection age of 9-14 days and a challenge dose of 107 HEV genome copies (GC). Both spleen and liver should be harvested for vaccine production which results in significantly lower recovery of virus than predicted by qPCR of the organs and their weights. There is significant scope for future optimisation of this process.</p> <p>Chapter 3 presents the results of four experiments investigating the ability of HEV to infect and replicate in SPF chicken embryos at 10 days of incubation. Infection via the allantoic sac proved most practical but recovery of HEV GC in allantoic fluid 7 days post infection was low, approximating the amount of virus inoculated. However, it was shown that inactive virus was cleared from embryos within 3 days so the virus detected at 7 days post infection represented live virus. Viral replication in SPF eggs proved insufficient to consider this as a method of viral propagation for vaccine production, but it was shown to be an effective method of titrating the infectivity of various HEV preparations, revealing that different preparations had very different infectivity for a given GC content, mostly reflecting loss of infectivity, but not GC during storage of samples at -22˚C.</p> <p>Chapter 4 presents the results of a single preliminary experiment in which a number of HEV strains or passages were administered to turkey poults to ascertain their infectivity and safety and for the putative vaccine HEV086 P1, the effects of administration at 3 different doses and two routes (oral and in drinking water). The profile of decline in maternal antibody directed against HEV out to 6 weeks of age was also determined. The results showed some possible mild adverse effects of the HEV vaccine although no clinical HE was induced. There was no difference between water and oral administration of vaccine. Passage of HEV in chickens did not affect the ability of the virus to infect and replicate in turkeys and the passaged virus appeared safe. HEV from splenic homogenates of two field samples did not induce clinical HE although turkeys infected with one field “strain” and observed marked diarrhea. The study confirmed that protective levels of HEV antibody are present out to 4 weeks of age in commercial poults, but the levels fall below protective levels in most birds at weeks 5 and 6. Vaccination should thus be delayed until later than 4 weeks to prevent interference from maternal antibodies.</p> <p>Chapter 5 provides a brief general discussion, summarises the practical applications and implications of the work and points to further work required.</p> <p>In summary the work of this thesis had developed practical methods for the production and titration of HEV vaccine in Australia in the absence of permissive cell lines or SPF turkeys. The putative vaccine appears to be safe for turkeys but confirmation is required in larger studies.</p>