Growth and Titration of Haemorrhagic Enteritis Virus of Turkeys In Chick Embryos

Haemorrhagic enteritis virus (HEV) is an immunosuppressive adenovirus that causes haemorrhagic enteritis in young turkey poults with increased incidence of secondary bacterial infections, such as colibacillosis (Pierson and Fitzgerald, 2013). Worldwide live vaccines propagated in cell culture or turkeys (crude spleen homogenates) are used to prevent the disease. In Australia, there is currently no licensed HEV vaccine due to inability to import the only cell line known to support HEV propagation (RP19) and to the unavailability of specific pathogen free (SPF) turkeys. The use of a vaccine to confer appropriate flock immunity could decrease the incidence of colibacillosis associated with HEV infection in Australia (Gerber et al., 2017). The main goal of this study was to investigate the feasibility of propagating and titrating HEV in SPF chicken embryos.

A total of 308 SPF viable eggs was used. In experiment 1 the susceptibility of embryos to infection was studied. A total of 127 eggs at 10 days of embryonic age was inoculated with saline (sham-inoculated), non-heat-treated (live) or heat-inactivated (dead) HEV. Allantoic fluid was retrieved at 0, 1, 3, 5 and 7 days post-investigation (dpi) and tested for HEV DNA by a quantitative PCR (qPCR) (Shah et al., 2013). In experiment 2, five HEV stocks were titrated using 181 fertile eggs inoculated with a 10-fold dilution of HEV virus stocks with 5 replicates per dilution/virus.

Inoculation with HEV did not cause visible growth impairment nor lesions in the chicken embryos and chorioallantoic membrane. Overall, there was no difference in the postinoculation mortality rates among groups sham-inoculated (6/30, 20.0%) or inoculated with live (34/252, 13.4%) or dead (3/26, 6.9%) HEV (P = 0.58). The amount of virus recovered in allantoic fluid at 7 dpi of eggs inoculated with live HEV was similar to the inoculated dose, indicating that HEV propagation in chicken embryos is not efficient. Viral DNA was detected in all samples from eggs inoculated with live HEV while no HEV DNA was detected after 3 dpi in eggs inoculated with dead virus. This indicates that it is possible to differentiate between live and dead HEV from virus stocks using a combination of egg inoculation followed by qPCR of allantoic fluid at 7 dpi. Preliminary data from experiment 2 shows that this method is suitable for titration of HEV infectivity in stocks.

In conclusion, HEV infects chicken embryos with low levels of replication without causing gross pathology. The total virus recovery at 7 dpi is similar to the inoculated dose, making the process unsuitable for vaccine production. However, since DNA from heat-treated virus is cleared from chicken embryos within 3 dpi, this method is suitable for titrating HEV infectivity when combined with qPCR detection of viral nucleic acids as the endpoint measurement.