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Wäckerlin, Regula (2009): Untersuchungen zu Infektionsverlauf und Ausbildung einer protektiven Immunität im Haushuhn (Gallus gallus) nach in ovo Infektion mit einem apathogenen aviären Paramyxovirusisolat vom Serotyp 1. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

The vaccination against avian paramyxovirus of the serotype 1 (APMV-1), the highly contagious agent of Newcastle Disease, is usually conducted with live attenuated vaccine strains within the first two weeks after hatch. Multiple efforts have been made to develop an in ovo vaccine, yet none of the approaches proved suitable for an application in commercial poultry. The aim of the performed study was to evaluate the strain APMV-1 599, which had proven apathogenic for day-old chicks, for its progress of infection and the induction of protective immunity after inoculation of spf-chicken eggs. The inoculation of embryonated chicken eggs with 106 EID50 of the APMV-1 599 strain at incubation day 18 caused an infection in virtually all hatched chicks at a maintained hatch rate of 81,7%, which was not substantially lower than that the 60% hatch rate of a group of uninfected sentinel animals that hatched in the same incubator and the hatch rate of 95% of an uninfected and separately hatched control group. Although the hatch rate was not reduced, the animals that were infected in ovo with APMV-1 599 showed pronounced clinical signs during the first ten days after hatch. These manifested themselves in the form of respiratory symptoms, apathy, diarrhea and in sporadic cases central nervous impairments, which led to a mortality rate of 54,1% within the first eight days after hatch. Opposed to this, the affiliated sentinel animals showed neither distinct clinical signs nor a substantially elevated mortality rate despite the proven transmission of the virus by the animals of the in ovo infected goup. To determine the spread of the virus to numerous organs, a novel Real-Time RT-PCR protocol targeting an amplificate located at the 5’ end of the nucleoprotein gene was established in addition to the published and validated method targeting a sequence on the matrix protein gene (WISE et al, 2004). Due to low fluorescence levels and irregular fluorescence curves, six of the conventional nucleotides incorporated into the 5’ nuclease probe were replaced by “locked nucleic acid” (LNA) nucleotides. This led to a shortening of the probe from 25 to 17 bases and thus an improvement of probe hybridization. The newly developed assay showed a comparable performance to the previously established protocol. During the first eight days after hatch, viral RNA was detectable in multiple organs with a predilection for lung and cecum / cecal tonsil. Viral RNA could also be detected in twelve of the liver samples as well as nine of the brain samples. Following the period of clinical impairment, no viral RNA was detectable in the samples from days 14 and 21. Specific antibodies could be detected by hemagglutination inhibition test from day seven and from day 21 by commercial ELISA. Additionally, the evaluation of cellular immune response indicates the formation of t-memory cells in the in ovo infected group and the sentinel animals. One single inoculation of APMV-1 599 in ovo was sufficient to induce a humoral and cellular immune response to protect all in ovo infected and most of the sentinel animals upon challenge with velogenic NDV Herts (Weybridge 33 / 56) at day 21. Shedding of the challenge virus was not noted in the in ovo infected group, while three of the ten sampled sentinel animals as well as all the animals in the control group shed virus two and four days after challenge, respectively. Due to the visible clinical impairment of the chicks, an in ovo vaccination with APMV-1 599 in the applied mode is not to be considered. The performed experiments demonstrated a generalization of the infection and servere clinical symptoms compared to the vaccination of animals after hatch. These results are indicative of embryonic developmental processes within the last three days before hatch, which are essential for the immunologic defense of very young chicks. A complete understanding of these ongoing immunological processes would not only be of importance for the development of in ovo vaccines against Newcastle Disease, but also in regard to potential early infections with other infectious agents.