Untersuchungen zu Infektionsverlauf und Ausbildung einer protektiven Immunität im Haushuhn (Gallus gallus) nach in ovo Infektion mit einem apathogenen aviären Paramyxovirusisolat vom Serotyp 1

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.