Ovine listeriosis: development of novel serological assays for diagnosis, experimental validation and field investigations into the epidemiology of infection
A panel of anti-Listeria monoclonal antibodies (mAbs) were produced following the immunization of mice with
heat-killed Listeria monocytogenes serovar 4b. Five of the mAbs recognised an antigen irregularly present in a
number of species and serovars of the genera Listeria and Bacillus. The antigen spontaneously adhered to sheep
erythrocytes and was putatively identified as lipoteichoic acid (LTA). A competitive sandwich ELISA was
developed which revealed that three of the mAbs recognised the same or overlapping antigenic epitopes.A protein of 58,000 Da molecular mass was purified from the supernatant fluid of a dialysis sac culture of L.
monocytogenes serovar 4b by cation exchange chromatography. The purified protein, homogeneous on SDSPAGE,
was identified as listeriolysin O (LLO) and used to develop an indirect ELISA for the measurement of
anti-LLO antibodies in sheep sera.Oral dosing of lambs with L. monocytogenes serovar 4b daily for three days produced no clinical signs but
conferred protection against bacteraemia following subsequent homologous subcutaneous challenges. Signs of
systemic illness were unremarkable after the subcutaneous injections but between two and 61 days later
neurological symptoms developed in six lambs. Histopathological lesions of listeric encephalomyelitis were
demonstrated and it seems probable these cases developed as the result of infection ascending via the spinal
nerves.Antibodies to whole cell antigens were detected in the sera of challenged animals using serum agglutination tests
and ELISA, and antibodies to LLO were detectable by immunoblotting and indirect ELISA. The subclass of
antibody involved in the anti-LLO response was shown to be predominantly IgG. However, the competitive
sandwich ELISA for the detection of anti-LTA antibodies was an unreliable indicator of infection. Seroconversion
after oral dosing was apparently a consequence of host invasion since no antibody responses were detected when
lambs were dosed with heat-killed L. monocytogenes. After oral dosing with viable L. innocua no antibody
response to L. monocytogenes somatic antigens could be recognised.In field studies the detection of anti-LLO antibodies by ELISA was shown to be useful for diagnosis of both
septicaemic and abortion forms of listeriosis. However, anti-LLO antibodies were detected in clinically normal
ewes from silage fed flocks suggesting that animals may be exposed to infection yet remain clinically normal as is
consistent with the experimental studies. In confirmed cases of listeric encephalitis it was impracticable to
diagnose the condition by the measurement of anti-LLO antibody titres or by the detection of LLO in CSF
samples. Although unsuitable for the diagnosis of this form of infection the indirect ELISA and the measurement
of anti-LLO antibodies may be useful in clarifying the pathogenesis of listeric encephalitis and the circumstances in
which listeriosis occurs in sheep flocks.In an examination of Listeria isolates from clinical cases of listeriosis nine isolates, all recovered from abortion
cases, were identified as L. ivanovii and the remaining 108 as L. monocytogenes, with serovar l/2a predominating.
The majority of isolates were from cases of encephalitis and serovar l/2b strains were confined to this form of
disease
