Jaagsiekte sheep retrovirus (JSRV) is the aetiological agent of ovine pulmonary
adenocarcinoma (OPA), a contagious bronchioloalveolar carcinoma, which imposes
a serious economic burden on the sheep farming industry. This study evaluated the
immunological responses during experimentally induced JSRV infection and
tumorigenesis in conventionally housed and specific pathogen free (SPF) neonatal
lambs, and in adult field cases in the terminal stages of OPA.
This study identified, for the first time, changes in cellular function in the
peripheral blood ofJSRV-infected animals by measuring the in vitro
lymphoproliferative response to various mitogens. The presence ofJSRV did not
affect the level of response to phytohaemagglutinin (PHA) and pokeweed mitogen
(PWM) stimulation, but a reduced response to concanavalin A (ConA) was
demonstrated in the JSRV - infected animals. The reduced response to ConA was
detected prior to the diagnosis of clinical symptoms and was also evident in the
terminal stages of OPA. The presence ofJSRV also affected the mitogenicity of the
mannose- specific, monocotyledonous Narcissus pseudonarcissus lectin (NPA). The
level of proliferation was comparable between JSRV -infected and control lambs, but
post -stimulation phenotyping revealed an altered phenotypic profile, with elevated
numbers of T and B lymphocytes from JSRV -infected animals. Furthermore, the
addition of exogenous marnose completely inhibited NPA mitogenicity in control
but not in JSRV infected lambs.
It has been established that NPA possesses insecticidal properties, which
could potentially increase pest resistance in transgenic crops. During this study we
revealed that NPA mitogenicity is age -dependent in sheep, with no
lymphoproliferative response detected in adult animals. This research was extended
to include human subjects, and we have shown that NPA is slightly mitogenic for
adult lymphocytes but that mitogenicity is increased more than sevenfold for
lymphocytes purified from umbilical cord blood. These findings indicate possible
physiological implications as a result of introducing foreign lectins into human and
animal food sources.
The peripheral blood mononuclear cells (PBMC) phenotypic profile was
monitored during acute experimental JSRV infection, and also the immune status of
natural OPA cases, in the terminal stage of the disease, was assessed. The PBMC
were labeled using a panel of mouse monoclonal antibodies recognising sheep
PBMC subsets, and results determined by flow cytometry. Neutrophils were counted
also. The results confirmed the CD4 lymphocytopaenia and neutrophilia previously
demonstrated in the blood of terminal OPA field cases. In contrast, no PBMC subset
frequency alterations were detected, at any time in JSRV - infected lambs, which
could have indicated an immunological response to JSRV infection. Moreover, the
results showed that lymphocytopaenia and neutrophilia do not occur during
experimentally induced infection leading to OPA. Hence, they are not an early event
of tumorigenesis and may not be in direct response to JSRV infection but a
consequence of secondary infections encountered during declining health.
At necropsy, samples of lung and tumour were collected from JSRV -infected
and control SPF lambs for immunohistochemical analysis. A novel aldehyde -free,
zinc salts fixative (ZSF) originally produced for immunolabeling human samples
was adapted and developed for ovine tissues. Antigen- retrieval steps were omitted
with ZSF and immunolabeling of otherwise fixation- sensitive antigens was possible.
This method also preserved the cellular morphology and delicate architecture of the
lung tissue. No difference in the distribution of immune cells was identified in the
non -affected area of the OPA lung when compared with the uninfected lung.
Immunolabeling tumour samples revealed unexpectedly that very few tumour
infiltrating lymphocytes were present, located to the periphery of the tumour
nodules.