MOLECULAR BASED DIAGNOSIS ALONG WITH HISTOMORPHOLOGICAL, IMMUNOHISTOCHEMICAL AND ULTRASTRUCTURAL STUDIES ON CLASSICAL SWINE FEVER VIRUS INFECTION IN PIGS
Loading...
Date
2013-07
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Assam Agricultural University, Khanapara, Guwahati
Abstract
Classical swine fever (CSF) is a fatal viral disease affecting pigs and wild boars causing severe economic loss mainly in countries with dense pig populations. The disease is prevalent in almost all pig producing states in the country, and more particularly in the north eastern states of India. Besides clinical signs, gross and histopathological examinations, a precise laboratory diagnosis is required to confirm and to take the early decision for control of the disease during large outbreaks.
In the present study, a total of six outbreaks were attended from January 2011 to January 2013. One hundred sixty-nine clinical and post mortem samples were collected from CSF suspected animals of which seventy eight samples were tested to detect E2 gene region of CSFV by nested RT-PCR and 5´ NTR in Real time RT-PCR. Nested RT-PCR showed 73% positivity and Real time RT-PCR showed 83% positivity in various clinical and post mortem samples. A total of 25 samples were tested for insitu detection of CSF glycoprotein 55(Gp 55) by Indirect fluorescence antibody tests(I-FAT) in cryosections as well as in paraffin embedded tissue sections and 18(72%) samples showed positivity in cryosections, and only 8(32%) samples showed positive result in paraffin sections. However, a total of 25 samples tested by Indirect immunoperoxidase test ( I-IPT) in paraffin sections, 18(72%)samples were found positive.
Haematological study revealed significant decrease in haemoglobin percent, TEC, PCV, TLC and platelet in the CSF affected animals. In DLC, significant decrease in lymphocyte percent was recorded along with relative increase in the granulocyte percent.
The clinical signs of classical swine fever were inactiveness, off fed, high rise of temperature, huddling, erythematous skin lesions, conjunctivitis and posterior weakness. The gross changes recorded were turkey egg kidney, enlarged and haemorrhagic lymph nodes and button ulcers in large intestine. Histopathological alteration recorded were interstitial and glomerular nephritis, lymphoid follicular depletion in all the lymphoid organs, perivascular cuffing, degeneration and necrosis of purkinje’s cells in the brain and ulcerative lesions and necrotic enteritis in large intestine.
Enzyme histochemical study revealed weak ATPase activity in the lymphoid organs indicating B cell depletion and weak nonspecific esterase activity in the lymphoid organs indicating T cell depletion in CSF affected animals.
The distribution of apoptotic cells in different lymphoid tissues were detected by TUNEL staining. There was significant increase in the number of apoptotic cells, mostly lymphocytes and smaller number of macrophages in the lymphoid follicles and interfollicular areas in lymph node, spleen, palatine tonsil and ileum.
Transmission electron microscopic study (TEM) revealed, thickening of glomerular basement membrane in the glomeruli, glomerular mesangial cell proliferation, shortening and swelling of foot processes along with spherical electron
dense virus particle within the dilated cytoplasmic vesicles of podocytes in the kidney. In the lymph node, TEM revealed proliferation and dilatation of rough endoplasmic reticulum cisternae and loss of cristae in the swollen mitochondria in macrophages. Lymphocyte apoptosis was observed in the lymphoid follicle characterized by condensation and margination of chromatin and fragmentation of lymphocyte nuclei and cytoplasm.
In the present study, nucleic acid based technique (RT-PCR) targeting E2 gene of CSFV in various clinical and post mortem samples showed a highly sensitive and satisfactory result indicated that RT PCR is a very reliable diagnostic tool with minimum time requirement for detection of CSFV during CSF outbreaks from antimortem as well as post mortem samples. However, monoclonal antibody based in-situ immunodiagnostic assays like indirect FAT and indirect IPT used in the present study could efficiently demonstrate CSFV antigen in different lymphoid and non lymphoid organs.
Description
Keywords
null