PATHOLOGY AND MOLECULAR DIAGNOSIS OF NECROTIC ENTERITIS IN CHICKEN
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Date
2019-07
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College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati
Abstract
The present research work was carried out with an aim to study the pathology
necrotic enteritis with isolation and molecular detection of C. perfringens and experimental
production of the NE in chicken to compare between the C. perfringens type A and C in
terms of hematological, biochemical and pathomorphological alterations. Total 320
numbers of samples based on different clinical signs/pathological conditions were
collected from 15 districts of Assam. Isolation and identification of C. perfringens was
done by cultural and morphological characteristics and confirmation was done by detection
of cpa gene of C. perfringens by PCR. In this study 20 (15.03%) intestinal content and 9
(4.81%) cloaca swabs were found to be positive for cpa gene of C. perfringens. Isolation of
bacteria from the samples collected during monsoon was found to be highest in
comparison to other seasons. Study showed around 80% of the total isolates of C.
perfringens was from the birds of 4-6 weeks of age. The C. perfringens isolated from the
enteritis samples were found to be highest. Total 29 samples were found to be positive for
cpa gene (324 bp) encoding for alpha gene and cpb gene (180 bp) encoding for beta gene
was detected in 11 isolates. The additional virulence toxin genes of C. perfringens like
TpeL and NetB were also detected. The gross lesions of NE in field condition revealed
haemorrhagic, eroded, detached dead mucosal tissues in intestine. Formations of
diphtheritic membrane, distention of intestine were also observed in intestine. Liver,
kidneys and lungs showed congestion, haemorrhage and focal areas of necrosis. Spleen and
Bursa of Fabricious in some birds was found to be moderately enlarged. The gross lesion
of brain was found to be limited to mild congestion of meningeal blood vessels.
Histopathology of NE in chickens revealed congestion of blood vessels in the lamina
propria and submucosa with vacuolation of epithelial cells of intestinal villi along with
necrosis making the villi broader and shorter. Different developmental stages of coccidia
were also seen in the mucosal epithelial cells. In other organs such as liver, kidneys, heart,
lungs, spleen, bursa of Fabricius and brain showed variable nature of histopathological
lesions like congestion, haemorrhage and focal areas of coagulative necrosis. Experimental
production of NE was done in chicken by infecting C. perfringens isolate Type A and type
C with and without coccidia in separate groups. The clinical signs shown by the
experimentally infected birds were diarrhoea, dehydration, depression, reluctance to move,
loss of appetite, ruffled feathers, drooping of wings and head and huddling. The clinical
pathology of experimental birds showed, significant decrease in TEC level, hemoglobin
(g/dl) level as well as in PCV (%) and significant increase in TLC in the birds of infected
group in comparison to the control. Serum ALT and AST both showed a significant
increase (P<0.01) and total protein showed a significantly decreased (P<0.05) level. The
gross lesion revealed congestion and haemorrhage and focal areas necrosis of mucosa of
intestine. Enlargement, congestion, haemorrhage with focal areas of necrosis of the liver
were common gross findings in all the experimentally infected groups. This might be due
to damage to RBC in entero-hepatic circulation by α toxin of Clostridium perfringens.
Kidneys, heart, lungs, spleen and Bursa of Fabricius revealed moderate degree of
congestion and haemorrhage. Variable degrees of vascular changes in terms of gross
lesions were observed in all most all the infected groups. The histopathological lesions
revealed developmental stages of coccidia (schizonts & merozoites) and infiltration of large no of mononuclear cells and few polymorhonuclear cells in intestine. The intestinal
villi have undergone necrosis and necrosed cells were sloughed off from the mucosa. Liver
revealed marked fatty change in hepatocytes, congestion in the sinusoids. Kidneys showed
focal areas of inter tubular congestion. Heart and lungs revealed focal areas of
mononuclear cell infiltration as well as congestion and haemorrhage. Spleen and Bursa of
Fabricius showed depletion of follicles and brain showed neuronal degeneration and
necrosis with neuronophagia. Based upon the clinical signs, gross and histopathology, the
present study revealed the groups infected by both coccidia and clostridial isolate showed
distinctly more pronounced qualitatively and quantitatively in terms of clinical signs and
pathological lesions. It has been also observed in this study that C. perfringens type A was
found to be more virulent in terms of pathogenesis and pathomorphology in comparison to
C. perfringens type C. TEM evaluation of experimentally infected birds showed disruption
of intercellular junctional complexes, formation of gaps between enterocytes and
delimitation of boundaries of individual enterocytes. Disintegration of nuclear material,
dilatation of endoplasmic reticulum, disruption of cristae of mitochondria, increase
intracytoplasmic vacuolizations and membrane bound vesicles were also prominent
ultrastructural alterations in this study. Data were subjected to statistical analysis and
analyzed by SAS System ('Local', X64_7PRO) using one way analysis of variance
(ANOVA). Means were presented as mean ± standard error (SE) and were compared by
the Duncan test at the 0.05 level of probability.