Geeta Devi Leishangthem

Histopathological and immunohistochemical diagnosis of infectious bursal disease in poultry birds

Aim: The aim of the present study was to diagnose infectious bursal disease (IBD) using gross, histopathological, and immunopathological approaches and to compare efficacy of immunohistochemical techniques with conventional diagnostic techniques. Materials and Methods: A total of 33 samples were collected from the six different poultry farms from Ludhiana and the nearby districts. Upon gross analysis of the necropsied birds, the relevant tissue samples such as bursa, kidney, junction of proventriculus and gizzard, heart, and muscles were then processed for histopathological and immunohistochemical studies. Results: Varied macroscopic changes were noted in bursa, characterized as swollen, hemorrhages to atrophy in size. Nonetheless, hemorrhages over thigh muscles were rarely seen. Histologically, the bursa showed prominent fibrotic and atrophic changes. Rarefaction of bursal follicles with intermittent infiltration of lympho-mononuclear cells with chronic cystic changes was additional changes, considered to be paramount for IBD. Expression and localization of IBD specific viral antigens were noticed mainly intracellular to the rarefied areas of bursal follicle section(s), in conjunction to inner lining of the cystic cavities of affected follicles. In addition, the junction of proventriculus and gizzard, the heart muscle, respiratory ciliated epithelium, and proventriculus also revealed positive expression to IBD virus (IBDV) antigen. Advanced immunopathological techniques, i.e., immunofluorescence further testified the evidence of antigen as positive green signal within affected follicles. Further consideration to the reliability of various techniques employed, positive correlation (r=0.64623) was emerged out with conventional pathological scoring. Conclusion: It is concluded that the bursa acts as an organ of choice for demonstrating IBDV antigen for specific diagnosis of disease using immunohistochemistry (IHC), and IHC staining is a precise, specific, rapid, and reliable method to demonstrate the IBDV antigen in the altered tissues due to IBDV infection.

Detection of Mycobacterium tuberculosis and Mycobacterium bovis in Sahiwal cattle from an organized farm using ante-mortem techniques.

Aim: The aim of this study was to investigate the prevalence of bovine tuberculosis (TB) and detection of Mycobacterium bovis in cattle from an organized dairy farm. Materials and Methods: A total of 121 animals (93 females and 28 males) of 1 year and above were studied for the prevalence of bovine TB using single intradermal comparative cervical tuberculin (SICCT) test, bovine gamma-interferon (γ-IFN) enzyme immunoassay, and polymerase chain reactions (PCRs). Results: Out of total 121 animals, 17 (14.04%) animals were positive reactors to SICCT test while only one (0.82%) animal for γ-IFN assay. By PCR, Mycobacterium TB complex was detected in 19 (15.70%) animals out of which 4 (3.30%) animal were also positive for M. bovis. Conclusions: Diagnosis of bovine TB can be done in early stage in live animals with multiple approaches like skin test followed by a molecular technique like PCR which showed promising results.

Histopathological and immunohistochemical approaches for the diagnosis of Pasteurellosis in swine population of Punjab.

Aim: Infectious porcine bronchopneumonia, caused by Pasteurella multocida, is a widespread disease of major economic significance. Thus, the aim of the present study was to diagnose swine Pasteurellosis using gross, histopathological, and immunopathological approaches in the swine population of Punjab and to compare the efficacy of immunohistochemical (IHC) techniques with conventional diagnostic techniques. Materials and Methods: A total of 71 adult swine lung samples showing gross pneumonic changes were collected along with the associated lymph nodes to carry out the study. The collected samples were then processed for histopathological and IHC studies. Results: Out of the total 71 lung samples, 26 samples were found to be suspected for Pasteurellosis as per the microscopic changes observed, and out of these 26 samples, 16 cases were confirmed to be positive for Pasteurellosis by IHC. Varied macroscopic changes noted in lungs were pneumonic patches with consolidation of many lobes, congestion, and focal hemorrhages. Main lesions associated with lymph nodes were its enlargement and hemorrhages. Histologically, the lung showed fibrinous and suppurative bronchopneumonia, multifocal suppuration, thickening of septa with fibrin combined with cellular infiltration and edema. The higher IHC expression of P. multocida was seen in the bronchial epithelium besides in alveolar and bronchial exudate. Moreover, on comparing the histopathological and IHC scores which were calculated on the basis of characteristic microscopic lesions and number of antigen positive cells, respectively, a significant positive correlation (r=0.4234) was found. Conclusion: It was concluded that swine population of Punjab is having P. multocida infection. The gross and histopathological lesions can be helpful in the preliminary diagnosis of Pasteurellosis but needs to be supplemented by other immunodiagnostic tests. Moreover, IHC technique proved to be a specific, reliable, precise, and rapid technique to supplement these conventional methods of diagnosis for Pasteurellosis.

Localization of classical swine fever virus antigen in young piglets by immunohistochemistry

The present study investigated the diagnosis of classical swine fever in piglets of four to six months using gross lesions, histopathology and immunohistochemistry. Gross examination revealed hemorrhagic kidneys, lymph nodes and spleen along with button shaped ulcer in intestine. Histopathological examination revealed focal to diffuse haemorrhages in the red pulp of spleen with depletion of the lymphocytes and multiple ulcerative lesions with necrosis of the epithelial mucosa in large intestine. Immunohistochemically, the viral antigen was demonstrated in the bronchial epithelial cells, mononuclear cells in spleen, intestine and Purkinje cells of the brain.

Diagnosis of Newcastle disease in broiler by histopathology and immunohistochemistry

Newcastle disease (ND) is an OIE notifiable, highly contagious, generalized viral disease of poultry. It remains a constant threat to the poultry industry and is a limiting disease for poultry producers worldwide. The present study report a severe enteric form of ND virus infections in backyard poultry birds by using histopathology and immunohistochemistry. ND viral antigens were detected in the lesions of various organs. ND viral antigen were mainly localized in the cytoplasm and nuclei of necrotic cells of epithelium of proventriculus, gizzard and intestines, Kupffer cells and circulating monocytes in the liver, tracheal epithelial cells, bronchiolar and parabronchiolar epithelial cells and alveolar macrophages in the lungs. Thus, immunohistochemistry serve as rapid and reliable tools to study the tropism and distribution of ND viral antigen in the tissues of infected birds and hence its diagnosis.

Epidemiological and immunopathological studies on Porcine parvovirus infection in Punjab

Aim: The aim of this study was to get the first-hand knowledge about the seroprevalence of Porcine parvovirus (PPV) in Punjab and a diagnosis of PPV from abortion cases of swine using gross, histopathological, and immunohistopathological techniques to observe the tissue tropism of the virus strain. Materials and Methods: Tissue samples from the reproductive tract of pig (n=32), placental tissue (n=10), and aborted fetuses (n=18) were collected from Postmortem Hall of the Department of Veterinary Pathology, GADVASU, field outbreaks and from butcher houses in and around Ludhiana. These samples were processed for histopathological and immunohistochemical (IHC) studies. For seroprevalence study, 90 serum samples of different sex and age were collected from 15 swine farms of Punjab and were subjected to indirect enzyme linked immunosorbent assay using commercial kit. Results: Overall, seroprevalence of PPV was found to be 41.1%. Sex and age related difference in the prevalence was noted. In abortion cases grossly congested and emphysematous lungs, congested internal organs with fluid in abdominal cavity and congestion in brain, changes were noted in fetuses, while diffuse hemorrhages and edema was observed in placental tissue. Histopathologically, the most frequent fetal lesions in aborted fetuses were noted in lungs, liver, and brain. IHC staining revealed PPV antigens in sections of heart, liver, lung, spleen, brain, lymph node of fetuses, placenta, and uterus of sow. Gross, histopathological, and IHC examination of the samples confirmed 5 fetus, 2 placenta and 3 female reproductive samples positive for parvovirus infection. Conclusions: Seroprevalence results may serve as a support either in prevention or control of the disease. IHC is the sensitive technique for diagnosis of PPV associated with the reproductive tract of swine and was found to supplement the gross and histopathological alterations, respectively, associated with the disease.

Ascaridia galli induced ulcerative proventriculitis in a poultry bird.

Various possible causes of proventriculitis include virus, bacteria, fungus, protozoans, nematodes, biogenic amines and excessive copper sulphate. In the present case, parasites were found in the lumen of the proventriculus, gizzard and duodenum of a poultry bird. Characteristic features of the parasite were studied and confirmed as Ascaridia galli. An ulcerative proventriculitis evident as denuded superficial epithelium, sub-epithelial hemorrhages, infiltration of the inflammatory cells and fibrosis were seen at histopathology. Proventriculitis caused by A. galli has not been reported till date. Here, we report a case of ulcerative proventriculitis in a poultry bird caused by nematode, A. galli.

Diagnosis of Bovine Tuberculosis in Lactating Cattle and Buffaloes by Comparative Intradermal Tuberculin Test and Bovine Gamma-Interferon Immunoassay

Two hundred lactating animals (158 cattle and 42 buffaloes) of organized and unorganized farms were investigated for the bovine TB using comparative intradermal tuberculin test (CITT) and IFN-γ assay. CITT was performed using avian and bovine PPD and IFN-γ assay by Mycobacterium bovis gamma interferon test kit. Overall, 14.5% and 11.5% animals were found positive by CITT and IFN-γ assay, respectively. However, 22.5% animals were detected TB positive through combination of both the tests. So, both CITT and IFN-γ assay, when used together lead to more accurate screening for bovine TB in dairy herd.

Molecular and immunohistochemical detection of Mycobacterium bovis in formalin-fixed tissues from animals with spontaneous bovine tuberculosis

Bovine tuberculosis (TB) is an infectious and emerging but neglected disease of cattle, caused by Mycobacterium bovis. Diagnostic techniques including histopathology, acid fast staining, immunohistochemistry, polymerase chain reaction (PCR) and real-time PCR were compared for extent of accurate diagnosis of bovine TB. Study was conducted on 16 archival tissue samples collected from suspected cases of bovine tuberculosis. Histopathological studies revealed both well-organized and poorly-organized granulomas in the lungs and lymph nodes. Acid fast bacilli were detected by Ziehl Neelsens staining in 13 (81.25%) of the 16 suspected cases. Immunohistochemistry using specific polyclonal antibody against M. bovis detected mycobacterial antigen extracellularly in caseous areas as well as intracellularly in macrophages and giant cells. DNA was extracted from formalin-fixed, paraffin-embedded tissues and subjected to IS6110 PCR using T4/T5 and INS1/INS2 primers specific for Mycobacterium tuberculosis complex (MTC) with an expected band size of 123 bp and 245 bp, respectively. The IS6110 PCR assay was positive in 12 out of 16 cases (75%). IS6110 real-time PCR using IS6110_T primers confirmed 6 (37.5%) samples positive for M. bovis. The present investigation indicated that real-time PCR using IS6110_T primers had increased specificity over conventional IS6110 PCR assay and can be used for detecting specific M. bovis infection in animals suspected for bovine tuberculosis.