Molecular detection and patho-morphological study of enteric Escherichia coli pathotypes in diarrheic neonatal calves.

To understand the pathology of natural cases of E. coli pathotypes infection in bovine calves, 45 cases of bovine calves, below one month of age, died due to enteritis were studied. Total seventeen cases (37.77%) turned positive for different pathotypes of E. coli by RT-PCR. Out of seventeen positive samples for E. coli, six cases (35.29%) were positive for eae gene, three cases (17.64%) for bfp gene and eight cases (47.05) for fimA gene of E. coli. Gross lesions in these cases showed pin-point to ecchymotic hemorrhages in the mucosa of jejunum, ileum and colon. The draining mesenteric lymph nodes were swollen, enlarged and showed cord -like structure. Histopathology of small intestine showed, villi lining cells were sloughed off, tips of villi capillary plexus were congested and hemorrhagic, and skipping lesions of microabscesses in the crypts of mucosa were observed. In the duodenum, necrosis of crypts and infiltration of mononuclear cells in the lamina propria and around Brunner's gland. In mesenteric lymph nodes the subscapular space were infiltrated with mononuclear cells with depletion of lymphoid follicles in cortical area. Peri-trabecular and medullary sinuses of mesenteric lymph nodes were necrosed.

Pathology and molecular characterization of chicken infectious anemia virus and in silico antigen prediction.

The present study investigated five poultry flocks (size 142-600 birds) suspected of chicken infectious anemia (CIA) from Maharashtra, India. The necropsy of dead birds revealed severe atrophy of the thymus, gelatinization of bone marrow, subcutaneous hemorrhages, growth impairment, and severe anemia. Specific PCR targeting, 1390 bp fragment of the CIAV, VP1 gene was used in this study. Sequence analysis revealed that CIAV sequences of this study were grouped in genotype A. At the nucleotide level identity of 99.6% or more was seen between field sequences. At the amino acid level identity of 100% was seen between field sequences and NGP-1. Also, VP1 protein sequences of this study showed high identity with TJBD40, GD-K-12 strains from China and AB046590 strain from Japan. Further, the protein sequences of field CIAV had 0.7% to 2.5% divergence from VP1 sequences of vaccine strains. Antigenic epitopes of VP1 protein were predicted by SVMTriPtool and the field CIAV presented substitutions in two epitopes. To conclude, present study confirms the circulation of genotype A of CIAV in Maharashtra, India and predicted VP1 proteins of field CIAV revealed changes in two epitopes compared to vaccine strains.

Patho-morphological and immunohistochemical diagnosis of fibrous tissue pulmonary hamartoma in young buffaloes.

Hamartoma is a developmental disorder characterized by the presence of well-differentiated tissue, of multiple types, in excess at its normal location. The present study describes a fibrous tissue pulmonary hamartoma in two buffaloes. Grossly, there were large pale white masses present on the left diaphragmatic lobe in both cases. Microscopic examination of the lungs revealed that the bronchioles were dilated and showed many terminal respiratory bronchioles lined by a single layer of cuboidal epithelium supported by thick bands of collagenous tissue. Alveoli were distorted and separated by wide bands of connective tissues. Interalveolar septa were greatly expanded by collagen fiber proliferation and subsequent fibrosis. Fibrous connective tissues were demonstrated by immunohistochemistry (IHC) and Masson's Trichrome staining (MTS). Based on gross and histological examination, coupled with IHC and MTS, it was possible to confirm a case of hamartoma, which is rarely reported worldwide and is the first reported case from India.

Immunofluorescence and molecular diagnosis of bovine respiratory syncytial virus and bovine parainfluenza virus in the naturally infected young cattle and buffaloes from India.

Pneumonia in bovines is a multifactorial disease manifestation leading to heavy economic losses. Infections of bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus-3 (BPI-3) are among the important contributing factors for the development of pneumonia in young animals. These viral agents either primarily cause pneumonia or predispose animals to the development of pneumonia. Although, the role of BRSV and BPI-3 in the pathogenesis of pneumonia is well established, there are no reports of involvement of BRSV and BPI-3 from Indian cattle and buffaloes suffering from pneumonia. In the present investigation, we performed postmortem examinations of 406 cattle and buffaloes, which were below twelve months of age. Out of 406 cases, twelve (2.95%) cases were positive for BRSV and fifteen (3.69%) cases were positive for BPI-3, screened by reverse transcriptase polymerase chain reaction (RT-PCR). Further, positive cases were confirmed by sequence analysis of RT-PCR amplicons and direct immunofluorescence antibody test (d-FAT) in paraffin-embedded lung tissue sections. BRSV positive cases revealed characteristic findings of bronchiolar epithelial necrosis, thickened alveolar septa by mononuclear cells infiltration and edema; alveolar lumens were filled with mononuclear cells and numerous syncytial cells were seen having intracytoplasmic inclusions. The BRSV antigen distribution was found to be in bronchiolar and alveolar epithelium and syncytial cells in the lung sections. In fifteen cases, where BPI-3 was detected, bronchointerstitial pneumonia in the majority of cases with thickened alveolar septa by mild macrophage infiltration, hyperplasia of type-II pneumocytes and bronchiolar necrosis along with syncytial cells having intracytoplasmic inclusions in the majority of cases were observed. The BPI-3 antigen distribution was found to be in bronchiolar and alveolar epithelium and syncytial cells in the lung sections. RT-PCR amplicons of BRSV and BPI-3 obtained were sequenced and their analysis showed homology with already available sequences in the NCBI database. It is the first report of detection of BRSV and BPI-3 from pneumonic cases by RT-PCR and d-FAT from cattle and buffaloes of India, indicating the need for more epidemiological studies.

Immunohistochemical and molecular detection of natural cases of bovine rotavirus and coronavirus infection causing enteritis in dairy calves.

Bovine rotavirus (BRoV) and bovine coronavirus (BCoV) are major enteric viral pathogens responsible for calve diarrhoea. They are widespread both in dairy and beef cattle throughout the world and causing huge economic losses. The diagnosis of these agents is very difficult due to non-specific nature of lesions and the involvement of some intrinsic and extrinsic risk factors. We performed postmortem of 45 calves, which was below three months of age. Out of 45 necropscid calves, three (6.66%) cases were positive for BRoV and four (8.88%) cases were found positive for BCoV, screened by reverse transcriptase polymerase chain reaction (RT-PCR). Further RT-PCR positive cases were confirmed by immunohistochemistry (IHC) in paraffin-embedded intestinal tissue sections. Three cases of enteritis caused by BRoV showed the hallmark lesions of the shortening and fusion of villi, denudation and infiltration of mononuclear cells in the lamina propria. The BRoV antigen distribution was prominent within the lining epithelium of the villi, peyer's patches in the ileum and strong immunoreactions in the lymphocytes and some macrophages of the mesenteric lymph nodes. Four cases in which BCoV was detected, grossly lesions characterized by colonic mucosa covered with thick, fibrinous and diphtheritic membrane. Histopathologically, jejunum showed skipping lesion of micro-abscesses in crypts. The BCoV antigen distribution was prominent within the necrotic crypts in the jejunum and cryptic micro-abscesses in the colon and ileum. It is the first report of BRoV and BCoV antigen demonstration in the jejunum, colon, ileum, Peyer's patches and mesenteric lymph nodes of naturally infected calves from India by using IHC.

Molecular characterization of chicken anemia virus outbreaks in Nagpur province, India from 2012 to 2015.

Chicken anemia virus (CAV) is one of the important poultry pathogen. CAV infection can cause immunosuppression, aggravation of co-infections, vaccination failures and mortality. We are reporting the CAV outbreaks from the Nagpur province of India, between the years 2012-2015. The breeds included cockerel and Black Australorp of age varying from 29 to 50 days. The mortality rate observed among poultry was from 20% to 62.5%. Clinical symptoms like anemia, subcutaneous hemorrhages, growth retardation, abnormal feathers and hind limb paralysis suggested CAV infection. Postmortem analysis showed hemorrhages in thigh muscle and atrophy of the thymus and bone marrow. Seven out of 11 samples showed positive amplification of the CAV genome upon PCR. Phylogenetic analysis of all the seven isolates based on VP1 gene nucleotide sequence suggested circulation of genotype A strains in Maharashtra. The study will help us understand the circulating genotype of CAV in India and formulate its diagnosis and vaccination.