key: cord-0875176-tykltj8j authors: Kamdi, Bhupesh; Singh, Rajendra; Singh, Vidya; Singh, Shailendra; Kumar, Pawan; Singh, Karam Pal; George, Neethu; Dhama, Kuldeep title: Immunofluorescence and molecular diagnosis of bovine respiratory syncytial virus and bovine parainfluenza virus in the naturally infected young cattle and buffaloes from India date: 2020-03-20 journal: Microb Pathog DOI: 10.1016/j.micpath.2020.104165 sha: 384bc4d5a50cfa38152d31878d01073554988e8b doc_id: 875176 cord_uid: tykltj8j 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. Livestock production plays a key role in India's agricultural economy as it offers bovine rhinovirus, bovine reovirus and Influenza A viruses have also been reported occasionally 89 to be associated with pneumonia in calves (Sreenivasan et al., 2019; Hodgins et al., 2002) . suppression of the local immune defense mechanism, which may facilitate rapid bacterial 97 multiplication leading to acute to chronic pneumonia in a synergistic manner (Caswell, 2014) . Processing of tissues for histopathology 140 After proper fixation in 10% NBF, tissues were cut into small sections with a thickness of Belgium, UK and the USA in NCBI database. Further, the isolates were distantly related to the 230 sequences of the isolates reported from Denmark, France and the Czech Republic (Fig. 3) . Sequence distance of the BRSV isolates with respect to those reported in the NCBI database is 232 presented in Fig. 3 . 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Veterinary Clinics: Food Animal 462 Practice Comparison of DNA application methods to reduce 465 BRSV shedding in cattle Pathogenesis and pathology of bovine pneumonia Neonatal 469 calf infection with respiratory syncytial virus: drawing parallels to the disease in human 470 infants s 472 Pathology of Domestic Animals. 5 th ed A retrospective search for bovine respiratory syncytial virus 475 (BRSV) antigens in histological specimens by immunofluorescence and 476 immunohistochemistry Histological Staining Methods of the Armed Forces Institute of Pathology Identification and genome characterization 505 of genotype B and genotype C bovine parainfluenza type 3 viruses isolated in the United 506 Surveillance, isolation and complete genome sequence of bovine parainfluenza virus type 509 Genomic characterization of 512 bovine parainfluenza virus type 3 in Hokkaido and development of rapid diagnostic 513 method Bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus-3 (BPI-3) are responsible for the development of pneumonia in young animals BPI-3 revealed characteristic findings of bronchiolar epithelial necrosis and numerous syncytial cells having intracytoplasmic inclusions in the lungs