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

Abstract

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.

Keywords: BPI-3; BRSV; Bovine; Immunofluorescence; Pneumonia; Syncytia.

Fig. 1

Ethidium bromide stained agarose gel electrophoresis (2% agarose): positive samples (S1. Sn) for BRSV (a) and BPI-3 (b) with negative control (NTC), positive control (PC), 100bp DNA ladder (M).

Fig. 2

Pathology of BRSV pneumonia: Dark brown areas of consolidation at right cranial, middle and anterior part of the diaphragmatic lobe with severe edema and multifocal hemorrhages (a); BRSV pneumonia: Photomicrographs of lung showing thickened alveolar septa, and alveolar lumen filled with macrophages and syncytia (b, 400x); alveolar lumen filled with macrophages and syncytial cells having intracytoplasmic inclusion (arrow) (c, 400x). H&E; granular apple green fluorescence in the epithelium cells and macrophages (d, 400x). dFAT; Photomicrographs of trachea showing hemorrhages in the lamina propria and necrosis of sub mucosal gland (e, 200x); medullary area of lymph node showing fibrinous exudate with moderate infiltration of plasma cells, lymphocytes and macrophages (f, 200x). H&E. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Phylogenetic analysis, sequence divergence and percent identity of BRSV based on G Gene amplicons (246bp) using Megalign (DNA star).

Fig. 4

Pathology of BPI-3 pneumonia: Light brown areas of consolidation in lobular pattern and pale inflated areas of emphysema (a); photomicrograph showing thickened alveolar septa and alveolar lumen filled with proteinacious fluid and few syncytia (b, 200x); thickened alveolar septa, few foamy macrophages and intracytoplasmic inclusion in alveolar macrophage (c, 400x). H&E; Lung section showing, granular apple green fluorescence in alveolar macrophages and pneumocytes (d, 100x). dFAT. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Phylogenetic analysis and sequence divergence and percent identity of BPI-3 based on N Gene amplicons (127bp) using Megalign (DNA star).