title: Isolation and adaptation of bovine herpes virus Type 1 in embryonated chicken eggs and in Madin-Darby bovine kidney cell line.
abstract: OBJECTIVE
  Objective of the present study was to isolate bovine herpes virus Type 1 (BHV-1) from semen of infected bull and to adapt it onto embryonated eggs and Madin-Darby bovine kidney (MDBK) cell line. Further, the virus was identified by agar gel immunodiffusion (AGID) test.
  
  
  METHODS
  Semen samples were collected from five BHV-1 positive bulls previously confirmed for the presence of antibodies against BHV-1 using avidin-biotin enzyme linked immunosorbent assay test. The virus from semen samples was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cell line. The presence of BHV-1 in infected CAM and cell culture fluid was confirmed by AGID test.
  
  
  RESULTS
  Virus infected CAM showed edema, congestion and thickening at first passage level. Small foci ranged from 1 to 2 mm in diameter, scattered all over the membrane were observed at first passage. More severe changes were observed in CAM after serial passaging. The large pock lesions, round in shape with opaque raised edge and depressed gray central area of necrosis ranged from 3 to 5 mm in diameter were developed at fourth passage. Blind passages in MDBK cell culture were made. The MDBK cell line at second passage level showed characteristic cytopathic effect viz. rounding of cells with shrinkage, followed by aggregation or clumping of cells which progressed rapidly and appeared as "bunch of grapes" at 72 h post inoculation. Few cells become elongated when compared with uninfected controls. A homogenate of CAM with distinct pock lesions and infected cell culture fluid developed precipitation line within 48 h against specific anti-BHV-1 immune serum by AGID test.
  
  
  CONCLUSIONS
  BHV-1 was easily adapted in CAM of chicken embryos and in MDBK cell line. Virus infected CAM and cell culture fluid showed precipitin band by AGID test.
authors.affiliation: Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Anjora Durg, Chhattisgarh, India.
authors.forename: Devprabha
authors.initials: D
authors.lastname: Samrath
authors.affiliation: Department of Veterinary Public Health, College of Veterinary Science and Animal Husbandry, Anjora Durg, Chhattisgarh, India.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Shakya
authors.affiliation: Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Anjora Durg, Chhattisgarh, India.
authors.forename: Nidhi
authors.initials: N
authors.lastname: Rawat
authors.affiliation: Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Anjora Durg, Chhattisgarh, India.
authors.forename: Varsha Rani
authors.initials: VR
authors.lastname: Gilhare
authors.affiliation: Division of Animal Health, ICAR-Central Sheep and Wool Research Institute, Avikanagar-304501, Rajasthan, India.
authors.forename: Fateh
authors.initials: F
authors.lastname: Singh
keywords: Bovine herpes virus Type 1; Madin–Darby bovine kidney cell line; chorioallantoic membrane; cytopathic effect; pock