title: Elimination of toxicity and enhanced detection of lumpy skin disease virus on cell culture from experimentally infected bovine semen samples.
abstract: Lumpy skin disease virus (LSDV), a poxvirus of the genus Capripoxvirus, is shed in the semen of infected bulls. The screening of semen for infectious virus requires a sensitive diagnostic method. The isolation of the virus on cell cultures and/or the polymerase chain reaction (PCR) are sensitive diagnostic tests which may be used to screen semen for LSD viral DNA prior to artificial insemination. Although cell culture detects infectious virus and is a sensitive method, there are major difficulties in using this method due to the toxic effect of semen on the cells. The aim of this study was to find a method that decreases the toxic effect of semen and enhances the isolation of LSDV on cell culture. Semen samples from LSDV sero-negative bulls were collected and infected with a field isolate of LSDV, strain V248/93, with a titre of 6.5 log TCID50. The semen samples were treated with one of four different methods: centrifugation, serial dilution, filtration and chemical treatment with kaolin. The samples subjected to centrifugation, serial dilution and filtration were supplemented with gentamycin. Semen toxicity on cell cultures was eliminated when supernatants of semen samples centrifuged at 2000 rpm for 1, 3 and 5 min and serially diluted were used to inoculate confluent monolayer bovine dermis cells. The toxicity recorded when the pellet fractions of semen samples centrifuged for 5 min at 2000 rpm was comparable to results obtained from serially diluted samples supplemented with gentamycin. Filtration and kaolin treatment of semen samples did not remove the toxic effect.
authors.affiliation: Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
authors.forename: V P
authors.initials: VP
authors.lastname: Bagla
authors.forename: U I
authors.initials: UI
authors.lastname: Osuagwuh
authors.forename: C H
authors.initials: CH
authors.lastname: Annandale
authors.forename: P C
authors.initials: PC
authors.lastname: Irons
authors.forename: E H
authors.initials: EH
authors.lastname: Venter
mesh_terms: D000818:Animals; D002417:Cattle; D002498:Centrifugation / Q000379:methods / Q000662:veterinary; D004847:Epithelial Cells / Q000166:cytology*; D005374:Filtration / Q000379:methods / Q000662:veterinary; D005839:Gentamicins / Q000494:pharmacology; D007616:Kaolin / Q000494:pharmacology; D008166:Lumpy Skin Disease / Q000175:diagnosis*; D008167:Lumpy skin disease virus / Q000302:isolation & purification*; D008297:Male; D015203:Reproducibility of Results; D012661:Semen / Q000502:physiology / Q000821:virology*; D012680:Sensitivity and Specificity; D012867:Skin; D017201:Virus Shedding
chemical_list: D005839:Gentamicins; D007616:Kaolin