Title page for ETD etd-05272008-120446


Document Type Master's Dissertation
Author Bagla, Victor Patrick
Email drvictorb@yahoo.com
URN etd-05272008-120446
Document Title The demonstration of lumpy skin disease virus in semen of experimentally infected bulls using different diagnostic techniques
Degree MSc (Veterinary Science)
Department Veterinary Tropical Diseases
Supervisor
Advisor Name Title
Prof E H Venter Supervisor
Keywords
  • lumpy skin disease virus
  • viral nucleic acid
  • polymerase chain reaction
Date 2006-05-05
Availability unrestricted
Abstract
Lumpy skin disease virus (LSDV), a poxvirus that belongs to the genus Capripoxvirus is an important pathogen that can be shed in the semen of infected bulls. The screening of semen for infectious virus prior to artificial insemination requires a sensitive diagnostic method. The isolation of the virus on cell cultures and/or the use of polymerase chain reaction (PCR) are sensitive diagnostic tests which can be used to screen semen for LSD viral DNA prior to artificial insemination. Although cell culture is a sensitive method and detects infectious virus, its use has major limitations due to the toxic effect of semen on the cells. This study was therefore aimed at finding a method that decreases the toxic effect of semen on cell culture and enhances LSDV isolation. Secondly, the efficiency of this method in enhancing the isolation of LSDV in field samples was tested.

In order to eliminate the toxic effect of semen on cell culture, a pilot study was conducted in which semen samples from LSDV sero-negative bulls were collected and infected with a field isolate of LSDV, strain 248/93 with a titre of 6.5 log TCID50. The semen samples were subjected to one of four different methods, viz centrifugation, serial dilution, filtration and chemical treatment with kaolin. The centrifugation, serial dilution, and filtration methods were supplemented with additional amounts of gentamycin.

The toxic effects of semen on cell culture were completely eliminated when supernatants of semen samples, centrifuged at 2000 rpm for 1, 3 and 5 mins and serial diluted was used to inoculate confluent monolayers of bovine dermis cells. Semen diluted in MEM with or without additional antibiotics was the most sensitive method of demonstrating virus at higher dilutions, followed by pellets of samples centrifuged for 1 and 3 minutes. The toxicity recorded when the pellet fraction of semen samples were centrifuged for 5 mins at 2000 rpm was comparable to results obtained from serially diluted samples supplemented with gentamycin. The use of filtration and kaolin treatment of semen samples could not remove the toxic effect of semen on cells.

To evaluate the presence of LSDV in semen of experimentally infected bulls, six seronegative post-pubertal bulls housed in an insect proof facility were infected with LSDV via the intravenous route. The experimentally infected bulls were monitored for clinical sign of the disease. Two bulls showed severe, two a mild and two an inapparent infection. Blood samples were collected for virus isolation and semen samples for virus isolation and PCR. Vesicular fluid and preputial washes were also investigated for the presence of LSD viral nucleic acid using PCR. The infectious titre of the virus shed in semen of these bulls was also calculated.

The incubation period in infected bulls varied from 7 to 14 days. The length of viraemia varied between groups and did not correlate with the severity of clinical disease. The virus was isolated from blood samples of bulls in the severely infected group on several occasions. Bulls in the mildly infected group had the lowest rate of isolated virus when compared to those with inapparent infection.

The use of supernatants of centrifuged serial diluted semen samples, as shown in the pilot study, have considerably reduced the toxic effect of semen on cell culture. This method was used to test field samples for its sensitivity to isolated LSDV in semen of experimentally infected bulls with PCR as a gold standard.

In all the semen samples tested using supernatants of semen samples LSDV was isolated in 53.1% of the samples on cell culture while in the serial diluted samples, only 28.1% of samples were positive with a median time of detection on cell culture of 4 and 8 days, respectively. The use of the supernatant fraction was able to detect infectious LSDV in semen samples for prolonged periods with reduced time of development of cytopathic effect, than previously reported.

In order to compare the sensitivity of PCR and virus isolation, PCR positive and a few negative samples were subjected to virus isolation using the centrifugation method developed in the pilot study. The PCR was able to detect LSD viral nucleic acids in some semen samples even when virus could not be isolated on cell culture.

The PCR was also able to detect viral nucleic acid in vesicular fluid and preputial washes of infected bulls. The titre of the virus shed in the semen at a certain stage of the infection was calculated to be 3 log TCID50.

In conclusion, this study provides evidence of a complete reduction of the toxic effect of semen on cell culture and increase chances of LSDV isolation with reduced detection time when semen samples are processed using the centrifugation method as described in the pilot study. Furthermore, it showed PCR was more sensitive than virus isolation in the detection of LSD viral nucleic acid in semen samples and can be used for routine diagnosis. However, virus isolation must be used when the infective nature of virus shed in semen is desirable. This study provides the first evidence of the shedding of LSDV nucleic acid in vesicular fluid and preputial washes of experimentally infected bulls. It also represents the first report that a considerable amount of LSDV is shed in semen of experimentally infected bulls, which may be infective at certain stages of clinical disease.

Lumpy skin disease virus (LSDV), a poxvirus that belongs to the genus Capripoxvirus is an important pathogen that can be shed in the semen of infected bulls. The screening of semen for infectious virus prior to artificial insemination requires a sensitive diagnostic method. The isolation of the virus on cell cultures and/or the use of polymerase chain reaction (PCR) are sensitive diagnostic tests which can be used to screen semen for LSD viral DNA prior to artificial insemination. Although cell culture is a sensitive method and detects infectious virus, its use has major limitations due to the toxic effect of semen on the cells. This study was therefore aimed at finding a method that decreases the toxic effect of semen on cell culture and enhances LSDV isolation. Secondly, the efficiency of this method in enhancing the isolation of LSDV in field samples was tested.

In order to eliminate the toxic effect of semen on cell culture, a pilot study was conducted in which semen samples from LSDV sero-negative bulls were collected and infected with a field isolate of LSDV, strain 248/93 with a titre of 6.5 log TCID50. The semen samples were subjected to one of four different methods, viz centrifugation, serial dilution, filtration and chemical treatment with kaolin. The centrifugation, serial dilution, and filtration methods were supplemented with additional amounts of gentamycin.

The toxic effects of semen on cell culture were completely eliminated when supernatants of semen samples, centrifuged at 2000 rpm for 1, 3 and 5 mins and serial diluted was used to inoculate confluent monolayers of bovine dermis cells. Semen diluted in MEM with or without additional antibiotics was the most sensitive method of demonstrating virus at higher dilutions, followed by pellets of samples centrifuged for 1 and 3 minutes. The toxicity recorded when the pellet fraction of semen samples were centrifuged for 5 mins at 2000 rpm was comparable to results obtained from serially diluted samples supplemented with gentamycin. The use of filtration and kaolin treatment of semen samples could not remove the toxic effect of semen on cells.

To evaluate the presence of LSDV in semen of experimentally infected bulls, six seronegative post-pubertal bulls housed in an insect proof facility were infected with LSDV via the intravenous route. The experimentally infected bulls were monitored for clinical sign of the disease. Two bulls showed severe, two a mild and two an inapparent infection. Blood samples were collected for virus isolation and semen samples for virus isolation and PCR. Vesicular fluid and preputial washes were also investigated for the presence of LSD viral nucleic acid using PCR. The infectious titre of the virus shed in semen of these bulls was also calculated.

The incubation period in infected bulls varied from 7 to 14 days. The length of viraemia varied between groups and did not correlate with the severity of clinical disease. The virus was isolated from blood samples of bulls in the severely infected group on several occasions. Bulls in the mildly infected group had the lowest rate of isolated virus when compared to those with inapparent infection.

The use of supernatants of centrifuged serial diluted semen samples, as shown in the pilot study, have considerably reduced the toxic effect of semen on cell culture. This method was used to test field samples for its sensitivity to isolated LSDV in semen of experimentally infected bulls with PCR as a gold standard.

In all the semen samples tested using supernatants of semen samples LSDV was isolated in 53.1% of the samples on cell culture while in the serial diluted samples, only 28.1% of samples were positive with a median time of detection on cell culture of 4 and 8 days, respectively. The use of the supernatant fraction was able to detect infectious LSDV in semen samples for prolonged periods with reduced time of development of cytopathic effect, than previously reported.

In order to compare the sensitivity of PCR and virus isolation, PCR positive and a few negative samples were subjected to virus isolation using the centrifugation method developed in the pilot study. The PCR was able to detect LSD viral nucleic acids in some semen samples even when virus could not be isolated on cell culture.

The PCR was also able to detect viral nucleic acid in vesicular fluid and preputial washes of infected bulls. The titre of the virus shed in the semen at a certain stage of the infection was calculated to be 3 log TCID50.

In conclusion, this study provides evidence of a complete reduction of the toxic effect of semen on cell culture and increase chances of LSDV isolation with reduced detection time when semen samples are processed using the centrifugation method as described in the pilot study. Furthermore, it showed PCR was more sensitive than virus isolation in the detection of LSD viral nucleic acid in semen samples and can be used for routine diagnosis. However, virus isolation must be used when the infective nature of virus shed in semen is desirable. This study provides the first evidence of the shedding of LSDV nucleic acid in vesicular fluid and preputial washes of experimentally infected bulls. It also represents the first report that a considerable amount of LSDV is shed in semen of experimentally infected bulls, which may be infective at certain stages of clinical disease.

2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.

Please cite as follows:

Bagla, VP 2006, The demonstration of lumpy skin disease virus in semen of experimentally infected bulls using different diagnostic techniques, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-05272008-120446 / >

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