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Doctoral thesis, 1997

Diagnostic application of the polymerase chain reaction (PCR) in veterinary microbiology

Ros Bascunana, Carlos;

Abstract

The practical applicability of the polymerase chain reaction (PCR) in veterinary diagnostics was evaluated in the present studies. Various PCR-based detection procedures were developed in order to improve the diagnosis of several infectious diseases of importance in veterinary medicine.
A PCR system was developed for the detection and identification of Mycoplasma capricolum subsp. capripneumoniae, which causes a respiratory disease in goats, called contagious caprine pleuropneumonia (CCPP). A specific PCR was developed for the detection of Mycoplasma bovis. which causes mastitis, arthritis and respiratory disease in cows, and of Mycoplasma agalactiae. which is responsible for mastitis in goats and sheep. In this study the PCR proved to be an expeditious way to detect Mycoplasma bovis in nasal swabs of infected animals. Mycobacterium bovis and Mycobacterium qvium-intracellulare, which are the most important agents of animal mycobacteriosis, were detected and identified by a PCR/REA assay. The system was applied to fresh and formalin-fixed, paraffin-embedded tissue specimens (PET). An internal control of amplification was constructed to recognize the false negative results.
During the Aujeszky’s disease eradication programme established in Sweden, several peculiar cases emerged in which only a single animal was found seropositive on certain farms. These animals were called ‘single reactors’ (SR). Apart from the serological result, the SR animals had no other signs of infection. The PCR technology was applied to ascertain the infectious status of the SR animals. While virus isolation proved negative, three different nested PCR assays showed that Aujeszky’s disease virus sequences were present in these particular, apparently uninfected individuals.
​​​​​​​The technology of the PCR was also used to improve the diagnosis of the closely related caliciviruses rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV). Reverse transcription-PCR (RT-PCR) was applied to amplify a segment of the VP60 gene from a large number of RHDV and EBHSV isolates. The sequence data obtained from the PCR products were used to infer phylogenetic relationships. The results clearly separatedRHDV and EBHSV as two distinct members ofthe Caliciviridae family. The sequence data were also used to develop two specific and consistent RT-PCR assays by locating the primers in highly conserved sequence motifs. The diagnostic value of the assays was evaluated in a large number of fresh and fixed specimens from various geographic regions and years of collection. The phylogenetic information and the specific RT-PCR systems obtained established a basis for the molecular epidemiology of the rabbit caliciviruses.

Keywords

PCR; diagnostic; bacteria; virus; animal; sequencing; phylogeny; REA

Published in

Acta Universitatis Agriculturae Sueciae. Veterinaria

1997, number: 13
ISBN: 91-576-5247-3
Publisher: Swedish University of Agricultural Sciences

Authors' information

Ros Bascunana, Carlos
Swedish University of Agricultural Sciences, Department of Veterinary Microbiology
Ros Bascunana, Carlos
National Veterinary Institute (SVA)

UKÄ Subject classification

Clinical Science

URI (permanent link to this page)

https://res.slu.se/id/publ/117378