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Pigs serve as major reservoirs of H1N1 and H3N2 influenza viruses which are endemic in pig populations worldwide and are responsible for one of the most prevalent respiratory diseases in pigs. Therefore the early detection and identification of such events are paramount in monitoring the spread of influenza viruses. Transfer of influenza A viruses from animal hosts to man may lead to the emergence of new human pandemic strains. The aim of the study was to prove the matrix gene useful for reverse transcription nested-PCR as sensitive and specific for the detection of swine influenza A viruses in clinical samples. 75 RNA-virus positive samples out of 235 samples, including nasal swabs, lung tissues and whole blood samples, were detected by RT-n-PCR. Using conventional virology method we isolated 33 SIV strains in embryonated chicken SPF eggs. The results of PCR were 100% in agreement with those of virus isolation. The limit of detection of SIV was 10-7 EID50/0.1 ml. These results demonstrate the usefulness of RT-n-PCR for the detection and identification of influenza A virus in clinical material.
This paper discusses important issues that have to be taken into account in the laboratory diagnosis of infectious animal diseases, particularly swine diseases. Some of the most fundamental issues are standardization and harmonization of diagnostic tests in the international scale. Different approaches to test selection are related to the etiology of the infectious disease. The paper mentions diseases caused by one pathogen, diseases of multifactorial etiology and subclinical infections. The importance of a disease-free herd is underlined - particularly for eradication and the possibility of exporting animals. The difference between qualitative and quantitative results is defined. The number of samples used and the gold standard, as well as the sensitivity and specificity of diagnostic tests are characterized. The DIVA strategy is mentioned. The value of laboratory diagnosis in dealing with disease syndromes and infections caused by facultativly pathogenic microorganisms is described. Limitations of laboratory tests in the diagnosis of infectious swine diseases are also enumerated.
Atrophic rhinitis is a disease of high economic impact for pig production, caused by Pasteurella multocida (P.m.) strains able to produce dermonecrotoxin (DNT). In general the diagnosis of pigs’ infection with P.m. is based on the direct detection of bacteria from nasal swabs and their identification as well as on the detection of DNT specific antibodies by using an ELISA test. The aim of the study was the elaboration of a PCR test for the detection of dermonecrotic strains of P.m. directly in nasal swabs. The optimalization of the process includes: estimation of Mg²⁺ concentration, the temperature of primer hybridization and the number of cycles. The specificity and sensitivity of the test was estimated for both bacterial culture and nasal swabs. The optimal conditions were as follow: Mg²⁺ concentration - 2.5 m M/reaction; the temperature of primers hybridization - 56°C; and the number of cycles - 50. The sensitivity of the test for both the culture and nasal swabs was 2.5 × 10³ cfu/ml. The test was also highly specific - the product of 501 bp was detected only in the samples containing DNA of P.m. Summarizing, the elaborated test was specific and sensitive enough to be used for routine detection of P.m. DNT strains in clinical samples and for the elimination of the carriers from the herd.
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