Application of the polymerase chain reaction in the Hungarian veterinary diagnostics
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The polymerase chain reaction (PCR) has been widely used in basic research and in diagnostics as well since its invention. Based on its unique sensitivity, specificity and speed the routine diagnostic application of the technique is steadily growing. However, the PCR has not been used for the purpose of veterinary diagnostics in Hungary till now. Thus, the aim of my work was to introduce this technique into those fields of the diagnostic work where it is reasonable and the advantages of it could properly be taken. We established our PCR laboratory in 1994 and settled to work with model experiences in animal adenoviruses of different origin. We found that after the modification of a primer pair that was originally designed for the universal detection of human adenovirus (HAV) serotypes of all subgenera it can adequately be used for the detection of different bovine, ovine, and porcine adenovirus (BAV, OAV and PAV, respectively) serotypes. Apparently, in the examined viruses, parts of the DNA sequence coding for the basal part of the hexon protein are conserved enough for being applicable in polymerase chain reaction (PCR) as primers. Positive amplification could be obtained even from the so-called subgroup 2 BAVs, which viruses do not cross react with HAVs or subgroup 1 BAVs in Southern hybridization. In order to test the primers and the method for the detection of the causative agent of the Rubarth's disease of dogs (canine adenovirus type 1, CAV-1) in the infected liver tissues we elaborated a simple, effective sample processing method and used it successfully not only for the exposition of CAV-1 DNA, but for goose parvovirus (GPV) DNA or mycoplasma genomic DNA as well in the ensuing protocols. The isolation and identification of mycoplasmas has its well-known difficulties, such as the long incubation period of these microorganisms, the cross reactivity of certain antigens, 78 PCR as veterinary diagnostics summary and occasionally the low sensitivity of the serological assays. Therefore, it seemed obvious to introduce the PCR in this process, where the improved speed of the identification presents the most valuable account of the technique - from practical point of view. Primers complementary to the 16S rRNA genes was used to detect avian mycoplasmas. This primer pair was designed for the detection of human and rodent mycoplasmal species and we examined their ability to detect the most important avian mycoplasmas. After testing the respective reference strains we found, that Mycoplasma iowae, M. meleagridis, and M. synoviae could be detected by PCR with this primer pair, and distinction could be made among them by restriction fragment length polymorphism (RFLP) assay with two restriction enzymes (BamUl, and Rsal). For the detection of M. gallisepticum by PCR we needed species specific primers. The results of the PCR and RFLP-based identification procedures of seventeen different field isolates agreed with those obtained by conventional methods. Thus, our experiments showed that the primer pair originally designed for the detection of human and rodent mycoplasmas is suitable for amplifying a similar product in size from other, avian mycoplasmas. It has been clear from our data that the method based on PCR and RFLP can serve as a simple, rapid, and easy technique in routine mycoplasma detection-identification procedure. Specific result can be achieved with samples taken by swabbing different organs, within one working day in positive cases, and significantly shorter time is sufficient for definitive positive and negative results combining PCR with culture than culture alone. We have been applying the PCR for the detection of the GPV DNA in clinical samples since the early summer of 1996. We have already investigated as many as 57 samples and we found sometimes that the PCR gave the decisive result for the diagnosis of the disease. During the introduction of the PCR into the routine tools for the diagnosis of Derzsy's disease the positive results were confirmed further by virus isolation in embryonated goose eggs. 79 PCR as veterinary diagnostics summary However, the isolation of GPV in embryonated eggs is quite difficult under the recent circumstances, i.e., most of the eggs contain maternal antibodies against GPV, the SPF eggs are virtually non-existent in the Hungarian market or they are very expensive, the availability of the goose eggs is seasonable, and last but not least, these are time consuming investigations. Therefore, as the results of both methods agreed in all cases we have been continuing only with the much faster and simpler PCR to prove the presence of GPV in the samples examined since the spring of 1997. The close antigenic relationship of the Pestiviruses and their ability to cross-infect ruminants and suidae can lead to diagnostic problems. Taking into account both the aforementioned fact and the great economical importance of classical swine fever virus infections it is reasonable to use the highly specific PCR (combined with reverse transcription) to differentiate these viruses. In our approach two sets of primers are used for the detection and identification of CSFV and BVDV. One is a general pestivirus-primer that flanks to a highly conserved region of the virus genome (the 5' UTR) and detects all the known pestiviruses in a very sensitive way. For the species specific detection a nested primer set in the genome region coding for the E2 (main envelope glycoprotein) is used for CSFV while a primer pair within the p80 (nonstructural protein) coding region is used for BVDV identification. BDV has not been targeted in our experiments since it is virtually non-existent in our investigating area. We tested our PCR protocol using different samples and we could detect the presence of CSFV in the buffy coat of suspected animals being alive, therefore the necessary measures could be carried out in good time. On the other hand, we found that samples that are not accessible for other diagnostic methods like some day-old thus autolyzed corpses of wild boars could even be used as targets for the PCR. Taking into account all these aspects we state 80 PCR as veterinary diagnostics summary that PCR is a really powerful tool for diagnosing or excluding CSF in a quick and precise way. We found the BVDV specific PCR system reliable as well, and we performed retrospective analyses on stored samples originating from four BVD suspected animals and our findings confirmed the diagnoses which were based on pathological examinations. Beyond the detection of particular pathogens PCR serves as a very useful tool for epidemiological investigations where different scales of the identification can be gained through the analysis of the PCR product, i.e. by restriction fragment length polymorphism assay, by single-strand conformational polymorphism assay, or by the most informative way, the nucleotide sequencing. Our experiences showed that the PCR can be a valuable tool for the veterinary diagnostics based on its most important features like sensitivity, specificity and speed. However, these are the very characteristics of PCR that present the potential danger of contamination. Therefore, the maximum care should be taken during the PCR experiments to prevent either false positive or negative results. It should be emphasized too, that the PCR does not replace the conventional isolation techniques. Nevertheless, we have successfully extended our facilities with this technique for the detection of microorganisms that are difficult to isolate, or for situations when rapid diagnosis is necessary.