The thesis is devoted to the of African (ASF) and Classical swine fever (CSF) epidemiological monitoring in the world and in Ukraine as well as the improvement of laboratory diagnostics of these diseases through the development of a national test kit based on quantitative reverse transcription polymerase chain reaction (RT-qPCR).
The results of the epidemiological monitoring have shown that in 2012–2018 ASF spread into Europe (14) and Asia countries (1). ASF is considered endemic in most African countries (25). Over ten years ASF epidemic in Eastern Europe indicates the potential formation of a new stable East-European nosoarea of ASF with the risk of further disease spreading into all continents. It has been found that the ASF epidemic situation in Ukraine is complex and tense. It is proved that in Ukraine, unlike most European countries, anthropogenic factor, not wild boars, plays significant role in ASF spreading.
In 2012–2018 most outbreaks of CSF were registered in Asia (16 countries), sporadic outbreaks — in Central and South America (9), Eastern Europe (3) and Africa (1). The North America, most part of Europe (22), Australia, New Zealand, New Caledonia are officially free from CSF according to OIE. In Ukraine, the current situation of CSF is relatively stable. Over the past 7 years, only one outbreak was reported in wild boars, but there is still a risk of CSF virus (CSFV) introduction into the country and its spreading. The main factors that could lead to CSF spreading in our country are wild boar’s migration and their contacts with domestic pigs, total vaccination against CSF and international trade as well.
The RT-qPCR kit for ASF and CSF differential diagnosis has been developed. The proposed test kit allows simultaneous detection of three targets: ASFV DNA, CSFV RNA and an internal control (ITC). For the ASFV DNA detection specific primers and a TaqMan probe recommend by OIE (King et al., 2003) have been used. Primers and ROX-labeled TaqMan-probe specific for CSFV selected from 5′ untranslated region (5′UTR) as well as primers and R6G-labeled TaqMan-probe specific for ITC selected from conserved regions of PRP gene have been designed using Primer Express Software. Conditions for both reverse transcription and amplification steps in one tube were optimized to decrease the risk of contamination.
For evaluation of the RT-qPCR kit intralaboratory validation, international validation and interdepartmental evaluation studies have been performed in compliance with OIE Validation Guidelines according to analytical and diagnostic sensitivity, specificity and repeatability and reproducibility. The RT-qPCR kit sensitivity was determined by testing 10-fold serial dilutions of the ASFV DNA and CSFV RNA. For specificity determination reference samples of ASFV DNA different genotypes, ASF and CSF positive and negative field samples, as well as pathogens which cause similar to ASF and CSF clinical syndromes were used. As a result of accomplished evaluation high sensitivity, specificity and reproducibility of RT-qPCR kit were confirmed. In particular, it was determined that the limit of detection of the RT-qPCR kit was five copies of the ASFV and CSFV genomes per one reaction. High accuracy and reproducibility of the RT-qPCR assay (CV ranging from 0.9% to 1.89%) were demonstrated both within and between laboratories using different real-time PCR thermocyclers. The 100% specificity of the assay to ASFV (I, II, V, VIII, IX and X genotypes) and CSFV was confirmed. It was shown no cross-reactions with pathogens which cause similar to ASF and CSF clinical syndromes (porcine circoviruses (type 1 and 2), porcine reproductive and respiratory syndrome virus, virus of Aujeszky’s disease, Teschen virus, porcine epidemic diarrhea virus, porcine parvovirus, bovine viral diarrhea virus, Erysipelothrix rhusiopathiae, M. hyorhinis, M. gallisepticum, P. multocida, spirochetes of the genus Leptospira and E. coli). Test results on 121 ASF and 76 CSF positive samples have been shown 97.5% and 100% diagnostic sensitivity for ASFV and CSFV, respectively. In conclusion, all investigated analytical performance criteria of the RT-qPCR kit for differential diagnosis of ASF and CSF are in compliance with international standards, which ensures accurate and definite results.
The combined ASF and CSF monitoring strategy have been suggested. It is based on using of the developed test kit ‘ASF/CSF duo qPCR’ for the differential diagnosis of African and Classical swine fever by qPCR (№ ВВ-00871-06-18 of 27.04.2018) as a main tool for detection of infected animals.
