Uporedno ispitivanje antitela protiv virusa bolesti kvrgave kože imunoenzimskim i virus neutralizacionim testom

Abstract

Bolest kvrgave kože (lumpy skin disease – LSD) je virusno oboljenje goveda, veoma važno sa ekonomskog aspekta, zbog velikih gubitaka u govedarskoj proizvodnji. Brzi i pouzdani laboratorijski testovi su neophodni, kako za rano otkrivanje bolesti i utvrđivanje statusa zapata, tako i za praćenje imunološkog statusa jedinki nakon vakcinacije, koja je i dalje najsigurnija mera u borbi protiv ove bolesti. Radi sprovođenja efikasnih mera kontrole protiv LSD, kao što su pravovremena vakcinacija, naročito teladi i serološki nadzor zapata, neophodno je vršiti istraživanja vezana za postvakcinalni imunološki odgovor, kako kod odraslih jedinki, tako i kod teladi. Cilj ove doktorske distertacije je praćenje antitela protiv virusa bolesti kvrgave kože (lumpy skin disease virus – LSDV) kod vakcinisanih krava, praćenje perzistencije maternalnih antitela kod teladi poreklom od vakcinisanih krava, kao i sprovođenje postupka validacije i potvrde primene modifikovanog virus neutralizacionog testa razvijenog na Odeljenju za virusologiju Naučnog instituta za veterinarstvo „Novi Sad“. Materijal za ispitivanje korišćen u ovom istraživanju potiče od goveda sa područja u kojima nije registrovana bolest tokom epizootije u Srbiji 2016. godine (Južnobački, Sremski i Južnobanatski okrug), a vakcinacija goveda protiv bolesti kvrgave kože je počela da se sprovodi tokom jula meseca 2016. godine. Uporedno ispitivanje je izvršeno na ukupno 355 uzoraka krvnih seruma krava, 15 uzoraka kolostruma i 270 uzoraka krvnih seruma teladi. Za uporedno ispitivanje prisustva antitela protiv LSDV u uzorcima korišćene su metode ELISA i virus neutralizacioni test (VNT). ELISA test je izvođen korišćenjem komercijalnog kita ID Screen® Capripox Double Antigen Multispecies proizvođača IDvet (France), dok je VNT izvođen upotrebom kulture ćelija Madin-Darby bovine kidney (MDBK) i virusa bolesti kvrgave kože poreklom od klinički obolelog govečeta u trajanju od 3 dana. Prvo prisustvo specifičnih antitela protiv LSDV u krvnim serumima vakcinisanih krava utvrđeno je 20 dana nakon vakcinacije, najviši nivo serokonverzije utvrđen je 30 dana nakon vakcinacije, a prisustvo antitela je bilo moguće detektovati tokom četiri meseca nakon vakcinacije, modifikovanim VNT kod 34% krava, a ELISA testom kod 30% vakcinisanih krava. Prisustvo specifičnih antitela protiv LSDV ELISA metodom je bilo moguće utvrditi 90 dana nakon teljenja kod 16,67% teladi, 105 dana nakon teljenja kod 10% teladi, a u poslednjem terminu uzorkovanja, 120 dana nakon teljenja, kod svega 6,67% teladi. VNT je u istim terminima prisustvo specifičnih antitela bilo moguće utvrditi kod 10%, 6,67%, i 3,33% teladi. Ni u jednom terminu uzorkovanja nije bilo moguće utvrditi prisustvo antitela protiv LSDV kod svih ispitanih teladi. Na dan teljenja je prisustvo specifičnih antitela protiv LSDV utvrđeno u 63,33% uzoraka krvnih seruma krava ELISA metodom, u 73,33% uzoraka krvnih seruma krava metodom VNT, dok je obema metodama prisustvo antitela utvrđeno u 86,67% uzoraka kolostruma. Statističkom analizom je utvrđen značajno veći nivo antitela u kolostralnom u odnosu na krvni serum krava. Rezultati uporednog ispitivanja modifikovanog VNT i komercijalnog ELISA testa za detekciju antitela protiv LSDV u uzorcima krvnih seruma iz banke seruma i vakcinisanih krava pokazali su skoro savršenu saglasnost poređenih metoda (k=0,913). S druge strane, visoka saglasnost poređenih metoda (k=0,7239) je postignuta kod uporednog ispitivanja modifikovanog VNT i komercijalnog ELISA testa za detekciju antitela protiv LSDV u uzorcima krvnih seruma vakcinisanih krava i njihove teladi. Ispitivanjem uzoraka krvnih seruma iz banke seruma goveda pre pojave LSD u Republici Srbiji izračunata je specifičnost modifikovanog VNT od 100%, a komercijalnog ELISA testa od 99,2%. S obzirom da je VNT zlatni standard serološke dijagnostike LSD, izračunata je osetljivost komercijalnog ELISA testa u odnosu na modifikovani VNT za detekciju antitela u uzorcima krvnih seruma vakcinisanih krava od 88,24% i za detekciju antitela protiv LSDV u uzorcima krvnih seruma vakcinisanih krava i njihove teladi od 86,44%. Rezultati ovog istraživanja su pokazali da se modifikovani VNT i komercijalni ELISA test (proizvođača „IDvet“) mogu koristiti za detekciju antitela protiv LSDV. Modifikovani VNT se pokazao kao jednostavniji i brži za izvođenje u odnosu na preporučeni VNT od strane OIE. Datum

Lumpy skin disease (LSD) is a viral disease of cattle, very important from an economic aspect, due to significant losses that can cause in livestock production. Rapid and reliable laboratory tests are necessary, both for early detection of disease and determination of the status of the herds, as well as for monitoring of the immune status of individual animals after vaccination, which is still the most effective measure for controlling the spread of LSD. In order to implement effective LSD control measures, such as timely vaccination, particularly in calves and serological monitoring, it is necessary to carry out researches related to postvaccinal immune response, both in adult cattle and in their calves. The goal of this doctoral dissertation is to monitor the presence of antibodies against lumpy skin disease virus (LSDV) in vaccinated cows, to monitor the persistence of maternal antibodies in calves born to vaccinated cow, as well as to carry out the validation process and confirm the use of the modified virus neutralization test developed at the Department of virology of the Scientific veterinary institute "Novi Sad", Serbia. The material for testing used in this study originated from cattle in areas where no LSD outbreaks were registered during the epizootic in Serbia in 2016 (South Bačka, Srem and South Banat Districts), and the vaccination of cattle against LSD started in July 2016. A comparative examination was carried out on a total of 355 cow blood sera samples, 15 colostrum samples and 270 calf blood sera samples. ELISA and virus neutralization test (VNT) were used for comparative examination of samples on the presence of antibodies against LSDV. ELISA test was performed using the commercial set kit ID Screen® Capripox Double Antigen Multi-species manufactured by IDvet (France), while a 3-day VNT was performed using Madin-Darby bovine kidney (MDBK) cell line and LSDV isolated from clinically infected cow. The first presence of specific antibodies against LSDV in vaccinated cows was detected 20 days after vaccination, the highest seroconversion was determined 30 days after vaccination, and the presence of antibodies against LSDV could be detected during four months after vaccination by VNT and commercial ELISA in 34% and 30% of vaccinated cattle, respectively. The presence of specific antibodies against the LSDV by ELISA method could be determined 90 days after calving in 16.67% of calves, 105 days after calving in 10% of calves, and in the last sampling interval, 120 days after calving, in only 6.67% of calves. In above-mentioned sampling intervals, the presence of specific antibodies by VNT could be determined in 10%, 6.67%, and 3.33% of calves. In any sampling period we did not determined that all calves were seropositive to LSDV. On calving day, the presence of specific antibodies against LSDV was detected in 63.33% of cow blood sera samples by ELISA, in 73.33% of cow blood sera samples by VNT method, while the presence of antibodies was found in 86.67% of colostrum samples by both methods. A statistical analysis showed a significantly higher level of antibodies in the colostrum compared to the cow blood sera. The results of comparative examination of modified VNT and commercial ELISA test for the detection of antibodies against LSDV in blood sera samples from the sera bank and of vaccinated cows showed an almost perfect agreement of the compared methods (k = 0.913). On the other hand, the substantial agreement of the compared methods (k = 0.7239) was achieved in a comparative examination of modified VNT and commercial ELISA test for the detection of antibodies against LSD in blood sera samples of vaccinated cows and their calves. The specificity of modified VNT and commercial ELISA was 100% and 99.2% respectively. It was calculated by testing blood sera samples from sera bank before the occurrence of LSD in the Republic of Serbia. Since VNT is “the gold standard” serological test for LSDV, the sensitivity of commercial ELISA in relation to modified VNT was calculated for the detection of antibodies in blood sera samples of vaccinated cows of 88.24% and for the detection of antibodies against LSDV in blood sera samples of vaccinated cows and their calves of 86.44%. The results of this study showed that modified VNT and commercial ELISA manufactured by „IDvet“ can be used for the detection of antibodies against LSDV. Modified VNT proved to be simpler to perform and to take less time compared to the recommended VNT by the OIE.