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Respiratory diseases often correspond to primary infections with different pathogens of cattle, causing heavy economic losses in young stock and breeding herds. Between 2012 and 2014, nasal swab samples were collected from twenty-eight herds from 133 affected live cattle that were clinically suffering from symptoms of respiratory disease, pyrexia, cough, serous nasal and lacrimal discharge, increased respiratory rate, and breath sounds. Individual swab samples were tested in the laboratory using three commercial and one in-house real-time PCR methods, to detect nucleic acids of a total of ten different respiratory pathogens. Pasteurella multocida (P. multocida) was detected in 58.65% of samples, Mannheimia haemolytica (M. haemolytica) in 15.04%, while Mycoplasma bovis (M. bovis) and Histophilus somni (H. somni) were positive in 9.77% of nasal swab samples. Among viral pathogens, the highest prevalence (40.60%) was observed for bovine respiratory syncytial virus (BRSV), followed by bovine coronavirus (BCV) 12.03%, bovine para-influenza 3 (PI-3) 3.01%, and bovine viral diarrhea virus (BVDV) with 1.50% of positive samples. The less frequently detected viral pathogens were bovine herpes virus type 1 (BHV-1) and bovine adenovirus (BAdV) with 0.75% positive samples each. The new implemented molecular methods can be an important diagnostic tool for laboratories and farmers to improve the therapy, control, and prevention of respiratory disease in cattle herds. Key words: bovine respiratory disease; nasal swab samples; diagnostics; real-time PCR detection; cattle UGOTAVLJANJE PRISOTNOSTI DESETIH PATOGENOV Z METODO PCR V REALNEM ČASU V ODVZETIH VZORCIH NOSNIH BRISOV PRI ŽIVEM GOVEDU Z ZNAKI RESPIRATORNEGA OBOLENJA Bolezni dihal so pri govedu pogosto posledica primarne okužbe z različnimi patogeni, ki pri teletih in tudi v plemenski čredi povzročijo veliko gospodarsko škodo. V letih od 2012 do 2014 smo v 28 govejih čredah odvzeli vzorce nosnih brisov pri obolelih živih živalih. Vzorčenje smo izvedli pri 133 živalih, ki so klinično kazale enega ali več znakov obolenja dihal, povišano telesno temperaturo, kašelj, serozni nosni in očesni izcedek, pospešeno dihanje in povišan zvok ob pregledu pljuč. Vzorce nosnih brisov smo testirali s tremi komercialnimi in eno novo uvedeno laboratorijsko metodo PCR v realnem času na prisotnost nukleinskih kislin desetih različnih patogenov. Prisotnost bakterije Pasteurella multocida smo ugotovili v 58.65 % vseh vzorcev, bakterije Mannheimia haemolytica v 15.04 %, pristonost bakterij Mycoplasma bovis in Histophilus somni pa smo ugotovili v 9.77 % vzorcev. Med iskanimi virusi smo bovini respiratorni sincicialni virus ugotovili v 40.60 % vzorcev, bovini koronavirus v 12.03 % vzorcev, virus parainfluence 3 v 3 %, virus bovine virusne diareje v 1.5 % vzorcev; najmanj pogosto smo ugotovili prisotnost bovinega herpesvirusa 1 in bovinega adenovirusa (0.75 % vseh vzorcev). Novo uporabljene molekularne metode predstavljajo pomembno diagnostično orodje za laboratorije in rejce in v goveji čredi pomagajo pri izbiri ustreznejše terapije, nadzoru in preprečevanju bolezni dihal. Ključne besede: respiratorno oboljenje; vzorci nosnih brisov; diagnostika; PCR v realnem času; govedo

Summary: Malignant catarrhal fever (MCF) is a ubiquitous disease of cattle and other ruminants caused by ovine herpesvirus-2 (OvHV-2) in Europe and other continents and alcelaphine herpesvirus-1(AlHV-1) in Africa. In March 2010 MCF was recognized in small private zoological garden in three American bison (Bison bison) imported from Vienna. They were housed in an enclosure next to llamas, goats and domestic sheep, with direct contact between all mentioned species. Typical clinical signs of the acute head and eye form of MCF were observed, with especially inflammation of conjunctives, oral and nasal mucosa and marked depression. Mortality was 100%. Necropsy findings in one bison were consistent with MCF. Acute ulcerative abomasitis and omasitis, acute hemorrhagic enterotyflitis, acute purulent bronchitis and moderate emaciation were found. The histopathology revealed mixed lymphohistiocytic and neutrophilic vasculitis in the brain, meninges, liver, spleen, heart, lungs and mixed lymphohistiocytic and neutrophilic hepatitis and adrenalitis. OvHV-2 DNA was detected post-mortem in tissue samples by PCR. Direct sequencing of the PCR product confirmed 100% nucleotide identity to OvHV-2 strain BJI035 and closely related to two OvHV-2 positive sheep samples collected in 2007. These are the first case of MCF confirmed by laboratory diagnostic methods in Slovenia. Furthermore, these are also the first cases of MCF in American bison in Slovenia.

Serum and oral swab samples were collected from a persistently infected cow and her calf in a two-month period to test usefulness of oral swab samples for the detection of bovine viral diarrhea virus. Nucleic acids of the virus were detected by two molecular methods: conventional gel-based RT-PCR and commercial real-time RT-PCR. The bovine viral diarrhea virus genome was detected in serum and oral swab samples on days 0, 7, 15, 22, 23, 29, 36, 37, 43, 44, 46, 51, 52, 53, and 57. The dry cotton swabs showed a reduction of diagnostic sensitivity after three days when samples were stored at room temperature (+ 21 °C), but storage of oral swab samples at + 4 °C or in a freezer (< – 15 °C) for at least 10 days had no negative impact on the detection of the virus. No reduction of diagnostic sensitivity was observed when oral swab samples were collected in tubes with a liquid virus transport medium. Oral swabs provide an easy, reliable and cost-effective sampling tool for identification of PI animals, together with RT-PCR methods. The oral swab sampling could be especially useful for screening newborn calves during testing and removing PI animals from bovine viral diarrhea virus-infected herds.

Background: To study bats, as a reservoir for European bat lyssavirus (EBLV) in Slovenia, native bat samples were tested in year 2008. Bats were captured from diff erent locations in Slovenia and blood samples, mouth and brain swabs were collected from live and dead bats. 260 samples of oral swabs and 38 brain samples were tested by specifi c RT-PCR assay to detect lyssavirus genome. Results: 216 blood samples, collected from the same bats, were tested by FAVN (Fluorescent Antibody Virus Neutralization) test to detect the prevalence of lyssavirus antibodies among bats. Virus RNA was not detected in any of the samples, all blood samples werealso negative for specifi c antibodies. Conclusions: Despite the data from this study, EBL viruses can cause fatal infections in humans and all bats involved in contact incidents with humans should be tested to determine whether the victim was exposed to EBL virus. In order to prevent lyssavirus transmission from bats to humans, all bat handlers and laboratory personnel should be informed about the possible risk of lyssavirus exposure via bats and their vaccination against rabies is strongly recommended .