THE PREVALENCE OF TEN PATHOGENS DETECTED BY A REAL-TIME PCR METHOD IN NASAL SWAB SAMPLES COLLECTED FROM LIVE CATTLE WITH RESPIRATORY DISEASE
Abstract
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
References
(1) Garibaldi RA. Epidemiology of community-acquired respiratory tract infections in adults: incidence, etiology, and impact. Am J Med 1985; 78: 32–7.
(2) Snowder GD, Van Vleck LD, Cundiff LV, Bennett GL. Bovine respiratory disease in feedlot cattle: environmental, genetic and economic factors. J Anim Sci 2006; 84: 1999–2008.
(3) Jones C, Chowdhury S. A review of the biology of bovine herpesvirus type 1 (BHV-1), its role as a cofactor in the bovine respiratory disease complex and development of improved vaccines. Anim Health Res Rev 2007; 8: 187–205.
(4) Luzzago C, Bronzo V, Salvetti S, Frigerio M, Ferrari N. Bovine respiratory syncytial virus seroprevalence and risk factors in endemic dairy cattle herds. Vet Res Commun 2010; 34: 19–24.
(5) Ohlson A, Alenius S, Tråvén M, Emanuelson U. A longitudinal study of the dynamics of bovine coronavirus and respiratory syncytial virus infections in dairy herds. Vet J 2013; 197: 395–400.
(6) Büchen-Osmond C. Index of viruses – Adenoviridae. In: The Universal virus database of the International Committee on Taxonomy of Viruses, version 4. New York : Columbia University, 2006. http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fs_index.htm. (April 2016)
(7) Bierbaum S, Forster J, Berner R, et al. Detection of respiratory viruses using a multiplex PCR assay in Germany, 2009/10. Arch Virol 2014; 159: 669–76.
(8) Kim JK, Jeon JS, Kim JW, Rheem I. Epidemiology of respiratory viral infection using multiplex rt-PCR in Cheonan, Korea (2006–2010). J Microbiol Biotechnol 2013; 23: 267–73.
(9) Fukuda M, Kuga K, Miyazaki A, et al. Development and application of one-step multiplex reverse transcription PCR for simultaneous detection of five diarrheal viruses in adult cattle. Arch Virol 2012; 157: 1063–9.
(10) Wolf C, Emanuelson U, Ohlson A, Alenius S, Fall N. Bovine respiratory syncytial virus and bovine coronavirus in Swedish organic and conventional dairy herds. Acta Vet Scand 2015; 57: e2 (1–7). https://actavetscand.biomedcentral.com/articles/ 10.1186/s13028-014-0091-x
(11) Klem T, Gulliksen S, Lie KI, Løken T, Østerås O, Stokstad M. Bovine respiratory syncytial virus: infection dynamics within and between herds. Vet Rec 2013; 173: 476–82.
(12) Härtel H, Nikunen S, Neuvonen E, et al. Viral and bacterial pathogens in bovine respiratory disease in Finland. Acta Vet Scand 2004; 45: 193–200.
(13) Achenbach JE, Topliff CL, Vassilev VB, et al. Detection and quantitation of bovine respiratory syncytial virus using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays. J Virol Methods 2004; 121: 1–6.
(14) Železnik Z. Respiratorna obolenja v pitaliÅ¡Äih in zrejaliÅ¡Äih telet: poroÄilo . Ljubljana : Raziskovalna skupnost Slovenije, 1978.
(15) Železnik Z. Vloga virusov infekcioznega govejega rinotraheitisa in adenovirusov pri nastanku enzootske pljuÄnice pri govedu: poroÄilo. Ljubljana : Raziskovalna skupnost Slovenije, 1979: 10 str.
(16) Hostnik P, Železnik Z. Izbruh okužbe z virusom IBR/IPV. Zbornik Vet Fak 1992: 53–7.
(17) Toplak I, RihtariÄ D, Hostnik P, Paller T, PogaÄnik M. Genetska tipizacijga mikrobioloÅ¡kega druÅ¡tva: knjiga povzetkov. Bled, 2014: 48.
(18) Grom J, Hostnik P, Toplak I, BarliÄ-Maganja D. Molecular detection of BHV-1 in the artificially inoculated semen and the semen of latently infected bull treated with dexamethasone. Vet J 2006; 3: 539–44.
(19) Wong K, Xagoraraki I. Quantitative PCR assay to survey the bovine adenoirus levels in environmental samples. J Appl Microbiol 2010; 109: 605–1.
(20) Toplak I, Hostnik P, BarliÄ-Maganja D, Grom J. Study on prevalence of bovine viral diarrhoea virus (BVD) infections in breeding herds in Slovenia during 1997-2001. Vet Nov 2002; 10: 397–404.
(21) Baker JC, Werdin RE, Ames TR, et al. Study on etiologic role of bovine respiratory syncytial virus in pneumonia of dairy calves. J Am Vet Med Assoc 1986; 189: 66–70.
(22) West K, Bogdan J, Hamel A, et al. A comparison of diagnostic methods for the detection of bovine respiratory syncytial virus in experimental clinical specimens. Can J Vet Res 1998; 62: 245–50.
(23) Timsit E, Le Dréan E, Maingourd C, et al. Detection by real-time RT-PCR of a bovine respiratory syncytial virus vaccine in calves vaccinated intranasally. Vet Rec 2009; 165: 230–3.
(24) Sibley SD, Goldberg TL, Pedersen JA. Detection of known and novel adenoviruses in cattle wastes via broad-spectrum primers. Appl Environ Microb 2011; 77: 5001–8.
(25) Kirchhoff J, Uhlenbruck S, Goris K, Keil GM, Herrler G. Three viruses of the bovine respiratory disease complex apply different strategies to initiate infection. Vet Res 2014; 45: e20 (1–12) https://veterinaryresearch.biomedcentral.com/articles/10.1186/1297-9716-45-20
(26) Virtala AMK, Mechor GD, Gröhn YT, Erb HN, Dubovi EJ. Epidemiologic and pathologic characteristics of respiratory tract disease in dairy heifers during the first three months of life. J Am Vet Med Assoc 1996; 208: 2035–42.
(27) Toplak I, RihtariÄ D, Hostnik P, Mrkun J. The usefulness of two molecular methods for the detection of persistently infected cattle with bovine viral diarrhea virus using oral swab samples. Slov Vet Res 2015; 52: 23–30.
