The first case of Mycobacterium terrae infection in cattle in Bosnia and Herzegovina

Veterinaria, Vol. 64, No. 1, 2015 Research - Short Communications The first case of Mycobacterium terrae infection in cattle in Bosnia and Herzegovina Hajrudin Beširović 1 *, Amer Alić 1, Silvio Spičić 2, Senad Prašović 1, Mirsad Kadrić 1 Abstract We here describe a case of Mycobacterium terrae infection in a cow in a small dairy household. At necropsy of the cow otherwise positive on routine annual tuberculin skin testing, severe multifocal granulomatous dermatitis and panniculitis, and moderate granulomatous (parasitic) colitis were observed. Histopathology revealed granulomatous dermatitis. Bacteria isolated from regional and thoracic lymph nodes, and skin lesions were identified by colony morphology, biochemical testing and molecular methods as M. terrae. Our findings confirm the difficulties that non-tuberculous mycobacteria can cause in vivo diagnosis of mycobacterial infections, especially in conjunction with parasitic infestations. Keywords Cattle Mycobacterium terrae Granulomatous dermatitis Bosnia and Herzegovina 1 Department of Pathology, Faculty of Veterinary Medicine, University of Sarajevo, Sarajevo, Bosnia and Herzegovina 2 Laboratory for Bacterial Zoonosis and Molecular Diagnosis of Bacterial Diseases, Department of Bacteriology and Parasitology, Croatian Veterinary Institute, Zagreb, Croatia *Corresponding author:hajrudin.besirovic@vfs.unsa.ba Introduction Non-tuberculous mycobacteria (NTM) are group of atypical mycobacteria not belonging to those classified in the Mycobacterium tuberculosis complex. Their potential pathogenicity for humans and animals has long been recognized, especially in individuals with immunodeficiencies (1). Unlike in humans, the distribution and epidemiology of NTM in animals is poorly understood (7). However, many of NTM species were isolated from different animal hosts (1). It is assumed that natural infections with these mycobacteria may influence the development of tuberculosis in different animal hosts (3). Also, immune responses induced by NTM may interfere with current diagnostic tests (skin test and γ-interferon test) for bovine tuberculosis (12, 13), and with efficacy of the M. bovis BCG vaccine against tuberculosis (3). Mycobacterium terrae is a slow-growing, non-photochromogenic species of Mycobacterium (10). Although commonly regarded as non-pathogenic in humans, M. terrae isolates can cause serious skin infections (hand infections) as well as the infections of the joints, tendons, lung, gastrointestinal and genitourinary systems. These infections are regarded as relatively resistant to antibiotic therapy (4, 9). In animals, M. terrae was isolated in two out of 570 nasal and pharyngeal swab samples from cattle and buffaloes in South Africa, and represents one of four most frequently isolated NTM (7). It was isolated from tuberculosis-like skin lesions on the udder of a cow, but its pathologic role is still unknown (17). Little is known about cattle tuberculosis in Bosnia and Herzegovina. It was considered to be eradicated from the former Yugoslav Federation in 1973, with sporadic outbreaks recorded until 1990. No information is present about the war period (1992 1996), and only two outbreaks were documented between 1996 and 1999 (14). Recently, several outbreaks of bovine tuberculosis caused by M. caprae were recorded in three different municipalities (Livno, Tomislavgrad and Vitez) of Bosnia and Herzegovina (2). To the authors knowledge, there is no data about animal NTM in Bosnia and Herzegovina. Thus, the aim of the present report is to describe a case of M. terrae infection in a cow on a small dairy household in the municipality Vitez. Material and Methods A cow in a small household herd in the municipality Vitez in Central Bosnia and Herzegovina was tested positive on routine annual tuberculin skin test (TST). Later, comparative tuberculin skin test confirmed the previous result. Prior to euthanasia the cow was clinically examined. Later, complete necropsy of the cow was carried out near the farm. Samples collected for histopathology (subcutaneous granulomatous lesions, regional and thoracic lymph nodes, and colon) were fixed in 10% buffered neutral formalin for 24 hours, routinely processed and embedded in paraffin blocks. Multiple semithin sections (4-5 micrometers) were cut, stained with hematoxylin, eosin and Ziehl-Neelsen (Z-N) for microscopic evaluation. Following homogenization, decontamination and concentration, the material (subcutaneous granulomatous lesions and lymph nodes) was inoculated onto four Löwenstein-Jensen slants (two of them supplemented with glycerol) and one Stonebrink slant, which were checked for growth once a week for eight weeks (18). A PCR test targeting the heat shock protein 65 (hsp65) gene was employed in order to identify the colonies as the members of the genus Mycobacterium. Primer set TB1 (5 -GAG-ATC-GAG-CTG-GAG-GAT-CC- 3 ) and TB2 (5 -AGC-TGC-AGCCCA-AAG-GTG-TT-3 ) and the protocol described previously (8). Later, isolates were characterised to the species level. The identification 31

The first case of Mycobacterium terrae infection in cattle in Bosnia and Herzegovina 32/35 of isolated NTM was performed by means of DNA hybridization test (GenoType c CM/AS; Hain Lifescience) and by phenotypical methods. GenoType c CM/AS was performed according to manufacturer s instructions (18). Test procedures include DNA isolation from cultivated mycobacteria, amplification using biotin-labeled primers and reverse hybridisation. The final identification of these mycobacterial isolate at species level was done by niacin production, growth rate on different temperatures (25 C, 31 C, 37 C and 42 C), morphology of colonies, pigment production in the dark and after exposure to light, and standard biochemical tests like catalase, nitrate reduction, Tween 80 hydrolysis at 5 and 10 days, arylsulphatase at 3 and 21 days, growth on Lowenstein Jenson (L-J) medium containing 5% NaCl and utilization of carbon sources (citrate, mannitol, inositol) (11). Results The cow was healthy and in good body condition. No clinical signs of chronic illness were noted. No reactions were observed on auscultation and percussion of the chest cavity. At necropsy, severe multifocal granulomatous dermatitis and panniculitis were observed. Observed granulomatous foci were up to 2 cm in diameter and filled with granular, yellow caseous material (Fig. 1). Foci were surrounded with thin haemorrhagic rim. Furthermore, moderate to severe granulomatous (parasitic) colitis characterised by numerous up to 0.5 cm nodular foci protruding on the colonic serosal surface, was also noted. Histopathology revealed granulomatous dermatitis characterised with casesous necrosis, mineralization and thick inflammatory infiltrate (lymphocytes, macrophages and neutrophils) surrounding the foci (Fig.2). Z-N staining was negative for acid fast bacteria. In colon, multiple granulomatous foci with centrally located cross sections of nematode parasites (not identified, presumably Bunostomum sp.) were observed. After 32 days of incubation at 37 C on Löwenstein - Jensen slants supplemented with glycerol suspicious colonies were found from the thoracic lymph nodes and granulomatous foci from the skin material. Molecular identification by conventional PCR targeting the hsp65 gene was positive to Mycobacterium sp. Isolate was classified in NTM by positive result by specific hybridization with GenoType c CM/AS. The growth of the strain was examined on Löwenstein-Jensen (L-J) egg medium and 7H10 medium incubated at 25, 31, 37 and 42 C. The strain was growing at 25 C and 37 C but not at 42 C. According to the rate of growth and pigment production, it was classified as non-chromogenic mycobacteria. Due to the positive test for Tween 80 hydrolysis, 68 C and SQ catalase, nitrate reduction, β -galactosidase, and negative test for niacin production, growth in the presence of 5% NaCl, pyrazinamidase production, 3d arylsulphatase, and tellurite reduction it was further characterised as M. terrae. Discussion and conclusion Reports on M. terrae in animals are very scarce, and there is a lack of information on lesions and pathogenesis of Figure 1. Cow, skin, granulomatous focus filled with yellow, granular caseous material, caused by M. terrae. Bar = 1 cm. Krava, koža, granulom ispunjen žutim, granuliranim kazeoznim materijalom, uzrokovan sa M. terrae. Crtica = 1 cm. Figure 2. Cow, skin (subcutis), focal granuloma composed of caseous and mineralized center surrounded with thick infiltrates of lymphocytes, macrophages and neutrophils. Hematoxylin and eosin, 40x. Bar = 100 µm. Krava, koža (subkutis), fokalni granulom sastavljen od kazoznog i mineraliziranog centra ograničenog sa gustom infiltracijom limfocita, makrofaga i neutrofila. Hematoksilin i eozin. 40x. Crtica = 100 µm. infection in animals and humans (6). Also, the ecology and physiology of the NTM are significantly different from those of M. tuberculosis (11, 16). Our finding confirms that the M. terrae like other NTM infection in cattle can interfere with intradermal tuberculin test, especially in conjunction with parasitic infestations, and hamper the in vivo diagnosis of bovine tuberculosis resulting in significant economic losses due to unnecessary restrictions and culling of reactor animals. However, the majority of false positive reactions in this manner have been overcome by single intradermal comparative cutaneous test (SICCT) (5). Having in mind

The first case of Mycobacterium terrae infection in cattle in Bosnia and Herzegovina 33/35 the presence of HIV positive persons in Bosnia and Herzegovina and other parts of population at risk, the infections with NTM deserve more attention. The pathologies in humans reported to be caused by M. terrae contribute to this assumption (4, 9). Most countries in the European Union (EU) achieved bovine tuberculosis free status, while in some EU and non- EU member states tuberculosis remains non-eradicated, but with ongoing eradication programs. In developing countries tuberculosis still remains a public health concern (15). In Bosnia and Herzegovina, the epidemiological status of bovine tuberculosis is still unknown in animals and humans. Our finding represents the first report on isolation of NTM in cattle in Bosnia and Herzegovina. Absence of strict control measures such as annual TST testing, animal movement control, and trade control cause the continued presence of mycobacteria in animal populations. In addition, in most cases, tubercle-like and other granulomatous lesions found in animals at necropsies by local veterinarians and at abattoir inspections are not subjected to histopathology and bacterial culture to exclude tuberculosis or other infectious agents. Furthermore, there is obvious lack of communication between veterinary sector and human health sector on the prevalence of tuberculosis and non-tuberculous infections in animals and humans. Due to the economic problems, the poverty is particularly present in rural population that further contribute to spread of tuberculosis. For a better understanding of the epidemiology, better planning of control measures and eradication of bovine tuberculosis and NTM all TST positive animals should be submitted for necropsy, histopathology and microbiology for cultivation, identification and typisation of precise mycobacterial species involved. These measures will give a better insight into the epidemiological situation of bovine tuberculosis in Bosnia and Herzegovina and hopefully prevent further spread of the disease to the human population. Acknowledgments Authors would like to thank Samir Bogunić and Alma Jahić for technical support (Department of Pathology, Faculty of Veterinary Medicine in Sarajevo). References 1. Bercovier H, Vincent V. (2001): Mycobacterial infections in domestic and wild animals due to Mycobacterium marinum, M. fortuitum, M. chelonae, M. porci-num, M. farcinogenes, M. smegmatis, M. scrofulaceum, M. xenopi, M. kansasii, M. Simiae and M. genavense. Rev. Sci. Tech. Off. Int. 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(1996): Epidemiology of infection by nontuberculous mycobacteria.clin. Microbiol. Rev, 9:177-215. 7. Gcebe N., Rutten V., van Pittius N.C.G., a Michel A. (2013): Prevalence and Distribution of Non-Tuberculous Mycobacteria (NTM) in Cattle, African Buffaloes (Syncerus caffer) and their Environments in South Africa. Transbound. Emerg. Dis, 60;(Suppl. 1): 74 84. 8. Hance A.J., Grandchamp B., Lévy-Frébault V., Lecossier D., Rauzier J., Bocart D., Gicquel B. (1989): Detection and identification of mycobacteria by amplification of mycobacterial DNA. Mol. Microbiol, 7:843-849. 9. Henderson S.M., Baker J., Williams R., Gunn-Moore D.A. (2002): Opportunistic mycobacterial granuloma in a cat associated with a member of the Mycobacterium terrae complex. J. Feline Med. Surg, 5:37-41. 10. Kankya C., Muwonge A., Djone B., Munyeme M., Opuda-Asibo J., Skjerve E., Oloya J., Edvardsen V., Johansen B.T. (2011): Isolation of non-tuberculous mycobacteria from pastoral ecosystems of Uganda: Public Health Significance. BMC Public Health, 11:320. 11. Leao S.C., Martin A., Mejia G.I., Palomino J.C., Robledo J., Telles M.A.S., Portaels F. (2004): Practical handbook for the phenotypic and genotypic identification of mycobacteria. Brugges: Vanden BROELLE. 12. Michel A.L. (2008): Mycobacterium fortuitum infection interference with Mycobacterium bovis diagnostics: natural infection cases and a pilot experimental infection. J. Vet. Diagn. Invest, 20:501-503. 13. Michel A.L., Cooper D., Jooste J., de Klerk L.M., Jolles A. (2011): Approaches towards optimising the gamma interferon assay for diagnosing Mycobacterium bovis infection in African buffalo (Syncerus caffer). Prevent. Vet. Med. 98:142-151. 14. Pavlik I., Ayele W.Y., Parmova I, Melicharek I., Hanzlikova M., Kormendy B., Nagy G., Cvetnić Ž., Ocepek M., Fejzić N., Lipiec M. 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The first case of Mycobacterium terrae infection in cattle in Bosnia and Herzegovina 34/35 173:323-331. 16. Smith D.S., Lindholm-Levy P., Huitt G.A., Heifets L.B., Cook J.L. (2000): Mycobacterium terrae: case reports, literature review, and in vitro antibiotic susceptibility testing. Clin. Infect. Dis, 30:444-453. 17. Thorel M.F., Morand M., Fontaine J.J., Gourreau J.M. (1990). Bovine Nodular Thelitis: a Clinicopathological Study of 20 Cases. Vet. Dermatol, 1:165-170. 18. Špičić S. (2008): Molekularna epizootiologija vrsta Mycobacterium tuberculosis i Mycobacterium avium kompleksa izdvojenih iz ljudi, životinja i okoliša. Doktorska disertacija, Zagreb, Republika Hrvatska, pp. 54-55.

The first case of Mycobacterium terrae infection in cattle in Bosnia and Herzegovina 35/35 Prvi slučaj Mycobacterium terrae infekcije krave u Bosni i Hercegovini Sažetak Uvod i ciljevi Netuberkulozne mikobakterije (NTM) su grupa atipičnih mikobakterija koje ne pripadaju vrstama klasificiranim u Mycobacterium tuberculosis kompleksu. Njihova potencijalna patogenost za ljude i životinje je odavno bila prepoznata, posebno kod individua s različitim imunodeficijencijama. Nasuprot situaciji u humanoj medicini, distribucija i epidemiologija NTM kod životinja je slabo poznata. Medutim, mnoge NTM vrste su izolirane iz različitih životinja. Mycobacterium terrae je spororastuća, nefotohromogena vrsta mikobakterija. Kod životinja M. terrae je medu najčešće izoliranim vrstama NTM. Njena patogenost kod životinja je dokazana u granulomatoznim lezijama na koži kod goveda. Malo se znao tuberkulozi goveda u Bosni i Hercegovini. Od 1970., sa sporadičnim slučajevima do 1990., smatrala se eradiciranom. U posljeratnom periodu, dokumentirana su svega dva slučaja od 1996. do 1999. godine. Molekularnim istraživanjima u nekoliko žarišta u više regiona Bosne i Hercegovine, dobijeni izolati su identificirani kao M. caprae. Zbog nedostatka podataka o NTM kod životinja u Bosni i Hercegovini, ovaj rad ima za cilj prikazati slučaj M. terrae infekcije krave iz malog uzgoja u Općini Vitez. Materijal i metode Krava s malog posjeda u Općini Vitez u centralnoj Bosni je pozitivno reagirala na tuberkulinski kožni test (TKT) u sklopu godišnje akcije tuberkulinizacije goveda. Nakon kliničkog pregleda, životinja je eutanazirana i podvrgnuta obdukciji. Uzorci organa (torakalni regionalni limfni čvorovi i kolon) i ustanovljene lezije (subkutani granuloma) za histopatološku pretragu su fiksirani u 10% neutralnom puferizovanom formalinu u periodu od 24 sata. Nakon toga, tkiva su procesuirana u tkivnom procesoru i ukalupljena u parafinske blokove. Isječci tkiva debljine do 5 mikrona su obojeni hematoksilin i eozinom te Ziehl-Neeelsenom. Mikrobiološki, kultivacijom uzoraka (subkutani granuloma, regionalni i torakalni limfni čvorovi) dobijeni izolati su podvrgnuti klasičnom PCR-u, ciljajući hsp65 (engl. Heat Shock Protein 65) gen, kako bi se dokazala pripadnost rodu Mycobacterium. Nakon toga, specifičnom hibridizacijom DNK pomoću GenoType c CM/AS testa (Hain Lifescience) i fenotipskih i biohemijskih karakteristika, izolat je finalno identificiran. Rezultati i interpretacija Na obdukciji krave pozitivne na TKT ustanovljen je multifokalni teški granulomatozni dermatitis i panikulitis, i umjereni do teški granulomatozni (parazitarni) kolitis. Ustanovljena granulomatozna žarišta su bila ispunjena granuliranim kazeoznim sadržajem, s multiplim mineraliziranim fokusima, a periferno ograničena obilnim infiltratom limfocita, makrofaga i neutrofila. U središtima granuloma u zidu kolona ustanovljeni su multipli presjeci nematodnih parazita (neidentificirani, najvjerovatnije Bunostomum sp.). Identifikacijom hsp65 gena utvrdeno je da izolirane bakterije pripadaju rodu Mycobacterium. Specifičnom hibridizacijom pomoću GenoType c CM/AS testa, fenotipskih i biohemijskih testova, identificirana je vrsta M. terrae. Glavni zaključci Naši rezultati predstavljaju prvi izvještaj o izolaciji NTM iz tkiva goveda u Bosni i Hercegovini. Oni potvrduju da M. terrae, kao i ostale NTM infekcije kod goveda, mogu utjecati na rezultate intradermalnog tuberkulinskog testa, posebno u slučaju parazitarnih koinfekcija, time otežavajući in vivo dijagnostiku tuberkuloze goveda, što rezultira značajnim ekonomskim gubicima, zbog nepotrebnih restrikcija i uklanjanja životinja reaktora. U cilju boljeg razumijevanja epidemiologije, planiranja kontrolnih mjera i eradikacije tuberkuloze goveda i drugih NTM infekcija, sva TKT pozitivna grla trebalo bi podvrgnuti obdukciji, histopatološkoj i mikrobiološkoj pretrazi u svrhu izolacije, identifikacije i tipizacije prisutnih vrsta mikobakterija.