Učinkovitost sljedeće generacije sekvenciranja u dijagnostici bakterijskih zoonoza

Brucella, an extremely diverse but yet genetically highly homogenous genus of bacteria, has been a puzzle for scientists for many decades. These bacteria remain a prominent public health issue, particularly in the Balkan region. Correctly identifying and understanding the pathogen is a vital step in the epidemiology and epizootiology of any bacteria. Identification can be challenging, especially in the case of zoonotic species. This study aimed to implement fourth-generation sequencing in the typing of 11 Brucella suis strains kept in our archive and to compare this method to the classical and non-sequencing based molecular methods used to date. Classical biotyping is highly subjective and gave inconclusive results for 3 strains. Molecular methods used were multiplex PCR and RFLP methods since no one method can identify both species and biovar which is vital in the case of Brucella suis infections. Species and biovars of all the strains were successfully confirmed and in concordance with biotyping results. Oxford Nanopore long-read sequencing was used on a MinION device for next-generation sequencing (NGS). Various algorithms were implemented for genome assembly and BioNumerics 8.0 software was used for MLST identification and analysis. MLST 21 was used for biovar identification and epidemiological comparison of tested strains. The assembled genomes were 3,2 Mb in size and assembled into two chromosomes. MLST 21 analysis placed our strains into species and biovar clusters in concordance with other molecular tests used. To the extent of our knowledge, this is the first documented use of long-read sequencing in Brucella suis identification in this region. We conclude that bacteriological biotyping is outdated and host-specific identification in this genus is incorrect and that molecular characterisation is always the safer, faster and more suitable option. MinION sequencing proved to be a strong, accessible solution for species determination. Future study is required to determine how detailed genome information it can give, considering the error rate.Rod Brucella biološki je iznimno raznolik, ali genetski vrlo homogen rod bakterija te je već desetljećima znanstvenicima nepoznanica. Ove bakterije su veliki javno-zdravstveni problem, a osobito na Balkanu. Pravilno prepoznavanje i razumijevanje patogena ključan je korak u epidemiologiji i epizootiologiji bilo koje bakterijske vrste, čija identifikacija može biti izazovna, osobito u slučaju zoonotskih vrsta. Cilj je ovog rada bio implementirati sekvenciranje četvrte generacije u tipizaciji 11 sojeva Brucella suis koje se čuvaju u našoj arhivi te ovu metodu usporediti s klasičnim i molekularnim metodama koje se trenutačno primjenjuju, a ne zasnivaju se na sekvenciranju. Klasično je biotipiziranje vrlo subjektivno i dalo je podvojene rezultate za 3 soja. Od molekularnih metoda koristili smo višestruku lančanu reakciju polimerazom (engl. Polymerase Chain Reaction, PCR) i polimorfizam duljine restrikcijskih fragmenata (engl. Restriction Fragment Lenght Polymorphism, RFLP) budući da niti jedna od metoda ne može zasebno identificirati i vrstu i biovar, a što je važno u slučaju Brucella suis infekcije. Vrsta i biovar svih sojeva uspješno su potvrđene i u skladu s rezultatima biotipizacije. Sekvenciranje sljedeće generacije (engl. Next Generation Sequencing, NGS) provodili smo na Oxford Nanopore MinION uređaju koji sekvencira duge lance DNK. Za sastavljanje genoma rabljeni su različiti algoritmi, a za identifikaciju i analizu rezultata MLST-a korišten je softver BioNumerics 8.0. MLST 21 je korišten za identifikaciju biovara i epidemiološku usporedbu ispitivanih sojeva. Genomi su bili veličine 3,2 Mb i sastavljeni u dva kromosoma. Analiza MLST 21 smjestila je naše sojeve u vrsne i biovarne skupine u skladu s drugim korištenim molekularnim testovima. Koliko je nama poznato, ovo je prva dokumentirana uporaba sekvenciranja dugih lanaca DNK u identifikaciji Brucella suis u jugoistočnoj Europi. Zaključujemo da je bakteriološka biotipizacija zastarjela i da je identifikacija biovara u ovom rodu, ovisno o domaćinu, netočna te da je molekularna karakterizacija uvijek sigurnija, brža i prikladnija opcija. MinION sekvenciranje pokazalo se kao vrlo pristupačno rješenje za određivanje vrste i biovara Brucella suis. Daljnja su istraživanja potrebna da bi se ustvrdilo koliko detaljne informacije o genomu može dati, imajući u vidu značajniji postotak pogreške prilikom sekvenciranja

Sumnjivi i pozitivni slučajevi tuberkuloze goveda u Republici Hrvatskoj od 2017. do 2020. godine

During regular implementation of the bovine tuberculosis-free cattle herd certification programme in the period from 2017 to 2020, the Laboratory for Bacterial Zoonoses and Molecular Diagnostics of Bacterial Diseases of Croatian veterinary institute Zagreb, Croatia tested material from 161 cattle from 27 holdings in 11 counties. The material was submitted following findings of pathoanatomical changes detected in the slaughter line suggesting tuberculosis, or after a positive reaction of cows to the tuberculin comparative methods. Species from the M. tuberculosis complex (M. bovis and M. caprae) were isolated from samples of 58 bovines (36%) from 16 holdings in eight counties. M. caprae was confirmed in 55 bovines (34%) originating from 13 holdings in seven counties, and M. bovis in three bovines (2%), each from a different holding in a different county. Saprophytic mycobacteria were isolated from four bovine samples (2.5%) from three holdings in two counties, i.e., M. gordonae (1), M. celatum (1) and two unidentified species (M. sp.). Based on the obtained results, we can conclude that the main causative agent of bovine tuberculosis in the Republic of Croatia is M. caprae, which confirmed previous findings. Control of bovine tuberculosis in the Republic of Croatia is still needed and, in the future, should be further suppressed using tuberculinisation, controls on slaughter lines, depopulation of infected herds, and etiological determination of the causative agents.U razdoblju od 2017. do 2020. godine tijekom redovitog provođenja programa certifikacije stada goveda slobodnih od tuberkuloze goveda bakteriološki je u Laboratoriju za bakterijske zoonoze i molekularnu dijagnostiku bakterijskih bolesti Hrvatskog veterinarskog instituta u Zagrebu, Hrvatska bio pretražen materijal 161 goveda iz 27 različitih uzgoja u 11 županija. Materijal je dostavljen nakon nalaza patoanatomskih promjena na liniji klanja koje upućuju na tuberkulozu ili komparativnom metodom nakon pozitivne reakcije goveda na tuberkulin. Vrste iz M. tuberculosis kompleksa (M. bovis i M. caprae) su izdvojene iz materijala 58 goveda (36 %) iz 16 uzgoja u osam županija. M. caprae je ustvrđen u 55 goveda (34 %) podrijetlom iz 13 uzgoja u sedam županija, a M. bovis u tri goveda (2 %) iz dva uzgoja u dvije županije. Saprofitske mikobakterije izdvojene su iz četiri uzorka goveda (2,5 %) dostavljenih iz tri uzgoja (21 %) u dvije županije i to M. gordonae (1), M. celatum (1) i dvije neidentificirane vrste (M. sp.). Na temelju dobivenih rezultata možemo zaključiti da je glavni uzročnik tuberkuloze goveda u Republici Hrvatskoj M. caprae, a koji je bio uzročnik i u prijašnjim istraživanjima. Kontrola tuberkuloze goveda u Republici Hrvatskoj i dalje je potrebna te bi se i u budućnosti trebala suzbijati na temelju tuberkulinizacije, kontrolama na liniji klanja te depopulacijom inficiranih stada i etiološkim dokazom vrste uzročnika

MELITOCOCCOSIS IN THE REPUBLIC OF CROATIA

Background: Melitococcosis is one of the most widespread zoonoses worldwide. In the period from 2009 to 2013, comprehensive melitococcosis testing was conducted in the Republic of Croatia. Methods and results: During the testing, the Rose Bengal test was applied to 344019 blood samples of sheep and goats, and positive reactions were confirmed in 1143 (0.3%) of samples. The complement fixation test (confirmatory test) was conducted on 43428 samples, with positive reactions confirmed in 768 (1.8%) of samples. The organs and tissues of 336 sheep and goats were inspected bacteriologically, and Brucella sp. was isolated in 15 (4.5%) of samples. Positive serological and bacteriological reactions were confirmed in the Karlovac, Lika-Senj and Split-Dalmatia Counties. Bacteriological and molecular techniques (Bru-up/Bru-low and Bruce-Ladder) in isolates proved the presence of Brucella melitensis biovar 3. Conclusion: On the basis of this study, it can be concluded that Croatia has a favourable situation concerning the infection of ruminants with B. melitensis, and that ongoing controls of the disease are necessary

Molecular epidemiology of Brucella melitensis strains causing outbreaks in Croatia and Bosnia and Herzegovina

The most recent data on the incidence of brucellosis in Southeast Europe prove the persistence of this zoonosis in the area, regardless of constant efforts at controlling it as one of the most dangerous zoonoses. Forty-three Brucella melitensis strains were collected from cattle, sheep, goats and humans from Croatia as well as Bosnia and Herzegovina between 2009 and 2015. The strains were identified and genotyped in order to determine their epidemiological background. Standard biotyping methods and Bruce-ladder were used to identify the strains. Genotyping was done using multilocus variable number tandem repeats analysis (MLVA) on 16 and multilocus sequence typing analysis (MLST) on nine loci. Results were compared to each other and to internationally available data. Twenty- five novel genotypes and two sequence types were identified. All tested strains, apart from vaccine and reference strains, showed very close phylogenetic and geographic relationships. The genotyping results indicate the endemicity of brucellosis in this region. MLST showed no variation, confirming the stability of housekeeping genes. The results confirm already established routes of disease spread in this area, showing that a more detailed and vigorous control of this zoonosis is necessary

Seroprevalence of leptospirosis in cattle in the Republic of Croatia

Leptospiroza je re-emergentna zoonoza ra- sprostranjena po čitavom svijetu, s izuzetkom polarnih krajeva, a najčešća je u tropskim i suptropskim područjima s velikom količinom padalina. Ekonomsko značenje ove bolesti oči- tuje se pobačajima, mrtvorođenčadi te smanje- njenom proizvodnjom mlijeka. Zbog svega na- vedenog, provođenje nadzora i mjera kontrole od osobite je važnosti za suzbijanje te važne zoonoze. Sukladno Naredbi o mjerama zašti- te životinja od zaraznih i nametničkih bolesti i njihovom financiranju tijekom 2015. i 2016. godine na leptospirozu su pretražene krvi kra- va i junica kod pobačaja te serološka pretraga krvi svih živih životinja u zaraženom dvori- štu, odnosno zaraženom objektu dva puta u razmaku od 21 dan nakon završenog liječenja i provedbe svih naređenih mjera. Tijekom 2015. i 2016. godine na leptospirozu su pretražena 2 882 krvna seruma goveda pordrijetlom iz 19 županija u Republici Hrvatskoj. Pozitivne re- akcije u 2015. godini utvrđene su u 349 goveda (21,6%), a 2016. u 192 goveda (15,2%). Tijekom 2015. najviše pozitivnih uzoraka utvrđeno je u Sisačko-moslavačkoj 88 (53,7%), Bjelovarsko- bilogorskoj 47 (13,5%), Zagrebačkoj 39 (16,9%) i Ličko-senjskoj županiji 35 (46,1%). Najzastu- pljeniji serovarovi bili su sv. Saxkoebing 136 (19,4%), sv. Sejroe 131 (18,7%), sv. Hardjo 107 (15,3%), i sv. Hardjobovis 104 (14,8%). U 2016. godini pretraženo je 1 265 uzoraka, a najveća prevalencija zabilježena je u Osječko-baranj- skoj 39 (30,5%), Bjelovarsko-bilogorskoj 36 (17,4%), Vukovarsko-srijemskoj 31 (27,4%) i Zagrebačkoj županiji 27 (16,4%), a od serova- rova najzastupljeniji su bili: sv. Saxkoebing 67 (17,4%), sv. Sejroe 65 (16,9%), sv. Hardjobovis 61 (15,8%), i sv. Hardjo 60 (15,6%).Bovine leptospirosis occurs throughout the world, though it is most common in warm climates. This is an economically significant disease that results in abortions, stillbirths and losses in milk production. Therefore, the implementation of surveillance and control measures is particularly important for the suppression of this important zoonosis. According to annual orders issued by the Ministry of Agriculture for 2015 and 2016, all cases of abortion in cattle must be reported and the blood of each animal tested serologically for leptospirosis. Pursuant to the Croatian legislation, serological blood tests are also performed twice following treatment, at intervals of 21 days. In 2015, 1 617 cattle blood samples were tested, with 349 positive reactions, while in 2016, 1 265 cattle blood samples were tested with 192 positive reactions. All sera were tested by the microscopic agglutination test (MAT) for the presence of 12 leptospiral antigens. An initial seral dilution of 1:100 was considered the cut-off point for a positive reaction. During 2015, the highest prevalence was found in the following counties: Sisak-Moslavina 88 (53.7%), Bjelovar-Bilogora 47 (13.5%), Zagreb 39 (16.9%) and Lika-Senj 35 (46.1%). In 2016, the highest prevalence was reported in the counties: Osijek-Baranja 39 (30.5%), Bjelovar- Bilogora 36 (17.4%), Vukovar-Srijem 31 (27.4%) and Zagreb 27 (16.4%). Positive reactions for leptospirosis during 2015 and 2016 were not found in the counties: Istria, Primorje- Gorski Kotar, Zadar, Šibenik-Knin, Split- Dalmatia, Dubrovnik-Neretva or Međimurje. The most frequent serovars in cattle in 2015 and 2016 were sv. Saxkoebing (18.4%), sv. Sejroe (17.8%), sv. Hardjo (15.5%), and sv. Hardjobovis (15.3%). The results indicate that cattle in continental Croatia are more exposed to leptospiral infection than animals from coastal counties, due to natural foci of this disease (Lonjsko Polje and Kopački Rit wetlands) and the holding of cattle, pigs and horses together. In order to control the spread of the disease within and among holdings, it is necessary to continue to disease control measures, to treat holdings with the aim of reducing the carrier state and availability, and to alter management practices to minimise the possibility of re-infection

Identifikacija i MLVA genotipizacija vrste Chlamydia abortus iz slučajeva pobačaja malih preživača u Hrvatskoj

In addition to zoonotic potential, Chlamydia (C.) abortus is a very important bacterium causing serious disease in small ruminants. The main outcome of the disease is abortion in the late stages of pregnancy and the economic impact for farms is significant. During a three-year period (2015–2017), 191 vaginal swabs, 24 placentas, 210 foetal organs and 2 milk samples from small ruminant abortion cases were tested for C. abortus by real-time PCR. Positive samples were detected on eight sheep farms and two goat farms, with 8.4% of total samples testing positive samples. These samples were characterised using the MLVA method, and a single MLVA genotype (genotype [2]) was identified from sheep and goat samples, suggesting highly conserved C. abortus strains among the national flock. This study is the first description of C. abortus as a causative agent of abortion in goats in Croatia. More detailed study is required to recognize the epidemiological relevance of the abortion chlamydiosis. An open register of farms with defined health status should be established for each farm at the national level for better disease(s) control.Osim zoonotskog potencijala, vrsta Chlamydia (C.) abortus je vrlo važna bakterija koja može prouzročiti tešku bolest malih preživača. Dominantni klinički znak bolesti je pobačaj u kasnom stadiju graviditeta sa znatnim ekonomskim učinkom za farme. Tijekom trogodišnjeg razdoblja (2015.-2017.) metodom Real time (RT) PCR na C. abortus testiran je 191 vaginalni obrisak, 24 placente, 210 organa fetusa i 2 uzorka mlijeka iz slučajeva pobačaja malih preživača. Pozitivni uzorci ustvrđeni su u 8 farmi ovaca i 2 farme koza, što ukupno predstavlja 8,4 % pozitivnih uzoraka. Ovi uzorci su tipizirani metodom MLVA i identificiran je jedinstveni genotip MLVA (genotip [2]). Dobiveni rezultat ukazuje na visok stupanj podudarnosti sojeva C. abortus u nacionalnom stadu ovaca i koza. Ovo istraživanje prvi je opis vrste C. abortus kao uzročnika pobačaja u koza u Hrvatskoj. Temeljem ustvrđenog rezultata bilo bi uputno provesti daljnja, detaljnija istraživanja da bi se prepoznalo epidemiološko i ekonomsko značenje infekcija vrstom C. abortus. Radi kvalitetnijeg nadzora bolesti potrebno je uspostaviti otvoreni registar gospodarstava s definiranim zdravstvenim statusom za svaku pojedinu farmu

Mastitis u krava prouzročen brzorastućim mikobakterijama iz okoliša

Rapid-growth mycobacteria were isolated from two cases of cow mastitis with similar clinical appearance and within a narrow time frame. Mycobacteria were isolated on blood esculine agar. The isolated mycobacteria were Gram stained, Ziehl-Nielsen stained and tested for growth at 25°C, 37°C and 42°C, iron uptake, growth on Löwenstein-Jensen (LJ) agar with and without 5% NaCl, arylsulphatase (3 days), tween 80 hydrolysis, tellurite reduction, nitrate reductase and niacin synthesis. Molecular identification was performed using the Mycobacteria GenoType CM and AS tests (Hain Diagnostika, Nehren, Germany). One isolate was additionally sequenced for the hsp65, rpoB, 16S rRNA gene sequence and transcribed spacer sequence (ITS) DNA. Susceptibility testing of isolates was performed on the Sensititre Rapmycol plate (TREK Diagnostic Systems Ltd.) for trimethoprim/sulfamethoxasole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin / clavulanic acid, amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, tobramycine and clarythromycine. Gram-positive acid-resistant rods were observed in stained smears. Both strains grew at 25°C, 37°C and 42°C on LJ medium, and on LJ medium containing 5 % NaCl. The conventional biochemical tests for iron uptake, arylsulphatase (3 days), Tween 80 hydrolysis, tellurite reduction and nitrate reductase were positive, while the niacin test was negative. Both isolates were identified by the GenoType Mycobacterium CM as Mycobacterium fortuitum II/ Mycobacterium mageritense, while application of the GenoType Mycobacterium AS kit identified both isolates as belonging to the species Mycobacterium smegmatis. Analysis of the isolate sequences (strain DS) for 16S ribosomal RNA confirmed a 100% identical result with Mycobacterium smegmatis strain INHR2. According to the CLSI criteria, both strains were sensitive to sulfametoxazole/trimethoprim, linezolid, doxicycline, amikacin and tobramycin. The strains differed in their sensitivity to cefoxitim, and both strains were resistant to clarithromycin. There was a strong difference between the isolates in sensitivity toward cefoxitime and tigecycline.Brzo-rastuće bakterije iz roda Mycobacterium izdvojene se iz dva klinički slična slučaja mastitisa krava u relativno kratkom vremenu. Mikobakterije su izdvojene na krvnom agaru s dodatkom eskulina, obojene po Grammu i Ziehl-Nielsenu te im je provjerena sposobnost rasta pri 25 °C, 37 °C i 42 °C, korištenje željeza, sposobnost rasta na Löwenstein-Jensen agaru bez i s dodatkom 5 % NaCl, tvorba arilsulfataze, sposobnost hidrolize tween 80, redukcije telurita i tvorbe nitrat reduktaze i niacina. Molekularna identifikacija izolata provedena je korištenjem testova Mycobacteria GenoType CM i AS. Odsječci gena hsp65, rpoB, 16S rRNK te tzv. (engl. transcribed spacer sequence (ITS)) jednog izolata su sekvencirani. Osjetljivost izolata provjerili smo pomoću SENSITITRE RAPMYCOL ploča (TREK Diagnostic Systems Ltd.) prema trimetoprim/sulfametoksazolu, linezolidu, ciprofloksacinu, imipenemu, moksifloksacinu, cefepimu, cefoksitinu, amoksicilinu s klavulanskom kiselinom, amikacinu, ceftriaksonu, doksiciklinu, minociklinu, tigeciklinu, tobramicinu i klaritromicinu. Gram-pozitivne acido-rezistentne štapićaste bakterije uočili smo u obojenim razmascima. Oba soja rasla su pri temperaturama 25 °C, 37 °C i 42 °C te na podlozi LJ s i bez dodatka 5  % NaCl. U oba soja utvrdili smo sposobnost korištenja željeza, tvorbe arilsulfataze, hidrolize Tween 80, redukcije telurita i tvorbe reduktaze nitrata. Sojevi nisu tvorili niacin. Oba izolata su testom GenoType Mycobacterium CM identificirana kao Mycobacterium fortuitum II/ Mycobacterium mageritense dok su GenoType Mycobacterium AS testom identificirana kao Mycobacterium smegmatis. Sekvencijskom analizom odsječka gena 16S ribosomske RNK soja DS i usporedbom s javno dostupnim izolatima, utvrdili smo potpunu podudarnost sa sojem Mycobacterium smegmatis soj INHR2. Prema CLSI kriterijima, oba izolata su osjetljiva prema sulfametoksazolu s trimetoprimom, linezolidu, doksiciklinu, amikacinu i tobramicinu, a rezistentna prema klaritromicinu. Razlike između sojeva očitovale su se u osjetljivosti prema cefoksitimu i tigeciklinu

Whole-genome sequence of the first sequence type 27 Brucella ceti strain isolated from European waters

ABSTRACT Brucella spp. that cause marine brucellosis are becoming more important, as the disease appears to be more widespread than originally thought. Here, we report a whole and annotated genome sequence of Brucella ceti CRO350, a sequence type 27 strain isolated from a bottlenose dolphin carcass found in the Croatian part of the northern Adriatic Sea. </jats:p

Presence of Coxiella burnetii in raw milk from milk vending machines in the Republic of Croatia

C. burnetii uzročnik je Q-groznice u ljudi i predstavlja patogen od javno zdravstvenog značenja. Rezervoar bolesti su goveda, koze, ovce i druge životinje. Mlijeko iz mljekomata predstavlja jedan od mogućih izvora ovog patogena posebice u slučajevima kada se ono konzumira bez prethodne toplinske obrade. U nedostatku podataka o prisutnosti C. burnetii u sirovom mlijeku provedeno je prvo istraživanje prisutnosti ovog uzročnika u uzorcima sirovog mlijeka iz mljekomata u Hrvatskoj te je po prvi put uzročnik dokazan primjenom molekularne metode. Istraživanje je provedeno u dva ciklusa u veljači i lipnju te je utvrđena prevalencija od 17,54% (17,86 i 17,24%) uzoraka mlijeka, a kod tri proizvođača mlijeka ponovljeno u ovim uzorcima uzorkovanim u vremenskom razmaku od četiri mjeseca. Preporučuje se provoditi kontinuirani monitoring ovog patogena u stadima i provođenje preventivnih veterinarsko sanitarnih mjera prilikom mužnje, prerade i prodaje sirovog mlijeka s ciljem zadržavanja ove relativno niske prevalencije te sprječavanja širenja ovog patogena koji se kad jednom dospije u stado teško uklanja.C. burnetii is known as the cause of human Q-fever disease and as such is a pathogen of public health interest. Disease reservoirs are cattle, goats, sheep and other animals. Raw milk from vending machines is a potential source of this bacteria. For the first time in Croatia, raw milk from vending machines was sampled for molecular analyses on C. burnetii and this pathogen was confirmed for the first time. Due to a lack of evidence of the presence of C. burnetii in raw milk, this study included almost all milk vending machines in Croatia and was conducted in two sampling periods, in February and June. The mean prevalence of C. burnetii in raw milk was 17.54% (17.86 and 17.25% by sampling period). However, it is of particular concern that this pathogen was found in both samples from three producers. Monitoring of C. burnetii and veterinary and sanitary measures during milking, processing and selling raw milk are recommended. This would assist in maintaining this relatively low prevalence and preventing the spread to other herds, where it will be very difficult to eradicate after pathogen first herd introduction

Reservoirs of Mycobacterium tuberculosis complex and their significance in the infection of animals and humans

Patogene mikobakterije imaju svoje domaćine, a većina njih i prirodne rezervoare u prirodi. Tuberkuloza prouzročena vrstom M. tuberculosis i dalje ostaje glavni uzročnik tuberkuloze u svijetu. M. bovis ima najrašireniji raspon prirodnih rezervoara od svih poznatih patogena. Različite vrste divljih životinja predstavljaju trajan rezervoar infekcije vrstom M. bovis za domaće životinje i ljude. U novije vrijeme otkrivene su i nove vrste mikobakterija (M. caprae, M. pinnipedii, M. mungi, M. orygis i M. suricattae) koje po svojim karakteristikama pripadaju u kompleks M. tuberculosis. Sve navedene vrste mikobakterija imaju svoje rezervoare u prirodi i kao zoonoze predstavljaju stalnu prijetnju za infekciju ljudi. M. leprae prouzroči lepru u ljudi. Dugo vremena se mislilo da je samo čovjek domaćin i rezervoar uzročnika. Danas je poznato da su prirodni rezervoari lepre devetopojasni pasanci (Dasypus novemcinctus) u SAD i Južnoj Americi, crvene vjeverice (Sciurus vulgaris) u Velikoj Britaniji i različite vrste majmuna u Africi i Aziji.Pathogenic micobacteria have their hosts and most also have natural reservoirs. Tuberculosis caused by M. tuberculosis remains the main cause of tuberculosis in the world. M. bovis has the widest natural reservoir range of all known pathogens. Different wildlife species represent a permanent reservoir of M. bovis infection for domestic animals and humans. New species of mycobacteria have recently been discovered (M. caprae, M. pinnipedii, M. mungi, M. orygis and M. suricattae) which by their characteristics belong to the M. tuberculosis complex. Each of these mycobacteria species have their own reservoirs in nature, and as zoonoses, present a constant threat to humans. M. leprae causes leprosy in humans. It was previously thought that only men were the host and reservoir of the causative agent. Today, it is well-known that natural reservoirs are nine banded armadillo (Dasypus novemcinctus) in the United States and South America, red squirrels (Sciurus vulgaris) in the United Kingdom and various types of monkeys in Africa and Asia