Detection and molecular characterization of Mycobacterium microti in wild boar. from northern Italy

Transcription

1 JCM Accepts, published online ahead of print on 28 May 2014 J. Clin. Microbiol. doi: /jcm Copyright 2014, American Society for Microbiology. All Rights Reserved. Detection and molecular characterization of Mycobacterium microti in wild boar from northern Italy M. Beatrice Boniotti 1 #, Alessandra Gaffuri 1, Daniela Gelmetti 1, Silvia Tagliabue 1, Mario Chiari 1, Anna Mangeli 1, Matteo Spisani 2, Claudia Nassuato 3, Lucia Gibelli 1, Cristina Sacchi 1, Mariagrazia Zanoni 1 and M. Lodovica Pacciarini 1 1 Istituto Zooprofilattico Sperimentale della Lombardia e dell Emilia-Romagna: Centro Nazionale di Referenza per la Tubercolosi Bovina, Brescia, Italy. 2 Azienda Sanitaria Locale della provincia di Brescia, Brescia, Italy. 3 Unità Organizzativa Veterinaria, Direzione Generale Salute, Regione Lombardia, Piazza Città di Lombardia, Milano, Italy Keywords: Mycobacterium microti, wild boar, bovine tuberculosis, pathology, genotyping Running Head: Mycobacterium microti in wild boar # Corresponding author: mariabeatrice.boniotti@izsler.it

2 ABSTRACT Approximately 23,000 hunter-harvested wild boars from the pre-alpine area of northern Italy were examined for tuberculosis over a nine year period ( ). Retropharyngeal and mandibular lymph nodes from the wild boars were examined grossly, and 1,151 of the lymph nodes were analyzed in our laboratory by histology (728) and culture isolation (819). Mycobacterium tuberculosis complex (MtbC)-specific PCR (1,142) was used for molecular level detection in tissue samples, as was a gyrb-rflp assay (322). Lesions compatible with tuberculosis, and indistinguishable from those described in cases of Mycobacterium bovis infection, had been observed since Mycobacterium microti was identified directly in 256 tissue samples by the adopted molecular approaches. However, only 26 M. microti strains were obtained by culture isolation due to the well-known difficulties in isolating this slow-growing mycobacterium. During 2006, a prevalence study was performed in two provinces of the area, and the diffusion of M. microti was calculated to be 5.8% (CI P95% : ). Over the following years ( ) the presence of M. microti appeared stable. All isolates were genotyped by spoligotyping and exact tandem repeat analysis (ETR A-F). In addition to the typical vole type (SB0118), a new spoligotype lacking the 43 spacers was found. Spoligotyping was also applied directly to tissue samples, and a geographical cluster distribution of the two spoligotypes was observed. This is the first report studying the diffusion and genetic variability of M. microti in wild boar. INTRODUCTION Mycobacterium microti belongs to the Mycobacterium tuberculosis complex (MtbC), which also includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium caprae, Mycobacterium africanum I and II, Mycobacterium canetti and Mycobacterium pinnipedi. These species are closely related and difficult to distinguish using biochemical properties alone.

3 M. microti was first described in the 1930s as the causative agent of an epizoonotic disease in the wild English field vole (Microtus agrestis) (1). M. microti can infect a variety of mammalians species, including wood mice (Apodemus sylvaticus), bank voles (Clethrionomys glareolus) and shrews (Sorex araneus) (2, 3, 4, 5). Occasionally, it was also found in llamas, cats, pigs, cows and dogs (6, 7, 8, 3, 9, 10, 11, 12, 13). Recently, the widespread host range of M. microti seemed to be increasing with reports of an infected cat population in England (9, 14, 15) and with accounts of new hosts, such as squirrel monkeys (16), merkats (17) and South American camelids (18). In humans, infection by M. microti is infrequent, and to date only 27 cases have been reported in human immunosuppressed and/or immunocompetent patients (19, 20, 21, 22, 23). Identification of M. microti by traditional methods is difficult since the typical curved cell morphology may be lost upon subculturing (6). Moreover, it grows very slowly on solid media, and it shows variable biochemical properties for pyrazinamidase and urease activities as well as niacin accumulation (22, 24). Therefore, M. microti s prevalence, geographical distribution and host range has probably been underestimated. At present, the identification and characterization of M. microti can easily be determined by molecular methods. Spoligotyping (spacer oligotyping) was successfully applied to distinguish M. microti from the other members of the MtbC (9). Several genotypes of M. microti have been recognized by spoligotyping. The most common are the llama type (SB0112), and the vole type (SB0118); both types are involved in animal and human infections (3, 19, 20, 21, 22, 23). Additionally, informative chromosomal deletions, such as RD1 mic and MiD1 were identified as characteristic of M. microti strains (25). In particular, the deletion of MiD1 is responsible for the vole spoligotype, which only contains two spacers. Wild boar harbours many important infectious agents that are transmissible to livestock and other animal species, including humans (26). In particular, the presence of MtbC in the wild boar has been reported in several European countries, including Italy (27, 28, 29, 30, 31, 32, 33, 34).

4 However, the role of the wild boar in the epidemiology of bovine tuberculosis (btb) is still under discussion and can vary among regions. At present, wild boar is considered a reservoir for tuberculosis in Iberia (35, 29), whereas in Italy it is considered a spill over host (27, 36). Recently, a study conducted in Sicily of free-ranging domestic black pigs suggested a role in the maintenance of btb in this ecosystem (37). Additionally, the geographical range and population densities of the Eurasian wild boar are currently increasing in Europe (38), including Italy (39), reaching previously unrecorded levels. To control the population size, hunting has increased, resulting in a rise of wild boar meat consumption (40). The awareness of a greater risk of interspecies disease transmission and zoonosis led to the development of wildlife health-monitoring programs in many countries. In northern Italy, wildlife infections and parasitic diseases have been monitored since As a result of these examinations, the presence of Mycobacteria strains in wild boar was reported (27, 36, 41). Both M. bovis and M. microti were isolated from wild boar in this area, and recently, M. microti was also detected using direct real-time PCR in tissue samples of two wild boars from a neighbouring area in Switzerland (34). Our goal was to assess the diffusion of M. microti in the wild boar population in northern Italy and to provide baseline data for future investigations. Through this study we showed the critical points for detecting M. microti, and we developed a specific diagnostic procedure. Additionally, we molecularly characterized M. microti strains circulating in the area of study, providing new insights on the genomic features of this poorly studied mycobacterium. MATERIALS AND METHODS Study area and sample collection This study was carried out on a wild boar population of the Central Alps in northern Italy and in particular in the provinces of Brescia (BS), Bergamo (BG), Como (CO) and Varese (VA) (Fig. 1).

5 To detect tuberculosis infections in hunted wild boar, we conducted a monitoring program from 2003 to In the early stage, from 2003 to 2005, we collected retropharyngeal and mandibular lymph nodes (242), both with TB-like lesions and without visible lesions (NVL), from samples from BS and BG. On the basis of the results obtained during this period, the Preliminary Study, we carried out two field studies: Study 1. To evaluate the presence of M. microti in wild boar, all the hunted animals during 2006 (602) coming from BS and BG were analysed. This study was limited to two provinces because it was not financially feasible to analyse the number of hunted wild boars from the whole area (2,500 5,000/year). Study 2. During the period from 2007 to 2011, only wild boars (307) with TB compatible lesions were analysed. In this study, samples came from all four provinces, and the culture incubation time was prolonged to 18 weeks. Post mortem examination was performed on approximately 23,000 wild boars by trained vets. Retropharyngeal and/or mandibular lymph nodes coming from wild boars with TB-like lesions (444) and without visible lesion (707), were submitted to other diagnostic assays. Most of these samples were submitted to PCR, while histology and culture isolation were performed only on a part of them (Table 1). Histology was performed on a lymph node from each animal. Culture isolation and IS6110-PCR were carried out using samples of pooled lymph nodes from each animal. When the PCR analysis was positive, the gyrb-rflp assay was performed for confirmation and to identify the MtbC members present in the sample. Isolates were further characterized by biochemical methods and by molecular assays, such as spoligotyping and ETRA-F genotyping. Post mortem examination. Macroscopic inspection was performed on retropharyngeal and mandibular lymph nodes to detect nodular lesions. Nodular lesions, such as caseous, necrotic and calcified nodules, and abscesses were considered as potential tuberculosis lesions. Lymph nodes with nodules ranging from 1 to 3 cm in diameter as well as NVLs were divided into two sections. One section was submitted for cultural isolation and

6 molecular assays, and the other one was fixed in 10% neutral buffered formalin for histological investigation. Lymph nodes with nodules of less than 1 cm in diameter were wholly submitted for microbiological assays. Histology. Portions of formalin-fixed lymph nodes were paraffin-embedded and cut into three serial sections of 3 μm thickness. The first section was stained with haematoxylin-eosin and the next two with Ziehl-Neelsen (ZN), which is specific for acid-fast bacteria. Fifty fields of each section were examined by light microscopy (60X). We identify three different types of granuloma: a) Specific granuloma, which corresponded to the typical TB lesion that had a necrotic core, surrounded by a mixed population of inflammatory cells (lymphocytes, epithelioid cells, macrophages, and multinucleated giant cells) and several degrees of fibroplasia. The lesions ranged from initial granulomas to strongly necrotic-calcified granulomas with intense fibroplasia; b) Atypical granuloma, which was composed of a central necrotic zone with variable neutrophilic infiltrations and mineralizations, and a zone of foamy macrophages containing frequent multinucleated giants cells and fibroplasias; and c) Club-forming granuloma, which was characterized by the presence in the middle of the so-called sulphur granules. Additionally, the histological lesions were divided into five classes: 0) Negative: absence of granuloma; 1) Mycobacteriosis: specific granuloma and positive ZN staining; 2) Suspect mycobacteriosis: specific granuloma and negative ZN staining; 3) Atypical granuloma; and 4) Club-forming granuloma. Bacteriology. For each animal, lymph nodes were pooled and cultured. Approximately 3 to 5 g of tissue samples were homogenized with 3 ml of 1X PBS ph 6.8 (30 mm phosphate buffer ph 7.2, 150 mm NaCl, and 2 mm EDTA), for 120 seconds in a blender (Stomacher 80 Laboratory Blender). The homogenate was collected and decontaminated with an equal volume of NaOH 4% for 30 min at 37 C, neutralized with drops of 10% H 2 SO 4 and centrifuged at 3,000 g for 15 min at 4 C. The sediment was suspended in 1X PBS ph 6.8 and cultured at 37 C onto solid media, Löwenstein-Jensen (L-J) (Heipha, GmbH, Eppelheim, Germany) and Stonebrink (St) (Heipha,

7 GmbH, Eppelheim, Germany) plus pyruvate, and liquid media Modified Middlebrook 7H9 broth (BBL MGIT, Becton Dickinson) in the Bactec MGIT 960 system (Becton, Dickinson and Company, Sparks, MD). Growth on solid media was monitored every week while the Bactec MGIT 960 system checked liquid media every day. In study 2 the incubation time for the isolation of M. microti was extended from 6 to 18 weeks. After that time, the absence of growth or characteristic colonies was interpreted as negative. Identification of isolates. Phenotypic and biochemical identification. The isolates were evaluated for a panel of biochemical and cultural features by standard procedures (42, 43). The tests included an analysis of the growth at 43 C, pigment production, colony morphology, and biochemical tests, including nitrate reduction, niacin accumulation, arylsulfatase and urease activities, and tolerance to thiophene-2-carboxylic hydrazide and pyrazinamydase. Molecular identification. Mycobacterial isolates were identified by a multiplex PCR assay described by Kulski et al. (44), and by detection of the RD1 mic region as described by Smith et al. (9). Additionally, the identification of M. microti by PCR-RFLP of the gyrb gene was performed both on isolates and on DNA from tissue samples. This analysis was carried out by the amplification of a 765 nt fragment of the gyrb gene using the MTUBf primer (45) and the gyrb Seq primer (46). Five microliters of a heat inactivated bacterial suspension or DNA extracted from a lymph node was used in a 50-l reaction mixture containing 0.1 mm of each primer, 0.2 mm each dntp, 1X KAPA2G GC buffer and 0.25 l KAPA2G TM Robust HotStart DNA Polymerase (KAPA Biosystems). The amplification profile was as follows: 15 min at 95 C followed by 40 cycles of 60 sec at 95 C, 60 sec at 60 C, 70 sec at 72 C and a final elongation step for 7 min at 72 C. The PCR were run using a GeneAmp 9700 PCR System (Applied Biosystems). The amplicons were analysed by restriction with RsaI (Roche) (47). Restriction digests were separated in 2% agarose gel along with a 50 bp DNA ladder (Roche).

8 Non-tuberculous mycobacteria were characterized using the Microseq S rrna-based bacterial ID system (Applied Biosystems) following the manufacturer s recommendations. Sequencing was performed in an ABI Prism 3130 genetic analyser (Applied Biosystems). DNA extraction and IS6110 PCR. A 200 l aliquot of homogenized lymph node sample was submitted to mechanical lysis using 100 mg of glass beads (100 to 200 m) in a Tissue Lyser apparatus (Qiagen) for 5 min at 30 Hz. Samples were digested with 25 l of Proteinase K (25 mg/ml) for 1h at 65 C and purified using a Qiaamp DNA minikit (Qiagen) according to the manufacturer s instructions. DNA was eluted in a final volume of 200 l of TE buffer (Tris-HCl 10 mm and EDTA 1 mm, ph 8). For PCR analysis the primers Ext-1 (5 CCCGGACAGGCCGAGTTT3 ) and Int-1 (5 - CCCCATCGACCTACTACG-3 ) were used to generate a fragment (209 bp) of the IS6110 region. The PCR reaction was performed in a 50 l reaction mixture containing 5 l of DNA sample, 10 mm Tris-HCl (ph 8.3), 50 mm KCl, 1.5 mm MgCl 2, 0.2 mm each dntp, 0.4 mm primers and 1.25 l Taq Gold Polymerase (Applied Biosystems). DNA samples after 1 cycle at 95 C for 10 min, were amplified through 50 cycles of denaturation for 30 sec at 95 C, primer annealing for 30 sec at 58 C, and elongation for 45 sec at 72 C. In the last cycle the elongation step was extended for 10 min. Amplified products were detected by electrophoresis in a 2% agarose gel and TBE 1X buffer (100 mm Tris-base, 100 mm boric acid, and 2 mm EDTA, ph 8.3). For positive IS6110 PCR samples, differentiation of MtbC species was attempted by the PCR-RFLP of gyrb gene using the DNA of tissue samples as described above. Genotyping of M. microti isolates. The molecular characterization of 26 M. microti isolates collected from 2003 to 2011 was endeavoured using three different techniques: spoligotyping, sequencing of a portion of the direct repeat locus (DR) and variable-number tandem repeat (VNTR) typing. Spoligotyping was performed as described by Kamerbeek et al. (48). The spacer sequences contained in the DR locus were detected by hybridization onto a spoligotyping membrane (Isogen Bioscience BV, Maarssen, The Netherlands). For spoligotyping performed directly on tissue

9 samples, the PCR product of the DR locus was analysed by electrophoresis in a 2% agarose gel and TBE 1X buffer to detect the typical smeared pattern. Moreover, we quantified the MtbC DNA in the samples by the artus M. tuberculosis TM PCR kit (Qiagen), according to the manufacture s instructions. Sequencing of a portion of the DR region located 3 of the IS6110 element was done following amplification with the primers IM_dir, 5 -CTACTACGCTCAACGCCAG-3, and IM_rev, 5 -ATCGACTGCGACAACATCC-3. The PCR product was 573 bp or 387 bp depending on the presence or absence of spacers. The fragments obtained were sequenced using an ABI PRISM 3130 Instrument (Applied Biosystems). VNTR typing was assessed by amplification of five loci according to Frothingham and Meeker-O Connell (49) with modifications described by Boniotti et al. (50). Allelic diversity was calculated by the method of Selander et al. (51). Statistical analysis. A statistical analysis was performed with Excel 2010 (Microsoft). Data obtained from Study 1 were used to evaluate the prevalence of M. microti infections. In this study, animals were considered infected when the IS6110 PCR and/or the gyrb-rflp was positive. The 95% confidence intervals (CI P95% ) surrounding the estimated prevalences (p) were calculated using a binomial distribution. RESULTS Field studies Preliminary study. In the early stage of our study (2003 to 2005), 242 samples coming from BG and BS were randomly monitored (Table 1). Lesions consistent with TB infection were macroscopically detected in 89 head lymph nodes. Using IS6110 based PCR we found 61 MtbC positive samples and another eight were detected among NVL samples. By gyrb-rflp analysis, applied directly on tissue samples, 52 were confirmed to be M. microti and two were M. bovis. By culture isolation, performed with protocols optimized for M. bovis growth (42), we isolated one M. microti and two M. bovis strains.

10 Despite the inefficiency of culture isolation, these preliminary data showed a considerable presence of M. microti in the wild boar population, which was much more prevalent compared to M. bovis. This underlined the importance of designing a specific survey to determine the prevalence of infection and to standardize a set of molecular methods to identify and characterize M. microti strains. Study 1. During the hunting season of 2006, we analysed all the hunted wild boars (602) from two provinces located in northern Italy (BS and BG). Forty-eight samples showed tuberculous-like lesions, whereas 554 contained NVL (Table 2). The samples underwent IS6110 PCR and those that tested positive were analysed using the gyrb-rflp assay to confirm and identify the presence of a member of the MtbC. Culture isolation was attempted, using a protocol optimized for M. bovis growth, on 273 samples, 44 with macroscopic lesions and 229 with NVL, but only six Mycobacterium spp. were isolated. In the same year, two M. microti and two M. bovis strains were isolated from two provinces, CO and VA, not included in Study 1. Using IS6110 PCR, 24 positive results in samples with macroscopic lesions and 27 positive results in NVL samples were obtained. Using gyrb-rflp, the presence of M. microti was confirmed in 22 samples with macroscopic lesions and in 13 NVL samples. The presence of M. microti in the wild boar population of these regions is 8.5% (CI P95% : ) based on PCR detection and 5.8% (CI P95% : ) based on gyrb-rflp identification. Considering that 47% of the PCR positive samples were detected in the 48 samples showing lesions, compared to the 53% detected in 554 samples without lesions, we decided to focus our subsequent efforts on samples with lesions, thereby significantly reducing the experimental costs. Study 2. In this survey the area of study was extended to the whole sub-alpine territory of the Lombardy region, but only lymph nodes with macroscopic TB-like lesions were analysed. Three hundred and seven samples out of 15,922 were collected during the 2007 to 2011 hunting seasons and were submitted for culture isolation and analysed by the IS6110 PCR assay (Table 3).

11 The number of animals with TB lesions was stable from 2008 to 2011, and the amount of MtbC positive samples, 60 to 70%, was also stable. Of the samples positives by IS6110 PCR, 85% were confirmed to be M. microti by gyrb-rflp analysis. However, only 23 were isolated using culturing protocols with a prolonged incubation time (18 weeks). The number of culturable isolates was still very low compared to the number of M. microti positive samples detected by molecular techniques. Pathology Post mortem examination was performed on retropharyngeal and mandibular lymph nodes coming from approximately 23,000 wild boars during the whole period of study (2003 to 2011). Gross lesions were present in 444 wild boars as yellowish-white nodules from 0.5 to 3 cm in diameter. The nodules were single, rarely multiple, and always surrounded by a capsule. The centre of the lesion ranged from necrotic caseous or necrotic-calcified to purulent. Sometimes, the necrosis was so extensive as to occupy almost the entire lymph node. Histological examinations were performed on 728 samples (Tables 1 and 4). TB-specific granulomas were observed in 187 samples (class 1 and 2) (Table 4 and Fig. 2). Lesions were mostly multifocal and appeared as strongly necrotic-calcified granulomas with intense fibroplasia. Scattered eosinophils granulocytes cells were distributed close to the fibrous capsule. One hundred and twenty-seven samples stained ZN positive. The acid-fast bacteria were rare and observed in low numbers (1 to 3) in necrosis. Most often deposits of calcium fragments, intensely basophilic, interfered with the interpretation of the ZN staining. Rarely, acid fast bacilli were observed in the cytoplasm of the multinucleated giant cells. Sixty TB granuloma stained ZN negative (class 2). In this latter group, four samples had no gross lesions and the PCR analysis was positive for two samples. Seventy-seven samples were classified as club forming granuloma (class 4). Most of them had an intensely eosinophilic precipitate in the centre with marked giant cell response. The precipitates formed a radiating appearance ending with clubbed projections. Eleven were observed in NVL lymph node samples. Seventy-seven samples were classified as atypical granulomas (class

12 3). The histological appearance was variable (Fig. 2). Large granulomas frequently had numerous foamy macrophages around the central area of mineralization and numerous multinucleated giant cells. The smaller granulomas had caryoretic centres surrounded by macrophages and histiocytes. Sometimes, only focal or multifocal microscopic mineralizations surrounded by macrophages were present. Seven atypical granulomas were observed in NVL lymph node samples, and the ZN staining was always negative. Three hundred eighty-seven lymph nodes had no histological lesions, even though 86 had gross lesions and 30 were positive by IS6110 PCR analysis. Culture isolation and biochemical identification of strains. Table 5 summarizes the isolates obtained during the period of 2003 to All the M. microti as well as the M. bovis isolated during the preliminary study and study 1 were from samples with macroscopic lesions, while most of the M. avium complex (11/18) and M. nonchromogenicum (6/11) were from NVL samples. M. microti isolates took between 12 and 18 weeks to grow. Eleven isolates grew on the liquid medium Middlebrook 7H9 only, ten grew on solid media only, and five grew on both liquid and solid media. After the primary isolation, M. microti strains were analysed by phenotypic and biochemical tests (Table 6) (42, 43). Due to the slow growth and the low bacterial loads, subculturing was difficult to perform and the biochemical results were often weak or negative. Almost all the strains (25/26) showed urease activity, a characteristic shared by M. bovis and M. tuberculosis. Niacin accumulation, which discriminates M. microti from M. bovis, was present in 18 strains. None of the 26 strains showed nitrate reduction as had been reported by Magee and Ward (43), and only three strains showed pyrazinamidase activity (43, 20). Molecular identification and genotyping of M. microti strains. The 26 M. microti strains isolated during the period of 2003 to 2011, were identified by PCR-RFLP analysis of the gyrb gene. All the strains were also possessed a deleted RD1 mic region (Table 7). Spoligotyping was performed for all the M. microti isolates. Out of 26 isolates, 22 showed the characteristic vole type profile (SB0118), while the four remaining showed an absence of spacers.

13 The genome sequence of this region confirmed these results. In the SB0118 isolates, we found the spacers 51, 52, 58, 66, 67, and 68, as reported by van Embden et al. (52), where 51 and 52 correspond to spacers 37 and 38 (48). No spacers were found in the other four isolates, indicating a larger MiD1 deletion in this region. This profile was named S000 in this study. By VNTR analysis, 10 genotypeswere identified, which when combined with the two spoligotypes, results in 11 total genotypes (Table 7). Due to the low number of strains analysed, the allelic diversity value calculated for each locus is not reliable; however, a few differences are evident compared to M. bovis isolates. The ETR-E and -F loci were monomorphic as already observed by Smith et al. (9) in M. microti strains from cats. Additionally, ETR-D showed an increased allelic diversity (0.74) compared to the M. bovis population from cattle in Italy (0.22) (50). Geographical localization of genotypes. Spoligotyping was performed directly on DNA from tissue samples when M. microti was identified by gyrb-rflp analysis; however, the spoligotype profiles were not always readable. To exclude PCR amplification problems, M. microti DNA was quantified by the artus M. tuberculosis TM PCR kit (Qiagen). All the SB000 samples showed an amount of target DNA comparable to the SB0118 samples (data not shown). We could assign spoligotypes to 118 samples, with 84 showing spoligotype SB0118 while 34 did not show any spacers. The frequencies of the two spoligotypes in tissue samples (SB0118: 84/118=71%; S000: 34/118=29%) are consistent to those found among the isolates (SB0118: 19/23=83%; S000: 4/23=17%). By georeferencing the spoligotype profiles obtained from tissue samples, we noticed a geographical clustering of genotypes (Fig. 1). The profile SB000 is predominant in the area near Garda Lake. S000 was occasionally present also in BG and CO in 2007 but it was not detected in the following years. Conversely, SB0118 prevailed in CO, VA and BG, and only occasionally appears in BS. DISCUSSION

14 Our work shows a considerable and unexpected presence of M. microti in the wild boar population of the Central Alps in northern Italy with a prevalence estimate of approximately 5.8%. Since 2003, lesions compatible with tuberculosis infections have been observed in wild boar. At the macroscopic and microscopic levels, lesions present in retropharyngeal and mandibular lymph nodes were similar to those described for M. bovis infections, and no notable differences were observed in this study. However, the hunted wild boars appeared to be in good condition with no signs of clinical disease. Similarly to what happens in M. bovis infections, calcified granulomatous lesions predominated, indicating a prolonged and chronic infection. Histological examinations and ZN staining showed a paucity of bacilli as well. At the beginning of our study, culture isolation from visible TB lesions failed to identify the mycobacterium involved. Instead, an IS6110-based PCR analysis showed the presence of the MtbC in tissue samples. However, only by using a gyrb-rflp assay, which is able to discriminate the members of the MtbC, could we establish that the causative agent of the tuberculosis infection was M. microti. This molecular approach was extremely important to correctly identify the mycobacterium involved, which can be confused with M. bovis, and the assay is essential in detecting very slow growing and difficult to culture mycobacteria, such as M. microti, whose prevalence would otherwise be underestimated. This microorganism is generally thought to be a disease agent of small mammals, but occasional isolations have been reported from other species, such as pigs, llamas, dogs, cattle and humans (11, 6, 7, 12, 19, 20, 21, 22, 23). Abundant recent literature reports the presence of the MtbC in the Eurasian wild boar (Sus scrofa) in several European countries, including Italy (27, 28, 29, 30, 31, 32, 36, 33, 37, 34). M. bovis is the most frequently isolated agent but occasionally M. microti is also isolated from the wild boar (36, 34). In the area of study, the presence of M. bovis was marginal with only four positive samples isolated in 2005 (2) and 2006 (2) from CO and VA. The genotyping of isolates

15 was performed by spoligotyping and MIRU-VNTR analysis using the following 12 markers: ETR- A, -B, -C, -D, -E, MIRU-26, VNTR 2163a, 2163b, 4052, 3155, 1895 and 3232 (50). Three of the isolates had the same genetic profile (SB0120, / ) and the fourth had a similar genotype with a single variation at locus ETR-E. No related M. bovis strains have been detected in the same geographical area in cattle herds. It is noteworthy that the prevalence of bovine tuberculosis in cattle is very low in the study area. In fact, Lombardy has been designated an officially Tb-free region since 2010 with prevalence below 0.1% from In other Italian regions the situation is different. In Piemonte, Liguria (36), Sardegna and Marche, M. bovis isolated from wild boar possessed genotypes found also in local cattle herds (unpublished results). Recent studies (9) reported that M. microti is endemic in certain areas of Great Britain in cats and that the geographic areas where M. bovis and M. microti were recovered from domestic cats do not overlap. The authors suggested that exposure to M. microti could contribute to protection against M. bovis, working as a natural vaccine, reducing the presence of bovine tuberculosis even in other wild and domestic animals. Even though in the study area the Tb-free status was achieved thanks to the eradication campaign, we can t exclude that the presence of M. microti in wild boar could contribute to protection against M. bovis in the future. The attention to wild boar health has intensified in the last years mainly as a consequence of a considerable increase in the population density and geographical range (53, 39). In particular, in the Central Italian Prealps wild ungulate population has grown as a result of changes in land use and game management strategies (39, 54). Consequently, hunting and the consumption of wild boar meat have increased, making the chances of human exposure to wild boar diseases greater. Skinning and evisceration procedures might lead to infection by direct contact with the organs and tissues of infected animals (55). Tuberculosis caused by M. microti is uncommon in humans, even if it is considered able to cause clinical illness in both immunocompetent and immunosuppressed patients (19, 20, 21, 22, 23). However, the prevalence of human infection may have been

16 underestimated due to the difficulties in primary isolation and differentiation, and because pulmonary tuberculosis caused by M. microti and M. tuberculosis are clinically indistinguishable. Additionally, livestock partially shares pastures with wild animals and contact between wild boars and domestic animals may occasionally occur. Cattle might get in contact with M. microti infected wild boar or other environmental sources, and become false positive skin reactors interfering with the btb diagnosis. Monitoring activities were implemented in Italy in 1998 with the cooperation of hunting associations and the veterinary official service. In the frame of this control programme, meat inspection was performed on a total of approximately 23,000 wild boar head lymph nodes from hunted animals during the period of 2003 to During Study 1, the diffusion of M. microti infection in wild boar in two provinces of northern Italy was calculated by analysing the hunted wild boars coming from BS and BG. The prevalence of M. microti was 8.5% (CI P95% : ) based on PCR detection and 5.8% (CI P95% : ) based on gyrb-rflp identification. The discrepancy between IS6110 PCR and gyrb-rflp analyses is probably due to the different sensitivities of the two techniques. The first amplifies a target (209 bp) present in multiple copies in the genome of M. microti, while the second amplifies a product more than three times longer (765 bp). For these reasons the prevalence of M. microti in this wild boar sampling is likely to be closer to 8% than 6%. Based on this study, the probability of detecting MtbC in samples with macroscopic lesions (50%, 24/48) was higher compared to NVL samples (4%, 27/554). Therefore, in the following studies we analysed only lymph nodes with visible lesions. In study 2, the presence of M. microti in the Lombardia region over a five year period (2007 to 2011) was monitored. The presence of TB-like lesions and M. microti positive samples was essentially stable (Table 3). To increase the efficiency of culture isolation, the culture growth incubation period was increased to 18 weeks. Despite this, only 23 of 169 M. microti positive samples were isolated. It is possible that a different sample pre-treatment, culture medium

17 composition and a combination of liquid and solid media could be optimized for more efficient M. microti growth. Histological examination revealed the different evolutionary stages of the TB-specific lesions as well as of unspecific lesions (Fig. 1). Especially in NVL samples, the histological examination was important to discriminate unspecific granulomas, such as atypical granulomas and club forming granulomas. Granulomas are frequently observed in wild boar probably due to their feeding behaviours, such as rooting, which can induce micro-traumas to the oral mucosa, facilitating the entry of environmental bacteria that can colonize the retropharyngeal and mandibular lymph nodes. Histology was less efficient than PCR in detecting TB infections (Table 4). Since the culture isolation in a TB diagnostic laboratory takes priority, the most symptomatic portion of the sample is used for this purpose. The remaining portion may be less symptomatic, resulting in a disadvantage for the histological analysis. However, the combination of histopathology and PCR proved to be useful to reveal mixed infections (class 3 and 4). During the study period (2003 to 2011), a total of 26 M. microti strains were isolated and characterized by biochemical and molecular methods (Tables 6 and 7). Molecular characterization by spoligotyping discriminated two genotypes: the vole type profile (SB0118) and a new, never described before, profile where no spacers were found. This result, confirmed by sequencing of the DR region located at the 3-end of the IS6110 element, indicates a rearrangement of this region. Further genomic studies will be needed to understand the evolutionary origin of this strain and its association with the wild boar host. All the strains contained deletions of the RD1 mic region (Table 7), a common feature of M. microti strains. Genotyping by ETRA-F analysis identified 10 genotypes that, combined with the two spoligotypes, resulted in a total of 11 genotypes (Table 7). The ETR-E and -F loci were monomorphic as already observed in GB by Smith et al. (9) for M. microti strains from many sources.

18 Spoligotyping, performed directly on DNA from those tissue samples where M. microti was confirmed by gyrb-rflp analysis, showed a geographical clustering of genotypes (Fig. 1). Geographical clustering of M. microti genotypes was also observed by Smith et al (9) in GB. In wild boar, M. microti infections can be transmitted via direct contact with wild rodents and other small mammals favoured by their behaviour and eating habits (rooting and scavenging on carcasses). However, the high prevalence of M. microti in the wild boar population suggests an active role of the wild boar in maintaining M. microti in the environment and, on the basis of the current knowledge, an intra-species transmission cannot be excluded. The understanding of the wild boar s role in the epidemiological cycle of this Mycobacterium needs further investigation. In the meantime, constant monitoring is desirable since M. microti can interfere with the diagnosis of TB infections. ACKNOWLEDGMENTS We thank Daniela Loda, Andrea Moneta, Lidia Tonoli, Mario D Incau, Massimo Datteri and Laura Tassielli for their skilled technical assistance. Thanks also to Science Docs for the English language editing of this manuscript.

19 REFERENCES 1. Wells AQ, Oxon DM Tuberculosis in wild voles. Lancet I: Wayne LG, Kubica GP The Mycobacteria. The Williams and Wilkins Co., Baltimore, MD. 3. Kremer K, van Soolingen D, van Embden J, Hughes S, Inwald J, Hewinson G Mycobacterium microti: more widespread than previously thought. J. Clin. Microbiol. 36: Cavanagh R, Begon M, Bennett M, Ergo T, Graham IM, De Haas PE, Hart CA, Koedam M, Kremer K, Lambin X, Roholl P, van Soolingen D Mycobacterium microti infection (vole tuberculosis) in wild rodent populations. J. Clin. Microbiol. 40: Burthe S, Bennett M, Kipar A, Lambin X, Smith A, Telfer S, Begon M Tuberculosis (Mycobacterium microti) in wild field vole populations. Parasitology. 135: Pattyn SR, Portaels FA, Kegeruka P, Gigase P Further studies on African strains of Mycobacterium tuberculosis. Comparison with M. bovis and M. microti. Ann. Soc. Belge Med. Trop. 50: Oevermann A, Pfyffer GE, Zanolari P, Meylan M, Robert N Generalized tuberculosis in llamas (Lama glama) due to Mycobacterium microti. J. Clin. Microbiol. 42: Gunn-Moore DA, Jenkins PA, Lucke VM Feline tuberculosis: a literature review and discussion of 19 cases caused by an unusual mycobacterial variant. Vet. Rec. 138: Smith NH, Crawshaw T, Parry J, Birtles RJ Mycobacterium microti: More diverse than previously thought. J. Clin. Microbiol. 47: Huitema H, Jaartsveld FH Mycobacterium microti infection in a cat and some pigs. Antonie Van Leeuwenhoek. 33:

20 11. Taylor C, Jahans K, Palmer S, Okker M, Brown J, Steer K Mycobacterium microti isolated from two pigs. Vet. Rec. 159: Jahans K, Palmer S, Inwald J, Brown J, Abayakoon S Isolation of Mycobacterium microti from a male Charolais-Hereford cross. Vet. Rec. 155: Deforges L, Boulouis HJ, Thibaud JL, Boulouha L, Sougakoff W, Blot S, Hewinson G, Truffot-Pernot C, Haddad N First isolation of Mycobacterium microti (Llama-type) from a dog. Vet. Microbiol. 103: Gunn-Moore DA, McFarland SE, Brewer JI, Crawshaw TR, Clifton-Hadley RS, Kovalik, M, Shaw DJ Mycobacterial disease in cats in Great Britain: I. Culture results, geographical distribution and clinical presentation of 339 cases. J. Feline Med. Surg. 13: Gunn-Moore DA, McFarland SE, Schock A, Brewer JI, Crawshaw TR, Clifton-Hadley RS, Shaw DJ Mycobacterial disease in a population of 339 cats in Great Britain: II. Histopathology of 225 cases, and treatment and outcome of 184 cases. J. Feline Med. Surg. 13: Henrich M, Moser I, Weiss A, Reinacher M Multiple granulomas in three squirrel monkeys (Saimiri sciureus) caused by Mycobacterium microti. J. Comp. Pathol. 137: Palgrave CJ, Benato L, Eatwell K, Laurenson IF and Smith NJ Mycobacterium microti infection in two meerkats (Suricata suricatta). J. Comp. Path. 146: Zanolari P, Robert N, Lyashchenko KP, Pfyffer GE, Greenwald R, Esfandiari J, Meylan M Tuberculosis caused by Mycobacterium microti in South American camelids. J. Vet. Intern. Med. 23: Panteix G, Gutierrez MC, Boschiroli ML, Rouviere M, Plaidy A, Pressac D, Porcheret H, Chyderiotis G, Ponsada M, Van Oortegem K, Salloum S, Cabuzel S, Banuls AL, Van de Perre P, Godreuil S Pulmonary tuberculosis due to Mycobacterium microti: a study of six recent cases in France. J. Med. Microbiol. 59:

21 20. van Soolingen D, van der Zanden AG, de Haas PE, Noordhoek GT, Kiers A, Foudraine NA, Portaels F, Kolk AHJ, Kremer K, van Embden JD Diagnosis of Mycobacterium microti infections among humans by using novel genetic markers. J Clin Microbiol. 36: Niemann S, Richter E, Dalugge-Tamm H, Schlesinger H, Graupner D, Konigstein B, Gurath G, Greinert U, Rusch-Gerdes S Two cases of Mycobacterium microti derived tuberculosis in HIV-negative immunocompetent patients. Emerg. Infect. Dis. 6: Horstkotte MA, Sobottka I, Schewe CK, Schafer P, Laufs R, Rusch-Gerdes S, Niemann S Mycobacterium microti llama-type infection presenting as pulmonary tuberculosis in a human immunodeficiency virus-positive patient. J. Clin. Microbiol. 39: Xavier Emmanuel F, Seagar AL, Doig C, Rayner A, Claxton P, Laurenson I Human and animal infections with Mycobacterium microti, Scotland. Emerg. Infect. Dis. 13: Levy-Frebault VV, Portaels F Proposed minimal standards for the genus Mycobacterium and for the description of new slowly growing Mycobacterium spp. Int J Syst Bacteriol. 42: Brodin P, Majlessi L, Brosch R, Smith D, Bancroft G, Clark S, Williams A, Leclerc C, Cole ST Enhanced protection against tuberculosis by vaccination with recombinant Mycobacterium microti vaccine that induces T cell immunity against region of difference 1 antigens. J. Infect. Dis. 190: Meng XJ, Lindsay DS, Sriranganathan N Wild boars as sources for infectious diseases in livestock and humans. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 364: Serraino A, Marchetti G, Sanguinetti V, Rossi MC, Zanoni RG, Catozzi L, Bandera A, Dini W, Mignone W, Franzetti F, Gori A Monitoring of transmission of tuberculosis

22 between wild boars and cattle: genotypical analysis of strains by molecular epidemiology techniques. J. Clin. Microbiol. 37: Richomme C, Boschiroli ML, Hars J, Casabianca F, Ducrot C Bovine tuberculosis in livestock and wild boar on the Mediterranean island, Corsica. J. Wildl. Dis. 46: Naranjo V, Gortazar C, Vicente J, de la Fuente J Evidence of the role of European wild boar as a reservoir of Mycobacterium tuberculosis complex. Vet. Microbiol. 127: Parra A, Garcia A, Inglis NF, Tato A, Alonso JM, Hermoso de Mendoza M, Hermoso de Mendoza J, Larrasa J An epidemiological evaluation of Mycobacterium bovis infections in wild game animals of the Spanish Mediterranean ecosystem. Res. Vet. Sci. 80: Vicente J, Hofle U, Garrido JM, Fernandez-De-Mera IG, Juste R, Barral M, Gortazar C Wild boar and red deer display high prevalences of tuberculosis-like lesions in Spain. Vet. Res. 37: Zanella G, Durand B, Hars J, Moutou F, Garin-Bastuji B, Duvauchelle A, Ferme M, Karoui,C, Boschiroli ML Mycobacterium bovis in wildlife in France. J. Wildl. Dis. 44: Santos N, Correia-Neves M, Ghebremichael S, Kallenius G, Svenson SB, Almeida V Epidemiology of Mycobacterium bovis infection in wild boar (Sus scrofa) from Portugal. J. Wildl. Dis. 45: Schoning JM, Cerny N, Prohaska S, Wittenbrink MM, Smith NH, Bloemberg G, Pewsner M, Schiller I, Origgi FC, Ryser-Degiorgis MP Surveillance of bovine tuberculosis and risk estimation of a future reservoir formation in wildlife in Switzerland and Liechtenstein. PLoS One. 8:e Gortázar C, Ferroglio E, Höfle U, Frölich K, Vicente J Diseases shared between wildlife and livestock: a European perspective. Eur. J. Wildl. Res. 53:

23 36. Dondo A, Zoppi S, Rossi F, Chiavacci L, Barbaro A, Garrone A, Benedetto A, Goria M Mycobacteriosis in wild boar: results of activity in North-Western Italy. Epidémiol. et santé anim. 51: Di Marco V, Mazzone P, Capucchio MT, Boniotti MB, Aronica V, Russo M, Fiasconaro M, Cifani N, Corneli S, Biasibetti E, Biagetti M, Pacciarini ML, Cagiola M, Pasquali P, Marianelli C Epidemiological significance of the domestic black pig (Sus scrofa) in maintenance of bovine tuberculosis in Sicily. J. Clin. Microbiol. 50: Melis C, Szafranska PA, Jedrzejewska B, Barton K Biogeographical variation in the population density of wild boar (Sus scrofa) in western Eurasia. J. Biogeogr. 33: Carnevali L, Pedrotti L, Riga F, Toso S Banca Dati Ungulati: Status, distribuzione, consistenza, gestione e prelievo venatorio delle popolazioni di Ungulati in Italia. Biologia e Conservazione della Fauna-Rapporto Eds. ISPRA 117: Ramanzin M, Amici A, Casoli C, Esposito L, Lupi P, Marsico G, Mattiello S, Olivieri O, Ponzetta MP, Russo C, Trabalza Marinucci M Meat from wild ungulates: ensuring quality and hygiene of an increasing resource. Ital. J. Anim. Sci. 9(3): Magnino S, Frasnelli M, Fabbi M, Bianchi A, Zanoni M, Merialdi G, Pacciarini ML, Gaffuri A The monitoring of selected zoonotic diseases of wildlife in Lombardy and Emilia-Romagna, northern Italy. Wageningen Academic Publisher 123: Metchock BG, Nolte FS, Wallace RJ Mycobacterium, p In Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, Manual of Clinical Microbiology, 7 th Edition, ASM Press, Washington, D.C. 43. Magee JG, Ward AC Family III. Mycobacteriaceae Chester 1897, 63 AL emend. p Bergey s manual of systematic bacteriology, 2nd ed, vol 5. Springer, New York, NY.

24 44. Kulski JK, Khinsoe C, Pryce T, Christiansen K Use of a multiplex PCR to detect and identify Mycobacterium avium and M. intracellulare in blood culture fluids of AIDS patients. J. Clin. Microbiol. 33: Kasai H, Ezaki T, Harayama S Differentiation of phylogenetically related slowly growing Mycobacteria by their gyrb sequences. J. Clin. Microbiol. 38: Huard RC, Lazzarini LC, Butler WR, van Soolingen D, Ho JL PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions. J. Clin. Microbiol. 41: Niemann S, Harmsen D, Rusch-Gerdes S, Richter E Differentiation of Mycobacterium tuberculosis complex isolates by gyrb DNA sequence polymorphism analysis. J. Clin. Microbiol. 38: Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, Bunschoten A, Molhuizen H, Shaw R, Goyal M, van Embden J Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35: Frothingham R, Meeker-O'Connell WA Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats. Microbiol. 144 (Pt 5): Boniotti MB, Goria M, Loda D, Garrone A, Benedetto A, Mondo A, Tisato E, Zanoni M, Zoppi S, Dondo A, Tagliabue S, Bonora S, Zanardi G, Pacciarini ML Molecular typing of Mycobacterium bovis strains isolated in Italy from 2000 to 2006 and evaluation of variable-number tandem repeats for geographically optimized genotyping. J. Clin. Microbiol. 47: Selander RK, Caugant DA, Ochman H, Musser JM, Gilmour MN, Whittan TS Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. Environ. Microbiol. 51:

25 52. van Embden JD, van Gorkom T, Kremer K, Jansen R, van Der Zeijst BA, Schouls LM Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria. J. Bacteriol. 182: Monaco A, Carnevali L, Riga F, Toso S Il cinghiale sull arco alpino: status e gestione delle popolazioni. Report Centro Ecologia Alpina. 38: Acevedo P, Farfan MA, Marquez AL, Delibes-Mateos M, Real R, Vargas JM Past, present and future of wild ungulates in relation to changes in land use. Landscape Ecology 26: De la Rua-Domenech R Human Mycobacterium bovis infection in the United Kingdom: Incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis. Tuberculosis (Edinb). 86: Downloaded from on October 22, 2018 by guest

26 FIGURE LEGENDS FIG 1. Geographical distribution of spoligotype profiles detected in wild boar tissue samples from the pre-alpine area of northern Italy. FIG 2. Histopathology in wild boar lymph nodes. Panels A to F show a NVL lymph node (A) and nodal lesions (B,C, D, E and F), stained by hematoxylin and eosin (A, B, C, E and F) and Ziehl-Neelsen (D). (A)Class 2 granuloma (suspected mycobacteriosis): cluster of multinucleated Langhans type giant cells in the paracortex region (20X). (B) Class 2 granuloma: granulomas in the cortical area with a necrotic centre, minimal calcification surrounded by epiteliodi cells, multinucleated giant cells and macrophages (20X). (C) Class 2 granuloma: extensively necrotic and calcified granuloma surrounded by a wide connective capsule (20X). (D) Class 1 mycobacteriosis: rare acid fast bacteria in necrotic debris (100X). (E) Class 3 atypical granuloma: granuloma in the medullary zone with minimal necrosis, neutrophilic infiltration, foamy macrophages and fibroplasia (20X). (F) Class 4 Club-forming granuloma: colonies with radiating clubs surrounded by a granulomatous reaction with giant cells (20X).

27 Tables Table 1. Summary of diagnostic tests performed during the period (N=1,151) Preliminary Diagnostic test study Study1 Study2 TOT Colture isolation Histology IS6110 PCR gyrb-rflp Wild boars Table 2. Study1: TBL and M. microti molecular detection (N=602) Post mortem examination MtbC detection by IS6110 PCR M. microti identification by gyrb- RFLP TBL (48) NVL (554) TOT (602) TBL, tuberculous-like lesions; NVL, no visible lesions. Table 3. Study2: Positive results of diagnostic tests (N=307) Year TBlesions MtbC (IS6110 PCR) M. microti (gyrb-rflp) Culture isolates M. microti isolates TOT

28 Table 4. Histological Results (N=728) Lesion class N samples Gross lesions (405) NVL (323) MtbC (IS6110 PCR) M. microti (gyrb-rflp) N samples MtbC (IS6110 PCR) M. microti (gyrb-rflp) TOT Table 5. Isolates from wild boar from 2003 to 2011 Preliminary Study1 Study2 TOT study M.microti 1 1+1* M. bovis 2 2* 4 M. avium complex M. nonchromogenicum M. hiberniae 1 1 M. engabaekii 1 1 Nocardia 1 1 Hafnia alvei 1 1 Actinomadura Micromonospora 2 2 Streptomyces 1 1 TOT *Coming from CO and VA provinces, not included in Study1 Table 6. Phenotypic and biochemical characteristics of M. microti isolates Result for the following isolates: Phenotypic or biochemical test Expected results (ref.) Niacin Accumulation (M.W.,Levy) Nitrate Reduction /- (M.W.,Levy)a Urease (M.W,Levy) +/- (M.W, Pyrazinamidase nd Soolingen)b TCH Tolerance (M.W,Levy) 14-day Arylsulfatase nd nd nd (M.W,Levy) Growth at 43 C (M.W,Levy) Pigmentation (M.W,Levy) nd, not determined; +, weak; ++ medium a Levy reported absence of nitrate reduction in M.microti strains, while Magee and Ward reported that 11-89% of the strains showed nitrate reduction b Magee and Ward reported positive rection to pyrazinamidase, van Soolingen reported 2 positive results among 8 strains

29 Table 7. Genotyping of M. microti strains and allelic diversity at each locus Strain ETR Year ID RD1 mic Spoligotype A B C D E F A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB A SB Allelic diversity 0,28 0,59 0,11 0,11 0,74 0 0

30

31