DOI: 10.7589/2013-06-135
Journal of Wildlife Diseases, 50(2), 2014, pp. 330–333 # Wildlife Disease Association 2014
Isolation of Mycobacterium caprae (Lechtal Genotype) from Red Deer (Cervus elaphus) in Italy Mario Chiari,1,2 M. Zanoni,1 L. G. Alborali,1 G. Zanardi,1 D. Avisani,1 S. Tagliabue,1 A. Gaffuri,1 M. L. Pacciarini,1 and M. B. Boniotti1 1IZSLER—Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna ‘‘Bruno Ubertini,’’ Brescia, Italy – Via Bianchi, 9 – 25124 Brescia, Italy; 2Corresponding author (email: [email protected])
The assessment of the epidemiologic role of wildlife is crucial for the implementation of effective control measures to eradicate TB in cattle (Prodinger et al. 2005; Gorta´zar et al. 2011). Recently, red deer was revealed to be a maintenance host for M. caprae infections in the Tyrol region (Austria), where TB prevalence ranged from 0% to 23% in a population with a density of 5.6 animals/km2 (Schoepf et al. 2012). Mycobacterium caprae is responsible for over 10% of TB outbreaks in cattle herds in Italy; nevertheless, most of the northern Italian regions were officially declared TB-free 10 yr ago. During the same period, wild ungulate populations have increased significantly, but M. caprae has been detected only in cattle (Boniotti et al. 2009). Using targeted surveillance, we determined whether TB infections were present in free-ranging red deer sampled from an Italian alpine area. Calculation of required sample size (n553) was based on estimated total population size of about 930 deer derived from the official hunting bags and performed using WinEpiscopeH 2.0 software (Thrusfield et al. 2001), assuming a prevalence of 5% and a 95% confidence level. The area (46u149N, 10u239E) is located in Valcamonica (Brescia Province), an alpine valley characterized by high red deer population density (16 deer/km2) as estimated from a kilometric abundance index obtained by spotlight data (Whipple et al. 1994). This area was declared officially free of bovine tuberculosis (officially tuberculosis free; OTF) in cattle in 2010. Mycobacterium caprae was previously identified in this region from cattle during TB outbreaks involving eight herds in 2001 and one herd
ABSTRACT: During tuberculosis (TB) surveillance, 53 hunted red deer (Cervus elaphus) were collected to determine whether TB was present in free-ranging animals from an Italian alpine area. Samples (lungs, liver, intestine, and lymph nodes) were cultured and analyzed by real-time PCR assay carried out directly on tissue. Mycobacterium caprae was isolated from small granulomatous, tuberculosis-like lesions in the liver of a 12-yr-old female. Identification of suspect colonies was done by PCR restriction fragment length polymorphism analysis of the gyrb gene, and genotyping was performed by spoligotyping and mycobacterial interspersed repetitive unit variable number tandem repeat analysis. The isolated strain was genetically identical to strains isolated in the study area in 2001 from dairy cows imported from Austria and in 2010 from an indigenous cow. The genotype, called ‘‘Lechtal,’’ is the most frequently detected in the TB outbreaks in Austria and Germany. The possibility that red deer act as a maintenance host of M. caprae between TB outbreaks could be not excluded. Despite the high red deer population density, the detection of only one infected red deer could suggest that the wildlife management measures applied in the study area (prohibition of artificial feeding and secure removal of offal from hunted animals) may reduce the risk of TB spreading. Key words: Italy, Mycobacterium caprae, red deer, tuberculosis.
Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex (MtbC), is a zoonotic pathogen that causes tuberculosis (TB) in livestock and wild animals (Aranaz et al. 2003). It has been isolated mainly in central Europe from humans (Kubica et al. 2003), cattle (Bos primigenius; Prodinger et al. 2002; Erler et al. 2004; Boniotti et al. 2009), goats (Capra aegagrus hircus; Aranaz et al. 1999), and wildlife, including red deer (Cervus elaphus; Prodinger et al. 2002; Rodrı´guez et al. 2011; Schoepf et al. 2012) and wild boars (Sus scrofa; Garcı´a-Jime´nez et al. 2013). 330
SHORT COMMUNICATIONS
in 2010 (Boniotti et al. 2007). The last outbreak has not compromised the OTF legal status of the area. As shown by epidemiologic investigations and confirmed by molecular typing of the isolates, the first TB outbreak cluster of 2001 was linked to the introduction of infected dairy cows from Austria and Germany through a livestock collection center (Boniotti et al. 2007). The local trade of those animals from the collection center and the grazing of cattle on common pastures during the summers facilitated the spread of TB into the eight affected herds. In 2010, M. caprae of the same genotype was reisolated in the same area from TB lesions observed in an indigenous dairy cow of a different herd. This case was not connected to the introduction of the TB-positive animals from endemic countries, and no other risk factors were discovered (Zanella et al. 2012). Between August and December 2011, the presence of M. caprae in red deer was examined from the viscera (lungs, liver, and intestine) and lymph nodes (Lnn) of 53 hunted deer. Samples were obtained from eight calves, 23 female (seven females ,2 yr old and 16 females .2 yr old) and 22 males (11 males ,5 yr old and 11 males 5–9 yr old). At postmortem examination, we observed small granulomatous, tuberculosis-like lesions in the liver of a 12-yr-old female (Fig. 1). This organ and two Lnn pools for each deer were subjected to a bacteriologic examination and real-time PCR analyses. The first Lnn pool was composed of retropharyngeales Lnn and the second of tracheobronchialis, mesenteriales, and mediastinales Lnn. Standard mycobacterial culture was used. Briefly, the samples were homogenized, decontaminated, and inoculated onto solid selective media, Lo¨wenstein-Jensen and Stonebrink (Heipha Diagnostika, Heidelberg, Germany), and a liquid medium, Modified Middlebrook 7H9 broth (BBL MGIT, Becton Dickinson and Company, Milan, Italy). Bacteriologic cultures were positive only
331
FIGURE 1. Small granulomatous tuberculosis-like lesion (about 1 cm diameter) in the liver of a 12-yrold red deer (Cervus elaphus) from Italy, 2011.
from the liver and the first Lnn pool of the deer with lesions. We employed real-time PCR using the Artus M. tuberculosis TM PCR kit (Qiagen, Hilden, Germany) for the direct detection of MtbC mycobacteria. DNA was extracted and purified by mechanical lysis with glass beads followed by affinity column purification using the PureLinkTM Genomic DNA mini Kit (Invitrogen, Paisley, UK). The real-time PCR assay detected MtbC bacteria only in the liver of the deer with TB lesions. Identification of the isolates was confirmed by PCR restriction fragment length polymorphism (RFLP) analysis of the gyrb gene through RsaI and SacII restriction enzymes. Genotyping of isolates was performed by spoligotyping and mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) analysis using the following 12 markers: ETR-A, -B, -C, -D, and -E; MIRU-26; and VNTR 2163a, 2163b, 4052, 3155, 1895, and 3232 (Boniotti et al. 2009). The isolate was identified as M. caprae by gyrb-RFLP analysis. The genotype profile, according to spoligotyping and MIRU-VNTR analyses, was SB0418, 5-35-2-3-4-8-5-3-4-3-11. This profile corresponds to the ‘‘Lechtal genotype,’’ which was the most frequently detected in the
332
JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 2, APRIL 2014
TB outbreaks in Austria and Germany (Prodinger et al. 2002; Erler et al. 2004). Moreover, it was the same genotype identified in the 2001 and 2010 cattle herd TB outbreaks reported in the sampling area. During this period, neither tuberculin skin testing (done every 2 yr for all cattle in the region) nor surveillance at the abattoirs, performed in agreement with the Italian and EU legislation for TB eradication program (DM 592/95 and DLgs 196/99, and Council Directive 64/ 432/EEC, Annexes A, I, and B, respectively), revealed any TB infections in cattle in the study area. The absence of TB reports in cattle suggests the lengthy undisclosed persistence of the Lechtal genotype in the study area. The absence of classical TB lesions and the presence of only one lesion in the liver did not allow us to evaluate the stage of infection. The possible movement of the red deer to the Tyrol infected areas is unlikely considering the distance (.200 km) between the two areas, which is much more than the natural migratory behavior of deer (20– 40 km). Therefore, since no likely sources of infection were identified, and considering that cattle graze on common pastures with red deer during the summers, the possibility that red deer act as a maintenance host of M. caprae in the environment from the first to the last TB outbreak in cattle could not be excluded. In contrast to the TB epidemiologic situation of neighboring alpine countries (Schoepf et al. 2012), the detection in northern Italy of only one infected red deer, despite the high-density red deer population, could suggest that the wildlife management measures applied in the study area reduced the risk of TB spreading. Management options were designed to improve local wildlife management and hunting rules. In particular, the two main factors influencing the spread of TB were addressed (Schoepf et al. 2012; Zanella et al. 2012). First, unlike normal practice in Tyrol (Schoepf et al. 2012), artificial feeding and salt licks for
wildlife were not permitted, limiting aggregation that occurs especially during winter at the feeding sites. Second, hunters securely disposed of offal from hunted animals, after evisceration at the slaughterhouse, to reduce the presence of infected material in the field (Zanella et al. 2012). To our knowledge, this is the first report of a M. caprae infection in wildlife in Italy. Targeted surveillance should be performed to monitor the presence and spread of TB, as well as the efficacy of applied control measures. This work is part of the ‘‘TB Alpine Wildlife’’ project supported by FP7 EMIDA ERA-Net 2007. We thank Elena Bonavetti and the hunters of CA1-Brescia for their collaboration and Lesley Benyon (ScienceDocs Inc.) for editing this manuscript. LITERATURE CITED Aranaz A, Liebana E, Gomez Mampaso E, Galan J, Cousins D, Ortega A, Blazquez J, Baquero F, Mateos A, Suarez G, Dominguez L. 1999. Mycobacterium tuberculosis subsp. caprae subsp. nov.: A taxonomic study of a new member of the Mycobacterium tuberculosis complex isolated from goats in Spain. Int J Syst Bacteriol 49:1263–1273. Aranaz A, Cousins D, Mateos A, Dominguez L. 2003. Elevation of Mycobacterium tuberculosis subsp. caprae Aranaz et al. 1999 to species rank as Mycobacterium caprae comb. nov., sp. nov. Int J Syst Evol Microbiol 53:1785–1789. Boniotti MB, Pacciarini ML, Zanoni M, Tagliabue S, Bonazza V, Avisani D, Gaffuri A, Zanardi G. 2007. Descrizione di quattro cluster di M. bovis isolate in Lombardia. L’Osservatorio 5:4–9. Boniotti MB, Goria M, Loda D, Garrone A, Benedetto A, Mondo A, Tisato E, Zanoni M, Zoppi S, Dondo A, et al. 2009. Molecular typing of Mycobacterium bovis strains isolated in Italy from 2000 to 2006 and evaluation of variablenumber-tandem repeats for a geographic optimized genotyping. J Clin Microbiol 47:636–644. Erler W, Martin G, Sachse K, Naumann L, Kahlau D, Beer J, Bartos M, Nagy G, Cvetnic Z, ZolnirDovc M, Pavlik I. 2004. Molecular fingerprinting of Mycobacterium bovis subsp. caprae isolates from central Europe. J Clin Microbiol 42:2234– 2238. Garcı´a-Jime´nez WL, Benı´tez-Medina JM, Ferna´ndez-Llario P, Abecia JA, Garcı´a-Sa´nchez A,
SHORT COMMUNICATIONS
Martı´nez R, Risco D, Ortiz-Pela´ez A, Salguero FJ, Smith NH, et al. 2013. Comparative pathology of the natural infections by Mycobacterium bovis and by Mycobacterium caprae in wild boar (Sus scrofa). Transbound Emerg Dis 60:102–109. Gorta´zar C, Delahay RJ, McDonald RA, Boadella M, Wilson GJ, Gavier-Widen D, Acevedo P. 2011. The status of tuberculosis in European wild mammals. Mammal Rev 42:193–206. Kubica T, Rusch-Gerdes S, Niemann S. 2003. Mycobacterium bovis subsp. caprae caused one-third of human M. bovis–associated tuberculosis cases reported in Germany between 1999 and 2001. J Clin Microbiol 41:3070–3077. Prodinger WM, Eigentler A, Allerberger F, Schonbauer M, Glawischnig W. 2002. Infection of red deer, cattle, and humans with Mycobacterium bovis subsp. caprae in western Austria. J Clin Microbiol 40:2270–2272. Prodinger WM, Brandsta¨tter A, Naumann L, Pacciarini M, Kubica T, Boschiroli ML, Aranaz A, Nagy G, Cvetnic Z, Ocepek M, et al. 2005. Characterization of Mycobacterium caprae isolates from Europe by mycobacterial interspersed repetitive unit genotyping. J Clin Microbiol 43:4984–4992.
333
Rodrı´guez S, Bezos J, Romero B, De Juan L, A´lvarez J, Castellanos E, Moya N, Lozano F, Javed MT, Sa´ez-Llorente JL, et al. 2011. Mycobacterium caprae infection in livestock and wildlife, Spain. Emerg Infect Dis 17:532–535. Schoepf K, Prodinger WM, Glawischnig W, Hofer E, Revilla-Fernandez S, Hofrichter J, Fritz J, Ko¨fer J, Schmoll F. 2012. A two-years’ survey on the prevalence of tuberculosis caused by Mycobacterium caprae in red deer (Cervus elaphus) in the Tyrol, Austria. ISRN Vet Sci 2012. doi: 10.5402/2012/245138. Thrusfield M, Ortega C, de Blas I, Noordhuizen JP, Frankena K. 2001. WIN EPISCOPE 2.0: Improved epidemiological software for veterinary medicine. Vet Rec 148:567–572. Whipple DJ, Rollins D, Schacht WH. 1994. A field simulation for assessing accuracy of spotlight deer surveys. Wildl Soc B 22:667–673. Zanella G, Bar-Hen A, Boschiroli ML, Hars J, Moutou F, Garin-Bastuji B, Durand B. 2012. Modelling transmission of bovine tuberculosis in red deer and wild boar in Normandy, France. Zoonoses Public Health 59:170–178. Submitted for publication 10 May 2013. Accepted 30 August 2013.
Journal of Wildlife Diseases, 50(2), 2014, pp. 330–333 # Wildlife Disease Association 2014
Isolation of Mycobacterium caprae (Lechtal Genotype) from Red Deer (Cervus elaphus) in Italy Mario Chiari,1,2 M. Zanoni,1 L. G. Alborali,1 G. Zanardi,1 D. Avisani,1 S. Tagliabue,1 A. Gaffuri,1 M. L. Pacciarini,1 and M. B. Boniotti1 1IZSLER—Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna ‘‘Bruno Ubertini,’’ Brescia, Italy – Via Bianchi, 9 – 25124 Brescia, Italy; 2Corresponding author (email: [email protected])
The assessment of the epidemiologic role of wildlife is crucial for the implementation of effective control measures to eradicate TB in cattle (Prodinger et al. 2005; Gorta´zar et al. 2011). Recently, red deer was revealed to be a maintenance host for M. caprae infections in the Tyrol region (Austria), where TB prevalence ranged from 0% to 23% in a population with a density of 5.6 animals/km2 (Schoepf et al. 2012). Mycobacterium caprae is responsible for over 10% of TB outbreaks in cattle herds in Italy; nevertheless, most of the northern Italian regions were officially declared TB-free 10 yr ago. During the same period, wild ungulate populations have increased significantly, but M. caprae has been detected only in cattle (Boniotti et al. 2009). Using targeted surveillance, we determined whether TB infections were present in free-ranging red deer sampled from an Italian alpine area. Calculation of required sample size (n553) was based on estimated total population size of about 930 deer derived from the official hunting bags and performed using WinEpiscopeH 2.0 software (Thrusfield et al. 2001), assuming a prevalence of 5% and a 95% confidence level. The area (46u149N, 10u239E) is located in Valcamonica (Brescia Province), an alpine valley characterized by high red deer population density (16 deer/km2) as estimated from a kilometric abundance index obtained by spotlight data (Whipple et al. 1994). This area was declared officially free of bovine tuberculosis (officially tuberculosis free; OTF) in cattle in 2010. Mycobacterium caprae was previously identified in this region from cattle during TB outbreaks involving eight herds in 2001 and one herd
ABSTRACT: During tuberculosis (TB) surveillance, 53 hunted red deer (Cervus elaphus) were collected to determine whether TB was present in free-ranging animals from an Italian alpine area. Samples (lungs, liver, intestine, and lymph nodes) were cultured and analyzed by real-time PCR assay carried out directly on tissue. Mycobacterium caprae was isolated from small granulomatous, tuberculosis-like lesions in the liver of a 12-yr-old female. Identification of suspect colonies was done by PCR restriction fragment length polymorphism analysis of the gyrb gene, and genotyping was performed by spoligotyping and mycobacterial interspersed repetitive unit variable number tandem repeat analysis. The isolated strain was genetically identical to strains isolated in the study area in 2001 from dairy cows imported from Austria and in 2010 from an indigenous cow. The genotype, called ‘‘Lechtal,’’ is the most frequently detected in the TB outbreaks in Austria and Germany. The possibility that red deer act as a maintenance host of M. caprae between TB outbreaks could be not excluded. Despite the high red deer population density, the detection of only one infected red deer could suggest that the wildlife management measures applied in the study area (prohibition of artificial feeding and secure removal of offal from hunted animals) may reduce the risk of TB spreading. Key words: Italy, Mycobacterium caprae, red deer, tuberculosis.
Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex (MtbC), is a zoonotic pathogen that causes tuberculosis (TB) in livestock and wild animals (Aranaz et al. 2003). It has been isolated mainly in central Europe from humans (Kubica et al. 2003), cattle (Bos primigenius; Prodinger et al. 2002; Erler et al. 2004; Boniotti et al. 2009), goats (Capra aegagrus hircus; Aranaz et al. 1999), and wildlife, including red deer (Cervus elaphus; Prodinger et al. 2002; Rodrı´guez et al. 2011; Schoepf et al. 2012) and wild boars (Sus scrofa; Garcı´a-Jime´nez et al. 2013). 330
SHORT COMMUNICATIONS
in 2010 (Boniotti et al. 2007). The last outbreak has not compromised the OTF legal status of the area. As shown by epidemiologic investigations and confirmed by molecular typing of the isolates, the first TB outbreak cluster of 2001 was linked to the introduction of infected dairy cows from Austria and Germany through a livestock collection center (Boniotti et al. 2007). The local trade of those animals from the collection center and the grazing of cattle on common pastures during the summers facilitated the spread of TB into the eight affected herds. In 2010, M. caprae of the same genotype was reisolated in the same area from TB lesions observed in an indigenous dairy cow of a different herd. This case was not connected to the introduction of the TB-positive animals from endemic countries, and no other risk factors were discovered (Zanella et al. 2012). Between August and December 2011, the presence of M. caprae in red deer was examined from the viscera (lungs, liver, and intestine) and lymph nodes (Lnn) of 53 hunted deer. Samples were obtained from eight calves, 23 female (seven females ,2 yr old and 16 females .2 yr old) and 22 males (11 males ,5 yr old and 11 males 5–9 yr old). At postmortem examination, we observed small granulomatous, tuberculosis-like lesions in the liver of a 12-yr-old female (Fig. 1). This organ and two Lnn pools for each deer were subjected to a bacteriologic examination and real-time PCR analyses. The first Lnn pool was composed of retropharyngeales Lnn and the second of tracheobronchialis, mesenteriales, and mediastinales Lnn. Standard mycobacterial culture was used. Briefly, the samples were homogenized, decontaminated, and inoculated onto solid selective media, Lo¨wenstein-Jensen and Stonebrink (Heipha Diagnostika, Heidelberg, Germany), and a liquid medium, Modified Middlebrook 7H9 broth (BBL MGIT, Becton Dickinson and Company, Milan, Italy). Bacteriologic cultures were positive only
331
FIGURE 1. Small granulomatous tuberculosis-like lesion (about 1 cm diameter) in the liver of a 12-yrold red deer (Cervus elaphus) from Italy, 2011.
from the liver and the first Lnn pool of the deer with lesions. We employed real-time PCR using the Artus M. tuberculosis TM PCR kit (Qiagen, Hilden, Germany) for the direct detection of MtbC mycobacteria. DNA was extracted and purified by mechanical lysis with glass beads followed by affinity column purification using the PureLinkTM Genomic DNA mini Kit (Invitrogen, Paisley, UK). The real-time PCR assay detected MtbC bacteria only in the liver of the deer with TB lesions. Identification of the isolates was confirmed by PCR restriction fragment length polymorphism (RFLP) analysis of the gyrb gene through RsaI and SacII restriction enzymes. Genotyping of isolates was performed by spoligotyping and mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) analysis using the following 12 markers: ETR-A, -B, -C, -D, and -E; MIRU-26; and VNTR 2163a, 2163b, 4052, 3155, 1895, and 3232 (Boniotti et al. 2009). The isolate was identified as M. caprae by gyrb-RFLP analysis. The genotype profile, according to spoligotyping and MIRU-VNTR analyses, was SB0418, 5-35-2-3-4-8-5-3-4-3-11. This profile corresponds to the ‘‘Lechtal genotype,’’ which was the most frequently detected in the
332
JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 2, APRIL 2014
TB outbreaks in Austria and Germany (Prodinger et al. 2002; Erler et al. 2004). Moreover, it was the same genotype identified in the 2001 and 2010 cattle herd TB outbreaks reported in the sampling area. During this period, neither tuberculin skin testing (done every 2 yr for all cattle in the region) nor surveillance at the abattoirs, performed in agreement with the Italian and EU legislation for TB eradication program (DM 592/95 and DLgs 196/99, and Council Directive 64/ 432/EEC, Annexes A, I, and B, respectively), revealed any TB infections in cattle in the study area. The absence of TB reports in cattle suggests the lengthy undisclosed persistence of the Lechtal genotype in the study area. The absence of classical TB lesions and the presence of only one lesion in the liver did not allow us to evaluate the stage of infection. The possible movement of the red deer to the Tyrol infected areas is unlikely considering the distance (.200 km) between the two areas, which is much more than the natural migratory behavior of deer (20– 40 km). Therefore, since no likely sources of infection were identified, and considering that cattle graze on common pastures with red deer during the summers, the possibility that red deer act as a maintenance host of M. caprae in the environment from the first to the last TB outbreak in cattle could not be excluded. In contrast to the TB epidemiologic situation of neighboring alpine countries (Schoepf et al. 2012), the detection in northern Italy of only one infected red deer, despite the high-density red deer population, could suggest that the wildlife management measures applied in the study area reduced the risk of TB spreading. Management options were designed to improve local wildlife management and hunting rules. In particular, the two main factors influencing the spread of TB were addressed (Schoepf et al. 2012; Zanella et al. 2012). First, unlike normal practice in Tyrol (Schoepf et al. 2012), artificial feeding and salt licks for
wildlife were not permitted, limiting aggregation that occurs especially during winter at the feeding sites. Second, hunters securely disposed of offal from hunted animals, after evisceration at the slaughterhouse, to reduce the presence of infected material in the field (Zanella et al. 2012). To our knowledge, this is the first report of a M. caprae infection in wildlife in Italy. Targeted surveillance should be performed to monitor the presence and spread of TB, as well as the efficacy of applied control measures. This work is part of the ‘‘TB Alpine Wildlife’’ project supported by FP7 EMIDA ERA-Net 2007. We thank Elena Bonavetti and the hunters of CA1-Brescia for their collaboration and Lesley Benyon (ScienceDocs Inc.) for editing this manuscript. LITERATURE CITED Aranaz A, Liebana E, Gomez Mampaso E, Galan J, Cousins D, Ortega A, Blazquez J, Baquero F, Mateos A, Suarez G, Dominguez L. 1999. Mycobacterium tuberculosis subsp. caprae subsp. nov.: A taxonomic study of a new member of the Mycobacterium tuberculosis complex isolated from goats in Spain. Int J Syst Bacteriol 49:1263–1273. Aranaz A, Cousins D, Mateos A, Dominguez L. 2003. Elevation of Mycobacterium tuberculosis subsp. caprae Aranaz et al. 1999 to species rank as Mycobacterium caprae comb. nov., sp. nov. Int J Syst Evol Microbiol 53:1785–1789. Boniotti MB, Pacciarini ML, Zanoni M, Tagliabue S, Bonazza V, Avisani D, Gaffuri A, Zanardi G. 2007. Descrizione di quattro cluster di M. bovis isolate in Lombardia. L’Osservatorio 5:4–9. Boniotti MB, Goria M, Loda D, Garrone A, Benedetto A, Mondo A, Tisato E, Zanoni M, Zoppi S, Dondo A, et al. 2009. Molecular typing of Mycobacterium bovis strains isolated in Italy from 2000 to 2006 and evaluation of variablenumber-tandem repeats for a geographic optimized genotyping. J Clin Microbiol 47:636–644. Erler W, Martin G, Sachse K, Naumann L, Kahlau D, Beer J, Bartos M, Nagy G, Cvetnic Z, ZolnirDovc M, Pavlik I. 2004. Molecular fingerprinting of Mycobacterium bovis subsp. caprae isolates from central Europe. J Clin Microbiol 42:2234– 2238. Garcı´a-Jime´nez WL, Benı´tez-Medina JM, Ferna´ndez-Llario P, Abecia JA, Garcı´a-Sa´nchez A,
SHORT COMMUNICATIONS
Martı´nez R, Risco D, Ortiz-Pela´ez A, Salguero FJ, Smith NH, et al. 2013. Comparative pathology of the natural infections by Mycobacterium bovis and by Mycobacterium caprae in wild boar (Sus scrofa). Transbound Emerg Dis 60:102–109. Gorta´zar C, Delahay RJ, McDonald RA, Boadella M, Wilson GJ, Gavier-Widen D, Acevedo P. 2011. The status of tuberculosis in European wild mammals. Mammal Rev 42:193–206. Kubica T, Rusch-Gerdes S, Niemann S. 2003. Mycobacterium bovis subsp. caprae caused one-third of human M. bovis–associated tuberculosis cases reported in Germany between 1999 and 2001. J Clin Microbiol 41:3070–3077. Prodinger WM, Eigentler A, Allerberger F, Schonbauer M, Glawischnig W. 2002. Infection of red deer, cattle, and humans with Mycobacterium bovis subsp. caprae in western Austria. J Clin Microbiol 40:2270–2272. Prodinger WM, Brandsta¨tter A, Naumann L, Pacciarini M, Kubica T, Boschiroli ML, Aranaz A, Nagy G, Cvetnic Z, Ocepek M, et al. 2005. Characterization of Mycobacterium caprae isolates from Europe by mycobacterial interspersed repetitive unit genotyping. J Clin Microbiol 43:4984–4992.
333
Rodrı´guez S, Bezos J, Romero B, De Juan L, A´lvarez J, Castellanos E, Moya N, Lozano F, Javed MT, Sa´ez-Llorente JL, et al. 2011. Mycobacterium caprae infection in livestock and wildlife, Spain. Emerg Infect Dis 17:532–535. Schoepf K, Prodinger WM, Glawischnig W, Hofer E, Revilla-Fernandez S, Hofrichter J, Fritz J, Ko¨fer J, Schmoll F. 2012. A two-years’ survey on the prevalence of tuberculosis caused by Mycobacterium caprae in red deer (Cervus elaphus) in the Tyrol, Austria. ISRN Vet Sci 2012. doi: 10.5402/2012/245138. Thrusfield M, Ortega C, de Blas I, Noordhuizen JP, Frankena K. 2001. WIN EPISCOPE 2.0: Improved epidemiological software for veterinary medicine. Vet Rec 148:567–572. Whipple DJ, Rollins D, Schacht WH. 1994. A field simulation for assessing accuracy of spotlight deer surveys. Wildl Soc B 22:667–673. Zanella G, Bar-Hen A, Boschiroli ML, Hars J, Moutou F, Garin-Bastuji B, Durand B. 2012. Modelling transmission of bovine tuberculosis in red deer and wild boar in Normandy, France. Zoonoses Public Health 59:170–178. Submitted for publication 10 May 2013. Accepted 30 August 2013.
