European Journal of Wildlife Research (2023) 69:111 https://doi.org/10.1007/s10344-023-01738-3 BRIEF REPORT Lack of detection of Mycobacterium microti infection in wild rodents from a free‑ranging wild boar outbreak area Enric Vidal1,2 · Johan Espunyes3 · Maria Puig Ribas3 · Cristian Melgarejo1 · Laura Martino1 · Lorraine Michelet4 · Maria Laura Boschiroli4 · Albert Sanz5 · Alberto Allepuz6 · Oscar Cabezón3 · Bernat Pérez de Val1,2 Received: 4 August 2023 / Revised: 21 September 2023 / Accepted: 27 September 2023 © The Author(s) 2023 Abstract Wild small rodents are considered the natural reservoirs of Mycobacterium microti, a member of the Mycobacterium tubercu- losis complex (MTBC) that can cause tuberculosis (TB) in humans and animals, as well as interfere with current tuberculosis eradication plans in livestock. A cross-sectional study was carried out in the Catalan Pyrenees (Iberian Peninsula) in an area where M. microti was previously isolated from wild boars, to evaluate the role of micromammals in the epidemiology of this outbreak. A total of 350 wild rodents were necropsied (306 Murinae and 44 Arvicolinae) in spring and autumn during two consecutive natural years. Tissues were analyzed by histopathology to look for TB-like lesions and by qPCR and culture to detect MTBC. Sera were analyzed by MTBC-specific ELISA. No evidence of TB infection in wild rodents was confirmed. Results suggest that small rodents did not play a role in the epidemiology of M. microti in the area. The source of this myco- bacterium remains unknown, but previous detections of M. microti in various species in southern France suggest the move- ments of wild boars across the French Pyrenees as the most likely origin of the outbreak detected in the Iberian Peninsula. Keywords Tuberculosis · Mycobacterium tuberculosis complex · Vole · Micromammals · Pyrenees · Wildlife Background Wild small rodents are considered the natural reservoirs * Johan Espunyes johan.espunyes@gmail.com of Mycobacterium microti (Wells 1937; Kipar et al. 2014), a member of the Mycobacterium tuberculosis complex 1 IRTA. Programa de Sanitat Animal. Centre de Recerca (MTBC). Field voles (Microtus agrestis), bank voles (Myo- en Sanitat Animal (CReSA), Campus de la Universitat des glareolus), yellow-necked mice (Apodemus flavicol- Autònoma de Barcelona (UAB), 08193 Bellaterra, Catalonia, Spain lis), wood mice (Apodemus silvaticus) and shrews (Sorex 2 araneus) suffer tuberculosis (TB) after the infection with Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal. Centre de Recerca en Sanitat Animal M. microti and can transmit the disease within their own (CReSA). Campus de la Universitat Autònoma de Barcelona populations (Cavanagh et al. 2002; Smith et al. 2009; Kipar (UAB), 08193 Bellaterra, Catalonia, Spain et al. 2014; Tagliapietra et al. 2021). Besides, M. microti also 3 Wildlife Conservation Medicine Research Group causes infection in livestock (Michelet et al. 2016, 2017), (WildCoM), Departament de Medicina i Cirurgia Animals, large wild animals (Boniotti et al. 2014; Michelet et al. Universitat Autònoma de Barcelona, 08193 Bellaterra, 2015a; De Val et al. 2019) and even humans (Frota et al. Catalonia, Spain 4 2004; Panteix et al. 2010), although it is generally consid- National Reference Laboratory for Tuberculosis, ered low-pathogenic in large mammals. However, since M. Paris-Est University, Animal Health Laboratory, Anses, Maisons‑Alfort, France microti exposure may induce positive results to tuberculin 5 skin testing, this bacteria interferes with bovine TB eradi- Departament d’Acció Climàtica, Agricultura i Agenda Rural de la Generalitat de Catalunya, Barcelona, Catalonia, Spain cation programs focused on other members of the MTBC 6 such as M. bovis, M. caprae or M. tuberculosis infections Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain (Michelet et al. 2020). 13 Vol.:(0123456789) 111 Page 2 of 6 European Journal of Wildlife Research (2023) 69:111 Studies conducted in England reported relatively high adiaspiromycosis presumably caused by Emmonsia crescens. prevalence of vole TB due to M. microti (Kremer et al. Microscopically, multifocal small granulomatous lesions 1998; Cavanagh et al. 2002; Smith et al. 2009; Kipar et al. compatible with TB were identified in 111 animals, mostly 2014), while in France this pathogen has been isolated in a in the liver and the lung (Fig. 1). None of them had AFB number of large-mammal species including cattle (Michelet upon Ziehl Neelsen’s staining. Such lesions were observed in et al. 2017), goats (Michelet et al. 2016), lama (Michelet 101 murids (33%, 101/302) and 10 arvicolines (21%, 10/48). et al. 2015a), wild boars (Michelet et al. 2015a, b), badgers None of the mycobacterial cultures showed growth identi- (Michelet et al. 2020), foxes (Michelet et al. 2021), dogs fied as MTBC, and qPCR tests were all negative. The 347 (Deforges et al. 2004), and cats (Rüfenacht et al. 2011). In analyzed sera were negative to MTBC-specific IgG. addition, closely related strains of M. microti have been Therefore, infection by TB was not confirmed in any of recently isolated in both wild boars and wild rodents in the 302 murines and 48 arvicolines tested. The estimated Northern Italy strongly suggesting interspecies transmission TB prevalence was 0% [95% Confidence Interval ­(CI95%): (Tagliapietra et al. 2021). 0 – 1.3] in murines and 0% ­[CI95%: 0 – 7.4] in arvicolines, Recently, an outbreak of M. microti was detected in the while the overall prevalence was 0% [­ CI95%: 0 – 1.1]. The Catalan Pyrenees (north-eastern Iberian Peninsula) involv- ­CI95% of these prevalence were calculated in VassarStats ing wild boars during 2017–2019 (De Val et al. 2019). The website (Lowry 1998). strain involved presented the same genotype (genogroup D) to that previously isolated in the French side of the Pyrenees (Michelet et al. 2015a). This finding raised the question of Discussion whether M. microti was introduced from France via wild boars or was endemic in the outbreak area and whether its In the present study, M. microti infection was not detected in natural reservoir, namely wild rodents, played a role in the the community of wild rodents (302 murines and 48 arvico- epidemiology of the outbreak. The aim of this study was to lines) inhabiting a presumptive endemic area where infected investigate the presence and prevalence of M. microti among wild boars were previously identified. Similarly, no evidence wild rodents in different epidemiological scenarios within of M. microti in over 300 trapped wild mice was found in a the outbreak area. region of Switzerland with several cases of feline TB due to M. microti in domestic cats (Peterhans et al. 2020). On the contrary, M. microti was confirmed in wild rodents in Results a region of Northern Italy where infected wild boars were previously found (Tagliapietra et al. 2021). None of the macroscopical lesions observed were sub- These findings highlight different roles of wild rodents in sequently identified microscopically as TB compatible the epidemiology of M. microti and point to their apparent lesions, the most frequent being parasitic granulomatous negligible role in the area studied in the present work. Given hepatitis presumably caused by Capillaria spp., and lung that the maximum possible prevalence in micromammals in a b Fig. 1  a Liver granuloma with multinucleated giant cells in an Apodemus sylvaticus b Lung granuloma Apodemus sylvaticus 13 European Journal of Wildlife Research (2023) 69:111 Page 3 of 6 111 the study area was estimated to be 0% [­ CI95%: 0 – 1.1], TB indistinguishable to that observed in M. bovis or M. caprae is most likely not endemic in the micromammal population infections (De Val et al. 2019), i.e. granulomatous lym- of the outbreak area, and they cannot be considered as the phadenopathy, particularly in the submandibular lymph natural reservoir (De Val et al. 2019). However, the maxi- nodes (the sample that is most frequently obtained through mum possible prevalence in arvicolines was higher (7.4%) the surveillance program). In addition, recent studies indi- since less animals were captured, and thus the prevalence in cated that they can play a role in the maintenance and this subfamily may have been underestimated. Field voles transmission of M. microti (Chiari et al. 2016; De Val et al. and bank voles, the main described reservoirs of M. microti 2019; Michelet et al. 2020) and eventually participate, (Cavanagh et al. 2002), are members of this subfamily. together with wild rodents, in a maintenance community Therefore, the 44 analyzed animals might fall short to prop- of this pathogen (Tagliapietra et al. 2021). Indeed, TB due erly detect the infection in their populations. to M. bovis and M. caprae is considered a multi-host dis- In endemic regions, the prevalence of M. microti in wild ease and different susceptible species may play a role in rodents can fluctuate depending on the sampling season or the epidemiology constituting a maintenance community other ecological factors (Cavanagh et al. 2002). However, to of the infection (Santos et al. 2020) or acting as spillovers avoid this factor, the present study was designed including depending on the local epidemiological conditions. samples in two different seasons (spring and autumn) of two consecutive years in two different valleys and in areas with two different ecological conditions (farm surroundings and Conclusion areas distant from human influence). None of the captured wild rodents presented the TB During the hunting seasons (2020–2022) following the pathological phenotype described in field M. microti infec- detection of the positive wild boar, even though the sam- tions which includes skin granulomatous lesions as well as pling has been irregular or scant in some areas, no further granulomatous lesions of viscera (Kipar et al. 2014). Even M. microti cases have been detected in this species through though numerous microscopical granulomatous lesions were the Catalan wildlife health surveillance program. This sug- observed in liver and lung that showed similar features to gests that the most likely origin of the outbreak are wild those observed in the field (Kipar et al. 2014) and in experi- boar movements form across the French border where M. mental infections (Vidal et al. 2022), these lesions were not microti is endemic in wild boar populations (Michelet confirmed by Ziehl Neelsen’s stain, PCR, nor microbiologi- et al. 2015a, b, 2020), indeed, in the Ariège region, iso- cal culture in any instance. lates with the same spoligotype (SB0423 and its closely The M. microti previously isolated in wild boars in the related SB0112) have been found in wild boars and badg- studied area presented the spoligotype pattern SB0423 ers (Source: ANSES unpublished findings). However, if (De Val et al. 2019), phylogenomically associated with further cases were diagnosed, the hypothesis of the ability the SB0112, commonly called “llama-type” (Deforges of wild boars to maintain the infection without the involve- et al. 2004), and both of them characterized by the pres- ment of wild rodents will need to be considered. ence of the spacers 4, 5, 6, 7, 23 and 24 (www.​Mbovis.​ Therefore, wild boar TB must be carefully monitored in org). This subtype has been previously isolated in a wide areas where a case is detected in the species to prevent it range of large-mammals such as badgers, wild boars, cats, from spreading to domestic species or interfering with live- dogs and llamas (Michelet et al. 2015a). Nevertheless, to stock TB control measures. Likewise, the role of micromam- our knowledge, it has never been reported to be isolated in mals should be assessed in areas with wild boar M. microti wild rodents, in which the most frequently described are cases, such as the Central French Pyrenees, to investigate the “vole type” strains (e.g. SB0118, SB2277), character- their involvement in the disease maintenance community. ized by the absence of the spacers mentioned above (Van Soolingen et al. 1998; Tagliapietra et al. 2021). This suggest that micromammals could be less susceptible to the infection Methods by “llama type” strains compared to other mammals. How- ever, a recent experimental infection of bank voles with M. Area of study, animal trapping and sampling microti SB0423 (“llama-type”) induced similar pathologi- cal features (Vidal et al. 2022) to those described in voles Four capture campaigns were planned over a 2-year period naturally infected with “vole-type” strains in the field (Kipar in spring and autumn, from autumn 2020 to spring 2022 et al. 2014), indicating that the infection is potentially feasi- in the Central Pyrenees, Catalonia (Spain). Each campaign ble regardless of the natural susceptibility. was composed of two weeks of sampling, with one week in In any case, wild boars are newly identified susceptible Bagergue Valley (Naut Aran, N42.73 E0.91), and the other hosts to both M. microti subtypes that cause TB pathology in Isil Valley (Alt Àneu, N42.68 E1.08), corresponding to 13 111 Page 4 of 6 European Journal of Wildlife Research (2023) 69:111 the areas where the positive wild boars were previously Mycobacterial culture detected (De Val et al. 2019). In both valleys, 40 baited Heslinga™ traps (Groningen, The Netherlands) were placed After gross pathology examination, tissue samples around the farms where livestock spend the colder months, (~ 0.1 g) from spleen, liver, and lungs were pooled and as well as in the fields where they spend the summer months. sliced with sterile scissors and mechanically homogenized During these campaigns, wild rodents belonging to the in 1.5 ml of sterile distilled water. An aliquot of 0.5 ml of subfamilies Arvicolinae (n = 48, including Microtus arvalis, each homogenate was processed for mycobacterial culture Myodes glareolus, Microtus gerbei and Chiononys nivalis) in Löwenstein-Jensen with pyruvate and Coletsos solid and Murinae (n = 302, including Apodemus sylvaticus and media (BD diagnostics Sparks, MD, USA) and BAC- Mus musculus) were captured alive (See Table 1). Species TEC MGIT 320 system (BD diagnostics) as previously were identified, based on their morphological character- described (Vidal et al. 2022). istics using Gosàlbez and Noguera(Gosàlbez i Noguera DNA extraction from one milliliter of tissue homogen- 1988) as reference guide. After capture, mice and voles ates was performed by using the ID Gene™ spin universal were humanely euthanized in situ using an isoflurane over- extraction kit (ID.vet, Grabels, France) according to the dose, following the American Veterinary Medical Asso- manufacturer procedure. Afterwards, DNA samples were ciation (AVMA) guidelines for the euthanasia of animals amplified in a 7500 fast real-time PCR system (Applied (AVMA 2020). Immediately after, blood was collected Biosystems, Walham, MA, USA) using a MTBC-specific from all animals (except two arvicolines and one murine) real time PCR (ID Gene™ Mycobacterium tuberculosis by intracardiac puncture and were stored in a cooler with complex Duplex, ID.vet), following the manufacturer frozen ice packs until return to the base camp (maximum instructions. DNA samples were additionally tested with two hours later). At base camp, necropsies were performed IS6110 and IS1081 PCR system to detect MTBC (Lesellier on the animals, and the presence of macroscopic changes et al. 2019). Reactions were carried out in a 25 μl reac- was recorded. A fragment of spleen, liver, and lungs were tion mix containing TaqMan™ Fast Advanced Master Mix collected and stored at -20 °C. Submandibular and inguinal (ThermoFisher Scientific, Villebon sur Yvette, France), lymph nodes, as well as all thoracic and abdominal viscera, 300 nM forward and reverse primers, 250 nM probes, were removed from the carcasses, placed in cassettes, and sterile water, and 5 μl of DNA template. Thermocycling immersed in a 4% formaldehyde solution for histopathologi- conditions were 50 °C for 2 min (1 cycle), followed by cal examination. Blood was centrifuged and sera was col- one cycle of 20 s at 95 °C and 40 cycles of 3 s at 95 °C lected and stored at -20 °C until analyses. and 30 s at 60 °C. Pathological assessment IgG to MTBC detection by ELISA Formalin fixed tissues were sectioned embedded in paraf- Sera from 347 captured animals (48 of arvicolines and 302 fin wax. Four micrometer thick sections were obtained of murines) were analyzed for MTBC-specific antibodies and routinely stained with Hematoxylin and eosin for as previously described (Pérez de Val et al. 2017). Briefly, microscopical observation. When TB compatible lesions serum samples were thawed and analyzed by a multispecies were observed, additional sections were stained with indirect IgG ELISA to the MPB83 antigen (Lionex, Braun- Ziehl–Neelsen’s stain to visualize acid fast bacilli (AFB). schweig, Germany), using a Protein A/G mixture conjugated Table 1  Distribution of Municipality Sampling unit Subfamily Autumn 2020 Spring 2021 Autumn 2021 Spring 2022 wild rodents’ captures to investigate the prevalence of Naut Aran Farm Murinae 28 38 22 15 Mycobacterium microti, Central Arvicolinae 6 6 0 0 Pyrenees, Catalonia Field Murinae 13 18 22 10 Arvicolinae 1 5 0 0 Alt Àneu Farm Murinae 4 22 28 31 Arvicolinae 0 1 1 2 Field Murinae 7 10 10 24 Arvicolinae 11 7 6 2 13 European Journal of Wildlife Research (2023) 69:111 Page 5 of 6 111 with peroxidase (Merck, Darmstadt, Germany) for IgG need to obtain permission directly from the copyright holder. To view a detection. A sample was considered positive when ΔOD copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. (mean Optical Density -OD- at 450 nm of wells coated with MBP83 minus the OD of the uncoated well) ≥ 0.2. References Abbreviations AFB: Acid fast bacilli; AVMA: American Veterinary Medical Association; CI: Confidence interval; MTBC: Mycobacterium AVMA (2020) AVMA guidelines for the euthanasia of animals: 2020 tuberculosis Complex; OD: Optical Density; TB: Tuberculosis edition. American Veterinary Medical Association, Schaumburg, IL Acknowledgements We are grateful to Judit Burgaya, and the teams Boniotti MB, Gaffuri A, Gelmetti D et al (2014) Detection and molecu- of the pathology laboratory (Mònica Pérez, Rosa López and Anna lar characterization of Mycobacterium microti isolates in wild Barceló) and mycobacteria laboratory (Zoraida Cervera, Abel Muñoz boar from northern Italy. J Clin Microbiol 52:2834–2843. https://​ and Maite Martín) of IRTA-CReSA, the French National reference lab- doi.​org/​10.​1128/​JCM.​00440-​14 oratory for bovine TB (Anses) and WildCom Research Group (UAB) Cavanagh R, Begon M, Bennett M et al (2002) Mycobacterium microti for their excellent technical assistance. infection (vole tuberculosis) in wild rodent populations. J Clin Microbiol 40:3281–3285. https://d​ oi.o​ rg/1​ 0.1​ 128/J​ CM.4​ 0.9.3​ 281-​ Authors' contributions EV, JE, MP, OC and BP conceived and planned 3285.​2002 the study. EV, JE, MP, CM, LaM, LoM and MB performed the experi- Chiari M, Ferrari N, Giardiello D et al (2016) Spatiotemporal and ments and contributed to data collection. EV, JE, AA and BP analyzed Ecological Patterns of Mycobacterium microti Infection in Wild and interpreted the data. BP and AA acquired funding and were in Boar (Sus scrofa). Transbound Emerg Dis 63:e381–e388. https://​ charge of project administration. AS provided resources. EV and BP doi.​org/​10.​1111/​TBED.​12313 wrote the original draft. JE, MP and OC contributed to writing, review- De Val BP, Sanz A, Soler M et al (2019) Mycobacterium microti infec- ing, and editing the final version of the manuscript. All authors read tion in Free-Ranging Wild Boar, Spain, 2017–2019. Emerg Infect and approved the final manuscript. Dis 25:2152–2154 Deforges L, Boulouis HJ, Thibaud JL et al (2004) First isolation of Funding Open Access Funding provided by Universitat Autonoma Mycobacterium microti (Llama-type) from a dog. Vet Microbiol de Barcelona. This work was supported by the Grant EFA357/INNO- 103:249–253. https://​doi.​org/​10.​1016/j.​vetmic.​2004.​06.​016 TUB (Program Interreg POCTEFA 2004–2020) and the Department Frota CC, Hunt DM, Buxton RS et al (2004) Genome structure in the of Climate Action, Food, and Rural Agenda (DACC) of the Govern- vole bacillus, Mycobacterium microti, a member of the Mycobacte- ment of Catalonia and. IRTA is supported byCentres de Recerca de rium tuberculosis complex with a low virulence for humans. Micro- Catalunya(CERCA) Programme /Generalitat de Catalunya(www.cerca. biology 150:1519–1527. https://​doi.​org/​10.​1099/​MIC.0.​26660-0 cat). M. P. R. was funded through the 2021 FI Scholarship, Departa- Gosàlbez i Noguera J (1988) Insectívors i rosegadors de Catalunya: ment de Recerca i Universitats, Generalitat de Catalunya, Spain (FI_B metodologia d’estudi i catàleg faunístic 00171). C. M. is recipient of a pre-doctoral grant of the program “Don Kipar A, Burthe SJ, Hetzel U et al (2014) Mycobacterium microti Carlos Antonio López” of the Republic of Paraguay (Ref. 88/2020). Tuberculosis in Its Maintenance Host, the Field Vole (Microtus agrestis): Characterization of the Disease and Possible Routes of Availability of data and materials The datasets used and/or analysed Transmission. 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