Mycobacterium microti is a member of the Mycobacterium tuberculosis complex (MTBC), which also includes M. tuberculosis and M. bovis, the main causes of human and animal tuberculosis (TB), respectively. Even though voles and other wild small rodents were initially identified as its natural hosts (1), M. microti can cause pathology in a wide range of mammals, including pets, livestock, wildlife (2–5), and humans (6). M. microti infections have been previously reported in several countries in Europe, including Switzerland, Italy, and France (3,7–9). We report an outbreak of tuberculosis caused by M. microti in free-ranging wild boars in the Iberian Peninsula in Spain.

During June 2017–March 2019, a total of 9 free-ranging wild boars with lesions associated with TB were detected in the outbreak area, covering ≈3,000 hectares in the Catalan Pyrenees (Figure). TB was confirmed histologically, first by hematoxylin and eosin staining (9/9) and then by Ziehl-Neelsen staining of acid-fast bacilli (7/9). In all cases, submandibular lymph nodes showed granulomatous necrotizing lymphadenitis, sometimes with scant acid-fast bacilli, similar to that found in M. microti infections previously described in wild boar, which were generally confined to lymph nodes in the head (7,8).

To confirm the causative agent for these infections, we extracted DNA from tissue samples (DNAExtract-VK, Vacunek, http://vacunek.com) and performed real-time PCR (TBC-VK, Vacunek), which confirmed MTBC in 6 of 9 suspected cases. DVR spoligotyping at VISAVET Health Surveillance Centre, Universidad Complutense de Madrid, identified the pathogen in 4 of the 6 confirmed MTBC cases as M. microti (spoligopattern SB0423; Mycobacterium bovis spoligotype database, http://www.mbovis.org). In the other 2 cases, the laboratory was unable to determine the species because of low DNA load from the sample. This result likely was due to the slow in vitro growth rate of M. microti in infected animals, which makes it difficult to isolate in routine diagnostic laboratories.

Spoligopattern SB0423 is included in a phylogenetic cluster with spoligopattern SB0112, also associated with M. microti, on the basis of neighbor joining. Both spoligotypes are localized in the eastern French Pyrenees, close to the borders with Spain and Andorra (3). Most M. microti cases in France, found in cats, dogs, and llamas during 2005–2016 and, since 2015, in wild boars and badgers, have been found within 50 km of the outbreak area in Spain. The 2017 M. microti cases described in this report were found closer to the border with France; the remaining 2 cases, detected in 2019, were localized near the southern limit of the outbreak area (Figure).

In the outbreak area, up to 18 animals in 3 cattle herds showed positive results for a single intradermal tuberculin skin test (Figure). However, none of these animals showed gross lesions in target tissues (i.e., lungs, pulmonary, and retropharyngeal lymph nodes) at a slaughterhouse or positive results to mycobacterial culture and PCR. Similarly, in a recent case in France, a cow reacting to a TB skin test did not show TB-like lesions in respiratory tissues and returned negative results from cultures, but M. microti DNA was finally detected in retropharyngeal lymph nodes only by using advanced molecular techniques (5). These results indicate that cattle exposed to M. microti may induce positive results to diagnostic tests performed in bovine TB eradication campaigns. M. microti infection can sometimes cause visible lesions in cattle (2), but the fact that M. microti are natural knockouts for the virulence-related RD1^mic genomic region (10) may indicate a lower pathogenicity compared with other MTBC species and account for these negative test results.

We tested additional wild ungulates in the outbreak area and found that 6 (2 red deer, 1 fallow deer, and 3 chamois) were seropositive for MTBC using a MPB83-specific IgG indirect ELISA test (Figure). Unfortunately, no tissue samples were submitted to examine for lesions or to detect and identify mycobacteria. However, overall positive results for M. microti and the absence of other MTBC strains during the 2017–2019 period in the outbreak area suggest a multihost circulation of M. microti. Because voles are known maintenance hosts of M. microti (1), further investigation of wild small rodent populations in the Outbreak area could determine the epidemiology of this outbreak in greater detail.

These findings, together with previously reported cases nearer the border between France and Spain, indicate a transboundary circulation of M. microti across the Pyrenean border that should be taken into account for wildlife TB surveillance. Coordinated action between animal health authorities and laboratories in Spain and France is required, as well as the improvement of livestock management and biosecurity practices.