*Article* **Spatial and Temporal Distribution of** *Mycobacterium tuberculosis* **Complex Infection in Eurasian Badger (***Meles meles***) and Cattle in Asturias, Spain**

**Cristina Blanco Vázquez 1 , Thiago Doria Barral 2 , Beatriz Romero 3 , Manuel Queipo 4 , Isabel Merediz 5 , Pablo Quirós 6 , José Ángel Armenteros 6 , Ramón Juste 7 , Lucas Domínguez 3,8 , Mercedes Domínguez 9 , Rosa Casais <sup>1</sup> and Ana Balseiro 10,11, \***


**Simple Summary:** The aim of the present work was to investigate the prevalence, spatial distribution, and temporal distribution of tuberculosis in 673 free-ranging Eurasian badgers (*Meles meles*) and cattle from Asturias (Atlantic Spain) during a 13-year follow-up. The study objective was to assess the role of badgers as a reservoir of tuberculosis for cattle and other sympatric wild species in the region. During the follow-up, 27/639 badgers (4.23%) were positive for the *Mycobacterium tuberculosis* complex based on bacterial isolation, while 160/673 (23.77%) were positive based on P22 ELISA. Badger infection was spatially and temporally associated with cattle herd infection.

**Abstract:** The present work investigated the prevalence, spatial distribution, and temporal distribution of tuberculosis (TB) in free-ranging Eurasian badgers (*Meles meles*) and cattle in Asturias (Atlantic Spain) during a 13-year follow-up. The study objective was to assess the role of badgers as a TB reservoir for cattle and other sympatric wild species in the region. Between 2008 and 2020, 673 badgers (98 trapped and 575 killed in road traffic accidents) in Asturias were necropsied, and their tissue samples were cultured for the *Mycobacterium tuberculosis* complex (MTC) isolation. Serum samples were tested in an in-house indirect P22 ELISA to detect antibodies against the MTC. In parallel, data on MTC isolation and single intradermal tuberculin test results were extracted for cattle that were tested and culled as part of the Spanish National Program for the Eradication of Bovine TB. A total of 27/639 badgers (4.23%) were positive for MTC based on bacterial isolation, while 160/673 badgers (23.77%) were found to be positive with the P22 ELISA. The rate of seropositivity was higher among adult badgers than subadults. Badger TB status was spatially and temporally associated with cattle TB status. Our results cannot determine the direction of possible interspecies transmission, but they are consistent with the idea that the two hosts may exert infection pressure on

**Citation:** Blanco Vázquez, C.; Barral, T.D.; Romero, B.; Queipo, M.; Merediz, I.; Quirós, P.; Armenteros, J.Á.; Juste, R.; Domínguez, L.; Domínguez, M.; et al. Spatial and Temporal Distribution of *Mycobacterium tuberculosis* Complex Infection in Eurasian Badger (*Meles meles*) and Cattle in Asturias, Spain. *Animals* **2021**, *11*, 1294. https://doi.org/10.3390/ani11051294

Academic Editor: Nicole Gottdenker

Received: 1 March 2021 Accepted: 28 April 2021 Published: 30 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

each other. This study highlights the importance of the wildlife monitoring of infection and disease during epidemiological interventions in order to optimize outcomes.

**Keywords:** *Meles meles*; badger; tuberculosis; *Mycobacterium tuberculosis* complex; P22 ELISA; isolation; serology; cattle; Atlantic Spain

#### **1. Introduction**

Animal tuberculosis (TB) is caused by infection with members of the *Mycobacterium tuberculosis* complex (MTC), mainly *M. bovis* and, to a lesser extent, *M. caprae*. TB is a zoonotic disease that subjects livestock worldwide and can cause substantial economic losses [1]. Its main domestic reservoir is cattle, and eradication campaigns spanning more than four decades in developed countries including Spain have been quite but not totally successful [2]. Wildlife hosts are also susceptible to *M. bovis* and can act as reservoirs of the infection for livestock. Badgers (*Meles meles*) and wild boars (*Sus scrofa*) are major wildlife reservoirs of *M. bovis* for bovine TB in several European countries. *M. bovis*-infected badgers have been found in Ireland [3], the United Kingdom (UK) [4], France [5,6], and Spain [7,8]; in Ireland and the UK, badgers are recognized as major reservoirs with the potential to transmit infection to local cattle herds [9,10]. Infection in wild boars has been described in France and Italy, as well as the southern and central Iberian Peninsula [5,11].

In Spain, the southern and central regions, which have a Mediterranean climate, show a TB cattle herd-prevalence as high as 12.3%, while the northwestern region with an Atlantic climate shows an overall prevalence of 0.02–0.08% but with hotspots where prevalence can be as high as 5% [2,12]. This variability may reflect several factors. One is climate: drier, hotter areas in the south favor animal aggregation and therefore disease spread [13]. Another factor is type of cattle: dairy herds, in which TB prevalence is lower, concentrate in the north, while beef and bullfighting cattle are more abundant in the central and southern regions [13]. A third factor is cattle management: beef and bullfighting cattle herds are extensively managed with a lower biosecurity and are therefore at higher risk of infection due to contact with animals in other herds, as well as with other potentially infected domestic (goat/sheep) or wildlife species [14–16].

Recent studies have suggested that badgers may be a reservoir of *M. bovis* infection in hotspots in Atlantic Spain, as reported in Ireland and the UK [12,17]. In addition, wild boar, red deer (*Cervus elaphus*), and, to a lesser extent, fallow deer (*Dama dama*) are major wildlife reservoirs in continental Mediterranean habitats, where wild boars can show a TB prevalence of >50% [14]. In contrast, wild boars in Atlantic habitats show a TB prevalence of around only 5% [16]. Mid- and long-term studies of wildlife reservoirs such as badgers and wild boar can shed light on the spatial and temporal dynamics of TB, as well as on risk to local livestock [14,18,19].

Along these lines, the present work investigated the prevalence, spatial distribution, and temporal distribution of TB in free-ranging Eurasian badgers and cattle from Asturias (Atlantic Spain) during a 13-year follow-up. The study objective was to assess the potential role of badgers as a TB reservoir for cattle and other sympatric wild species such as wild boar in the region.

#### **2. Materials and Methods**

#### *2.1. Ethics Statement*

All methods were carried out in accordance with relevant guidelines and regulations. All experimental procedures with trapped badgers were approved by the Government of the Principality of Asturias (010/07-01-2011, PROAE 20/2015, PROAE 47/2018).

#### *2.2. Study Area*

The study was carried out in the region of Asturias in northwestern Spain. This region has an Atlantic climate, and the temperature ranges from −4 to 8 ◦C in the coldest months; precipitation is abundant throughout the year, annually reaching 1400–2100 mm [20]. More than 30% of the territory is forest and mainly comprises oaks, beech, and birch woods. Asturias is bordered to the north by the Cantabrian Sea and to the south by the Cantabrian Range. Nowadays, the cattle population is 391,797 animals and 15,856 herds, and the well-studied badger population shows intermediate density of 3.81 adults/km<sup>2</sup> [21].
