1 INTRODUCTION
Bovine tuberculosis (bTB) is an OIE (World Organisation for Animal Health) notifiable disease with major impact on livestock and wildlife (OIE, 2011). The disease is present in most countries, but with variable prevalence (Cousins, 2018). While certain developed nations have significantly reduced or eradicated the disease, developing countries with absent or inefficient control programs struggle to contain it, leading to a significant toll on livestock producers and/or devastating effects on wildlife (O’Reilly, Daborn, 1995; Ayele et al. 2004; Ferreira Neto, 2019). bTB can also be transmitted to humans through close contact with infected animals or the consumption of unpasteurized milk (Olea-Popelka et al., 2017). It is estimated that 147,000 new cases and 12,500 deaths occur due to zoonotic TB every year (WHO, 2017). Recently, the OIE, WHO (World Health Organization), The Union, and FAO (Food and Agriculture Organization) launched the roadmap for zoonotic TB, urging stakeholders to apply the One Health approach to tackle zoonotic TB and contribute to achieve the sustainable development goal of ending the global TB epidemic by 2030 (WHO, 2017).
Brazil is the second largest beef producer in the world and the number one exporter. In 2001, the Brazilian government launched the National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), setting structured strategies of bTB detection and control through tuberculin skin testing (TST) and slaughterhouse and trade surveillance in cattle and buffalo herds. Fifteen years later, the prevalence of bTB in 13 states was assessed through TST, representing 75% of the Brazilian bovine herd (Ferreira Neto et al., 2016). The epidemiologic status was described as heterogeneous within and among states, with the prevalence varying from 0 to 2.5% in cattle and from 0 and 13.9% in herds (Ferreira Neto et al., 2016). In contrast to few other countries, a wildlife reservoir of Mycobacterium bovis , the main causative pathogen of bTB, has not been identified in Brazil until now, hence current bTB control efforts are mostly concerned with cattle transit and introduction of infected cattle into uninfected herds.
Molecular epidemiology has been a resourceful tool to understandM. bovis populational structure and transmission dynamics in livestock and multi-host systems (Guimaraes & Zimpel, 2020). More recently, whole genome sequencing (WGS) of M. bovis has allowed a more precise and in depth understanding of these topics. A recent study of M. bovis genomes from multiple countries showed that the historically used clonal complexes (i.e. European 1 and 2, African 1 and 2) (Smith et al., 2011; Müller et al., 2009; Berg et al., 2011; Rodriguez-Campos et al. 2012) do not represent the whole genetic diversity of this pathogen, and the existence of at least four lineages (Lb1-Lb4) and three “unknown groups” of M. bovis was suggested (Zimpel et al., 2020). On the other hand, numerous other studies have used WGS of M. bovis to evaluate pathogen transmission in cattle herds and wildlife [reviewed by (Guimaraes & Zimpel, 2020)], showing the usefulness of this technique for bTB surveillance systems. WGS allows, for instance, to detect how many M. bovis strains are circulating in a particular herd, when these strains were introduced, and the transmission links among animals or farms (Guimaraes & Zimpel, 2020).
In Brazil, despite the rare bTB detection in free-ranging wildlife (Maciel et al., 2018), reports of M. bovis infecting captive wild animals are frequent (Zimpel et al.,2017; Rocha et al., 2011, Murakami et al. 2012, Ikuta et al. 2018). The source of infection to these zoo animals, however, remains elusive. In 1977, a privately-owned safari park was inaugurated in the state of Rio Grande do Sul, Southern Brazil for public entertainment and environmental education. This safari comprised an area of 320 acres and harbored many different species of animals, both exotic and native, including deer, capybaras, llamas, camels, hippopotamus, different bird species, horses, non-human primates, among others. This man-made multi-host system posed various sanitary challenges over the years, culminating with its closure as defined by a court order in 2013. Among these challenges, several animals died of TB, with reports dating back to 2003. Therefore, the aims of this study were to compile the TB history of this safari park through official records and to evaluate M. bovis strain diversity, time of bTB introduction in the park, and path of transmission using WGS data. To achieve these objectives, we sequencedM. bovis genomes of 19 deer culled in 2018 and gathered other twoM. bovis genomes obtained from llamas in 2012, sequenced in a previous study (Zimpel et al., 2020). The events that took place in the safari park described in this study highlight many regulatory gaps of the current bTB control and animal welfare legislations in the country. While we were able to access the data presented herein only after many years that these events ensued, we take this opportunity to launch a call for action for the control of TB in captive wild animals in Brazil.