4 Discussion
In this study, we describe an outbreak of tuberculosis caused byM. bovis in a Brazilian safari park that lasted for at least 15
years. This outbreak was a result of multiple pathogen introductions and
culminated with the death of many animal specimens. There are two main
reasons that can explain why this outbreak was so severe. First,
starting in 2005 the park passed through management changes and
financial hurdles that led to animal overcrowding and malnutrition. Many
animals of different species shared a small enclosure and feeding
practices were not adequate to serve them all. Overcrowding and
malnutrition are known risk factors of bTB (Pollock & Neill, 2002).
Second, the park owners often acquired and introduced animals of unknown
bTB status (i.e. without diagnostic testing) into the premises,
particularly cattle and buffalo. Collectively, these factors may have
facilitated pathogen introduction and spread in the property as well as
the development of clinical disease by the wild animals.
Our results suggest two possible timeline scenarios for the dynamics of
this bTB outbreak. One scenario involves at least three differentM. bovis introductions occurring in the time period between the
park inauguration in 1977 up until around 2003. The founding M.
bovis subsequently evolved into many different strains over time,
fueled by persistent animal to animal transmission. These strains were
likely circulating at low levels or asymptomatically until the
above-described conditions of overcrowding and malnutrition became
conducive of a deadly outbreak. As M. bovis is endemic in the
region (5.2% of infected herd prevalence (Queiroz et al., 2016)), a
second scenario involves the introduction of many different strains over
the years, possibly through cattle, contributing to the high M.
bovis diversity observed in the property. Although cattle and wildlife
were separated by a fence, escape and mixing of animals in both
enclosures were reportedly common events. Alternatively, pathogen
introduction may have occurred with the acquisition of specimens of
infected wild animals, since TB cases occur in wild animals in captivity
in Brazil (Zimpel et al., 2017; Rocha et al., 2011; Murakami et al.,
2018; Ikuta et al., 2018; Murakami et al., 2012).
As a wild animal reservoir of bTB has not been identified in Brazil so
far, in its inception the PNCEBT was designed to cover cattle and
buffalo only. Hence notifications of TB cases in wildlife are not
mandatory, diagnostics are not standardized, and there are no official
guidelines for outbreak resolution or prevention in wildlife. These
factors likely influenced the course of the outbreak described herein.
Thus, we believe the inclusion of wildlife in the PNCEBT can bring great
benefits in terms of animal welfare, while increasing biosafety in zoos
and parks. In addition, regulation of the diagnosis of tuberculosis in
wild animals would have an impact on reducing outbreaks that may occur
through sale or trade of infected animals. The many outbreaks seen in
Brazilian captive populations over the years (Zimpel et al., 2017; Rocha
et al., 2011; Murakami et al., 2018; Ikuta et al., 2018; Murakami et
al., 2012), along with the one reported herein, should form the
rationale necessary to develop this nationwide plan to control and
report TB in wildlife, as also suggested previously (Valvassoura &
Ferreira Neto, 2014). Notwithstanding, for these policies to become
effective, it is necessary to integrate federal and state surveillance
services and environmental regulatory agencies.
Our study adds to the importance of evaluating potential paths of
transmission to understand bTB outbreaks. Using phylogenetic analysis
and M. tuberculosis -based cut-offs for the number of SNPs, many
different strains of M. bovis were found circulating in the park,
with four recent transmission events involving five deer. The low SNP
distance between these five genomes suggests that the same M.
bovis strain was transmitted from one animal to another, and the amount
of accumulated genetic changes is just a reflection of microevolution.
In addition, one of the animals was co-infected with multiple strains,
as it presented many heterogeneous SNPs. This mixed infection occurs
when the individual is exposed to a single infection event carrying
multiple strains, or to several infection events with different strains
throughout its life, resulting in a superinfection. Such condition is
normally seen in conditions of high disease endemicity, as described in
this park.
Using the SNP-cutoff approach, recent transmission events between the
surveyed cattle farms and the safari park were not observed in this
study. It is possible that the sampling of cattle farms was not
comprehensive enough to capture the true M. bovis diversity of
Rio Grande do Sul state. The origin of the cattle maintained at the
safari park was unknown to the authors of this study; thus, it is also
possible that the region from which they originated was not covered in
the cattle sampling. Alternatively, the M. bovis strains
infecting deer and llamas have been introduced a long time ago in the
park (as suggested by the dating analysis) and evolved in geographic
isolation. Nevertheless, the phylogenetic relatedness among strains
indicates that M. bovis was introduced in the park from somewhere
in the region at some point in time.
Although commonly used as a measure of M. tuberculosis orM. bovis transmission between individuals, the SNP cutoff
approach was never standardized for M. bovis , is sensitive to the
pipeline used to detect SNPs (Guimaraes & Zimpel, 2020), and also a
simplistic approach to follow the complex evolution of tuberculous
mycobacteria (Menardo et al., 2019). The BEAST analysis performed herein
rejected a strict clock evolution model of M. bovis ,
corroborating previous studies on M. bovis (Zimpel et al., 2020;
Crispell et al., 2019; Salvador, 2019) and M. tuberculosis(Menardo et al., 2019; Rutaihwa et al., 2019). We are also unaware if
evolutionary rates of M. bovis are influenced by the disease form
(e.g. latent versus clinical disease) or host species. Out of the
32 possible transmission links, we observed discordant spoligotype
patterns in five, from which three were due to loss of spacers, but two
either gained or lost spacers (Appendix Figure 5). Therefore, results
reported regarding recent transmission events should be interpreted with
caution; and further studies should be conducted to address how we can
best use genomics to infer transmission of M. bovis strains and
how these can be compared to traditional genotyping techniques such as
spoligotyping and MIRU-VNTR.
This study has limitations. Unfortunately, M. bovis isolates from
all other animals that died during the outbreak were not obtained,
precluding our ability to evaluate the true genetic diversity of the
pathogen in the park. We were also unable to control sample collection
at the slaughterhouse where deer were culled, thus preventing a better
description of the lesions found, number of affected animals, and host
species. And finally, we did not have access to records of animal
introductions that occurred in the park, impeding a more comprehensive
analysis of pathogen introduction.