DISCUSSION
It should be noted that the state of Pernambuco occupies a prominent
place in milk production in the Northeast region, and the municipality
of Garanhuns and its microregion is recognized as the state’s milk basin
(Penaforte Júnior et al., 2009). Dairy cattle and buffaloes are
considered more vulnerable to M. tuberculosis var. bovisinfection, as they have a longer life expectancy, stay longer on the
properties and are subjected to the rearing semi-intensive and intensive
system, very common in the region. During milking and other common
management practices, animals cohabit, therefore increasing their
likelihood of contact and the transmission of tuberculosis (Lima et al.,
2016; Veloso et al., 2016), considered endemic in the State of
Pernambuco (Izael et al., 2009; Lima et al., 2016).The constant transit
of animals between the properties within and between neighboring
municipalities, the interstate cattle trade and the absence of an
effective sanitary control of the herds are factors that contribute to
the spread of the disease in the region (Lima et al., 2016; Veloso et
al., 2016).
In the present study, all animals presented clinical symptoms of
tuberculosis with predominating respiratory impairment. In dairy farms,
female animals generally remain for longer periods depending on the
reproductive period, and this could be the main reason for having
observed in this study the predominance of females over the age of five
years to be exposed to M. tuberculosis var. bovis when compared
to young cattle (Veloso et al., 2016). Nonetheless, young animals also
contract the infection and develop disease, as demonstrated by M.
tuberculosis var. bovis isolation from a seven-month-old calf. The
frequency of tuberculosis in cattle aged less than 12 months is
generally associated with the ingestion of colostrum/milk from infected
cows or transplacental infection (Konradt et al., 2016; Silveira et al.,
2018). The most evident clinical signs were observed in the advanced
stages of the disease, as described by Izael et al. (2009) and Waters
(2015), except for the calf that manifested the disease earlier in the
form of cerebral tuberculosis combined with depression and paresis of
the limbs. In addition to the predominant respiratory impairment in the
animals in this study, two animals showed clinical changes in the
mammary gland result similar to described by Waters (2015). This
observation reinforces the potential risk of the disease to public
health due to the consumption of raw milk and non-pasteurized
derivatives, mainly observed in inland cities and rural areas, such as
Garanhuns and the microregion (Penaforte Júnior et al., 2009).
The generalized form of the disease was predominant both in cattle and
the two buffaloes, with lesions that had disseminated to several organs.
All animals had granulomatous injuries in the thoracic organs (lungs,
pleura, tracheobronchial and mediastinal lymph nodes), causing
respiratory impairment. This result is similar to those described by
Ramos et al. (2018), who reported a higher prevalence of lesions
compatible with tuberculosis in tracheobronchial, mediastinal lymph
nodes and lungs; such typical predominance of lesions in the respiratory
tract is indicative for air-borne transmission. On the other hand,
Alzamora Filho et al. (2014) identified the most evident lesions in the
lymph nodes of the head (retropharyngeal and parotid) with pulmonary
parenchyma. These results corroborate with the findings of the present
study, due to the typical predominance of lesions in the respiratory
tract, suggesting the airway, as the main gateway for M.
tuberculosis var. bovis in bovines. The lower occurrence of mesenteric
lymph node involvement here observed was also described by Ramos et al.
(2018) and justified by the fact that oral route infection is secondary
to the respiratory route in adult cattle.
The granulomatous lesions observed in the mammary gland and uterus
common to two animals in this study reinforce the potential risk of
transmission of M. tuberculosis var. bovis to humans due to the
consumption of raw milk and its products (Cezar et al., 2016; Siala et
al., 2019). On the other hand, the granulomatous lesions located in the
central nervous system in young cattle is probably related to the
ingestion of colostrum/milk from infected cows and can be justified by
ascending infection via hematogenic route. This form of cerebral
tuberculosis in cattle was also reported by Konradt et al. (2016) and
Silveira et al. (2018).
The histopathological characterization of lesions present in granulomas
was similar to the findings described by França et al. (2013) who found
in some samples a marked process of calcification with mineralization,
differing from the lesions observed by Ramos et al. (2018) and Silva et
al. (2018) who presented a more caseous aspect, suggesting that the
animals that had been slaughtered were suffering from a recent infection
or disease development.
The frequency of isolation, of M. tuberculosis var. bovis, of
57% observed presently in animals, with clinical tuberculosis. It has
been described that some factors can interfere with the success of
mycobacterial isolation and in particular of M. tuberculosis var.
bovis , including the rigorous decontamination process of samples and
the chronic character of the disease that confers intense calcification
of the lesions and leading to low concentration or absence of viable
bacilli (Ambrosio et al., 2008). That might have been influenced the low
isolation of M. bovis in the present sampling.
Besides Mycobacterium spp ., we also observed bacteria belonging
to other genera such as Trueperella pyogenes and Nocardia
spp . It is worth mentioning that some microorganisms besides these,
such as Actinomyces spp and Actinobacillus spp , are also
responsible for causing granulomatous lesions similar to tuberculosis
lesions (Mendes et al., 2013).
In the present study, 17 isolates compatible with Mycobacterium
spp . Were subjected to molecular diagnostics by RFLP of the gyr B
gene. However, the analysis classified only 14/17 isolates as M.
tuberculosis var. bovis , different from the study carried out by Franco
et al. (2017) that obtained 100% compatibility between the isolation ofMycobacterium spp and the gyr B analysis. The result
obtained in the RFLP is probably related to factors that interfere with
molecular tests, such as the presence of inhibitors of PCR reactions,
low amount of viable bacilli due to chronic lesions, contaminants in the
samples, failures in extraction processing or DNA degradation (Carel et
al., 2014).
Spoligotype SB0121, the most frequently encountered was described
as the most prevalent in national territory with a frequency of 29.1%
in a study conducted in Latin American countries (Zumárraga et al.,
2013). The fact that we identified this spoligotype in the three
defined geographical regions studies here could be caused by the
constant movement of animals, due to the practice of interstate cattle
trade and also strongly suggestive for recent infections (Rodríguez et
al., 2010; Zumárraga et al., 2013).
The SB0295 profile was the second most prevalent spoligotype in
this study (29%) and has been referenced in Brazil with a prevalence of
24% (Zumárraga et al., 2013). This is similar to that in the Midwest
Region of the country, being identified in 16.2% of the total isolates
(Carvalho et al., 2016). The two buffalo isolates in this study also
presented spoligotype SB0295, a profile typical to those
identified in buffaloes in the Amazon region (Carneiro et al., 2019), it
is common to breed mixed buffalo and cattle under the same management
conditions.
Spoligotype SB0852 was identified in two isolates. According to
the international database, SB0852 has only been registered in Italy
(Boniotti et al., 2009), suggesting a process of natural selection of
these strains between geographic locations (Carvalho et al., 2016) or
convergent evolution (Zumárraga et al., 2013).
Finally, two spoligotypes were observed in this study single
isolates only, being the case for SB0120, similar to the low frequency
of occurrence in other regions of the country (Parreiras et al. 2012;
Zumárraga et al., 2013; Franco et al., 2017). The other was from a
bovine that presented a spoligotype not present in the
international database; this could be due to some microevolutionary
events in the DR regions of a strain with an existing pattern (Adesokan
et al., 2019).
In the region of development of the study, bovine tuberculosis is
characterized as endemic, and the practice of commercialization and
consumption of milk and fresh products increases the risk of zoonotic
transmission, increasing the risk of sharing M. tuberculosis var.
bovis isolates common among dairy cattle and the human population of
the region, as previously recorded in other studies in different areas
of the world. Genomic diversity in the M. tuberculosis complex
remains a significant factor in the pathogenesis of tuberculosis, which
can affect virulence, transmissibility, host response, and drug
resistance (Adesokan et al., 2019).
The genotyping performed in this study from the set of 24-lociMIRU-VNTR is recommended for the comparative study of M.
tuberculosis var. bovis profiles world wide (Supply et al., 2006).
Molecular genotyping identified 13 distinct genetic profiles, suggesting
a diversity of M. bovis within and between the regions studied
and considerable higher discriminatory power as compared toSpoligotyping . This is according to earlier results obtained both
in Brazil (Carvalho et al., 2016) and in other countries. This
demonstrated that although a large cluster was observed byspoligotyping alone, there exists genetic diversity among the
strains of M. tuberculosis var. bovis in Pernambuco,
probably due to the movement of animals between different regions,
states and rural properties (Zumárraga et al., 2013; Carvalho et al.,
2016).
The analysis of allelic diversity of the different MIRUs are similar to
those found by Souza Filho et al. (2013) and Carvalho et al. (2016) and
demonstrating that for this MTBC species, only six of 24 loci allowed
good discrimination, different from M . tuberculosis (Hilty
et al., 2005). The HGDI of 24-MIRU-VNTR and spoligotyping in this
study was 0.980 and 0.713, respectively, close to that observed by
Carvalho et al. (2016) with values 0.980 and 0.810 and the HGDI of 0.912
reported by Souza Filho et al. (2013). Therefore, it seems that
simultaneous consideration of both genotyping techniques for clustering
might be more accurate for M. bovis transmission studies, also in
the present study. However, the association between these techniques has
been considered the best strategy for the molecular typing of M.
tuberculosis var. bovis because they present better reproducibility and
reliability, aiming at the analysis of strains mycobacterial (Carvalho
et al., 2016).
This study is of great importance for the region as it is the first work
carried out on molecular genotyping through the association betweenSpoligotyping and MIRU-VNTR aiming at the molecular
characterization of M. tuberculosis var. bovis isolates and
identification of circulating genotypes in the state of Pernambuco. It
is worth mentioning the importance of M. bovis as a cause of
human tuberculosis, although sometimes neglected, especially in
developing countries. The consumption of raw milk and dairy products and
the constant exposure to reservoir animals are considered the main risk
factors in the epidemiological chain of infection.