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.