RESULTS
The 17 animals from which M. tuberculosis var. bovis was isolated came from ten municipalities in the state of Pernambuco (Alagoinha, Bom Conselho, Chã Grande, Garanhuns, Ibirajuba, Jurema, Pedra, Pesqueira, Ribeirão and Venturosa), which were mostly raised in the semi-intensive management system. These municipalities belong to three geographic regions of the state, namely Southern Agreste, Central Agreste and South Agreste. Among the animals diagnosed with the disease, females were the most affected (16/17) and 64.7% (11/17) were older than five years; one calf of seven months old also yielded positive culture.
The clinical examination of cattle and buffaloes revealed apathy, lack of appetite, low body mass score, seromucous nasal discharge, dry cough, dyspnea, tachypnea, polyps, crackles, and areas of silence in the lung fields. Upon evaluation of the mammary gland, two (2/17) bovines were diagnosed with hypertrophied lymph nodes: one of these presented an enlarged posterior breast of firm consistency, hyperemia and hyperthermia and physical changes in milk in one of the teats (lumps with serum).The other bovine had an anterior breast of firm consistency but with no visible changes of the milk. During rectal examination, some animals presented nodular structures of varying sizes and hardened consistency in the region of the mesentery, serous in the rumen and uterus.
Macroscopic observation of lesions seen during post-mortemexamination revealed that 12/17 animals (70.6%) had miliary or protruding tuberculosis, distributed mainly in the lungs, mediastinal and tracheobronchial lymph nodes, liver and mesenteric lymph nodes, and less frequently in the kidneys, spleen and greater omentum. Among the animals with generalized tuberculosis, two cattle also showed changes in the mammary gland and the uterus, characterized by granulomatous lesions with multifocal distribution and varied sizes, with areas of calcification and abscesses.
The granulomatous nodules observed in all animals were pleomorphic, had a caseous, thick, and yellowish content, with the formation of a fibrous capsule (Figure A). In buffaloes, granulomas had a more whitish color when compared to cattle (Figure B). In the young calf, in addition to lung lesions, small granulomas were observed in the central nervous system and lesions compatible with meningoencephalitis.
Histopathological analysis of the lesions revealed areas of central caseous necrosis and dystrophic calcification, intense inflammatory reaction in the regions adjacent to the necrosis areas, with a predominance of epithelioid macrophages and multinucleated giant cells, like Langhans .
Microbiological cultivation presented growth of colonies in 17/30 (57%) samples that were confirmed to be Mycobacterium spp and more specifically M. tuberculosis var. bovis by molecular techniques. In three samples, presence of Trueperella pyogenes and in a single animal, Nocardia spp was encountered. Of the 17 bacterial growths, 14 were classified by the enzymatic restriction analysis of thegyr B gene as M. bovis . However, due to the importance of bacterial isolation, recognized as a gold standard test, the 17 samples were submitted to molecular genotyping techniques bySpoligotyping and 24-loci MIRU-VNTR.
Spoligotyping revealed 17 patterns classified as belonging toM. tuberculosis var. bovis , including SB0121 (n=8), SB0295 (n=5), SB0852 (n=2), SB0120 (n=1) and a spoligotype that was not yet present in the Database (Table I).
The analysis of 24-loci MIRU-VNTR was identified 13 genetic profiles from the 17 isolates of M. tuberculosis var. bovis from 14 properties in the state of Pernambuco (Table II).
The analysis of the discriminatory power (HGDI) of MIRU-VNTR in this study was higher, as expected, than Spoligotyping , respectively (0.980) and (0.713). Distribution of the isolates according to the number of alleles in each locus and the analysis of the allelic diversity of the 24 loci is summarized in (Table III). Locus ETR A showed the highest discriminatory power ( ₌ 0.69), while five loci (ETR B, MIRU 16, ETR C, MIRU 27 and QUB 26) were classified as moderately discriminatory with h between 0.33 to 0.58. Eight loci (MIRU 20, MIRU 26, Mtub 04, Mtub 29, QUB 11b, QUB 4156, Mtub 21, Mtub 39) presented low discriminatory power (h ≤0.27) while ten loci showed absence of allelic diversity.
Isolates 1 and 10 showed failures in the amplification of someloci that are generally attributed to possible DNA mutations or degradation (Supply et al., 2006), thus preventing the primersfrom ringing. Given these results, the respective isolates started to be analyzed only in Spoligotyping, obtaining significant results.