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 (h ₌
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.