Zoonotic Tuberculosis - Evaluating The Status And Potential Hazards

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Date
2013
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Tamil Nadu Veterinary and Animal Sciences University
Abstract
The present study was attempted to identify the status of zoonotic tuberculosis in particular in different species including bovines, ovines and humans. The Postgraduate and Research Institute in Animal Sciences (PGRIAS), TANUVAS, Kattupakkam was the organised dairy unit chosen for the study where regular herd based survey for bovine tuberculosis was conducted and unorganised small holder dairy units sampled included cattle and buffaloes from Large Animal Clinics (LAC), Madras Veterinary College (MVC) Teaching Hospital, Chennai (urban setup) and representative samples from Dharmapuri and Krishnagiri districts (rural category). The human subjects for the study were chosen from Thiruwatteeswarar hospital for thoracic diseases, Chennai. The samples for the study included 504 bovine milk, 210 pre-scapular lymph gland (PSLG) fine needle aspirates, 510 post-mortem(PM) tissues, 100 processed milk samples and 357 sera samples from bovines, 35 PM tissues from ovines and 279 human clinical samples which included 269 sputum, five lung aspirates, three lymph gland fine needle aspirates and two urine samples. The diagnostic tests employed were staining for acid fast bacilli (AFB), auramine staining, culture, genus specific polymerase chain reaction (PCR) to identify the Mycobacterium tuberculosis complex (MTBC), multiplex PCR to differentiate Mycobacterium tuberculosis and Mycobacterium bovis, spoligotyping and sequence analysis of isolates. Risk factors for zoonotic tuberculosis were also analyzed with the database created. While AFB and auramine staining detected 4.33 per cent and 5.39 per cent respectively in bovines, PCR detected 4.51 per cent positivity on screening of 1224 samples. Ovines showed per cent positivity of 14.29 and 20.00 for AFB and auramine staining respectively while PCR identified 8.57 per cent positivity by screening of 35 samples. The positive percentage was 50.90, 60.57 and 65.95 in human subjects by AFB, auramine staining and PCR respectively and the study population included actually suspected or confirmed cases of tuberculosis. Out of 504 milk samples tested, 7 (1.39 per cent), 9 (1.79 per cent) and 18 (3.57 per cent) samples were positive for tuberculosis by acid-fast, auramine staining and PCR respectively and of the 210 PSLG aspirate samples tested, 10 (4.76 per cent), 13 (6.19 per cent) and 12 (5.71 per cent) samples were positive for tuberculosis for AFB, auramine staining and PCR respectively. Out of the 510 tissue samples tested, 35 (6.86 per cent), 42 (8.24 per cent) and 23 (4.51 per cent) were positive for tuberculosis for AFB, by auramine staining and PCR respectively. In a routine slaughter house examination of 2000 slaughtered heads of cattle examined during the study, 15.9 per cent were diagnosed with gross tuberculosis like lesions in various organs and of 100 sheep examined at slaughter, 35 per cent showed gross tuberculosis like lesions. The humoral antibody response was 4.48 per cent in bovine serum by ELISA in a representative unit of dairy cattle indicating progress in clinical tuberculosis. None of the 100 processed milk samples were found to be positive by PCR. Comparative evaluation of different tests used for diagnosis of bovine tuberculosis and human tuberculosis by statistical analysis using chi-square test indicated a significant difference between the various tests employed (AFB and auramine staining, auramine staining and PCR and AFB and PCR). Statistical analysis using chi-square test indicated a highly significant difference between the age groups and also among the species studied viz. non- descript animals, cross bred cattle and buffalo using both the ELISA and PCR positivity. Of the 1224 bovine samples screened by IS 6110 PCR, 53 were found positive for MTBC organisms. On differentiation by multiplex PCR, 48 (4.24 per cent) of 1224 samples were M. tuberculosis and only 5 (0.44 per cent) were M. bovis. Isolation of M. tuberculosis and M. bovis were attempted on samples positive for AFB or auramine staining or PCR. Totally 70 samples were subjected for culture of which 37 samples were found to show characteristic growth. Of the 37 culture positives, 34 were M. tuberculosis and three were M. bovis. In the present study there was higher number of isolations of M. tuberculosis in animals in comparison to M. bovis. Transmission Electron Microscopy (TEM) of an isolate of M. bovis was carried out for further confirmation to reveal mycobacteria with corresponding morphology. All the three tissue isolates of ovines were M. tuberculosis and the positive per cent was 8.57. However, one chimpanzee lung nodule sample suspected for tuberculosis tested negative for AFB and auramine staining but positive for both M. tuberculosis and M. bovis by culture and PCR. In human subjects suspected and positive for TB, out of 279 samples tested, M. tuberculosis and M. bovis were observed in 183 (65.59 per cent) and one (0.36 per cent) respectively. Sequence analysis plot and sequence similarity matrix of nucleotide sequences of hypothetical protein ‘Rv1506c’ (accession no. Z79701) region of eight M. tuberculosis and six M. bovis field isolates and genbank isolates had shown the difference in sequences between isolates vary from 0 to 2.3 per cent. The spoligopatterns of the bovines, ovines and a chimp isolate showed the following lineages: Manu1, U, EA15, EA13 and Beijing. While the lineages from various isolates were almost similar, two of them showed a spoligopattern that did not match with any existing pattern in the database (orphan). The M. bovis isolates from human and chimp matched with Bovis 1 type. The results of the binary logistic regression analysis factors associated the occurrence of tuberculosis infection in human beings with the variables presumed to be the determinants in human beings, viz., association with animals, raw milk consumption, family member with tuberculosis, age, education and socio-economic status were found to be statistically significant when all the data points were considered. Though the major objective of this study was to identify the extent of M. bovis in humans, M. tuberculosis infection established was at a higher level in the animal population studied. The occurrence of M. tuberculosis is an example of reverse zoonosis and these animals potentially constitute a grave public health hazard as virulent bacilli can be transmitted to humans.
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