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We evaluated by nested PCR reaction, different cow secretions from a herd with 48% of prevalence of bovine tuberculosis (BTB), seeking to determine niches where Mycobacterium bovis could be found. Postmortem examination of 18 (75%) tuberculin reacting cows allowed demonstrates BTB-compatible lesions in six, all of them PCR positives in milk and four in colostra samples. Our results showed that up to 62% of the colostra analysed contained M. bovis DNA, whereas only 18% of milk gave a positive reaction. Moreover, in bronchoalveolar lavages from cattle with compatible lesions in lungs or lymph nodes, where macrophages account up to 90% of cells, we did not find evidences of M. bovis. Altogether, these results suggest that differences in the anti-bacterial capacity of bovine macrophages, dependent upon microenvironment and organ-specific factors, exist. Alternatively, we hypothesize that hypoxic conditions that are encountered in mammary glands macrophages could induce M. bovis entrance into a 'dormancy-like' state, and that the high number of colostra samples were M. bovis was detected, could be an indicator of reactivation during 'peripartum'.

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ByWestern blotting, we demonstrate high-level expression of NRAMP1 proteins in peripheral blood cells and granulomas of Mycobacterium bovis-infected bovines. Immunohistochemistry of granulomatous lesions showed heavily labeled epithelioid macrophages and Langhans cells. These data suggest that M. bovis infection enhances NRAMP1 expression and that active tuberculosis can occur despite this response.Mycobacterium bovis is the main causative agent of bovine tuberculosis (TB), a disease of great concern in developing countries such as Mexico for its detrimental economic impact and its effect on human beings (7). To control bovine TB, most programs rely on tuberculin tests to identify infected cattle, which are then sacrificed. Tuberculin tests, however, are not entirely reliable, and false-negative and false-positive results are not uncommon (8,19,20,22). To fight this disease, it would be a great asset to develop resistant breeds (17), a goal at present unattainable, as little is known about genetic factors of bovine TB (18,28).During the last decade, the gene coding for the so-called natural resistance-associated macrophage protein (NRAMP1) has been the center of much attention. Nramp1 was first described for the mouse as a gene conferring resistance to Salmonella enterica serovar Typhimurium, Leishmania donovani, and M. bovis BCG. A single recessive point mutation in Nramp1 was shown previously to occur in BCG-susceptible mouse strains (11). In human beings, eight NRAMP1 alleles have been identified (13, 24), some of them apparently associated with susceptibility to TB (4, 14). In bovines, two Nramp1 alleles have been cloned (16), the predicted polypeptide being a transmembrane molecule with a conserved transporter motif and multiple phosphorylation sites (9).To gain information on the role of NRAMP1 in bovine TB, we investigated the expression of NRAMP1 in peripheral blood cells (PBC) and tuberculous granulomas of M. bovisinfected bovines. A polyclonal antiserum was kindly donated by Jennifer Blackwell (University of Cambridge Medical School) for this purpose. The antiserum was raised in rabbits against a recombinant polypeptide spanning 35 residues of the carboxyl end of mouse NRAMP1 (1). Tested in our laboratory, this antiserum reacted with a 65-kDa band on phorbol myristate acetate-activated cells of the murine monocyte line J774A (data not shown). It showed no reactivity with M. bovis proteins by Western blotting (data not shown). PBC were obtained from 20 animals from a herd with 50% TB prevalence; 10 cattle were purified protein derivative (PPD) positive, and 10 were PPD negative. For a control, PBC from five PPD-negative cattle belonging to a TB-free herd were employed. Tracheobronchial and mediastinal lymph nodes (LN) with TB granulomas were isolated at autopsy from five cattle with M. bovis infection confirmed by histology, Ziehl-Neelsen stains, and bacteriological cultures (data not shown). For the Western blot, cells and tissues were treated with RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% s...

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