Cross reactive immune responses in cattle arising from exposure to Mycobacterium bovis and non-tuberculous mycobacteria.

Accurate diagnosis of tuberculosis in cattle may be compromised in areas where there are high rates of exposure to environmental/non-tuberculous mycobacteria (NTM). This cross reaction of immune responses to Mycobacterium bovis antigens shared with NTMs can result in reduced specificity of commonly used diagnostic tests including tuberculin skin tests and the interferon gamma assay (IFN-É£). In this study we assessed the cross-reactive immune responses of M. bovis (infected) and NTM exposed animals to M. bovis and M. avium tuberculin, the ESAT6/CFP10 cocktail antigen, tuberculin derived from cultures of selected NTMs, and a panel of recombinant mycobacterium tuberculosis complex (MTBC) antigens sharing homology with orthologues in NTM. Gamma interferon (IFN-É£) responses were measured in whole blood cultures using the IFN-É£ assay and the IFN-É£ elispot assay on purified peripheral blood mononuclear cells (PBMC). We observed the expected strong IFN-É£ response to PPD-B in the M. bovis infected animals that distinguished this group from non-infected NTM exposed cattle. The IFN-É£ responses to PPD-N (M. nonchromogenicum), were relatively high in both infected and non-infected NTM exposed cattle, but were not significantly different to classify the true infection status of each group. The results indicated that the cross-reactive responses to PPD-B and/or PPD-A with PPD-N, likely arose from prior exposure to environmental non-tuberculous mycobacteria. The IFN-É£ immune responses to the 10 R-Mag measured by the IFN-É£ elispot assay revealed that three of the selected antigens, Rv3615 (ESpC), Rv0287 (esxG) and the ESAT6/CFP10, were immunogenic in the infected cattle, and distinguished the infected cattle from the non-infected NTM exposed animals. The combined data of PPDs and R-Mags derived from NTM mycobacteria may prove useful in future development of novel bTB diagnostic tests.

Original Mycobacterial Sin, a consequence of highly homologous antigens?

The role of antigens shared between Mycobacteria in in-vivo cross-reactive immune responses in host animals, have been reported to be responsible for reduced BCG vaccination efficacy as well reduced specificity of routine immunological diagnostic tests. This presents with significant disease control challenges in humans and animals. The present review highlights the results of previous studies on the effect of pre-sensitization to environmental mycobacteria on either pathogenic mycobacteria and/or M. bovis BCG, in experimental animals. It also takes an in-depth view into assessing the genetic similarities and relationships between atypical mycobacteria and Mycobacterium tuberculosis complex (MTBC) and how they might explain the immunological imprint of environmental mycobacteria in directing the hosts' immune response upon subsequent exposure to other classes of mycobacteria. The outcome of this review suggests that genetic closeness between particular atypical mycobacteria and MTBC usually indicate a higher level of homology for certain shared protective antigens. This ultimately results in a higher level of cross reactive immune responses as compared with other atypical mycobacteria that are further away genetically. This would explain the different effects of environmental mycobacteria on MTBC that have been reported in the different studies. In other words the direction of the host immune system in response to exposure to MTBC would depend on the type of environmental mycobacteria that was encountered in the initial exposure. We also explain these mycobacterial interactions in the context of the phenomenon of "Original Mycobacterial Sin". The effects of these inevitable mycobacterial interactions on field diagnosis and control by vaccination and how to circumvent them are discussed.

Field application of immunoassays for the detection of Mycobacterium bovis infection in the African buffalo (Syncerus caffer).

The African buffalo (Syncerus caffer) is considered the most important maintenance host of bovine tuberculosis (BTB) in wildlife in Southern Africa. The diagnosis of Mycobacterium bovis infection in this species mostly relies on the single intradermal comparative tuberculin test (SICTT). As an alternative, the BOVIGAM® 1G, an interferon-gamma (IFN-γ) release assay, is frequently used. The test performance of cell-mediated immunity (CMI-) and humoral immunity (HI-) based assays for the detection of M. bovis infections in buffaloes was compared to identify the test or test combination that provided the highest sensitivity in the study. Buffaloes were sampled during the annual BTB SICTT testing in the Hluhluwe-iMfolozi-Park (KwaZulu-Natal, South Africa) during June 2013. A total of 35 animals were subjected to the SICTT, 13 of these tested positive and one showed an inconclusive reaction. CMI-based assays (BOVIGAM® 1G (B1G) and BOVIGAM® 2G (B2G)) as well as a serological assay (IDEXX TB ELISA) were used to further investigate and compare immune responsiveness. Thirteen SICTT positive buffaloes and one inconclusive reactor were slaughtered and a post-mortem (PM) examination was conducted to confirm BTB. Lesions characteristic of BTB were found in 8/14 animals (57.1%). Test results of individual assays were compared with serial and parallel test interpretation and the sensitivity was calculated as a percentage of test positives out of the number of SICTT positive animals with granulomatous lesions (relative sensitivity). The B1G assay showed the highest individual sensitivity (100%; 8/8) followed by the B2G assay (75%; 6/8) and the IDEXX TB ELISA (37.5%; 3/8). Therefore, using in parallel interpretation, any combination with the B1G showed a sensitivity of 100% (8/8), whereas combinations with the B2G showed a 75% sensitivity (6/8). Out of the 21 SICTT negative animals, 7 animals showed responsiveness in the B2G or IDEXX TB ELISA. In conclusion, this study has shown that the BOVIGAM® IFN-γ assay had the highest test performance.

Mycobacterium bovis infection in livestock workers in Ibadan, Nigeria: evidence of occupational exposure.

SETTING: Bovine tuberculosis (TB) is endemic in the cattle population in Nigeria. Livestock workers are at risk of Mycobacterium bovis infection and unaware of their health status. OBJECTIVE: To determine the occurrence of pulmonary M. bovis infection among livestock workers. DESIGN: A cross-sectional study of livestock traders was conducted for TB through screening of sputum samples using a simple random sampling method coupled with oral interview on the assumption of sub-clinical pulmonary TB infection. Specimens were cultured, and the isolates analysed using molecular typing techniques. RESULTS: Overall, 10% (7/70) of the livestock traders had a positive culture indicative of M. tuberculosis complex, which were differentiated into M. bovis (n = 2) and M. tuberculosis (n = 5) using deletion typing. Further spoligotyping analyses of the M. bovis and two available M. tuberculosis isolates classified the strains as SB1432 and SB09444 and LAM_10 CAM and T1 using respectively www.mbovis.org and spotclust databases. Prolonged cough and >3 years in the livestock trade were risk factors for infection. CONCLUSION: We confirm that there is undetected pulmonary M. bovis infection among livestock traders in Nigeria. Further studies on the role of occupationally exposed workers in the transmission of M. bovis infection to the larger community are required.

Molecular epidemiology of human and animal tuberculosis in Ibadan, Southwestern Nigeria.

From 2005 to 2007, Mycobacterium tuberculosis complex (MTC) strains were isolated from cattle, goats and pigs samples collected at the Bodija abattoir and from human samples from tuberculosis patients and livestock traders at the Akinyele cattle market in Ibadan, Southwestern Nigeria. Seventy four isolates obtained from humans (24) and livestock (50) were identified as MTC strains. Thirty two isolates were spoligotyped. Nineteen of these 32 isolates were identified as M. tuberculosis whilst 13 were identified as Mycobacterium bovis. M. bovis was isolated from two humans, whereas M. tuberculosis was isolated from a bovine, a pig and a goat. All the M. bovis isolates identified in this study belonged to the Africa 1 clonal complex. Multiple locus VNTR [variable number of tandem repeats] analysis (MLVA) was carried out on the 74 isolates. Three major clusters were defined. Group A consisted of 24 M. tuberculosis isolates (MLVA genotypes 1-18). One strain was isolated from a bovine and one from a pig. Group B consisted of 49 M. bovis strains (MLVA genotypes 19-48), mainly of cattle origin but also included four goat, nine pig and two human isolates. Group C consisted of a single M. tuberculosis isolate (MLVA genotype 49) obtained from a goat. Spoligotyping and MLVA confirmed it as clustering with the East Africa Indian clade found in humans in Sudan and the Republic of Djibouti. The isolation of three M. tuberculosis strains from livestock raises the question of their epidemiological importance as a source of infection for humans.

Comparison of the capillary and agarose electrophoresis based multiple locus VNTR (variable number of tandem repeats) analysis (MLVA) on Mycobacterium bovis isolates.

Electrophoretic techniques that can be used for genotyping of bacterial pathogens ranges from manual, low-cost, agarose gels to high-throughput capillary electrophoresis sequencing machines. These two methods are currently employed in the electrophoresis of PCR products used in multiple locus VNTR (variable number of tandem repeats) analysis (MLVA), i.e. the agarose electrophoresis (AE) and the capillary electrophoresis (CE). Some authors have suggested that clusters generated by AE are less reliable than those generated by CE and that the latter is a more sensitive technique than the former when typing Mycobacterium tuberculosis complex (MTC) isolates. Because such a claim could have significant consequences for investigators in this field, a comparison was made on 19 Belgian Mycobacterium bovis strains which had previously been genotyped using CE VNTR analysis. The VNTR profiles of the CE VNTR analysis were compared with those obtained by AE VNTR analysis at 14 VNTR loci. Our results indicated that there were no differences in copy numbers at all loci tested when the copy numbers obtained by the AE VNTR analysis were compared with those obtained by CE VNTR analysis. The use of AE VNTR analysis in mycobacterial genotyping does not alter the sensitivity of the MLVA technique compared with the CE VNTR analysis. The AE VNTR can therefore be regarded as a viable alternative in moderately equipped laboratories that cannot afford the expensive equipment required for CE VNTR analysis and data obtained by AE VNTR analysis can be shared between laboratories which use the CE VNTR method.

Mycobacterium tuberculosis and Mycobacterium africanum in stools from children attending an immunization clinic in Ibadan, Nigeria.

BACKGROUND: Tuberculosis is a major cause of childhood morbidity and mortality in Nigeria. Diagnosis of childhood tuberculosis is a global challenge making early treatment a mirage. In this study we investigated the stools of children for the presence of mycobacteria. METHODS: Stool samples from children aged 3 days to 3 years who presented for postnatal immunization at a large university-based clinic in Nigeria, were subjected to Ziehl-Neelsen staining. Samples with acid-fast bacilli were further processed using mycobacterial culture, spoligotyping, and deletion typing. RESULTS: One hundred and ninety-two stool samples from different children were collected and processed. Thirty (15.6%) had acid-fast bacilli. Of these, eight had Mycobacterium tuberculosis and one had Mycobacterium africanum. CONCLUSIONS: Approximately 5% (9/192) of apparently well children had evidence of potentially serious tuberculosis infection. The usefulness of stool specimens for diagnosing pediatric tuberculosis warrants further investigation.

Altered neuronal distribution of parvalbumin in anterior cingulate cortex of rabbits exposed in utero to cocaine.

Previous studies of rabbits exposed in utero to cocaine have revealed an increase in the number of neurons which are GABA immunoreactive in the anterior cingulate cortex (ACC), suggesting a cocaine-elicited modification in the balance of excitatory and inhibitory interactions. Of the major calcium binding proteins expressed by different subgroups of GABAergic neurons, parvalbumin has been observed in conditions involving excess excitation, and may serve to protect neurons from excitotoxicity. In the present study, we used immunocytochemistry to compare the effects of prenatal cocaine exposure on the postnatal development of parvalbumin immunoreactivity in interneurons of the visual cortex (VC) and ACC. We determined the number and laminar distribution of parvalbumin immunoreactive neurons, and we also assessed the distribution of parvalbumin immunoreactivity within primary, secondary and tertiary dendrites of neurons in these two cortical areas. In both ACC and VC, parvalbumin immunoreactive neurons were first observed around postnatal day 10 (P10) and their number increased rapidly from P10 to P20. At all ages studied (P10 to P60) there was no difference between cocaine-exposed and saline control animals in the number or laminar distribution of parvalbumin immunoreactive neurons in either cortical area. However, the distribution of parvalbumin immunoreactivity within dendrites revealed a significant difference between cocaine-exposed and saline control animals in ACC but not in VC. In ACC, at all ages studied, there was an increase in the number of primary, secondary and tertiary dendrites which were parvalbumin immunoreactive in cocaine-exposed animals compared with saline controls. This difference was most striking in secondary dendrites, and in laminae V and VI. The effect was observed at doses of 4 and 3 mg/kg per injection but not at 2 mg/kg per injection. In contrast to ACC, in VC there was no difference in the number of immunoreactive dendrites in cocaine-exposed animals compared with saline controls. These observations are consistent with the hypothesis that the ACC of rabbits exposed in utero to cocaine is characterized by altered excitatory/inhibitory interactions. ACC receives a dense dopaminergic input, but VC receives minimal dopaminergic innervation. Mechanisms by which the action of cocaine on the developing dopaminergic system may modify the balance of excitation and inhibition in ACC are discussed.