Glyn Hewinson

A new evolutionary scenario for the Mycobacterium tuberculosis complex

The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and “modern” strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum→M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

High Prevalence and Increased Severity of Pathology of Bovine Tuberculosis in Holsteins Compared to Zebu Breeds under Field Cattle Husbandry in Central Ethiopia

ABSTRACT A comparative study on the prevalence and pathology of bovine tuberculosis (TB) was conducted on 5,424 cattle (2,578 zebus, 1,921 crosses, and 925 Holsteins), which were kept on pasture in the central highlands of Ethiopia, using a comparative intradermal tuberculin test, postmortem examination, and bacteriology. The overall prevalence of bovine TB was 13.5%; prevalence was higher in Holsteins than either zebus (22.2% versus 11.6%, χ2 = 61.8; P < 0.001) or crosses (22.2% versus 11.9%, χ2 = 50.7; P < 0.001). Moreover, the severity of pathology in Holsteins (mean ± standard error of the mean [SEM], 6.84 ± 0.79) was significantly higher (P = 0.018) than the severity of pathology in zebus (5.21 ± 0.30). In addition, the risk of TB in Holsteins was more than twice (odds ratio [OR] = 2.32; 95% confidence interval [CI] = 1.89, 2.85) that in zebus. Animals between 5 and 9 years of age were at higher (OR = 2.37; 95% CI = 1.80, 3.12) risk of bovine TB than those 2 years of age or below. A significant difference (χ2 = 351; P < 0.001) in the occurrence of TB lesions in lymph nodes was recorded; the mesenteric lymph node (mean pathology score ± SEM, 1.95 ± 0.08) was most severely affected, followed by the retropharyngeal (0.80 ± 0.05) and caudal mediastinal (0.8 ± 0.06) lymph nodes. Fifty-six percent (n = 145) of the animals with gross TB lesions were culture positive; the lowest culture positivity was recorded in the skin lesions (27.3%) and the lesions of the mesenteric lymph node (31.5%). Both the skin test response and the postmortem findings suggested a higher susceptibility to bovine TB in Holsteins than zebus under identical field husbandry conditions (on pasture). In the light of increased numbers of Holstein cattle introduced into this area to raise milk production to satisfy the needs of Addis Ababas growing population, these findings highlight the need for a control program in these herds.

DNA Injection in Combination with Electroporation: a Novel Method for Vaccination of Farmed Ruminants

Injection of plasmid DNA encoding antigens into rodents followed by electroporation improved the immune response when compared with injection without electroporation (Widera et al. J Immunol 2000;164:4635–40; Zucchelli et al. J Virol 2000;74:11598–607; Kadowaki et al. Vaccine 2000;18:2779–88). The present study describes the extension of this technology to farm animals, by injecting plasmid DNA encoding mycobacterial antigens (MPB70, Ag85B and Hsp65) into the muscles of goats and cattle using two different types of electrodes, both allowing DNA delivery at the site of electroporation. The animals were vaccinated under local anaesthesia without any observed immediate or long‐term distress or discomfort, or any behavioural signs of muscle damage or pathological changes after the electroporation. DNA‐injected and electroporated goats showed increased humoral response after the primary vaccination when compared with nonelectroporated animals. Improved T‐cell responses following electroporation were observed in hsp65 DNA‐vaccinated cattle. DNA injection with or without electroporation did not compromise the specificity of the tuberculin skin test. In conclusion, a protocol applying in vivo electroporation free of side effects to farmed ruminants was established. In addition, we show that DNA vaccination in combination with electroporation can improve the primary immune responses to the encoded antigens.

Mycobacterial Lineages Causing Pulmonary and Extrapulmonary Tuberculosis, Ethiopia

Molecular typing of 964 specimens from patients in Ethiopia with lymph node or pulmonary tuberculosis showed a similar distribution of Mycobacterium tuberculosis strains between the 2 disease manifestations and a minimal role for M. bovis. We report a novel phylogenetic lineage of M. tuberculosis strongly associated with the Horn of Africa.

Molecular Epidemiology of Disease Due to Mycobacterium bovis in Humans in the United Kingdom

ABSTRACT Mycobacterium bovis is the causative agent of bovine tuberculosis, with a wide host range. Fifty human M. bovis isolates were typed using spoligotyping and variable number tandem repeats (VNTR). Fifteen of these spoligotypes have not yet been recorded in cattle. The predominant spoligotype in humans and cattle was subdivided by VNTR.

Development of a Skin Test for Bovine Tuberculosis for Differentiating Infected from Vaccinated Animals

ABSTRACT The tuberculin skin test has been used for the diagnosis of bovine and human tuberculosis (TB) for over a hundred years. However, the specificity of the test is compromised by vaccination with the Mycobacterium bovis-derived vaccine strain bacille Calmette-Guérin (BCG). Since current promising vaccines against bovine TB are based on heterologous prime-boost combinations that include BCG, there is a need for diagnostic tests for differentiating infected from vaccinated animals (DIVA). The application of antigens such as ESAT-6 and CFP-10 for DIVA has so far been realized largely through their application in the blood-based gamma interferon release assay. In the current study, we have reassessed the potential of such antigens as skin test reagents for DIVA in cattle. A cocktail of the Mycobacterium tuberculosis complex recombinant protein antigens ESAT-6, CFP-10, MPB70, and MPB83 elicited delayed-type hypersensitivity (DTH) skin test responses in 78% of naturally infected tuberculin-positive cattle. Importantly, this cocktail induced no skin responses in BCG-vaccinated cattle despite them being sensitized for strong tuberculin responses. Further optimization of skin test antigen combinations identified that the inclusion of Rv3615c (Mb3645c) enhanced skin test sensitivity in naturally infected cattle without compromising specificity. In addition, we demonstrate for the first time the utility of synthetic peptides as promising skin test antigens for bovine TB for DIVA. Our data provide a promising basis for the future development of skin tests for DIVA with practical relevance for TB diagnosis in both veterinary and clinical settings.

Improved cellular and humoral immune responses against Mycobacterium tuberculosis antigens after intramuscular DNA immunisation combined with muscle electroporation

New delivery methods are needed to improve the efficiency of existing DNA vaccines. We have measured the immune response to Mycobacterium tuberculosis antigens following intramuscular DNA injection in combination with or without electroporation. Three to 6-fold increase in the number of antigen specific CD4(+) and CD8(+) T cells, measured by IFN-gamma-producing cells in an ELISPOT assay, was found in mice DNA injected and electroporated compared with non-electroporated mice. Similarly, 5 to 10-fold increase in antigen specific IgG1, IgG2a and IgG2b antibodies were found in an immunoglobulin subclass specific ELISA. A 100-fold reduction in DNA dose could be used without loss of efficiency when immunisation was combined with electroporation. A single injection of 1 microg of antigen 85b (ag85b) plasmid DNA was sufficient to elicit a higher and long lasting level of IgG2a antibodies against antigen 85B (Ag85B) compared to standard BCG vaccination. We conclude that DNA immunisation in combination with electroporation can significantly improve the immunogenicity of plasmid-based DNA vaccines.

Molecular Typing of Mycobacterium bovis Isolates from Cameroon

ABSTRACT In order to gain a better understanding of the molecular epidemiology of Mycobacterium bovis isolates in Cameroon, 75 isolates of M. bovis collected in three provinces of northern Cameroon were studied by spoligotyping. For 65 of these isolates, typing was also carried out by pulsed-field gel electrophoresis (PFGE) with DraI, and 18 of the isolates were also typed by restriction fragment length polymorphism (RFLP) analysis with probe IS6110-RHS. Molecular typing of the isolates by these techniques revealed a high degree of homogeneity, with 10 spoligotypes for 75 isolates, four PFGE profiles for 65 isolates, and three RFLP types for 18 isolates. Some types were present in the three different provinces, while some were confined to one or two areas. These results suggest that geographical mapping of M. bovis strains could be helpful for the control of bovine tuberculosis at the regional level. An interesting feature of all the spoligotypes was the absence of spacer 30, suggesting a common origin for all of the Cameroon isolates tested; an evolutionary scenario for the isolates is discussed. In addition, a comparison of the three techniques showed that for M. bovis strain differentiation in Cameroon and in surrounding countries, spoligotyping would be a more discriminating and practical tool for molecular typing than the other two techniques used in this study.

Mycobacterium bovis isolates from tuberculous lesions in Chadian zebu carcasses.

This slaughterhouse study in Chad shows higher proportions of Mycobacterium bovis isolates among Mbororo than Arabe zebu cattle. Spoligotyping shows a homogenetic population structure for M. bovis and lack of spacer 30, as were found in neighboring Cameroon and Nigeria. This finding suggests transborder and ongoing transmission between cattle.

Appraisal of Interpretation Criteria for the Comparative Intradermal Tuberculin Test for Diagnosis of Tuberculosis in Cattle in Central Ethiopia

ABSTRACT Accurate detection and removal of infected cattle, using immunodiagnostic tests such as the comparative intradermal tuberculin (CIDT) test, are the basis of control strategies for bovine tuberculosis (TB). According to the Office des Internationale Epizooties recommendation, the cutoff point for positivity of the CIDT test, calculated as the difference between skin thicknesses after bovine tuberculin (B) and avian tuberculin (A) injections (B − A), is >4 mm. This cutoff point is used worldwide, although it is likely that local conditions influence test performance. Thus, this study was formulated to determine CIDT test cutoff points applicable to cattle in central Ethiopia. Receiver operating characteristic analysis was performed for the CIDT test, using data from 186 Bos indicus (zebu) and Bos taurus (Holstein) cattle. Detailed postmortem examination for the presence of TB lesions was used to define disease status. At a cutoff of >2 mm, CIDT test sensitivity was 69% (95% confidence interval [95% CI], 58.5 to 79%), while it was 59% (95% CI, 49 to 69%) at a cutoff of >4 mm. In contrast, specificities of the CIDT test at these two cutoff values were identical, at 97% (95% CI, 89 to 100%). Thus, the maximum sensitivity of the CIDT test can be realized using a >2-mm cutoff without affecting specificity. The apparent prevalence was significantly (χ2 = 13.56; P < 0.001) higher at a cutoff of >2 mm (16.0%; n = 5,424) than at a >4-mm cutoff (13.5%; n = 5,424). Nonetheless, no significant difference (χ2 = 2.15; P = 0.14) in true prevalence was observed at a cutoff of >2 mm (19.6%) and at a cutoff of >4 mm (18.5%). Thus, our study demonstrates the importance of defining local, relevant cutoff values to maximize test sensitivity, and we suggest the application of the >2-mm cutoff for testing of cattle in central Ethiopia.