Om Surujballi

European 1: a globally important clonal complex of Mycobacterium bovis.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.

Antibody Responses of Cervids ( Cervus elaphus ) following Experimental Mycobacterium bovis Infection and the Implications for Immunodiagnosis

ABSTRACT Captive and free-ranging wildlife animals are implicated in the maintenance and transmission of bovine tuberculosis and therefore pose a significant obstacle to eradication of the disease from domestic livestock. The current antemortem diagnostic method, the intradermal tuberculin skin test, is impractical for routine use with many wild animals. Antibody-based assays are particularly attractive because the animals are handled only once and immediate processing of the sample is not required. This report characterizes the antibody responses of red deer-elk hybrids (Cervus elaphus) against Mycobacterium bovis and subsequently evaluates the diagnostic performance of select antigens in a rapid-test format. Sequential serum samples were collected from 10 animals experimentally infected with M. bovis and 5 noninfected animals over a 7-month period postinfection (p.i.). Samples were evaluated by enzyme-linked immunosorbent assays, immunoblot analyses, and multiantigen print immunoassays for seroreactivity to mycobacterial antigens. Although all infected animals produced antibodies to M. bovis protein antigens, there was significant animal-to-animal variation in the kinetics and magnitudes of responses and the antigens recognized. The most frequently recognized antigens included MPB83, ESAT-6, CFP10, and MPB70. Responses to some antigens, such as MPB83, were consistently detected as early as 4 weeks after inoculation, whereas other antigens were detected only much later (>140 days p.i.). Antibody responses were boosted by injection of tuberculin for intradermal tuberculin skin testing. Comparison of single-antigen (fluorescence polarization assay) with multiantigen (CervidTB STAT-PAK) rapid tests demonstrated that a highly sensitive and specific serodiagnostic test for tuberculosis in cervids will require multiple and carefully selected seroreactive antigens covering a broad spectrum of antibody specificities.

Comparative assessment of fluorescence polarization and tuberculin skin testing for the diagnosis of bovine tuberculosis in Chadian cattle.

Effective surveillance of bovine tuberculosis (BTB) in developing countries where reliable data on disease prevalence is scarce or absent is a precondition for considering potential control options. We conducted a slaughterhouse survey to assess for the first time the burden of BTB in Southern Chad. Altogether, 954 slaughter animals were consecutively sampled and tested using the single intra-dermal comparative cervical tuberculin (SICCT) test, a recently developed fluorescence polarization assay (FPA) and routine abattoir meat inspection after slaughter. Gross visible lesions were detected in 11.3% (CI: 9.4-13.5%) of the animals examined and they were mostly located in the lymph nodes and the lung. Significantly more Mbororo zebus (15.0%) were affected by lesions than Arab zebus (9.9%; OR=2.20, CI: 1.41-3.41%; p<0.001). Of all animals tested, 7.7% (CI: 6.2-9.6%) reacted positively to SICCT if OIE guidelines were applied. However, receiver operating characteristic (ROC) analysis using Mycobacterium tuberculosis complex (MTBC) infected animals as the positive population and lesion negative animals as the negative population, revealed a better SICCT performance if the cut-off value was decreased to >2mm. SICCT reactor prevalence rose to 15.5% (CI: 13.3-18.0%) and FPA did not perform better than SICCT, when this setting adapted cut-off was applied.

Development and evaluation of a real-time reverse transcription-PCR assay for quantification of gamma interferon mRNA to diagnose tuberculosis in multiple animal species.

ABSTRACT Tuberculosis of free-ranging and captive wildlife, including species implicated in the maintenance and transmission of Mycobacterium bovis, is a difficult disease to diagnose and control. Historically, diagnosis of tuberculosis has relied largely upon assays of cell-mediated immunity (CMI), such as tuberculin skin testing. This approach, however, is problematic or impractical for use with many wildlife species. Increasingly, in vitro diagnostic tests, including gamma interferon (IFN-γ)-based assays, are replacing or complementing skin testing of cattle and humans. Analogous assays are unavailable for most wildlife because of a lack of species-specific immunological reagents. This report describes the development and validation of a whole-blood assay to quantify antigen-specific IFN-γ mRNA expression by quantitative real-time reverse transcription-PCR. Oligonucleotide primers and probes were designed and tested for reactivity towards several susceptible species of interest with respect to tuberculosis infection. The assay was subsequently optimized to quantify the IFN-γ mRNA expression in elk and red deer (Cervus elaphus) and was evaluated for its ability to detect mycobacterial antigen-specific responses of experimentally tuberculosis-infected animals. The assay was a simple, rapid, and sensitive measure of antigen-specific CMI. The IFN-γ mRNA responses correlated well with IFN-γ protein production and showed performance in determining an animals infection status superior to that of either lymphocyte proliferation or IFN-γ protein enzyme-linked immunosorbent assay methods. An additional advantage is the ease with which the assay can be modified to reliably quantify IFN-γ expression by using consensus sequences of closely related species or of other species for which IFN-γ sequence information is available.

CERVINE (CERVUS ELAPHUS) CYTOKINE mRNA QUANTIFICATION BY REAL-TIME POLYMERASE CHAIN REACTION

It has been difficult to perform cytokine studies for many wildlife and nontraditional species because of a lack of immunologic reagents at the protein level. Recently, simple and rapid assays for quantifying mRNA expression by real-time reverse transcription–polymerase chain reaction (RT-PCR) have been used for analysis of cytokine profiles in humans and other mammalian species. This report describes the development and application of real time RT-PCR to measure the expression of several important elk (Cervus elaphus) cytokine mRNAs, including interleukin (IL)-2, IL-4, IL-10, IL-12p40, interferon-gamma, tumor necrosis factor (TNF)- α, and the enzyme-inducible nitric oxide synthase, all of which are involved in immune responses and regulation. For the broadest potential application of the assay, primers and probes were designed using consensus sequences from several species of interest. To obtain standardized quantitative results, external controls consisting of a DNA template for each target gene were used to generate linear standard curves over a 6 to 8 log range with detection of as few as 10 copies of amplicon per reaction. Sample-to-sample variation in the efficiency of the RT, as well as in the quantity and quality of the starting RNA, was compensated for by normalizing the results to the endogenous housekeeping gene β2 microglobulin. The assay was evaluated by monitoring the kinetics of cytokine mRNA synthesis induced by mitogenic and antigenic stimulation of peripheral blood mononuclear cells (PBMCs) from Mycobacterium bovis–infected elk. Concanavalin A–stimulated PBMCs demonstrated a rapid but transient increase in cytokine mRNA expression following in vitro mitogenic activation with optimal mRNA induction observed after 4 to 16 hr. The PBMCs stimulated with the mycobacterial recall antigen, bovine-purified protein derivative (PPD-bovis), demonstrated variable mRNA induction kinetics for each cytokine. Whereas PPD-bovis optimally induced IL-2 mRNA after 8 hr of in vitro stimulation, longer in vitro stimulation times were necessary for the optimal induction of IL-4 and TNF- α mRNA (up to 48 hr). We demonstrate real-time RT-PCR to be a rapid, sensitive, and reproducible technique, which will make it a valuable tool in the study of immunologic responses and cytokine profiles of cervids and other nontraditional livestock and wildlife species.

Monoclonal antibodies suitable for incorporation into a competitive enzyme-linked immunosorbent assay (ELISA) for detection of specific antibodies to Leptospira interrogans serovar pomona

Monoclonal antibodies (mAb) were produced by fusing Sp2/0-Ag14 myeloma cells with spleen cells from BALB/c and ND4 mice that were immunized with killed Leptospira interrogans serovar pomona whole cells. Thirty hybridomas which produced antibodies (of the IgG1, IgG2a, IgG2b, or IgG3 isotype) that bound to epitopes on the serovar pomona whole cell antigen were identified by an indirect enzyme-linked immunosorbent assay (ELISA). Twenty-eight of these 30 mAbs cross-reacted in the indirect ELISA with at least one whole cell antigen prepared from 12 other pathogenic Leptospira serovars, and/or with whole cell antigen from the non-pathogenic Leptospira biflexa serovar patoc. The two serovar pomona-specific mAbs, which were designated M897 and M898, were obtained from the ND4 mouse and were both of the IgG1 isotype. In competitive ELISAs, M897 and M898 were inhibited from binding to the pomona antigen by bovine sera with anti-serovar pomona microscopic agglutination test (MAT) titres ranging from 100 to 6400. No significant inhibition was observed with pomona MAT-negative sera or with sera from animals experimentally infected with serovars canicola, copenhageni, grippotyphosa, hardjo type hardjobovis or sejroe. The epitopes recognized by M897 and M898 were both highly susceptible to sodium meta-periodate oxidation, indicating a carbohydrate composition. Neither of these mAbs reacted in immunoblots with the separated components of the serovar pomona whole cell antigen.

Contagious Ecthyma, Rangiferine Brucellosis, and Lungworm Infection in a Muskox (Ovibos moschatus) from the Canadian Arctic, 2014

Abstract An adult male muskox (Ovibos moschatus), harvested on 26 August 2014 on Victoria Island, Nunavut, in the Canadian Arctic, had proliferative dermatitis on the muzzle and fetlocks suggestive of contagious ecthyma or orf (Parapoxvirus). Histopathologic features of the lesions were consistent with this diagnosis. Orf virus DNA, phylogenetically similar to an isolate from a captive muskox of the Minnesota Zoo, US, was detected in the lesions by PCR using Parapoxvirus primers. Additionally, there was a metaphyseal abscess with a cortical fistula in the right metacarpus from which Brucella suis biovar 4 was isolated and identification supported by PCR. Brucella spp. antibodies were detected in serum. Finally, 212 nodules were dissected from the lungs. Fecal analysis and lung examination demonstrated co-infection with the lungworms Umingmakstrongylus pallikuukensis and Varestrongylus eleguneniensis. The zoonotic potential of orf and rangiferine brucellosis adds an important public health dimension to this case, particularly given that muskoxen are a valuable source of food for Arctic residents. Careful examination of these pathogens at a population level is needed as they may contribute to muskox population decline and potentially constitute a driver of food insecurity for local communities. This case underscores the importance of wildlife health surveillance as a management tool to conserve wildlife populations and maintain food security in subsistence-oriented communities.

COMPARISON OF TEST PERFORMANCE AND EVALUATION OF NOVEL IMMUNOASSAYS FOR TUBERCULOSIS IN A CAPTIVE HERD OF WOOD BISON NATURALLY INFECTED WITH MYCOBACTERIUM BOVIS

In 1996, the Hook Lake Wood Bison Recovery Project was initiated to establish a small, disease-free, captive, bison-breeding herd. Founders originated from wild bison herds in the Slave River Lowlands in northern Canada, which, like other bison herds in and around Wood Buffalo National Park, are endemically infected with bovine tuberculosis (caused by Mycobacterium bovis) and brucellosis (caused by Brucella abortus). After 9 yr of apparent disease freedom, tuberculosis was detected within the captive herd, leading to complete depopulation. This study examined the performance of antemortem tuberculosis diagnostic tests used during the project. Performances of the caudal-fold test, fluorescent polarization assay, multiantigen print immunoassay (MAPIA), and the rapid test (RT) were assessed by estimating sensitivity, specificity, positive predictive value, and negative predictive value for each test. Kappa values measuring agreement between tests were calculated. Overall, the tests did not differ with respect to sensitivities and specificities, which ranged from 50% to 92% and from 34% to 100%, respectively. The MAPIA tended to show high sensitivity, and there was significant agreement only between the MAPIA and RT. Serum collected from infected animals at slaughter produced highly variable results on the different assays, and one infected bison was negative on all antemortem tests. The results of this analysis suggest use of multiple antemortem tests in parallel, particularly those incorporating multiple antigens, to optimize sensitivity in detecting bovine tuberculosis in bison. However, as demonstrated in this herd, even a seemingly optimal antemortem testing regimen can fail to detect M. bovis–infected individuals.

Expression in Escherichia coli of flaB, the gene coding - for a periplasmic flagellin of Leptospira interrogans serovar pomona

A periplasmic flagellin gene, flaB, of Leptospira interrogans serovar pomona (strain Pomona) was expressed in Escherichia coli for the production and antigenic characterisation of the protein. The flaB structural gene, which was previously cloned into pUC118, was derived by PCR from the recombinant plasmid and used to generate an expression construct with the trc promoter-driven pProEx HT system. Under the conditions employed, the flaB was expressed as inclusion bodies formed within E. coli, yielding c. 120 mg of the recombinant protein/L of culture. A polyhistidine tag introduced at the amino-acid terminus of the FlaB protein allowed for the purification of the protein by nickel-chelate affinity chromatography. The expressed protein reacted with both mouse and bovine antisera to L. interrogans on Western blots, indicating that it could be of use in the diagnosis of leptospirosis. The recombinant leptospiral flagellin may also be of value in studying its role in the pathogenesis of leptospirosis.

Competitive Enzyme-Linked Immunosorbent Assay for Detection of Leptospira interrogans Serovar pomona Antibodies in Bovine Sera

ABSTRACT A competitive enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody (M898) was developed for detection of bovine antibodies to Leptospira interrogans serovar pomona. This assay was evaluated using field sera (n = 190) with serovar pomona microscopic agglutination test (MAT) titers of ≥100 as the positive population (group A); field sera (n = 1,445) which were negative in the MAT (1:100 dilution) for serovar pomona (group B); and sera (from a specific-pathogen-free cattle herd [n = 210]) which were negative in the MAT (1:100 dilution) for serovars canicola, copenhageni, grippotyphosa, hardjo, pomona, and sejroe (group C). At the cutoff point recommended by receiver operating characteristic (ROC) curve analysis of the combined ELISA results of serum groups A, B, and C, the sensitivity and specificity values were 93.7 and 96.3%, respectively. The value for the area under this ROC curve was 0.977, indicating a high level of accuracy for the ELISA. Similar results were obtained from the analysis of the combined results of serum groups A and B and from the analysis of the combined results of serum groups A and C.