Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded “echA12_2” in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2–7 months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.     

结核分枝杆菌rv3036c基因的原核表达及其多克隆抗体的制备

为制备结核分枝杆菌(Mtb) rv3036c基因的多克隆抗体,本研究以Mtb强毒株H37Rv基因组DNA为模板,扩增rv3036c基因,克隆至表达载体pET-28a(+)中,构建重组质粒pET-rv3036c,并转化大肠杆菌BL21(DE3),经1 mmol/L IPTG诱导表达组氨酸标签融合蛋白Rv3036c,采用Ni-NTA His-Bind Resin纯化目的蛋白,经western blot验证纯化蛋白.将Rv3036c蛋白纯化产物免疫新西兰大白兔,制备多克隆抗体.SDS-PAGE和western blot分析结果表明,Rv3036c以可溶形式表达,蛋白分子量大小为28 ku,并且具有良好的免疫原性.以上结果为进一步研究rv3036c基因在Mtb的致病性作用奠定了基础.     

Effects of intracellular expression of Mycobacterium tuberculosis CFP10-ESAT6 fusion protein on proliferation and apoptosis of mouse macrophages

AIM：To establish Mycobacterium tuberculosis culture filtrate protein 10 (CFP10)-early secretory antigenic target 6 (ESAT6) eukaryotic expression vector and investigate the effect of intracellular expression of CFP10-ESAT6 fusion protein on the proliferation and apoptosis of mouse macrophages (RAW264.7 cells). METHODS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was constructed by inserting CFP10-ESAT6 fusion gene into eukaryotic expression vector pEGFP-N1, and then transfected into RAW264.7 cells to express CFP10-ESAT6 fusion protein. The viability of RAW264.7 cells was measured by MTT assay. Flow cytometry was applied to measure the apoptotic rate and Toll-like receptor 2 (TLR2) expression in RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein or staurosporine. RESULTS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was successfully constructed and transfected into RAW264.7 cells. Compared with the control cells, intracellular expression of CFP10-ESAT6 fusion protein did not affect the viability of RAW264.7 cells, but could inhibit the apoptosis of RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein. Moreover, CFP10-ESAT6-expressing macrophages had markedly lower expression of TLR2 on the surface. CONCLUSION：Intracellular expression of CFP10-ESAT6 fusion protein has no cytotoxicity on mouse macrophages, but can inhibit the apoptosis of the macrophages treated with Mycobacterium tuberculosis 19 kD lipoprotein through down-regulating the expression of TLR2.     

A "down under" lesion on the muzzle of a dog

A 10-year-old, castrated, male Labrador Retriever was presented to a local veterinary practice for investigation of a firm, deeply pigmented, alopecic, subcutaneous mass (8 mm in diameter) on the left side of the muzzle. A fine-needle aspirate of the mass was submitted for cytologic evaluation to the University of Florida. Microscopically, the preparation contained a predominant population of histiocytes that contained variable numbers of intracytoplasmic, negative-staining, filamentous structures consistent with Mycobacterium sp. A presumptive diagnosis of canine leproid granuloma syndrome was based on the cytologic findings and location of the lesion. Acid-fast staining revealed bright pink, acid-fast organisms within the histiocytic cells, supporting the diagnosis. The bacteria were not detected in histopathologic sections or by a polymerase chain reaction (PCR) test 1 week later, however, possibly because of spontaneous remission. Canine leproid granuloma syndrome is a common disease in Australia, but is uncommon in dogs in North America. It is caused by a novel, unnamed Mycobacterium species and usually affects the skin and subcutaneous tissues of the head and ears. A diagnosis usually can be made in Wright's-Giemsa and acid-fast-stained cytologic specimens; however, definitive diagnosis requires PCR testing at a specialized laboratory.     

The impact of badger removal on the control of tuberculosis in cattle herds in Ireland

In Ireland, the herd prevalence of bovine tuberculosis has remained stable for several decades, and in common with several other countries, progress towards eradication has stalled. There is evidence in support of the potential role of infected badgers (Meles meles, a protected species) in bovine tuberculosis in Ireland and Britain. However, this evidence on its own has not been sufficient to prove disease causation. Field trials are likely to offer the best opportunity to define this role. Building on the earlier East Offaly project, our objectives were to assess the impact of badger removal on the control of tuberculosis in cattle herds in Ireland.

The study was conducted from September 1997 to August 2002 in matched removal and reference areas (average area of 245.1 km²) in four counties: Cork, Donegal, Kilkenny and Monaghan. Badger removal was intensive and proactive throughout the study period in the removal areas, but reactive (in response to severe tuberculosis outbreaks in cattle) in the reference areas. Removal intensity in the removal and reference areas during the first 2 years of the study averaged 0.57 and 0.07 badgers/km²/year, respectively.

The outcome of interest was restriction of cattle herds due to confirmed tuberculosis, where tuberculous lesions were detected in one or more animals. Data were analysed using logistic regression (modelling the probability of a confirmed herd restriction) and survival analysis (modelling time to a confirmed herd restriction).

During the study period, there was a significant difference between the removal and reference areas in all four counties in both the probability of and the time to a confirmed herd restriction due to tuberculosis. In the final year of the study, the odds of a confirmed herd restriction in the removal (as compared to the reference areas) were 0.25 in Cork, 0.04 in Donegal, 0.26 in Kilkenny and 0.43 in Monaghan. Further, the hazard ratios (removal over reference) ranged from 0.4 to 0.04 (a 60–96% decrease in the rate at which herds were becoming the subject of a confirmed restriction).     

Evaluation of surveillance strategies for bovine tuberculosis (Mycobacterium bovis) using an individual based epidemiological model

The Netherlands holds the bovine tuberculosis-free (BTB-free) status according to European Union standards, but in recent years small outbreaks of the infection have occurred. After the last outbreak in 1999 with 10 infected herds the question raised if the current surveillance system, visual inspection of carcasses at the slaughterhouse, is efficient enough to detect infected cattle in time and to maintain the official BTB-free status.

Through epidemiological modelling, the risk of a major outbreak is quantified, using one of six surveillance strategies. These are the currently used visual inspection of carcasses at the slaughterhouse (SL), the ELISA test on blood samples of carcasses at the slaughterhouse (ELISA-B), the γ-interferon test on blood samples of carcasses at the slaughterhouse (GAMMA-B), comparative tuberculination of the herd (CT), the combined method of single and comparative tuberculination of the herd (ST + CT) and the ELISA test on samples of bulk milk (ELISA-M). Test frequency of the last three methods was varied as well.

A stochastic individual based model (IBM) was developed to simulate a chain of infected herds, where each individual animal is followed in time. The model mimics the nation-wide situation after the introduction of one infected animal into one herd. BTB-transmission is simulated with an S-E₁-E₂-I state transition model. Output is time until detection of the infection, prevalence in the detected herd and the number of infected herds at the time of detection. For the assessment 500 simulations were used, representing 500 BTB-introductions. Model robustness to parameter values was analysed with Monte Carlo elasticity analysis, for which 1000 simulations were used.

Results of median time until detection and median number of infected farms at detection for SL (302 weeks and seven farms) were in agreement with estimates from an outbreak in the Netherlands in 1999. ELISA-B and GAMMA-B performed better than SL with a much lower median time until detection (189 and 97 weeks, respectively). The results for the tuberculination methods (ST + CT and CT) and ELISA-M depended heavily on the frequency in which the tests were performed. The tuberculination methods ST + CT and CT yield comparable results and detect the infection sooner than SL, also at the lowest tested frequency of once in 5 years. ELISA-M is comparable with SL at frequencies of once in 4 or 5 years, and this test works well at frequencies of once a year or higher. Our study results are used for an economical optimisation analysis of the six surveillance strategies.     

Ovine Johne's disease in Australia--the first 20 years

OBJECTIVE: To review the history of ovine Johne's disease in Australia. PROCEDURE: Relevant publications and reports were identified and reviewed to document the spread of ovine Johne's disease (OJD) from 1980 until the end of 2000, as well as the response of industry and government to the spread of this disease. RESULTS: OJD was first diagnosed in the central tablelands region of New South Wales in 1980. Since then it has spread, either from the initial focus or through separate introductions so that by December 2000 a total of 823 infected flocks had been identified. Cases have been confirmed in New South Wales, Victoria, the Australian Capital Territory, on Flinders Island in Tasmania, on Kangaroo Island in South Australia and in Western Australia. In early 1999, agreement was reached to fund and implement a 6-year, $40 million National OJD Control and Evaluation Program (NOJDP). This program is jointly funded by the sheep industries (national and state), and Commonwealth and State governments, and is managed by Animal Health Australia. CONCLUSION: A national program is now in place to support the control of OJD and research to determine the feasibility and cost-effectiveness of eradication. The development of new diagnostic techniques, such as abattoir surveillance and pooled faecal culture, provide opportunities to refine surveillance strategies and to define better the distribution and prevalence of this disease, as required by the national program. Effective control measures, combined with quality surveillance data, will enable informed decision making for the future national management of OJD.     

A Cross-sectional Study of Bovine Tuberculosis in Dairy Farms in Asmara,Eritrea

Bovine tuberculosis in dairy cattle in Asmara, Eritrea, was studied using a cross-sectional study to describe its prevalence and to identify factors associated with it. A total of 72 randomly selected herds were included in the study. The comparative intradermal tuberculin test was used for the diagnosis. Of 1813 individual animals tested, 14.5% were reactors. Thirty herds (41.7%) had at least one reactor but, by defining a reactor herd as any herd with two or more reactors, only 19 (26.4%) herds were classified as reactor herds. Based upon individual animal specificity of 98.5%, the calculated herd specificity was >99%. A multiple logistic model showed that the presence of exotic breeds was associated with a high risk (odds ratio = 5.70; 95% confidence interval 1.13–28.8). An increased risk was also linked to large herds. Keeping the animals always indoors reduced the risk, but could not be fitted to the model owing to empty cells.     

Experimental infection with Mycobacterium avium, serotype 2, 2. Oral infection with large doses of M. avium

Twelve pigs were inoculated orally with Mycobacterium avium. The doses used were 0.5, 2 or 10 mg daily for 5 days, or 10, 50 or 180 mg once (1 mg = 37 × 10⁶ viable units). Two pigs were used per dose, 1 of which was sacrificed 3 days, the other 28/31 days after the last inoculation (Table 1).Three days after inoculation, M. avium was found in the tonsils and in the intestinal mucosa of all 6 pigs, and in the mesenteric lymph nodes of 4. Viable unit counts for tonsils and intestinal mucosa were highest in pigs inoculated with 180 mg×1 and 10 mg×5. Histopathologically these pigs showed activation of the lymphoid tissue in the tonsils, Peyer patches and mesenteric lymph nodes. Twenty-eight/31 days after inoculation a spreading of the infection had taken place in all pigs, most often to the liver, less frequently to the spleen and the lungs. The kidneys and the musculature were not infected (Table 4). A correlation was apparent between the size of dose and the number of viable organisms in the tissues. Divided doses gave about 10 times higher viable counts than a single dose with the same total number of organisms (Table 5).No gross lesions were found 28/31 days after inoculation. Microscopic granulomatous lesions were found in the tonsils of 6 pigs, in the intestinal mucosa of 4 pigs, in the mandibular and mesenteric lymph nodes of 6 pigs, in the retropharyngeal lymph nodes of 3 pigs, and less frequently in the parotid and hepatic lymph nodes (Table 3).Five of 6 pigs were weakly sensitive to avian tuberculin PPD, 1000 t.u. per dose, when tested 22/25 days after inoculation; 1 of these pigs cross-reacted to human tuberculin (Table 2).     

Mycobacterium tuberculosis infection in a dog from Africa

A 4-year-old male Boxer dog with a history of vomiting, diarrhea, and weight loss moved from West Africa to Lyon, France, where it was further evaluated. Radiographs revealed pleural effusion and enlargement of tracheobronchial lymph nodes and liver. Cytologic examination of the pleural effusion and a fine needle aspirate specimen of the liver showed mixed mononuclear inflammation with nonstaining rod structures within epithelioid histiocytes. At necropsy, the main gross pathologic findings were exudative pleuritis, nodular hepatitis, and infarcts and caseous nodules in the kidneys. The main histologic lesions were granulomatous hepatitis, granulomatous pneumonia, fibrinous leukocytic pleuritis, necrotic and fibro-calcified granulomatous lymphadenitis, and granulomatous nephritis. A Ziehl-Neelsen stain applied to both cytologic and histologic samples was positive for acid-fast bacilli. Bacterial culture of the pleural fluid was positive for Mycobacterium tuberculosis. Cytology is a valuable tool in the diagnosis of this important zoonotic disease.