Evaluation of single and comparative intradermal tuberculin tests for tuberculosis eradication in caprine flocks in Castilla y León (Spain)

Goats can act as reservoirs for tuberculosis (TB) infection. The main etiological agents of TB in goats are Mycobacterium caprae and Mycobacterium bovis and they infect also a wide range of domestic and wild animals and humans. Control programmes based mainly on the application of single and comparative intradermal tuberculin (SIT and SCIT respectively) tests are being implemented in certain regions of Spain with a high density of caprine flocks as Castilla y León, including goats with epidemiological relationship with cattle. The aim of this study was to evaluate the performance of the intradermal tests in naturally TB-infected caprine flocks from this region. The study was performed using data from 17,450 goats in 54 different flocks that were classified as TB-infected in the control programmes executed in 2010 and 2011. Data from 1237 goats from 7 dairy flocks depopulated after the first intradermal testing were used to estimate the sensitivity (Se) using bacteriology as the gold-standard. Overall Se of the SIT test using the severe interpretation was 43.9% (CI 95%, 40.4–47.4) and decreased to 38.8% (CI 95%, 35.5–42.3) using the standard interpretation. Overall Se of the SCIT test ranged between 21.3% (CI 95%, 17.6–25.4) and 7% (CI 95%, 4.9–9.8) depending of the interpretation criteria. A significant weak positive correlation was found between age and skin fold thickness (Spearman’s test p < 0.05). Results from this study yielded, in general, low Se values probably due the systematic detection and slaughter of reactors as a consequence of the eradication programme in previous years or the presence of factors that may interfere in the diagnosis. Therefore, these results suggest the necessity of including ancillary diagnostic tools and/or strict interpretation criteria to maximize detection of positive animals in infected settings.     

牛源分枝杆菌变应原对免疫豚鼠变态反应试验

本文采用牛结核PPD检出阳性牛分离的分枝杆菌(包括有牛分枝杆菌、瘰疬分枝杆菌、乌胞内分枝杆菌2型及9型、副结核分枝杆菌、偶发分枝杆菌以及草分枝杆菌等),制成灭活免疫原进行豚鼠接种,并以各种分枝杆菌PPD进行变态反应检查,以观察其发生交叉反应的情况。试验结果表明:各种分枝杆菌除发生主反应外,均表现出明显的相互交叉的变态反应,尤以瘰疬分枝杆菌、乌胞内分杆菌2型及9型、副结核分枝杆菌以及牛分枝杆菌等交叉严重。提示:在牛结核及副结核的检疫中出现其它分枝杆菌干扰的假阳性反应。     

二维薄层色谱法分析分枝杆菌脂质成分

为探讨卡介苗巴氏德菌株(Bacillus Calmette-Guérin-pasteur,BCG-pasteur)和耻垢分枝杆菌(Mycobacterium smegma-tis,mc^2155)两种菌株的脂质成分,且通过比较脂质成分差异为研发新型牛结核病脂类疫苗佐剂提供试验基础。本文通过石油醚,氯仿,甲醇,丙酮等有机溶剂萃取BCG-pasteur和mc^2155两种菌株脂质,采用薄层色谱法分析其非极性脂质成分的差异。结果显示,BCG-pasteur中脂质成分富含三酰甘油,甲硫酚二角酰亚胺,PAT家族蛋白,甘油二酯,游离脂肪酸,游离分枝菌酸,葡萄糖单霉菌酸酯以及硫脂I类和硫脂III类等物质;mc^2155脂质成分除了硫脂I类,其他成分均有表达,但相对于BCG-pasteur其含量明显减少。本萃取方法简便稳定,适用于分枝杆菌的非极性脂质提取,为分枝杆菌脂质成分的分析提供基础;本试验通过薄层色谱法分析显示卡介苗和耻垢分枝杆菌细胞壁脂质成分差异明显,表明不同结核分枝杆菌的细胞壁脂质成分存在明显差异,从而可以引起不同的免疫反应,可为新型牛结核病疫苗提供方向。     

表达幽门螺杆菌外膜蛋白的重组耻垢分枝杆菌疫苗菌对小鼠的免疫保护作用

为研究含pLAO的重组耻垢分枝杆菌疫苗(Recombinant Mycobacterium smegmatis,rM.S)对小鼠的保护作用。将重组耻垢分枝杆菌疫苗pLAO(MS)2×107灌胃免疫接种BALB/c小鼠,同时设PBS组、耻垢分枝杆菌空菌组,免疫4周后,各组处死一批小鼠,取胃组织待用;余下的小鼠用幽门螺杆菌标准株(Helicobacter pylori SS1,Hp SS1)攻击2次,4周后处死小鼠,进行胃组织快速尿素酶试验、Hp培养、炎症程度及其炎症活动度评分,以评价疫苗对胃黏膜Hp感染的免疫保护作用,ELISA检测Hp特异性血清IgG和IgA水平。结果表明疫苗组Hp定植评分均明显低于PBS组和空菌组,疫苗组不仅可以降低Hp的定植,而且能减轻Hp造成的小鼠胃黏膜的局部慢性炎症反应。免疫后BALB/c小鼠特异性抗体显示rM.S疫苗诱导的Hp特异性血清IgG、IgA水平都升高。因此rM.S疫苗经灌胃接种BLAB/c小鼠能降低Hp定植,减轻胃黏膜的炎症反应,对Hp感染有明显的预防保护作用。     

Rapid identification of tissue micro-organisms in skin biopsy specimens from domestic animals using polyclonal BCG antibody

Immunostaining with polyclonal anti-Mycobacterium bovis (BCG) was evaluated as a single screening method for the histological identification of micro-organisms in skin biopsy specimens from various veterinary species. Confirmed archival cases infected with Mycobacteria, Nocardia, Actinobacillus, Actinomyces, Streptococcus/Staphylococcus, Dermatophilus, spirochetes, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, dermatophytes, Malassezia, Sporothrix, Leishmania, Pythium, phaeohyphomycetes and Prototheca organisms were selected. A total of 70 skin biopsy specimens from the dog, cat, horse, ox and llama were evaluated. The anti-BCG immunostain labelled bacteria and fungi with high sensitivity and minimal background staining but did not label spirochetes and protozoa (Leishmania). Differences were not noted between veterinary species. The results indicate that immunostaining with polyclonal anti-BCG is a suitable screening technique for the rapid identification of most common bacterial and fungal organisms in paraffin-embedded specimens. Also, mycobacterial and nocardial organisms were identified more readily with the anti-BCG immunostain in comparison to the histochemical stains.     

The effect of Johne's vaccination on tuberculin testing in farmed red deer (Cervus elaphus)

AIM: To assess the degree of interference with bovine tuberculin testing in farmed red deer that vaccination of young deer with an oil-adjuvanted vs aqueous formulation of live attenuated Mycobacterium paratuberculosis Strain 316F vaccines would be likely to cause, and to compare immunological responses between vaccine formulations.

METHODS: Five-month-old red deer (n=45) were randomly allocated to three treatment groups of 15 animals, which received either no vaccine, a single 2-ml dose of an oil-adjuvanted formulation or two 2-ml doses, 6 weeks apart, of an aqueous formulation of live attenuated M. paratuberculosis Strain 316F vaccine injected subcutaneously (S/C) in the neck (Control, Oil-adjuvant Ptb, and Aqueous Ptb groups, respectively). Injection-site reactions were described and measured on Weeks 3, 6 and 9. Animals were weighed and lymphocyte transformation tests (LTT) and antibody enzyme-linked immunosorbent assays (ELISA) using avian, bovine and Johnin tuberculin were conducted on blood samples collected at Weeks 0, 6, 12, 15, 24, 27, 36 and 39. A bovine mid-cervical skin test (MCT) was applied at Week 12, and comparative cervical skin tests (CCTs) at Weeks 24 and 36. At Week 42, the animals were slaughtered at a commercial deer slaughter premises and subjected to rigorous meat inspection.

RESULTS: Two animals were eliminated at the start of the trial due to a positive cross-reaction with bovine tuberculin in the initial LTT. Almost all animals reacted to the MCT at Week 12, with mean skin thicknesses of 3.9, 2.9 and 1.0 mm for the Oil- adjuvant Ptb, Aqueous Ptb and Control groups, respectively. When the CCT was conducted at Week 24, 2/15 Oil-adjuvant Ptb, 2/14 Aqueous Ptb and 1/14 Control animals were classified as CCT-positive to bovine tuberculin. By Week 36, all animals were CCT-negative. The Oil-adjuvant Ptb vaccination resulted in high persistent levels of antibody that reacted with bovine tuberculin, compared with negligible levels in the Aqueous Ptb group.

Overall, a single dose of the Oil-adjuvant Ptb vaccine in deer stimulated a vigorous, cross-reactive immune response, evidenced by high LTT, skin-test and antibody reactions to bovine tuberculin, with both cell-mediated and humoral characteristics. By comparison, two doses of the Aqueous Ptb vaccine produced less cross-reactivity and a bias towards a cell-mediated response. The Oil-adjuvant Ptb vaccine resulted in moderate injection- site lesions that were quite persistent, whereas the Aqueous Ptb vaccine resulted in smaller nodules that regressed more quickly.

CONCLUSIONS: Vaccination of farmed deer with an oil-adjuvanted Johne's vaccine has the potential to cause significant interference with routine tuberculin skin testing. The cross-reactivity should decline with time and the CCT should be able to clear MCT-positives, but there is a risk of false-positives to the blood test for tuberculosis (BTB), due to high persistent levels of antibody. The CCT could be used as a primary skin test in vaccinated deer on some farms.

The Aqueous Ptb caused fewer problems with skin testing and produced significantly less bovine antibody than the Oil-adjuvant Ptb, but stimulated persistent cell-mediated immune responses that may provide some protection against Johne's disease.     

Investigation of Zoonotic Infections Among Auckland Zoo Staff: 1991–2010

Investigation was undertaken to assess the occurrence of zoonotic infection among staff at Auckland Zoological Park, New Zealand, in 1991, 2002 and 2010. Serial cross‐sectional health surveys in 1991, 2002 and 2010 comprising a health questionnaire, and serological, immunological and microbiological analysis for a range of potential zoonotic infections were performed. Laboratory results for zoo animals were also reviewed for 2004–2010 to assess the occurrence of potential zoonotic infections. Veterinary clinic, animal handler, grounds, maintenance and administrative staff participated in the surveys, with 49, 42 and 46 participants in the 1991, 2002 and 2010 surveys, respectively (29% of total zoo staff in 2010). A small number of staff reported work‐related infections, including erysipelas (1), giardiasis (1) and campylobacteriosis (1). The seroprevalence of antibodies to hepatitis A virus and Toxoplasma gondii closely reflected those in the Auckland community. No carriage of hepatitis B virus (HBV) was detected, and most of those with anti‐HBV antibodies had been vaccinated. Few staff had serological evidence of past leptospiral infection. Three veterinary clinic staff had raised Chlamydophila psittaci antibodies, all <1 : 160 indicating past exposure. Two staff (in 1991) had asymptomatic carriage of Giardia lamblia and one person (in 2010) had a dermatophyte infection. After 1991, positive tests indicating exposure to Mycobacterium tuberculosis were <10%, comparable to the general New Zealand population. Zoo animals had infections with potential zoonotic agents, including G. lamblia, Salmonella spp., Campylobacter spp. and T. gondii, although the occurrence was low. Zoonotic agents pose an occupational risk to zoo workers. While there was evidence of some zoonotic transmission at Auckland Zoo, this was uncommon and risks appear to be adequately managed under current policies and procedures. Nevertheless, ongoing assessment of risk factors is needed as environmental, human and animal disease and management factors change. Policies and procedures should be reviewed periodically in conjunction with disease monitoring results for both animals and staff to minimise zoonotic transmission.     

鹿结核病的诊断

选取具有典型临床症状和结核菌素变态反应呈阳性的病鹿,处死剖检,采集有结节病变的肺、肠系膜、胸膜及肿胀的淋巴结等病料,进行细菌分离培养;将病料制作石蜡切片,HE染色镜检;PCR检测分支杆菌。结果表明,分离出的细菌菌落呈圆形、表面粗糙的颗粒状。经抗酸染色,见两端钝圆、平直或稍弯曲的红色杆菌。生化鉴定结果符合牛分支杆菌的生化特性;病理组织学分析,可见典型的增生性结核结节病变,中央为干酪样坏死,外周为上皮样细胞和朗罕氏细胞,最外层为淋巴样细胞浸润;经PCR检测,4份病料均扩增出与预期大小相符的441 bp核酸条带,证明分离的细菌为牛分支杆菌。     

Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton)

Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae , two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities.     

Mycobacterial nodular granulomas affecting the subcutis and skin of dogs (canine leproid granuloma syndrome)

Objective To obtain a better understanding of a disease affecting dogs in which nodular mycobacterial granulomas are present in the subcutis or skin. Design Retrospective survey Procedure A diagnosis of an unidentified mycobacterial infection was made in 45 dogs following detection of acid-fast bacilli surrounded by granulomatous or pyogranulomatous inflammation in tissue sections. Eight cases were identified from the records of our diagnostic laboratory. In all cases culture for mycobacteria was unsuccessful. Another 37 cases were identified by practitioners and veterinary pathologists in response to a survey mailed to veterinary clinics throughout Australia. Data from these 45 cases were entered into a data base. The data set was incomplete, as some respondents did not answer all questions. Results Over 90% of affected dogs had short coats and about half were Boxers or Boxer-cross dogs. A weak seasonal trend for the development of primary lesions in autumn and winter was identified. Cases were recorded from New South Wales (35 dogs), Western Australia (4), Queensland (4), Tasmania (1) and New Zealand (1). The subcutis and skin of the ears and head (including the ears) were involved in 64% and 85% of cases, respectively, for which lesion site was recorded. Culture was attempted but was unsuccessful in 19 cases, including 11 cases where material was submitted to our laboratory and/or a Mycobacteria Reference Laboratory. Lymph node enlargement, internal organ involvement or constitutional signs were not a feature of the syndrome, and most lesions did not worry the dogs. Although many practitioners recorded a favourable response to therapy with doxycycline (response rate 57%) or amoxycillin-clavulanate (63%), spontaneous resolution of infection was thought to have occurred in six of seven dogs (86%) not given systemic antimicrobials, and three dogs where antimicrobials had failed previously. A minority of cases failed to respond to antimicrobial therapy and continued to have chronic lesions. There was no discernible trend for dogs of a particular age or sex to be affected. Conclusion This syndrome is caused by saprophytic mycobacteria of limited pathogenicity that give rise to lesions restricted principally but not exclusively to the subcutis and skin of body extremities. Fastidious growth requirements have prevented their isolation on synthetic media used for culture of mycobacteria. Organisms presumably enter the subcutis following a breach in integrity of the epidermal barrier and produce self-limiting disease in immunocompetent dogs. Lesions tend to resolve spontaneously. The possibility of a public health threat from affected dogs is highly unlikely.