胞内分枝杆菌热休克蛋白65基因的克隆和原核表达

本研究以胞内分枝杆菌ATCC13950株基因组DNA为模板,应用巢氏PCR方法扩增热休克蛋白65(HSP65)基因,PCR产物经纯化后与PMD18-T载体连接构建克隆质粒PMD18-T-HSP65,并转化大肠杆菌感受态细胞DH5,筛选阳性克隆并测序,NdeⅠ和BamHⅠ双酶切PMD18-T-HSP65和表达载体PET15b,用T4连接酶4℃过夜连接,转化大肠杆菌感受态细胞BL21(DE3)pLysS中,筛选和构建了原核重组表达质粒PET15b-HSP65,双酶切鉴定正确后,以终浓度为1mmol/LIPTG诱导并进行SDS-PAGE分析。结果表明,获得约65KD大小的蛋白,与预期结果相符,为胞内分枝杆菌及HSP65基因功能的研究打下基础。     

Applying the One Health Concept to Mycobacterial Research – Overcoming Parochialism

Mycobacterial infections remain a public health problem. Historically important, globally ubiquitous and with a wide host range, we are still struggling to control mycobacterial infections in humans and animals. While previous reviews have focused on individual mycobacterial infections in either humans or animals, a comprehensive review of the zoonotic aspect of mycobacteria in the context of the One Health initiative is lacking. With the purpose of providing a concise and comprehensive resource, we have collated literature to address the zoonotic potential of different mycobacterial species and elaborate on the necessity for an inter‐sectorial approach to attain a new vision to combat mycobacterial infections.     

采用PCR法和限制性内切酶分析对12种分枝杆菌进行鉴定

采用６５ＫＤａ热激蛋白基因为模板,选择位于３９８－８３６基因序列处的一对分枝杆菌通用引物,扩增出４３９ｂｐ的基因片段。将１２种标准分枝杆菌的ＰＣＲ扩增产物即４３９ｂｐ用ＢｓｔＥⅡ限制性内切酶切分析,根据酶切带 大小,强弱以及带型数量进行分枝杆菌菌种鉴定。     

结核分支杆菌重组活疫苗研究进展

结核分支杆菌(MTB)引起的结核病 是一种人兽共患的慢性传染病,短程督导化 疗是治疗该病的主要措施,卡介苗是预防该 病的惟一疫苗,但其免疫效果极不稳定。分 子生物学技术的发展为研制新型MTB疫苗 提供了理想方法,人们利用鼠伤寒沙门氏菌 (Salmonellatyphimurium,St)减毒株作为 载体,将编码MTB保护性抗原的基因导入其 中,可构建重组St疫苗,如rSt Ag85B疫苗 和rSt ESAT 6疫苗;将编码MTB保护性抗 原的基因导入小鼠巨噬细胞或骨髓细胞可构 建MTB活细胞疫苗,如J774 HSP65疫苗和 BMC HSP65疫苗;利用基因诱变可构建 MTB减毒活疫苗,如MTB嘌呤营养缺陷株, ICL基因剔除株,VitB5营养缺陷株和十一萜 醇激酶基因变异株。文章介绍了结核分支杆 菌重组鼠伤寒沙门氏菌疫苗,活细胞疫苗和 减毒活疫苗的构建及其免疫机制等方面的研 究进展。     

Serological and cutaneous testing of bovine tuberculosis with the A60 antigen complex from Mycobacterium bovis, strain Calmette-Guérin

A60, a major thermostable macromolecular antigen complex of Mycobacterium bouis strain Calmette-Guérin (BCG), is immunodominant in tuberculosis and able to elicit both humoral and cellular immune reactions in infected hosts. A60-based ELISA and cutaneous tests have been used, in conjunction with the PPD-based skin reaction, in a control group of healthy animals, and in a herd including tuberculous animals. Cutaneous testing with A60 yielded results comparable with those with PPD: both were negative with control cattle and positive with infected animals. Moreover, comparative cutaneous testing with avian tuberculin yielded similar results with PPD and A60. When animals from the infected herd were tested with both avian and bovine sensitins, 54% of cattle were diagnosed as fully positive, 26% suspect, and 20% negative. Serological analysis with the A60-ELISA of part of the infected herd yielded 74% positive, 21 % suspect and 5% negative results. Thus, positivity was 74% for Serological analysis and 54% for cutaneous testing, whereas positive plus suspect results were 95% for serological analysis and 80% for cutaneous testing. It can be concluded that A60 can be used in place of PPD for cutaneous testing in cattle, and that the diagnostic value of the A60-ELISA is superior to that of the PPD-cutaneous test.     

Bovine Nodular Thelitis: a Clinicopathological Study of 20 Cases

Abstract— Bovine nodular thelitis in dairy cows is a chronic and enzootic infection. Four stages were distinguished: early stage, evolutive stage, ulcerative stage and final stage. Lesions were located mainly in the lower part of the udder or on the teat and recognized as solid papules and nodules. The histopathological study revealed a tuberculoid granulomatous pattern characterized by a subacute focal dermatitis with lymphoid cells, macrophages, epitheloid cells and a variable number of multinucleate giant cells (Langhans type) but no necrosis. Acid-fast bacilli were demonstrated in two of 14 cases. Three strains of acid fast bacilli were isolated but the only one identified was Mycobacterium terrae. M. terrae is a non-pathogenic slowly growing, non-photochromogenic mycobacterium. Its role in tuberculosis-like skin lesion is the bovine udder is uncertain. Résumé— La thélite nodulaire est une infection chronique et enzootique chez les vaches laitières. On distingue 4 phases: une phase précoce, une phase évolutive, une phase ulcérative et une phase finale. Vingt échantillons histologiques, mammelles et trayons, ont été examinés. Les lésions étaient localisées essentiellement à la partie basse de la mammelle ou sur le trayon et se présentaient sous l'aspect de papules solides ou de nodules. Les échantillons étaient divisés en deux parties: l'une destinée à l'isolement de mycobactéries, l'autre à l'examen histologique. Les lésions histologiques des trayons étaient des granulomes tuberculoïdes, caractérisés par une dermatite subaigue focale avec présence de lymphocytes, macrophages, cellules épithélioïdes et une nombre variable de cellules multinucléées géantes (type Langhans). Les infiltrats inflammatoires étaient principalement périvasculaires. Parfois, ils infiltraient toute la paroi du trayon. Aucune nécrose n'a été observée. Des bacilles acidoalcoolorésistants furent mis en évidence de manière inconstante (2 cas sur 14). Trois types de bacilles acidoalcoolorésistants ont été isolés raais, un seul a été identifié comme étant Mycobacterium terrae. M. terrae est une mycobactérie non pathogène, de croissance lente et non photochromogène. L'implication de cet organisme dans les lésions de type tuberculeux dans la mammelle de la vache est discutée. Zusammenfassung— Bei Milchkühen stellt die bovine noduläre Thelitis eine chronische und enzootische Infektion dar. Vier Stadien lassen sich unterscheiden: Früstadium evolutorisches Stadium, ulzeratives Stadium und Endstadium. 20 pathologische Proben von Euter und Zitzen wurden untersucht. Die Veränderungen waren hauptsächlich am unteren Teil des Euters oder an den Zitzen lokalisiert und ließen sich als solide Papeln oder Knoten ansprechen. Das Untersuchungsgul wurde für die Isolierung von Mykobakterien und für eine histopathologische Untersuchung geteilt. Die histopathologischen Veränderungen der Zitzen zeigten ein tuberkuloides granulomatöses Bild, das durch subakute fokale Dermatitis mit lymphyoiden Zellen, Makrophagen, Epitheloidzellen und einer variablen Anzahl von vielkernigen Riesenzellen (Langhans-Typ) gekennzeichnet war. Die entzündlichen Infiltrate befanden sich hauptsächlich perivaskulär. Manchmal ließen sie sich aber auch in der gesamten Zitzenwand nachweisen. Nekrosen bestaznden nicht. Säurefeste Bazillus-Keime waren inkonstant vorhanden (2 von 14 Fällen). Drei Arten von säurefesten Bazillus-Keimen wurden isoliert, aber nur eine wurde speziell identifiziert (Mycobacterium terrae). M. terrae ist ein nicht pathogenes, langsam wachsendes und nich photochromogenes Mykobakterium. Die Beteiligung dieses Organismus bei den tuberkulose-ähnlichen Hautveränderungen am bovinen Euter wird diskutiert. Resumen La Telitis Nodular Bovina es una infección enzoótica crónica de las vacas lecheras. Se distinguen cuatro etapas: etapa inicial, etapa evolutiva, etapa ulcerativa y etapa final. En el presente trabajo se examinaron 20 muestras de mama y de pezón. Las lesiones se localizaban principalmente en la parte inferior de la glándula mamaria o en el pezón y consistían en pápulas sólidas y en nódulos. Las muestras se dividían en dos partes, una para el aislamiento de micobacterias y la otra para los estudios histopatológicos. Las lesiones histopatológicas consistían en una reacción granulomatosa de tipo tuberculoide, caracterizada por una dermatitis focal subaguda con células linfoides, macrófagos, células epitelioides y un número variable de células gigantes multinucleadas (tipo Langhans). Los infiltrados inflamatorios estaban localizados predominantemente en la zone perivascular. En ocasiones, aparecían a lo largo de toda la pared de la glándula mamaria. No se observó necrosis. Se han aislado tres cepas de bacilos acido-alcohol resistentes, pero sólo una fue identificada específicamente como Mycobacterium terrae. M. Terrae es una micobacteria no fotocromogénica de crecimiento lento y no patógena. Se discute la posible participación de este microorganismo en la génesis de las lesiones pseudotuberculosas observadas en estos animales.     

Cross species transmission of ovine Johne's disease from sheep to cattle: an estimate of prevalence in exposed susceptible cattle

OBJECTIVE: To determine the prevalence of infection of cattle with the sheep strain of Mycobacterium avium subsp paratuberculosis at least two years after exposure at < 6 months old. DESIGN: Prospective survey One thousand seven hundred and seventy-four cattle from 12 properties (Farms A to L) were sampled by ELISA and faecal culture to detect evidence of infection with M a paratuberculosis. All properties had a known history of Johne's disease (JD) in sheep, and sampled cattle were likely to be susceptible to JD at the time they were first exposed, being at an age of 6 months or less. In addition, opportunistic investigations were undertaken of ELISA reactor cattle discovered during testing for the Australian Johne's Disease Market Assurance Program for Cattle (Farms M and N). RESULTS: All animals in the survey gave negative results on serology while one animal from a herd of 349 gave a positive faecal culture result. Follow-up faecal culture, post-mortem and histopathology on the latter animal were negative, suggesting that it was a passive faecal shedder or carrier. Two occurrences of OJD transmission to cattle were detected during the opportunistic investigations. CONCLUSION: These observations confirm existing beliefs about the risk of transmission of OJD to cattle, that the risk of transmission is low. However transmission occurs sporadically. An estimated upper limit of prevalence of S strain M a paratuberculosis infection in susceptible exposed cattle in the OJD high prevalence area of New South Wales is 0.8%, assuming a common prevalence within herds.     

Avian mycobacteriosis in companion birds: 20-year survey

The causative agents of avian mycobacteriosis in pet birds are rarely identified. The aim of this study is to add information about the etiology of avian mycobacteriosis. The identification of mycobacterium species in 27 cases of avian mycobacteriosis in pet birds was investigated by polymerase chain reaction (PCR) and sequencing of a rRNA hypervariable region. Avian mycobacteriosis appeared to be an infrequent diagnosis. Interestingly, a few cases of avian mycobacteriosis were recorded in very young birds. The most commonly affected species were the canary (Serinus canarius), the Eurasian goldfinch (Carduelis carduelis) and the red siskin (Spinus cucullatus). All but one bird were infected with Mycobacterium genavense. Mycobacterium avium was identified only in one case.     

Cytology and histopathology of canine leproid granuloma syndrome

OBJECTIVE: To describe the histopathology of canine leproid granuloma syndrome. DESIGN: Histological examination of biopsy specimens taken from dogs with leproid granuloma syndrome. Biopsies were acquired from four veterinary pathology practices after initial examination showed acid-fast bacilli or lesions suggestive of a mycobacterial aetiology. Tissue from 30 cases formed part of a retrospective survey while a further 7 cases were obtained prospectively. PROCEDURE: Tissue samples were fixed in formalin and paraffin embedded. Sections were stained with haematoxylin and eosin and Ziehl-Neelsen stains. Slides were evaluated for the type of inflammatory response and the number of bacteria. In a few cases smears made from crush preparations and needle aspirates were stained with DiffQuik and acid-fast stains. RESULTS: The common cytological feature seen in DiffQuik stained smears was numerous, often spindle-shaped, macrophages with variable numbers of lymphocytes and plasma cells and lower numbers of neutrophils. Usually few to moderate numbers of medium length bacilli were detected within macrophages or extracellularly. Histologically, lesions were chiefly pyogranulomatous within the subcutis and dermis. Pyogranulomas were composed chiefly of epithelioid macrophages and neutrophils but plasma cells and small lymphocytes were scattered throughout the lesions in which giant cells were seen. Lesions were pyogranulomatous in 36 cases and granulomatous with small suppurative foci in one. The numbers of acid-fast bacilli present were very low to low in 22 cases, moderate in 6 and regionally numerous to numerous in 9. Bacteria were pleomorphic, ranging from long, slender filaments in parallel sheaves to short and variably-beaded bacilli or highly beaded to coccoid organisms. The morphology was more uniform in DiffQuik stained smears than in fixed tissue sections. CONCLUSION: The pathology of canine leproid granuloma syndrome is highly uniform and is suggestive of saprophytic mycobacterial involvement. The morphological diversity of acid-fast bacilli probably results from differences in staining characteristics rather than indicating different species of Mycobacterium. While approximately half of the cases had only few organisms present, the veterinary practitioner using a Romanovsky stain such as DiffQuik on aspirated material might expect to obtain a rapid diagnosis in many cases. This would allow differentiation of the syndrome from neoplastic and other diseases of the skin and direct appropriate treatment at the initial presentation.     

分枝杆菌药物最低抑菌浓度的快速测定方法研究

为了建立快速测定分枝杆菌药物最低抑菌浓度的方法,同时了解耐多药结核分枝杆菌(MDR-TB)和非结核分枝杆菌(MOTT)对常规抗结核分枝杆菌以外的其他抗生素的敏感性。采用一种快速变色液体培养基系统,以微量法进行测定。结果表明:MDR-TB对CLA、AZI、ROX、LEV、SPA、CIP、OFL、CAP、AMI、TH1314和PTA高度敏感的菌株分别88.89％、72.2％、72.2％、55.6％、50％、66.7％、70％、40％、40％、70％和50％,未见对DOX和MIN高度敏感的MDR-TB菌株;CLA、LEV和TH1314对MDR-TB的MIC几何平均值分别为0.5、1和1μg／mL,显示出较好的活性;不同的MDR-TB和MOTT对抗生素的敏感性有差异;这种新型变色液体培养基检测系统具有快速、操作简便等特点,对多种一线抗结核药物耐药的多MDR-TB对CLA、LEV、AZI、ROX、CAP和TH1314敏感,可供首先选择用于MDR-TB的治疗;不同的MOTT应根据MIC结果选择抗生素进行治疗。