牛分枝杆菌esat-6和mpb70-mpb83基因DNA疫苗的免疫效果

用PCR技术和重叠延伸剪接技术获得牛分枝杆菌esat-6基因和mpb70-mpb83融合基因，连接真核表达载体pcDNA3．1（＋），构建了重组质粒pCE6和pC70-83-E6。分4组免疫小鼠：pCE6组、pC70—83-E6组、pcDNA3．1（＋）和PBS对照组，采用间接EI。ISA法检测免疫小鼠血清特异性抗体水平，MTT法检测免疫小鼠脾淋巴细胞增殖情况和IFN—y分泌情况。结果表明，2重组质粒免疫组小鼠的血清抗体水平持续上升，而2对照组始终维持在较低水平，且pC70-83-E6组小鼠的抗体水平高于pCE6组。经PPD刺激后，pCE6组和pC70—83-E6组小鼠的SI值与2对照组均差异显著（P〈0．05），2重组质粒免疫组间差异不显著（P〉0．05）；2重组质粒免疫小鼠脾细胞产生的IFN—y均显著高于2对照组（P〈0．05），且pC70-83-E6组明显高于其他3组（P〈0．05）。证实本试验构建的2种牛分枝杆菌DNA疫苗可有效诱导实验动物产生体液免疫和细胞免疫应答。     

CRISPR/Cas System and Its Application in the Study of Mycobacterium tuberculosis

Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-related protein (Cas) system is a heritable prokaryotic adaptive immune system against invading nucleic acids. It is an editing tool for many organisms that can generate highly specific gene double-strand breaks and repair them by non-homologous end joining (NHEJ) or homologous recombination (HR). Due to the complexity of its genetic manipulation, Mycobacterium tuberculosis genome research has many difficulties. Therefore, the emergence of the CRISPR/Cas system has a milestone significance for the research of Mycobacterium tuberculosis. In this review, we focus on the application of the CRISPR/Cas system in Mycobacterium tuberculosis and summarizes the present research progress in order to provide a reference for the selection of Mycobacterium tuberculosis research methods.     

The contribution of molecular biology to Mycobacteriumavium subspecies Paratuberculosis research

Molecular biology has contributed to our knowledge and understanding of the structure of Mycobacteriumavium subspecies paratuberculosis and has been particularly useful in determining those components that elicit immune responses in the host or discriminate M. avium paratuberculosis from other closely related environmental mycobacteria. As such, it has made a significant impact in the field of diagnosis, and has been instrumental in the development of specific and sensitive diagnostic tests. The next decade will see exciting new developments in paratuberculosis research as a consequence of substantial advances made in the construction of gene transfer systems in mycobacteria. These will provide opportunities for applying new strategies to determine the genetic basis for pathogenesis and the mechanisms of drug resistance and will offer new prospects for the rational design of efficient vaccines.     

Cutaneous Mycobacterium avium Infection in a Cat

Abstract— A 6-year-old spayed female domestic shorthair cat with a paranasal cutaneous mass caused by Mycobacterium avium is reported. Recurrence after cryosurgery and nonresponsiveness to dapsone necessitated surgical removal. The cat remained clinically normal for 14 months when the occurrence of multiple facial nodules with acid-fast bacilli present caused the owner to elect euthanasia. Résumé— Le cas d'une chatte européenne castrée de six ans présentant une masse périnasale due àMycobacterium avium est rapporté. La rechute après cryochirurgie et l'absence de réponse à la dapsone a nécessité une ablation chirurgicale. Le chat est resté apparemment sain 14 mois après l'opération, jusqu'à l'appartion de nodules multiples sur la face avec des baciles acid-alcolorésistants. L'animal a été euthanasié. Zusammenfassung— Beschrieben wird eine kurzhaarige Hauskatze, sechs Jahre alt, weiblich, kastriert, mit einer paranasalen kutanen Zubildung, die durch Mycobacterium avium verursacht wurde. Eine Wiederauftreten nach Kryochirurgie und ein Nichtansprechen auf Dapson zwang zu einer chirurgischen Entfernung. Die Katze blieb 14 Monate lang klinisch gesund bis das Auftreten von multiplen fazialen Knoten mit histologisch nachweisbaren säurefesten Bazillen dazu führte, daß der Besitzer sich für die Euthanasié des Tieres entschied. Resumen En este artículo se estudia una gata castrada de seis años de raza moméstica de pelo corto, con una masa cutánea paranasal causada por el Mycobacterium avium. La masa fue removida quirürgicamente después del tratamiento con dapsone y la aplicación de criocirugía con la consiguiente reaparición de la misma. Por un periodo de 14 meses la gata permaneció clínicamente normal, pero después el animal desarrolló nódulos faciales multiples con la aparición de bacilos que podian ser teñidos con tinturas ácidas, ante lo cual el dueño decidió terminar con la vida del animal.     

Experimental infection with Mycobacterium avium, serotype 2, in pigs. 1. Intravenous inoculations

A porcine strain of Mycobacterium avium, Serotype 2, was used for intravenous inoculation of pigs in doses 5, 1, 10⁻¹, 10⁻² and 10⁻³ mg (1 mg = 78 × 10⁶ viable units), 2 pigs per dose.Dose 5 mg proved fatal for both of the inoculated pigs, which were killed in extremis 64 and 69 days, respectively, after inoculation. Dose 1 mg caused clinical disease in 1 of 2 pigs, but was not lethal. Post mortem, the clinically affected pigs showed a generalized granulomatous tuberculosis. The other pig given 1 mg and the pigs given smaller doses, showed no clinical signs, and lesions and presence of acid-fasts were mostly limited to the lymph nodes of the lung, liver and digestive tract.All the pigs showed delayed hypersensitivity to avian PPD tuberculin (1000 t.u.) and some of them cross-reacted with human PPD tuberculin (1000 t.u.). The clinically affected pigs gave a very weak response to tuberculin, the others a strong response.The smallest dose capable of establishing an infection and producing tuberculous lesions was not determined, but seems to be less than 10⁻³ mg (78000 viable organisms).     

Serological Investigations of Mycobacterium Avium and M. Avium-Like Bacteria Isolated from Domestic and Wild Animals

One hundred and two Mycobacterium avium and M. avium-like strains isolated from domestic and wild animals were submitted to serological typing by means of the agglutination method developed by Schaefer. Of the 82 porcine strains M. intracellulare serotype 8 dominated, followed in order of frequency by serotypes 1, 4, 2, 10 and 6. Of the 20 strains originating from other animals 7 were typed as either serotype 2, 3, 8 or 10. The 3 strains isolated from wild birds were all serotype 2. As a considerable number of the wild animal strains were autoagglutinable, the suitability of the agglutination test for typing such strains is discussed.     

Molecular epidemiology of bovine tuberculosis in wild animals in Spain: a first approach to risk factor analysis

In human tuberculosis (Mycobacterium tuberculosis), molecular epidemiology has accurately indicated the risk factors involved in active transmission of the disease, by comparing individuals whose isolates belong to a cluster with patients whose strains are considered unique. Nevertheless, this application has not been used in bovine tuberculosis (Mycobacterium bovis). Our study describes the integration of epidemiological data into molecular classification data on M. bovis isolates. These were isolated from wild ungulates in Extremadura (western Spain) with the objective of detecting the risk factors linked to the association of strains in clades, which are indicators of the active spread of the disease. The molecular markers used were spoligotyping + VNTR typing (loci: VNTR 2165, VNTR 2461, VNTR 0577, VNTR 0580, VNTR 3192 VNTR 2163a and VNTR 2163b) on a population of 59 M. bovis strains isolated from deer (Cervus elaphus), 112 from wild boar (Sus scrofa), six from bovines, 28 from pigs and 2 from goats (n = 207). Epidemiological variables included the animal species from which the strain was isolated, pathological condition of the host (incipient lesion, early and late generalisation), date of sampling (during or after the reproductive period) and hunting season. Bivariant analysis was used to establish the risk factors connected to the association of strains and later, the variables were evaluated by means of logistic regression. Molecular typing grouped a total of 131 strains (64.21%) in 28 clusters and 76 isolates shows unique profiles. The association of strains was connected to the appearance of macroscopic lesions during the reproductive period (O.R. 4.80; 95% CI 1.09–22.99, P < 0.005), showing a possible higher transmission during the courting period. This happened mainly during the last hunting season analysed (2002–2003, O.R. 3.69; 95% CI 1.27–11.9, P < 0.05), clashing with the time of higher prevalence of the disease in wild ungulates. Active spread was not connected to any species in particular, or to any concrete pathological condition.     

Cultural and biochemical characteristics of Mycobacterium paratuberculosis isolated from goats in Norway.

In Norway a variant of Mycobacterium paratuberculosis occurs which causes disease in goats but very seldom in sheep and cattle. Cultural and biochemical characteristics of this variant are investigated by comparing different pre-treatment methods and culture media for primary isolation and by subjecting a number of strains to different enzymatic and biochemical tests. Decontamination of materials with 5% oxalic acid and 0.1% benzalkonium chloride and culture on Dubos, Finleyson’s and Herrold’s medium was tested. The investigations showed that the combination oxalic acid decontamination/Dubos’ medium is most suitable for isolation of the goat-pathogenic variant.The morphology of the colonies was also most easily studied after culture on Dubos’ medium from material pre-treated with oxalic acid. The biochemical tests were found to be poorly suitable for the identification of M. paratuberculosis and for its differentiation from other mycobacteria.Mycobactin dependence for growth seems not to be absolute as a few goat strains produced growth on Dubos’ medium without mycobactin. However, growth was in all cases far better in the presence of mycobactin.     

Isolation of Mycobacterium paratuberculosis from sheep and cattle in Iceland.

Isolation of Mycobacterium paratuberculosis from sheep and cattle in Iceland. Acta vet. scand. 1979, 20, 191–199. — Culture experiments concerning the Icelandic variant of Mycobacterium paratuberculosis are described. Various decontaminating agents and culture media were employed and the colonial morphology of freshly isolated strains on different media described. The growth rate and culture requirements are compared with those of the Norwegian goat-pathogenic variant of M. paratuberculosis. For primary isolation modified Herrold’s medium gave the best results. However, on all the various culture media used, the growth of the Icelandic variant was much more sporadic than that of the Norwegian goatpathogenic variant. It is concluded that bacteriological culture is not useful for the diagnosis of Johne’s disease caused by the Icelandic variant of M. paratuberculosis.     

Certification of herds as free of Mycobacterium paratuberculosis infection: actual pooled faecal results versus certification model predictions

Dutch dairy herds closed for at least 3 years with no history of paratuberculosis were recruited for a study on herd-certification. One hundred dairy herds were tested for Mycobacterium paratuberculosis at 6-month intervals by pooled faecal culture (five individual animal samples per pool) with solid media. Ninety of the herds completed 9 herd tests and 10 herds dropped out of the study for reasons other than a paratuberculosis diagnosis. Of the 90 herds completing the full study, 61% eventually were found to be M. paratuberculosis-infected. The number of infected herds detected decreased with each round of testing. Assuming that all infected herds had been detected by the ninth herd test, the observed percentage of herds that were truly noninfected (P-free) after each round of testing was calculated. The observed P-free was compared to the predicted P-free based on a previously reported herd-certification model. The P-free predicted by the model was significantly different from the observed P-free. When a single assumption in the model was changed and a diagnostic sensitivity of 40–50% was selected, the predicted P-free closely approximated the observed P-free for the 90 Dutch dairy herds studied. The critical assumption that was changed for Version 2.0 of the model was within-herd infection prevalence for infected but test-negative herds after each round of serial testing. Model Version 1.0 had assumed a 50% decrease in within-herd prevalence but Version 2.0 assumed a stable within-herd prevalence. Culture of pooled faecal samples provides a high-sensitive, high-specific, low-cost test for herd-certification programs.