Protective efficacy of intranasal vaccination with Mycobacterium bovis BCG against airway Mycobacterium tuberculosis challenge in mice

The effect of route of immunization on the protective efficacy of BCG against tuberculosis has been investigated. Immunoprotection was monitored by evaluating the bacterial burden in the lungs and spleen of mice challenged with Mycobacterium tuberculosis H(37)Rv after BCG immunization by intranasal (i.n.) and subcutaneous (s.c.) routes. Our results showed that as compared to s.c. BCG immunization, intranasal BCG vaccination induces significantly higher immune responses at local level (mediastinal lymph nodes, cervical lymph nodes and lung). Further, i.n. BCG vaccination induced significantly higher reduction in bacterial load in the lungs over s.c. BCG vaccination, whereas, the bacilli load in the spleen was comparable in both the groups. Hence, intranasal vaccination with BCG holds promise for pulmonary tuberculosis.     

Aerogenic vaccination of mice with Mycobacterium bovis BCG

The course of infection with Mycobacterium bovis BCG Pasteur was followed against time in groups of mice vaccinated by either the aerogenic or subcutaneous route. The generation of acquired protective immunity and immunological memory was determined in each group by adoptive immunisation procedures. In addition, subcutaneously vaccinated mice were tested for their ability to resist an aerogenic challenge with a lethal dose of M. tuberculosis. No overall qualitative differences in the magnitude or longevity of antituberculosis immunity in mice vaccinated by the two procedures were observed. It is concluded that aerogenic vaccination offers no immunological advantage over vaccination by the subcutaneous route.     

Mycobacterium bovis BCG vaccination modulates TNF-alpha production after pulmonary challenge with virulent Mycobacterium tuberculosis in guinea pigs

Tumor necrosis factor-alpha (TNF-alpha) plays critical and opposing roles in the pathogenesis of tuberculosis (TB). We examined the effects of Mycobacterium bovis BCG vaccination on TNF-alpha production in three distinct guinea pig leukocyte populations before and after pulmonary infection with M. tuberculosis H37Rv. Following BCG vaccination alone, and following challenge, bronchoalveolar lavage cells (BALC), resident peritoneal cells (PC), and splenocytes (SPC) were stimulated with purified protein derivative (PPD). Before virulent challenge, BCG vaccination clearly enhanced the ability of BALC, PC and SPC to produce TNF-alpha in response to PPD stimulation ex vivo. Following challenge, the TNF-alpha production of all three leukocyte populations from BCG-vaccinated animals remained relatively constant at pre-challenged levels. In sharp contrast, 5 weeks post-challenge, all three leukocyte populations from unvaccinated animals produced very high amounts of TNF-alpha in response to PPD. Three weeks post-challenge, SPC from one of the unvaccinated animals produced higher levels of TNF-alpha but the others produced lower levels of TNF-alpha than BCG-vaccinated animals. As expected, BCG vaccination reduced the levels of virulent mycobacteria in both the lungs and spleens. Thus, BCG vaccination allows guinea pigs to modulate TNF-alpha levels in conjunction with a reduction in bacillary loads in their tissues.     

Experimental pneumococcal meningitis in mice: a model of intranasal infection

Effective laboratory animal models of bacterial meningitis are needed to unravel the pathophysiology of this disease. Previous models have failed to simulate human meningitis by using a directly intracerebral route of infection. Hyaluronidase is a virulence factor of Streptococcus pneumoniae. In this study, a novel model of murine meningitis is described. Intranasal administration of S. pneumoniae with hyaluronidase induced meningitis in 50% of inoculated mice, as defined by a positive cerebrospinal fluid (CSF) culture and an inflammatory infiltrate in the meninges. None of the mice inoculated without hyaluronidase developed meningitis. Hyaluronidase was found to facilitate pneumococcal invasion of the bloodstream after colonization of the upper respiratory tract. Meningitis was characterized by pleocytosis of CSF and the induction of proinflammatory cytokines and CXC chemokines in brain tissue. These results indicate that this murine model mimics important features of human disease and allow for the use of this model for studying issues related to the pathophysiology and the treatment of pneumococcal meningitis.     

Isoniazid treatment of Mycobacterium bovis in cattle as a model for human tuberculosis

Cattle infected with Mycobacterium bovis spoligotype 9 were treated with Isoniazid (INH) from three to 14 weeks post infection, rested for four weeks to allow INH depletion and then challenged with M. bovis spoligotype 35. Post mortem examination (PME) 35 weeks after the initial infection showed partial protection against infectious challenge following INH-attenuated infection compared with the spoligotype 35 challenge controls. Antigen-specific IFN-γ responses decreased over time with INH therapy, following a similar pattern to that observed in the treatment of M. tuberculosis infection in humans. Following cessation of therapy, specific IFN-γ responses increased more strongly in those calves that were visibly lesioned at PME. IFN-γ responses were also used to identify two antigens, TB10.4 and Acr2, that induced anamnestic responses in INH-treated, re-challenged calves, suggesting a role for both antigens in protective immunity. Specific IL-10 responses were observed in all calves following treatment with INH suggesting a role for IL-10 in the resolution of infection.     

Cidofovir protects mice against lethal aerosol or intranasal cowpox virus challenge

The efficacy of cidofovir for treatment of cowpox virus infection in BALB/c mice was investigated in an effort to evaluate new therapies for virulent orthopoxvirus infections of the respiratory tract in a small animal model. Exposure to 2(-5)x10(6) pfu of cowpox virus by aerosol or intranasally (inl) was lethal in 3- to 7-week-old animals. One inoculation of 100 mg/kg cidofovir on day 0, 2, or 4, with respect to aerosol infection, resulted in 90%-100% survival. Treatment on day 0 reduced peak pulmonary virus titers 10- to 100-fold, reduced the severity of viral pneumonitis, and prevented pulmonary hemorrhage. The same dose on day -6 to 2 protected 80%-100% of inl infected mice, whereas 1 inoculation on day -16 to -8 or day 3 to 6 was partially protective. Cidofovir delayed but did not prevent the death of inl infected mice with severe combined immunodeficiency. Treatment at the time of tail scarification with vaccinia virus did not block vaccination efficacy.     

Establishment of a host-vector system in Mycobacterium bovis BCG

The recombinant plasmids, pYT72 and pYT92, were generated from a mycobacterial plasmid, pMSC262, and a Escherichia coli plasmid, pACYC177. These plasmids were capable of replication, and of stable maintainance in Mycobacterium bovis BCG when introduced by electroporation technique. Efficiency of transformation was about 10(4) transformants/micrograms DNA, and was the highest in the phage sensitive mutants (S-10, S-20) isolated from BCG Tokyo strain. We have also isolated transformable mutants from rapidly growing bacterium, M. smegmatis strains Jucho and TMC1533. By isolating deletion mutants from pYT72/92, we could determine the location of replication region of pMSC262 within a 2.3 kb Pst I-Hind III fragment. Using this fragment, we constructed "mini" shuttle plasmid pYT937 (5.9 kb in size) which possesses kanamycin and ampicillin resistance markers and replicates in both E. coli and Mycobacterium. Nucleotide sequence analysis of the replication region revealed that there are 2 potential coding regions which contain more than 200 amino acids. The largest one (ORF1) which codes 311 amino acids, however, lacks Shine-Dalgarno like sequence in the upstream and therefore may not be functional. The other coding region (ORF2) contains 260 amino acids and was preceded by Shine-Dalgarno like sequence. Upstream of the ORF2, there were several repeat sequences which may be important in the plasmid replication. GC content of the 2.3 kb fragment was 69.8%.     

鼻腔滴注法建立小鼠肺炎链球菌性脑膜炎模型

目的鼻腔滴注法建立小鼠肺炎链球菌性脑膜炎模型,为研究肺炎链球菌致脑膜炎的分子机制奠定基础。方法将冻存的TIGR4在新鲜TSA血平板上培养16 h～18 h后,刮取细菌并稀释成3种不同密度的菌液,每种密度分成2份,其中1份加入一定量的透明质酸酶,另1份不加。对小鼠行浅麻醉,用卡介苗注射器将上述菌悬液缓缓滴入小鼠鼻腔,待其自动吸入,每只小鼠50μl。每天观察小鼠情况,分别于感染后24、48和72 h处死小鼠,取心脏血和一半脑组织匀浆,做细菌计数,另一半脑组织用于组织病理学检查。结果加透明质酸酶的TIGR4 3个剂量组小鼠脑膜炎发生率分别为20.00%、65.00%和46.67%,对照组分别为13.33%、53.33%和66.67%,差异无统计学意义(P= 1.000 0);其中3×10~7CFU组和3×10~8CFU组差异无统计学意义(P=0.462 1),两组合并后与3×10~6CFU组差异有统计学意义(P=0.0196)。结论1)透明质酸酶不能促进鼻腔滴注TIGR4致小鼠脑膜炎的发生;2)小鼠脑膜炎的发生率与鼻腔感染TIGR4剂量有关,适宜剂量为3×10~7CFU,低于该剂量脑膜炎的发生率显著降低。     

Tuberculous pleuritis secondary to Mycobacterium bovis in a veterinarian

Mycobacterium bovis is a rare cause of tuberculosis in humans, but should be considered in individuals at risk secondary to medical comorbidities (notably immunocompromise) or occupational exposure. Most cases are secondary to reactivation of latent infection in elderly individuals although cases of primary infection still occur, usually involving animal‐to‐human transmission. Pleural fluid culture in the context of suspected tuberculous pleuritis is frequently negative and pleural biopsy significantly increases the likelihood of confirming the diagnosis histologically and microbiologically. Although thoracoscopic biopsies are the reference standard, closed pleural biopsies are an appropriate and more accessible alternative in the majority of cases – these should be done under direct ultrasound guidance to maximise diagnostic yield. Treatment for M. bovis infection is with prolonged combination anti‐tuberculous therapy, using an alternative to pyrazinamide as the organism is inherently resistant to this drug.     

Mycobacterium bovis hip bursitis in a lung transplant recipient

We present a report of extrapulmonary Mycobacterium bovis infection in a lung transplant recipient. M. bovis is acquired predominantly by zoonotic transmission, particularly from consumption of unpasteurized foods. We discuss epidemiologic exposure, especially as relates to the Mexico–US border, clinical characteristics, resistance profile, and treatment.     

Genomics of Mycobacterium bovis.

The imminent completion of the genome sequence of Mycobacterium bovis will reveal the genetic blueprint for this most successful pathogen. Comparative analysis with the genome sequences of M. tuberculosis and M. bovis BCG promises to expose the genetic basis for the phenotypic differences between the tubercle bacilli, offering unparalleled insight into the virulence factors of the M. tuberculosis complex. Initial analysis of the sequence data has already revealed a novel deletion from M. bovis, as well as identifying variation in members of the PPE family of proteins. As the study of bacterial pathogenicity enters the postgenomic phase, the genome sequence of M. bovis promises to serve as a cornerstone of mycobacterial genetics.     

结核分枝杆菌和牛分枝杆菌TaqMan荧光PCR检测的建立

目的对结核分枝杆菌和牛分枝杆菌分别建立荧光PCR快速检测方法。结核分枝杆菌荧光PCR对H37Rv标准菌株、结核分枝杆菌临床分离株的检测结果呈典型阳性反应，牛分枝杆菌荧光PCR对牛分枝杆菌标准菌株、BCG菌株的检测结果呈典型阳性反应，对其它分枝杆菌菌株以及常见微生物样品为阴性反应。两种荧光PCR对对应阳性重组质粒模板的检测灵敏度可达单个基因拷贝，对结核分枝杆菌或BCG标准菌株的检测灵敏度可达单个菌细胞。对临床样品的检测结果显示，荧光PCR对模拟感染奶样、血样的检测灵敏度可达单个菌细胞。所建立的方法，全过程包括血样、奶样、痰液等临床样品的前处理、核酸提取和荧光PCR检测，可在5～6h内完成。采用上述荧光PCR方法从临床采集的疑似病人痰液中检出多份结核分枝杆菌阳性样品。     

An intranasal challenge model for testing Japanese encephalitis vaccines in rhesus monkeys.

Placebo-controlled field efficacy trials of new Japanese encephalitis (JE) vaccines may be impractical. Therefore, an animal model to evaluate efficacy of candidate JE vaccines is sought. Previous work has shown that exposure of monkeys to JE virus (JEV) via the intranasal route results in encephalitis. Here we report the further development of this model and the availability of titered virus stocks to assess the protective efficacy of JE vaccines. To determine the effective dose of our JE challenge virus, dilutions of a stock JEV (KE-93 isolate) were inoculated into four groups of three rhesus monkeys. A dose-dependent response was observed and the 50% effective dose (ED50) was determined to be 6.0 x 10(7) plaque forming units (pfu). Among animals that developed encephalitis, clinical signs occurred 9-14 days postinoculation. Infection with JEV was confirmed by detection of JEV in nervous tissues and IgM to JEV in the cerebrospinal fluid. Viremia with JEV was also detected intermittently throughout infection. Validation of the model was performed using a known effective JE vaccine and saline control. One ED90 of virus (2.0 x 10(9) pfu) was used as a challenge dose. Four of four animals that received saline control developed encephalitis while one of four monkeys administered the JE vaccine did so. This study demonstrates that the virus strain, route of inoculation, dose, and the outcome measure (encephalitis) are suitable for assessment of protective efficacy of candidate JE vaccines.     

结核分枝杆菌和牛分枝杆菌的二重PCR快速检测

目的建立一种鉴别结核分枝杆菌和牛分枝杆菌的二重PCR快速检测方法。方法根据已发表的结核分枝杆菌23SrRNA和牛分枝杆菌特殊基因序列,设计并合成了两对可扩增结核分枝杆菌和牛分枝杆菌特异性基因片段的引物,建立二重PCR快速检测结核分枝杆菌和牛分枝杆菌的方法,测定其特异性和敏感性,并对238份牛临床样品的DNA分别进行了检测。结果该方法对牛分枝杆菌能扩增出838bp和631bp的特异性基因片段,结核分枝杆菌只扩增出838bp基因片段而扩增不出631bp的特异性基因片段,对参试的其它牛菌株的DNA扩增结果均为阴性。敏感度检测可达到50pg的DNA含量。临床样品检测结核分枝杆菌阳性有21份,牛分枝杆菌阳性有1份,其它临床样品扩增结果全部为阴性。结论本方法可作为牛分枝杆菌的快速检测和流行病学调查的工具。