Phage resistance and altered growth habit in a strain of Streptococcus bovis

Bacteriophage (phi Sb01) of Streptococcus bovis, isolated from pooled rumen fluid of cattle, was a small siphovirus of morphotype B1. It contained double-stranded DNA of length 30.9 kb, which was digested by the restriction endonucleases, EcoRI, HindIII, and PvuII. Bacteria which survived phi Sb01 infection (strain 2BAr) grew in long chains (100-200 cells), ultimately forming large clumps of cells. This growth habit was in distinct contrast to that of the parent host strain which grew predominantly in the form of single cells or diplococci. Strain 2BAr was genetically stable, resistant to phi Sb01 attack, and the observed differences in the growth characteristics of the parent strain and 2BAr indicated that cells of 2BAr were more adherent. In the rumen ecosystem, the selection of phage-resistant bacteria with altered growth characteristics may be a factor in modifying bacterial phenotypes, and thus increasing variability among bacteria which are closely related genetically.     

Adaptive surface antigen variation in Mycoplasma bovis to the host immune response

Abstract The variability of predominant Mycoplasma bovis surface antigens in the presence of specific immune pressure was analyzed in an in vitro assay to determine if M. bovis could escape immune destruction. We have shown that serum antibodies from immunized or experimentally infected calves and monoclonal antibodies which specifically react with previously characterized or as yet undefined major M. bovis membrane surface proteins cause repression of expression or shortening of the target protein, or induce switching to expression of an antigenically distinct variant protein. We have further demonstrated that removal of the inducing antibody results in reversion to the original phenotype. These results suggest that the level of expression and the length of M. bovis surface antigens in the host is modulated by cognate antibodies. According to the surface antigenic variation systems, random selection of preexisting variants resistant to antibody-mediated inhibition or direct regulation of gene expression may be means by which this organism evades host immune defences.     

Mating behavior and thermoregulation of the reindeer warble fly,Hypoderma tarandi L. (Diptera: Oestridae)

Hypoderma (=Oedemagena) tarandi L. (Diptera: Oestridae) is characterized by a mating strategy in which both sexes meet and mate at two types of distinct topographical landmarks. In the expansive, treeless vidda (= tundra-like) biome, mating places are unique, rocky areas located along rivers and streams or in rocky areas of drying river and stream beds. In wooded valleys below the vidda, flies mated at certain topographical areas along dirt road tracks/paths. Thermoregulatory activities of males occupying perches at mating places included selection of substratum at perch site, orientation of body to sun's rays, crouching, stilting, and flights into upper cooler air. On warm sunny days males perched for just 1–2 min before flying up into cooler air to promote cooling. Laboratory and field studies revealed that flies could not metabolically cool down when held at 25–38°C. Time spent at mating places depended on temperature, duration of sunshine, and wind velocity. Males were very aggressive in pursuing allHypoderma-sized objects that passed by them or that landed near them, but they did not defend specific perch sites. Males either pursued and caught females in flight, or they hopped onto females that landed near them. During 5 years, 74 males and 14 females were seen at mating places. Dissection of six females caught at mating places revealed them to be recently eclosed flies full of fat body and with all eggs intact; two not paired with males were non-inseminated. Three experimentally paired females remainedin copulo for 10, 13, and 19.5 min.     

Vaccination against Babesia bovis: T cells from protected and unprotected animals show different cytokine profiles

Vaccination of cattle against the haemoprotozoun parasite, Babesia bovis, with the recombinant antigen 11C5 resulted in 9 of 15 cattle being protected against challenge infection. The cellular immune responses of protected and unprotected cattle were compared in order to identify differences in response. No differences were observed in the pattern of change in various blood leukocyte populations throughout challenge infection. FACScan analysis revealed an increase in the proportion of cells bearing the CD2 marker in both protected and unprotected cattle over the course of infection. There were no observable differences in the frequency of various cell-surface markers between the unprotected and protected cattle. During the period of patent parasitaemia, in vitro cultures of peripheral blood mononuclear cells (PBMC) from protected cattle produced significantly more TNF- (P < 0.05) than cultures from unprotected cattle. TNF- concentrations remained at pre-challenge levels until day 10, when levels in the unvaccinated control and vaccinated/unprotected animals dropped. By peak parasitaemia, TNF- production in vitro was siguificantly greater (P < 0.05) in cultures of PBMCs from protected cattle. Interferon production showed an initial peak at day 5 in all cattle, followed by a decrease and a second peak at days 10–13 in protected cattle only, which coincided with resolution of the infection.     

不同牛分枝杆菌特异性基因PCR方法的比较

【背景】牛结核病是我国二类动物疫病,世界动物卫生组织将其列为法定报告的动物疫病。牛主要通过患病牛呼吸道分泌物和咳嗽所产生的气溶胶感染;人则主要通过食用未经高温处理的病牛的肉或奶感染。因此,经过病原学PCR检测对疑似患病牛牛奶或屠宰组织样品进行快速检验确诊,能够最大限度地减少奶牛养殖中乳品生产业的经济损失。【目的】研究并确定适宜的牛分枝杆菌PCR扩增引物及参数,为临床快速准确诊断牛结核病提供参考。【方法】对已报道的5对PCR引物,运用降落(touch down) PCR法确定适宜退火温度(Tm);运用梯度稀释的牛分枝杆菌C68001株(国内牛结核菌素生产用菌株)基因组DNA以及不同菌液含量的人工模拟临床样本(淋巴结、肺脏和牛奶),确定不同引物PCR方法的敏感性;同时以6种常见牛感染菌(牛种布鲁氏菌2308、羊种布鲁氏菌Rev.1、牛分枝杆菌C68001和AN5、禽分枝杆菌C68202、副结核分枝杆菌C68681和胞内分枝杆菌C68226)核酸样本,确定不同引物PCR方法的特异性。【结果】所有引物在53-63℃均含有目的条带,确定引物的最佳退火温度是60℃。在细菌核酸敏感性检验中,1号和3号引物的检测敏感性最高,达10-10 ng/μL;其次是2号和5号,达10-5 ng/μL。对于人工模拟感染样本,1号、3号和4号引物在淋巴结和肺脏中检测敏感性最高,其次是2号;而2号、3号、4号和5号引物对奶样检测敏感性最高。对于特异性检验,2号和5号引物特异性较好,可检测到明显的牛分枝杆菌特异性条带,对通常不引起牛结核病而只干扰免疫学诊断的禽分枝杆菌检测条带较微弱,而布鲁氏菌、副结核分枝杆菌和胞内分枝杆菌均无检测条带。【结论】2号引物及其反应参数的PCR方法敏感性、特异性良好,适合用于牛结核病的快速准确诊断。     

Membrane subproteomic analysis of Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been known for a long time to prevent tuberculosis (TB) worldwide since 1921. Nonetheless, we know little about BCG membrane proteome. In the present study, we utilized alkaline incubation and Triton X-114-based methods to enrich BCG membrane proteins and subsequently digested them using proteolytic enzyme. The recovered peptides were further separated by 2-D LC and identified by ESI-MS/MS. As a result, total 474 proteins were identified, including 78 integral membrane proteins (IMPs). Notably, 18 BCG IMPs were described for the first time in mycobacterium. Further analysis of the 78 IMPs indicated that the theoretical molecular mass distribution of them ranged from 8.06 to 167.86 kDa and pI scores ranged from 4.40 to 11.60. Functional classification revealed that a large proportion of the identified IMPs (67.9%, 53 out of 78) were involved in cell wall and cell processes functional group. In conclusion, here we reported a comprehensive profile of the BCG membrane subproteome. The present investigation may allow the identification of some valuable vaccine and drug target candidates and thus provide basement for future designing of preventive, diagnostic, and therapeutic strategies against TB.     

牛分枝杆菌单基因及双价融合基因DNA疫苗免疫效果的研究

利用PCR技术和SOE技术扩增牛分枝杆菌ag85b、esat-6、hsp65、mpb64基因和ag85b-esat-6、hsp65-esat-6和mpb64-esat-6融合基因,连接真核表达载体pCDNA3.1(+),构建重组质粒pCA、pCE6、pCH、pCM、pCAE、pCHE和pCME。转染SP2/0细胞,检测目的基因的表达。以各重组质粒和pCDNA3.1(+)及PBS免疫BALB/c小鼠后检测血清特异性抗体水平、脾淋巴细胞增殖情况和IFN~γ分泌情况。结果表明,七种重组质粒免疫后小鼠血清抗体水平持续上升,与 pCDNA3.1(+)对照组和PBS对照组相比差异显著 （P<0.05）,其中pCA组血清抗体水平明显高于其他六种DNA疫苗免疫组 （P<0.05）;三免两周后,融合基因免疫组的刺激值（SI值）与单基因免疫组相比差异显著（P<0.05）,其中以pCME组的SI值最高;PPD刺激后融合基因DNA疫苗免疫组小鼠脾细胞分泌的IFN~γ高于单基因DNA疫苗组（P<0.05）,而两对照组则未检测到IFN~γ的产生。本试验成功构建了牛分枝杆菌ag85b、esat-6、hsp65、mpb64单基因和ag85b-esat-6、hsp65-esat-6、mpb64-esat-6双价融合基因DNA疫苗,从而为牛结核病新型疫苗的研制奠定了基础。     

Characterization of Streptococcus bovis from the rumen of the dromedary camel and Rusa deer

AIMS: Isolation and characterization of Streptococcus bovis from the dromedary camel and Rusa deer. METHODS AND RESULTS: Bacteria were isolated from the rumen contents of four camels and two deer fed lucerne hay by culturing on the semi-selective medium MRS agar. Based on Gram morphology and RFLP analysis seven isolates, MPR1, MPR2, MPR3, MPR4, MPR5, RD09 and RD11 were selected and putatively identified as Streptococcus. The identity of these isolates was later confirmed by comparative DNA sequence analysis of the 16S rRNA gene with the homologous sequence from S. bovis strains, JB1, C14b1, NCFB2476, SbR1, SbR7 and Sb5, from cattle and sheep, and the Streptococcus equinus strain NCD01037T. The percentage similarity amongst all strains was >99%, confirming the identification of the camel isolates as S. bovis. The strains were further characterized by their ability to utilize a range of carbohydrates, the production of volatile fatty acids (VFA) and lactate and the determination of the doubling time in basal medium 10 supplemented with glucose. All the isolates produced l-lactate as a major fermentation end product, while four of five camel isolates produced VFA. The range of carbohydrates utilized by all the strains tested, including those from cattle and sheep were identical, except that all camel isolates and the deer isolate RD11 were additionally able to utilize arabinose. CONCLUSIONS: Streptococcus bovis was successfully isolated from the rumen of camels and deer, and shown by molecular and biochemical characterization to be almost identical to S. bovis isolates from cattle and sheep. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus bovis is considered a key lactic acid producing bacterium from the gastrointestinal tract of ruminants, and has been implicated as a causative agent of lactic acidosis. This study is the first report of the isolation and characterization of S. bovis from the dromedary camel and Rusa deer, and suggests a major contributive role of this bacterium to fermentative acidosis.     

Menadione-catalyzed luminol chemiluminescent assay for viability of Mycobacterium bovis

Stable luminol chemiluminescence was observed 10 min after the addition of menadione to a suspension of Mycobacterium bovis homogenized in Middlebrook 7H9 broth base including OADC enrichment. The chemiluminescence intensity was proportional to the absorbance of the bacterial suspension at 600 nm in a range of 0.005 to 0.15. Luminol chemiluminescence disappeared after 10 min incubation of M. bovis at over 60% of ethanol or 4 days of cultivation of M. bovis in the presence of 40 microg/ml of streptomycin. The bacterium showing the disappearance of chemiluminescence could not grow after being washed, suggesting that the inhibition concentration of the antimicrobials can be estimated on the basis of the disappearance of chemiluminescence. Menadione-catalyzed luminol chemiluminescent assay was rapid and sensitive in comparison to turbidimetry, tetrazolium (WST-8) reduction assay, and the assay using the Mycobacteria growth indicator tube (MGIT).