结核分枝杆菌分泌蛋白ESAT-6、CFP10基因的克隆、鉴定及其原核表达

以人型结核分枝杆菌 H37RV株基因组 DNA为模板 ,应用 PCR法对 ESAT- 6和 CFP10基因进行扩增 ,产物经纯化后与载体 PMD18- T连接、转化及酶切鉴定 ,亚克隆到原核表达载体 PGEX- 6 P- 1,构建原核重组表达质粒 ,转化入大肠杆菌 BL2 1中 ,以 1mmol/L IPTG诱导 ,进行 SDS- PAGE电泳。结果表明 ,ESAT- 6和 CFP10基因表达的融合蛋白相对分子质量分别为 32 0 0 0和 36 0 0 0 ,与实测相符。重组结核杆菌分泌蛋白 ESAT- 6和 CFP10的成功表达为结核病诊断及重组疫苗的构建打下了基础     

黄葵素对结核分枝杆菌的抑杀作用研究

Objective To evaluate the antituberculous activities of Huangkuisu (HISS). Meth-ods lOStandard human strains of mycobacterium tuberculosis (H37RV) and multi-drug resistant TBstrainswere inoculated in different concentrations of HISS, blank control and positive TB drug culture medium[()observe the growth of mycobacterium tuberculosis and evaluate the antibacterial activity in vitro.②Certain number of standard strains of Mycobacterium tuberculosis H37Rv and drug-resistant strains were injected into the tail vein[()establish a mouse model of mycobacterium tuberculosis infection. The mice were randomly divided into treatment group, rifampicin, HISS treatment group treated lasting for 6 weeks; the control group was given normal saline. The antibacterial activity in vivo were evaluated[()compare the treatment results according[()the pathological changes in lung and spleen tissues (HE staining and acid-fast staining) and charging amount of bacteria. Results lOthe bactreial colonies were grew normally in model group, but were not grew in     

应用变态反应方法诊断鹿结核病的研究

利用牛型新，旧结核菌素（ＰＰＤ，ＯＴ），禽型旧结核菌素（ＯＴ）为变应原，采用变态反应点眼法进行了鹿结核病的诊断研究。结果表明：变应原以牛型ＯＴ为最好，其敏感性高于禽型ＯＴ，检出率高于牛型ＰＤＤ；一次二回点眼的阳性率可达９２．３３％，且较一次一回的检出率高４４．６％；判定最佳时间为３，６，９ｈ。用牛型ＯＴ一次二回点眼法抽检了１１个鹿场２４５０余头成年鹿，其阳性率最高者为９９．８％，最低者为１．１％，     

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。     

结核分枝杆菌抗原的重组腺病毒载体的有效构建编码

以AdEasy腺病毒载体系统为基础,建立了一套简单有效构建编码结核分枝杆菌抗原的重组腺病毒载体的方法.依此方法挑取的重组腺病毒质粒阳性率高达90%,线性化的重组腺病毒质粒转染293A细胞24 h后可观察到报告基因GFP的表达.     

Mange in pigs: A survey

Extract

Mange in pigs caused by Sarcoptes scabiei var. suis is reported to be common in New Zealand, but, to the writer's knowledge, no investigation has been made of the number of infested farms in any area.     

Examination and control of lameness in dairy herds

Extract

Lameness is a significant problem in dairy herds in New Zealand. It affects production and causes cows to lose weight. If antibiotics are used, milk has to be withheld from the vat. Cows may go into extended anoestrus and some which fail to conceive have to be culled. Even in its mildest form lameness is an inconvenience to the normal management of the herd.     

The effect of fertility control on the transmission of bovine tuberculosis in wild brushtail possums

AIM: To determine the effect of fertility control on the rate of transmission of bovine tuberculosis (Tb), caused by Mycobacterium bovis, in brushtail possums (Trichosurus vulpecula).

METHODS: At two study sites with a history of Tb infection in the resident possum population, a sample of adult male and female possums (n=50), determined by palpation to be Tb-free, was surgically sterilised by gonadectomy. A sample of untreated Tb-free male and female possums (n=54) served as controls. Each study site was trapped every 2 months over a 3-year period, and the Tb status of the individuals in the trial assessed. At the conclusion of the trial, all remaining experimental individuals were killed, necropsied and examined for characteristic lesions typical of Tb. The rate of transmission of Tb infection was estimated using the incidence of tuberculous cases in the experimental animals and comparing it between sites, sex and sterilisation treatment.

RESULTS: Sterilisation by gonadectomy resulted in a reduction in the rate of transmission of Tb in male possums by 53%, but a corresponding increase in sterilised females for reasons that are still unclear. However, this interaction was statistically weak (p=0.10). When the sexes were combined, there was no overall effect of sterilisation on the rate of transmission of Tb (p=0.43). Sterility treatment notwithstanding, there was a highly significant difference in the rate of transmission between the sexes (p=0.01), being almost one order of magnitude higher in untreated males compared with untreated females.

CONCLUSIONS: Although lacking strong statistical support, these results suggest that fertility control that targets endocrine control of fertility may provide some additional benefits for disease control over that achieved by reductions in population density, by reducing the rate of transmission of M. bovis in male possums. However, additional studies are needed to confirm the validity of these results. The large difference in the rate of transmission of M. bovis in untreated males compared with untreated females suggests that transmission of Tb among males may be an important driver of the dynamics of Tb infection in possums.     

Oral vaccination of brushtail possums with BCG: Investigation into factors that may influence vaccine efficacy and determination of duration of protection

AIMS: To determine factors that may influence the efficacy of an oral pelleted vaccine containing Mycobacterium bovis bacille Calmette-Guérin (BCG) to induce protection of brushtail possums against tuberculosis. To determine the duration of protective immunity following oral administration of BCG.

METHODS: In Study 1, a group of possums (n=7) was immunised by feeding 10 pellets containing dead Pasteur BCG, followed 15 weeks later with a single pellet of live Pasteur BCG. At that time, four other groups of possums (n=7 per group) were given a single pellet of live Pasteur BCG orally, a single pellet of live Danish BCG orally, 10 pellets of live Pasteur BCG orally, or a subcutaneous injection of live Pasteur BCG. For the oral pelleted vaccines, BCG was formulated into a lipid matrix, and each pellet contained approximately 10⁷ colony forming units (cfu) of BCG, while the vaccine injected subcutaneously contained 10⁶ cfu of BCG. A sixth, non-vaccinated, group (n=7) served as a control. All possums were challenged by the aerosol route with a low dose of virulent M. bovis 7 weeks after vaccination, and killed 7–8 weeks after challenge. Protection against challenge with M. bovis was assessed from pathological and bacteriological findings.

In Study 2, lipid-formulated live Danish BCG was administered orally to three groups of possums (10–11 per group), and these possums were challenged with virulent M. bovis 8, 29 or 54 weeks later. The possums were killed 7 weeks after challenge, to assess protection in comparison to a non-vaccinated group.

RESULTS: The results from Study 1 showed that vaccine efficacy was not adversely affected by feeding dead BCG prior to live BCG. Feeding 10 vaccine pellets induced a level of protection similar to feeding a single pellet. Protection was similar when feeding possums a single pellet containing the Pasteur or Danish strains of BCG. All vaccinated groups had significantly reduced pathological changes or bacterial counts when compared to the non-vaccinated group. In Study 2, oral administration of Danish BCG induced protection against challenge with M. bovis, which persisted for at least 54 weeks after vaccination. Some protection was observed in possums challenged 54 weeks after vaccination, but this protection was significantly less than that observed in groups vaccinated 29 or 8 weeks prior to challenge. There was a strong relationship between the proportion of animals producing positive lymphocyte proliferation responses to M. bovis antigens and protection against challenge with M. bovis.

CONCLUSIONS: Factors considered potentially capable of interfering with vaccination, including feeding dead BCG to possums prior to feeding live BCG, feeding multiple doses of BCG at one time, and changing strains of BCG, were shown not to interfere with the acquisition of protective immune responses in possums. Protection against tuberculosis was undiminished up to 29 weeks after vaccination with BCG administered orally. It is concluded that vaccination of possums by feeding pellets containing BCG is a robust and efficient approach to enhance the resistance of these animals to tuberculosis.     

鹿结核病流行株与卡介苗差异基因原核表达质粒的构建及表达

以吉林农业大学经济动物疾病实验室保存的鹿体内分离并鉴定的结核病流行株的基因组为模板,应用PCR方法从已构建的鹿结核病野毒株与卡介苗差异基因文库中扩增RD1区的Rv3872、Rv3873、Rv3874、Rv3875和Rv3878 5段目的基因,分别与p MD18-T Simple Vector连接,经PCR、酶切鉴定后的阳性重组克隆质粒与原核表达载体p GEX-4T-1连接并进行原核表达,采用谷胱甘肽琼脂糖亲和层析方法对目的蛋白进行纯化,通过SDS-PAGE和Western blot对表达产物的反应原性进行初步研究。基因测序结果显示差异基因与期望的序列一致,并在大肠杆菌中成功诱导表达,获得以可溶形式表达的目的蛋白,经SDS-PAGE分析目的蛋白的相对分子量与理论值相符,经Western blot鉴定纯化好的目的蛋白能与鹿结核病的阳性血清发生反应,具有良好的反应原性,为其在鹿结核病血清诊断学中的应用奠定了试验基础。