牛瑟氏泰勒虫18S rRNA基因的克隆及分子分类学分析

为分析牛瑟氏泰勒虫延边株18SrRNA基因序列,根据GenBank上已发表的牛瑟氏泰勒虫18SrRNA基因序列设计2对特异性引物,通过PCR扩增目的基因片段,将扩增产物连接到pMD18一T载体中,经酶切鉴定和PCR鉴定为阳性的重组质粒进行测序,对测序结果用DNAMAN软件时其与GenBmlk上8个虫株相关序列进行同源性比较,建立系统发育树。结果显示,牛瑟氏泰勒虫中国延边株的18SrRNA基因大小为1744bp,瑟氏泰勒虫延边株、兰州株、日本株以及水牛泰勒虫中国株彼此之间亲缘关系最近;瑟氏泰勒虫延边株与突变泰勒虫亲缘关系较远：     

The Suppressive Effects of Dexamethasone on the Development of Pulmonary Oedema in East Coast Fever

Tropical Animal Health and Production -     

Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and Babesia caballi infections, respectively. These primer sets specifically amplified DNA of the respective parasites. Both primer sets amplified T. equi and B. caballi up to 10(-6) dilution of 10-fold serially diluted samples. Furthermore, DNA extracted from blood collected from a horse experimentally infected with T. equi was amplified by a T. equi LAMP primer set from days 2 to 35 post-infection, demonstrating the high sensitivity of these primers. Of 55 samples collected from China, 81.8% and 56.3% were positively detected by LAMP for T. equi and B. caballi infections, respectively. In contrast, 91.8% and 45.9% of the 37 samples collected from South Africa were LAMP positive for T. equi and B. caballi, respectively. These results suggest that LAMP could be a potential diagnostic tool for epidemiological studies of equine piroplasmosis.     

Investigation of Molecular Etiology of Theileria equi Infection in Three Herds

To explore the prevalence of Theileria equi (T.equi) infection in horse in Guizhou province, the antibody level and 18S rRNA gene were detected from blood samples of Guizhou pony, Southwest horse and Yili horse using competitive ELISA and PCR methods.Giemsa-stained blood smear was prepared to observe T.equi in red blood cells.Intact protozoans of T.equi were observed in red blood cells of horses at Giemsa-stained slide smears with a detection rate of 12.5%.The 18S rRNA gene fragment of T.equi was detected in Guizhou pony, Southwest horse and Yili horse, and the consistent rates with the known nucleotide sequence were 97% to 100%.The PCR result indicated that the positive rates of T.equi in Guizhou pony (76.62%) and Yili horse (73.81%) were similar, which were higher than that in Southwest horse (33.33%).Furthermore, the antibody levels against T.equi in Guizhou pony (24.68%) and Southwest horse (12.12%) were lower than that in Yili horse (31.71%).A weak correlation between the antibody level and the blood physicochemical indexes was calculated from Guizhou pony and Southwest horse, including weak positive correlations with neutrophils numbers, gamma-glutamyl transferase and creatine kinase levels, and weak negative correlations with the numbers of red blood cell, white blood cell, platelet and lymphocyte and contents of hemoglobin.It suggested that a higher proportion of T.equi infection present in three herds.     

Investigation of bovine haemoplasmas and their association with anaemia in New Zealand cattle

CASE HISTORY AND CLINICAL FINDINGS: A dairy cow, from a herd in the Waikato region of New Zealand, was reported with regenerative anaemia on 12 September 2014. Testing of blood from the animal using PCR assays for Theileria orientalis produced a negative result for both Chitose and Ikeda types.

LABORATORY FINDINGS: Using PCR and DNA sequencing, blood from the cow was positive for Candidatus Mycoplasma haemobos. Further testing of another 12 animals from the case herd, 27 days after the affected cow was first reported, showed 11 animals were positive for Candidatus M. haemobos or Mycoplasma wenyonii in the PCR. None of these cattle were clinically anaemic or positive for T. orientalis Ikeda type using PCR.

A convenience sample of 47 blood samples from cattle throughout New Zealand, submitted to the Investigation and Diagnostic Centre (Ministry for Primary Industries) for surveillance testing for T. orientalis Ikeda, was selected for further testing for bovine haemoplasmas. Of these samples, 6/47 (13%) and 13/47(28%) were positive for M. wenyonii and Candidatus M. haemobos, respectively. There was no difference in the proportion of samples positive for the bovine haemaplasmas between cattle with anaemia that were negative for T. orientalis (6/20, 33%), or without anaemia or T. orientalis (10/18, 56%), or from cattle herds experiencing anaemia and infection with T. orientalis Ikeda type (3/9, 33%).

DIAGNOSIS: Bovine haemoplasmosis.

CLINICAL RELEVANCE: The presence of bovine haemoplasmas in blood does not establish causality for anaemia in cattle. Diagnosis of anaemia associated with haemoplasmosis would require exclusion of other causes of regenerative anaemia and an association of the agent with anaemia in affected cattle herds. The data collected in this study did not provide evidence that bovine haemoplasmas were associated with a large number of outbreaks of anaemia in cattle in New Zealand.     

Concentrations of buparvaquone in milk and tissue of dairy cows

AIM: To determine the concentration of the anti-theilerial drug buparvaquone in the milk and tissue of dairy cattle following treatment with two different formulations, and to assess the effect of clinical theileriosis on the concentration of buparvaquone in milk.

METHODS: Healthy lactating dairy cows (n=25) were injected once (Day 0) I/M with 2.5?mg/kg of one of two formulations of buparvaquone (Butalex; n=12 or Bupaject; n=13). Milk samples were collected from all cows daily until Day 35. Five cows were slaughtered on each of Days 56, 119, 147, 203 and 328, and samples of liver, muscle and injection site tissue collected. Milk samples were also collected from cows (n=14) clinically affected with theileriosis for up to 21 days after treatment with buparvaquone. Milk and tissue samples were analysed by liquid chromatography-mass spectrometry; limits of detection (LOD) were 0.00018?mg/kg for muscle and 0.00023?mg/L for milk. Concentrations of buparvaquone in milk and tissues were log₁₀-transformed for analysis using multivariate models.

RESULTS: In healthy cows, concentrations of buparvaquone in milk declined with time post-treatment (p<0.001), but were above the LOD in 11 of 25 cows at Day 35. Concentration in milk was higher one day after treatment in cows treated with Butalex than in cows treated with Bupaject, but not different thereafter (p=0.007). Concentrations of buparvaquone in muscle were below the LOD for four of five animals at Day 119 and for all animals by Day 147, but were above the LOD at the injection site of one cow, and in the liver of three cows at Day 328. Tissue concentrations did not differ with formulation nor was there a formulation by time interaction (p>0.3).

Concentrations of buparvaquone in the milk of clinically affected animals were not different from those of healthy animals at 1 and 21 days post-treatment (p=0.72). Between 21 and 25 days post-treatment concentrations were below the LOD in 9/14 milk samples from clinically affected cows.

CONCLUSIONS: Detectable concentrations of buparvaquone were found in the milk of some cows for at least 35 days and in the liver and injection site of some cows until at least 328 days after injection. There were no biologically meaningful differences in milk or tissue concentrations between the formulations, or in the milk concentrations for cows that were clinically affected compared with those that were healthy at the time of treatment.     

新疆牛环形泰勒虫TamsⅠ基因原核表达条件的优化及蛋白纯化

为了提高新疆牛环形泰勒虫TamsⅠ基因在大肠杆菌中的表达量,研究了温度、诱导时间以及诱导剂（IPTG）浓度等不同条件对TamsⅠ融合蛋白表达量的影响。试验结果表明,大肠杆菌菌株BL21（DE3）在37℃培养2．5h后,使用终浓度为2mmol／L的IPTG在25℃振荡诱导培养8h时,GST—TamsⅠ融合蛋白表达量最高,达到2．5rmg／mL。用GST-Sepharose 4B亲和层析柱对GST—TamsⅠ融合蛋白进行纯化,SDS-PAGE检测表明GST-TamsⅠ融合蛋白大小约为56kl）,与预计的分子量大小一致。TamsⅠ基因的优化表达为牛环形泰勒虫病的分子免疫学诊断和哑单位疫苗的研制奠定了基础。     

牛含环形泰勒焦虫血源细胞的生物反应器培养条件的探索

利用生物反应器对牛含环形泰勒焦虫的血源细胞进行了培养条件的探索。结果证明,采用２５Ｌ培养罐、６片叶轮搅伴、转速１２０ｒ／ｍｉｎ、装量１２００ｍｌ、罐顶留一通气孔用沙布棉花封口与外界进行气体交换为最优培养条件。在此状态下,我们对原工艺要求的２０％新生牛血清用量减少至７％,水解乳蛋白也由国产产品替代了部分进口的ＤＩＦＣＯ公司产品,细胞接种密度４５×１０５ｃｅｌ／ｍｌ经３６小时培养后平均达２４×１０６ｃｅｌ／ｍｌ,收获细胞数与原工艺基本相同,但成本费用大幅下降。     

牛瑟氏泰勒虫P23基因的克隆与生物信息学分析

[目的]为研制牛瑟氏泰勒虫病基因工程苗和诊断试剂盒提供依据。[方法]通过PCR方法从牛瑟氏泰勒虫基因组DNA中克隆了1个P23基因,连接到pGEM-T-Easy载体中。运用生物信息学方法对该基因进行分析。[结果]P23基因全长为684 bp,包含1个长672 bp的开放阅读框,编码223个氨基酸,相对分子量是25.886 kD,等电点pI为9.22,包括1段19个氨基酸组成的信号肽和2段跨膜区。该序列在GenBank上的注册号为EU573168,与瑟氏泰勒虫Chitose型(D84446)和Ikeda型(D84447)的同源性分别为99%、90%。[结论]P23基因编码的蛋白有较好稳定性和免疫原性,可作为制备牛瑟氏泰勒虫基因疫苗的候补抗原。