【作者】 杨其清；

【导师】 侯加法； 周金林；

【作者基本信息】 南京农业大学 ， 兽医， 2013， 博士

【摘要】 吉氏巴贝斯虫病是由寄生在犬红细胞内一种蜱传吉氏巴贝斯虫原虫所致,犬表现发热、贫血、血红蛋白尿和消瘦,甚至死亡,是我国最常见的犬梨形虫病(焦虫)的病原之一。吉氏巴贝斯虫病在我国多个省市均有报道,但其系统流行病学和临床病理特征缺乏研究；诊断主要依赖于血液涂片镜检,但这种方法对于红细胞染虫率低的非急性感染诊断困难,也不适合样本数量大的流行病学研究。因此,开发一种特异性及敏感性较高的血清学诊断试剂,对巴贝西斯虫流行病学调查及小动物临床诊断具有重要的意义。本研究建立了基于截短型重组抗原(tBgTRAP)的酶联免疫吸附试验(ELISA)检测技术,应用该方法对来自南京、上海、无锡、杭州和郑州等地区不同犬血样进行检测,得出犬吉氏巴贝斯虫病流行病学的初步数据。此外,通过61例临床吉氏巴贝斯虫感染犬血液学及生化指标的分析,研究其临床症状、诊断及其病理学特征,为临床诊断和治疗提供支持。1犬吉氏巴贝斯BgTRAP蛋白重组表达及其ELISA诊断应用价值吉氏巴贝斯虫BgTRAP蛋白是一个重要的诊断抗原候选分子。为了基于该抗原建立实用的血清学诊断方法,选取C-末端跨膜区前,包含TSP功能区和抗原区的氨基酸所编码基因片段,重组表达了一个可溶性截短型BgTRAP抗原,解决了完整蛋白重组表达纯化的困难。免疫荧光试验表明,截短型抗原具有良好的抗原性。纯化的截短型抗原作为酶联免疫试验诊断抗原可清晰地区分阴性及阳性犬血清,并与其他病原感染无交叉反应,显示其诊断的特异性。犬感染吉氏巴贝斯虫系列血清检测表明,该抗原可检测早期感染(4d)和感染200d以后的样本,显示其适合感染早期和慢性持续感染的诊断。结果表明：重组BgTRAP截短型抗原可作为一种诊断制剂检测犬吉氏巴贝斯虫抗体。2南京、无锡、上海及杭州地区犬吉氏巴贝斯虫病血清学调查应用基于BgTRAP截短型抗原的ELISA方法,对来自南京地区、无锡地区、上海地区及杭州地区2011～2012年就诊犬的总计705份血样进行犬吉氏巴贝斯虫病的血清学调查。结果显示南京地区、无锡地区、上海地区及杭州地区门诊犬吉氏巴贝斯虫感染的血清学阳性率为分别为20.09%,3.03%,3.37%和0.00%,说明吉氏巴贝斯虫感染存在明显的地区差异。进一步对南京地区犬样本进行详细分析,我们发现巴贝西虫感染的临床发病率为仅为8.33%(17/204),显示本病存在一定的隐性感染率或漏诊率；工作犬的吉氏巴贝斯虫阳性率达47.05%,明显高于宠物犬20.09%的阳性率；在年龄上,阳性犬在2-6岁占70.73%,说明犬的活动性与感染有相关性；感染季节上,春、夏、秋、冬的阳性率分别为23.07%、16%、34.09%和10.34%,表明春秋二季是主要感染季节,这点与媒介硬蜱活动规律一致。使用tBgTRAP ELASA检测表明,南京地区吉氏巴贝斯虫阳性率明显高于上海、无锡地区；杭州地区未检测到阳性样本。南京地区工作犬吉氏巴贝斯虫阳性率明显高于普通犬,说明该寄生虫对工作犬危害高于普通犬。因此,应加强对宠物犬尤其工作犬的蜱虫预防和环境控制,降低巴贝斯虫对犬的危害,进而控制本病在南京地区的季节性流行。3郑州地区比特斗犬吉氏巴贝斯虫病的血清学调查应用基于BgTRAP截短型抗原的ELISA方法,检测了郑州地区三个养殖场共计171只比特犬样本,总的血清阳性率是38%(65/171),但阳性犬发病率仅为24.61%(16/65),表明与普通犬感染吉氏巴贝西类似,多数为亚临床感染。感染后康复犬复发率为43%(7/16)。1岁以下阳性率为4.6%(3/65),1-5岁比特犬阳性率为92.3%(60/65)；5岁以上阳性率为3.10%(2/65)。阳性犬中公犬占76.9%(50/65),母犬占23.1%(15/65)A犬场为大型比特犬养殖场,血清阳性率为26.72%(35/131),阳性犬发病率为17%(6/35),复发率为33.33%(2/6)。B、C犬场为斗犬爱好者养殖户,吉氏巴贝斯阳性率分别为85.7%(12/14)和69.2%(18/26),B、C犬场巴贝斯阳性犬发病率分别为21.42%(3/14)和22.22%(6/26),显示吉氏巴贝斯有较高的隐性感染率；B、C犬场吉氏巴贝斯复发率为66.66(2/3)和复发率为50%(3/6),表明部分病例不能根治,治疗方法还有待改进。96.9%(63/65)的阳性犬有咬斗史,98%(64/65)阳性犬有被咬伤疤痕,表明其主要传播途径为血液传播。吉氏巴贝斯虫在比特斗犬品种中广泛传播,阳性率、发病率及复发率都很高,说明吉氏巴贝斯虫在比特犬群中隐性感染或康复带虫率较高。其主要传播途径为咬伤后的血液传播,其他感染途径可能为经胎盘垂直传播。比特犬作为储虫宿主及其种群成为吉氏巴贝斯虫的储虫库在传播巴贝斯虫路径中扮演重要角色461个吉氏巴贝斯虫病例的临床特征、临床病理学特征及诊断分析对来自南京市三家动物医院的61例确诊为吉氏巴贝西斯虫感染病例的临床特征及血液学分析结果为：体温分布不一。体温升高的占48.78%(20/41),体温正常或偏低的占51.22%(21/41)。食欲减退的占100%,精神沉郁至嗜睡的占100%,黏膜苍白的占87.8%(37/41),重度到重度苍白的占80.48%(33/41)。黄疸表现为轻度黄疸,比例为39.02%(16/41)；脾肿大的95%(39/41),中度到重度肿大的为75(75%)。贫血的分布为：轻度贫血的(37<HCT>25)为9.7%、中度贫血的(25<HCT>15)为34%及严重贫血的(HCT<15)分别为9.7%、34%及46%。总的贫血比例为90.2%(37/41)。MCHC低于正常范围下限的为73%(30/41)。95%(39/41)的病例血小板数降低。血小板轻度降低(175<PLT>100)、血小板中度降低(100<PLT>50)及血小板严重降低(PLT<50)分别为21.95%(9/41),21.95%(9/41)及51.21%(21/41)。对30个吉氏巴贝西斯虫病血涂片的染虫率的统计结果为：染虫率为0.5%-0.8%的为占23.33%(7/30),染虫率1%--5%的占66.66%(20/30),染虫率5%--30%为占10%(3/30)。球形红细胞,多染红细胞,低染性大红细胞为血液细胞学主要特征。对20例确诊为吉氏巴贝西斯虫感染病例生化指标的结果进分析显示：65%(13/20)的病例总胆红素(T-BIL)升高,30%(6/20)的病例总胆红素超过正常范围的50%,主要与溶血有关；有45%病犬(9/20)ALT升高,有25%(5/20)ALT/AST/ALP值同时升高,表明1/5病例肝细胞受损；有20%(4/20)CRE,BUN同时高于正常范围,表明1/4病例出现氮血症,可能源自肾功能不全；有25%(5/20)例肌酸激酶CK升高,其中4例伴随LDH升高,提示吉氏巴贝西斯虫病可能使横纹肌受损。有75%(15/20)样本的血清球蛋白(GLOB)升高,有40%(8/20)例犬的血糖下降。本文最后从流行病学、病史、临床症状、血液学及临床生化及血清学诊断入手对吉氏巴贝西斯虫疑似病例总结鉴别诊断方法。更多还原

【Abstract】 Babesia gibsoni is a tick-borne protozoan parasite that lives in the erythrocyte, which causes babesiosis in domestic dogs and wild Canidae. Clinical manifestations are characterized by fever, anemia, hemoglobinuria, and marked emaciation as well as sometime death. Babesia gibsoni is one of the most important pathogen caused piroplasmosis in China. Dog babesiosis caused by Babesia gibsoni was reported widely in China, but the epidemiology and clinical characteristic are short of clear researches. The diagnostic method of B. gibsoni infection is practiced mainly by microscopic examination of Giemsa-stained thin blood smear films. However, it is difficult to detect B. gibsoni organisms in unapparent or chronic infections because of low levels of parasitemia, and it is not fit for epidemiology investigation because of large amount of samples. Therefore, it is desired for development of high specific and sensitive method of serological diagnosis of B. gibsoni infection, which will contribute to the epidemiology survey and clinical diagnosis. This paper will report the researches on establishing an enzyme-linked immunosorbent assay (ELISA) based on the recombinant truncated BgTRAP protein for serological diagnosis of B. gibsoni infection, and also on conducting a survey using a large number of field dog samples in Nanjing, Shanghai, Wuxi, Hangzhou and Zhengzhou for epidemiology investigation. Furthermore, we have observed and analyzed clinical signalment, blood and biochemistry character from the41dog samples infected with B. gibsoni, which will provide the important reference for clinical diagnosis and treatment for this disease.1. Expression of truncated Babesia gibsoni thrombospondin-related adhesive proteins in Escherichia coli and evaluation of their diagnostic potential by enzyme-linked immunosorbent assay (ELISA)BgTRAP from Babesia gibsoni is an important candidate for the development of a diagnostic reagent for canine babesiosis. In order to establish an effective diagnostic method for practical use based on the BgTRAP protein, the gene encoding truncated BgTRAP containing the TSP region and antigenic area ahead of transmembrane region was cloned and expressed in soluble form recombinant protein,which was easy for purification comparing with intact recombinant. The truncated protein showed a strong antigenicity by indirect fluorescent-antibody test. The recombinant truncated BgTRAP was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate between B. gibsoni-infected dog serum and B. canis-infected dog serum or non-infected dog serum. Furthermore, the antibody response against the recombinant protein was maintained from early time (4day after infection) until the chronic stage of infection (200day after infection) in dogs experimentally infected with B. gibsoni. These results demonstrate that the recombinant truncated BgTRAP protein might be a useful diagnostic reagent for detection of antibodies to B. gibsoni in dogs.2. Serological investigation on prevalence of Babesia gibsoni infection in clinical canine cases in Nanjing、Shanghai、Wuxi and Hangzhou of ChinaA total of705Sera collected from dogs in small animal clinic in Nanjing、Shanghai Wuxi and Hangzhou of China in2011-2012were tested for antibodies to B. gibsoni by ELISAs based on recombinant tBgTRAP.20.09%,3.03%,3.37%and0%of the tested samples were positive by ELISA in Nanjing、Shanghai、 Wuxi and Hangzhou respectively, it shows the obvious difference of B. gibsoni infection in dogs among different areas. Moreover, after analyzing all blood samples in Nanjing area, we found the clinical morbidity of dog is8.33%, which indicates most of dogs were asymptomatic chronic infections; in the tested dogs, work dogs is higher positive rate(47.05%) than that of pet dogs (20.09%);70.73%the age of positive dogs is2-6years old, which shows the activity is relative to B. gibsoni infection; the seasonal variation of dog infection rate is clear,23.07%,16%,34.09%and10.34%in spring, summer, autumn and winter respectively, the higher infection rate in spring and autumn seasons was also found in other investigated area.3. Serological investigation on prevalence of Babesia gibsoni infection in fighting dog samples in Zhengzhou of ChinaA total of171Sera collected from fighting dogs in in Zhengzhou of China were tested for antibodies to B. gibsoni by ELISAs based recombinant tBgTRAP.38%of the tested samples were positive by ELISA, however, morbidity of positive dog is only24.61%, which indicates most of dogs were asymptomatic as in ordinary dogs. Recurernce rate of treated dogs is43%, which shows the present treatments on B. gibsoni existed limitation.92.3%the age of positive dogs is1-5years old, which shows the activity is relative to B. gibsoni infection.76.9%of positive dogs is male,98%of positive dogs have the fighting experience and kept the scar in body, which indicates the fighting dogs might be transmitted by dog to dog by fight wound route.4. A total of61dogs infected with B. gibsoni were observed and analysed for clinical signalment, hematology and biochemistry result.(1)Clinical finding and hematological result of41samples from61dog cases infected by Babesia gibsoni, which were collected from three veterinary hospitals in Nanjing showed that, body temperature varies from nomal range to hyperthemia, with48.78%hyperthermia and51.22%normal. Anorexia, depression or even lethargy existed in all cases. Percentages of cases showing pale mucous membranes and splenomegaly are87.8%and95%, respectively. Hemoglobinuria was seen in all cases. In general,90.2%cases showed anemia with9.7%mild anemia (37<HCT>25),34%moderate anemia (25<HCT>15) and46%severe anemia (25<HCT>15).73%cases’ MCHC were lower than normal range.95%cases presented thrombocytopenia, including21.95%slight decrease (175<PLT>100),21.95%moderate decrease (100<HCT>50) and51.21%severe decrease (PLT<50).(2) The ratio of parasitized erythrocyte from0.5to0.8%took up23.33%;1-5%was66.66%;5-30%was10%. erythrocyte morphology included spherocytosis, polychromatic erythrocytes, hypochromic erythrocyte.(3)Biochemistry characteristic:75%of cases have a high serum globulin; the increase of ALT was seen in45%cases(9/20), Elevation of ALT/AST/ALP simultaneously were seen in 20%cases (4/20), indicating that there were1/5cases having hepatocytes damage.20%cases(5/20) had higher CREA and BUN than normal range, which indicated there were1/4cases had azotemia, which might be caused by renal insufficiency. Raised CK were seen in25%(5/20) cases, among which there were4cases also had elevated LDH, indicating that Babesia gibsoni infection may cause skeletal muscle cell damage. Significant increase of serum globin covered75%cases, and40%(8/20) cases present decreased blood glucose.At last, for suspected Babesia gibsoni infected cases, differential diagnosis should not only depend on the epidemiology, clinical signalment, hematology, biochemistry, but more importantly on serology.更多还原