吉氏巴贝斯虫单克隆抗体制备的研究

应用淋巴细胞杂交瘤技术制备杂交瘤细胞株 ,经间接萤光抗体法筛选和克隆 ,获得了 5株能稳定分泌吉氏巴贝斯虫特异性抗体的杂交瘤细胞株 ,分别命名为C3B5、M8B7、E9C5、G6 D8、H2 A7。经过鉴定 ,这 5株杂交瘤细胞分泌的单克隆抗体亚类及相对分子质量分别为IgG2b,1 8× 1 0 4 ;IgG2a,1 8× 1 0 4 ;IgG1 ,1 8× 1 0 4 ;IgM ,3 2× 1 0 4 ;IgM ,1 8× 1 0 4 。腹水效价为 1∶1 0 4 ～ 1∶1 0 5。其中E9C5杂交瘤细胞株分泌的单克隆抗体是一种保护性抗体 ,经对实验感染吉氏巴贝斯虫小鼠体内虫体的杀虫试验证明具有较强的杀灭作用。     

吉氏巴贝斯虫cDNA探针的制备及杂交试验

本实验将－６．６ｋｂ的吉氏巴贝斯虫ｃＤＮＡ片段以光照活化法标记光敏生物素，制备成光敏生物素探针。与吉氏巴贝斯虫基因组ＤＮＡ、伊氏锥虫基因组ＤＮＡ、犬白细胞ＤＮＡ的斑点杂交试验表明，该探针可与０．００１ｎｇ以上量的吉氏巴贝斯虫ＤＮＡ杂交，而不与任何浓度的伊氏锥虫ＤＮＡ和犬ＤＮＡ杂交，具有很高的敏感性和特异性。     

Babesia gibsoni infection in a dog from indiana

Abstract: A 10-year-old spayed female mixed-breed dog was presented to the Purdue University Veterinary Teaching Hospital (PUVTH) with complaints of persistent anemia with occasional exacerbations, anorexia, and lethargy. The dog had been presented to the referring veterinarian 2 months prior with multiple bite wounds received during a fight with 3 Pit Bull Terriers. The dog was discharged after the wounds were cleaned and surgically closed. Upon admission to the PUVTH, blood was collected for a complete blood count and biochemical analysis. Microscopic examination of peripheral blood smears revealed intraerythrocytic protozoal parasites consistent with Babesia gibsoni . Molecular analysis confirmed that the organism was B gibsoni and that its 18S ribosomal RNA sequence was identical to that of other B gibsoni isolates from Oklahoma, North Carolina, and Okinawa, Japan. Hematologic changes included moderately severe, regenerative, macrocytic, normochromic anemia, with poikilocytosis, polychromasia, anisocytosis, and a marked increase in nucleated RBCs. Biochemical changes included increased serum alanine aminotransferase, alkaline phosphatase, and gamma glutamyl-transferase activities. The dog was treated with imidocarb, but despite initial clinical improvement, the dog died 2 weeks after the first dose. A necropsy was not performed. The infection in this dog is the first reported case of B gibsoni infection in Indiana. Because of the widespread geographical distribution of the organism, veterinarians and veterinary clinical pathologists throughout the United States should carefully examine Romanowsky-stained blood smears from patients with acute hemolytic anemia for small intraerythrocytic babesial parasites.     

吉氏巴贝斯虫mRNA的磁性分离及cDNA合成

从感染吉氏巴贝斯虫的摘脾犬的红细胞内提纯虫体,用异硫氰酸胍法提取虫体总RNA,再以Poly(A)~ mRNA磁性分离系统提取mRNA.最后以NotⅠPrimer-Adaptor为引物反转录出cDNA片段.琼脂糖凝胶电泳显示,所合成的cDNA分子量范围由100到6500bP,且绝大部分大于500bP.     

一例犬吉氏巴贝斯虫感染引起的免疫介导溶血性贫血的诊治

犬免疫介导溶血性贫血是一种与自身红细胞抗体有关的溶血性贫血,发病原因可分为原发性和继发性,论文主要介绍了一例由吉氏巴贝斯虫感染后继发的犬自身免疫溶血性贫血的诊断、治疗和预后。     

The Effect of Diminazene Aceturate on Spienectomized Dogs with Babesia gibsoni Infection

The effect of diminazene aceturate on splenectomized and nonspienectomized dogs with Babesia gibsoni (B. gibsoni) infection was investigated. In splenectomized dogs, the fissional and multiplicational stages of B. gibsoni were observed in peripheral blood films, and hemoglobinuria was frequently observed. These findings were different from previous reports and were not changed by administration of diminazene aceturate. It is clear that the intramuscular administration of diminazene aceturate at the dose of 3 mg/kg body weight for 3 days is not effective against B. gibsoni infection in splenectomized dogs.     

吉布森巴贝虫顶膜抗原-1基因的表达及表达产物的生物学活性

通过免疫筛选法，从构建的犬吉布森巴贝虫cDNA基因表达文库中筛选到1个cDNA克隆，此cDNA核苷酸全长2108bp，包含1个完整的开放阅读框，编码蛋白的分子质量约为62ku。经过分析，此蛋白（BgAMA-1）与其他巴贝虫属的顶膜抗原-1具有较高的同源性。为了研究其生物学活性，以此cDNA为模板，利用PCR扩增BgAMA-1的全基因，并将其克隆到pGEX-4T-3载体上，构建了原核表达载体pGEX-4T-3／BgAMA-1。通过Western—blotting对表达并纯化的重组蛋白的生物学活性进行分析。结果显示，构建的pGEX-4T-3／BgAMA-1能在大肠杆菌DH5口中表达，所获的重组抗原BgAMA-1在Western—blotting上仅与吉布森巴贝虫感染犬血清发生反应，证实其具有种特异性，在诊断方面可能有一定的应用价值。     

Further epidemiological survey for atovaquone resistant related gene of Babesia gibsoni in Japan during 2015–2018

A single-nucleotide polymorphism causing the replacement of methionine with isoleucine (M121I) in cytochrome b of Babesia gibsoni has been reported to reduce the susceptibility to atovaquone (ATV) in B. gibsoni infection. In our previous study, B. gibsoni with M121I was suggested to exist in nature. Thus, further examinations were performed. In total, 105 genomic DNA samples from B. gibsoni-infected dogs were collected from western (98 samples from 15 prefectures) and eastern areas (7 samples from 4 prefectures) in Japan. The M121I variant population was identified using allele-specific real-time PCR: it was then detected in nine samples (8.57%), which was higher than that in the previous study (4.11%). Although there are unclear points, such as the history of ATV usage, careful attention should be given to emerging ATV resistance.     

一例犬吉氏巴贝斯虫病的诊治

3岁雄性贵宾犬,体重3.20 kg,免疫完全,未绝育,从未进行过体内外驱虫,院内散养。主诉最近2周发现犬的精神状态渐差,进食量逐渐减少。为了对其进行诊治,采用体格检查、血液学检查以及影像学检查的方法进行诊断,并根据诊断结果进行治疗,结果表明,该犬感染吉氏巴贝斯虫且并发肝脏损伤,通过采取输血、驱虫等治疗措施,患犬的临床症状消失,镜检外周血虫体消失,治疗后未见复发。说明临床上可通过血液学检测对犬的吉氏巴贝斯虫感染进行确诊,且输血结合驱虫治疗可有效治疗该病。     

Epidemiological survey of Babesia gibsoni infection in dogs in Japan by enzyme-linked immunosorbent assay using B. gibsoni thrombospondin-related adhesive protein antigen

A nationwide epidemiological survey of Babesia gibsoni infection in non-fighting dogs was conducted using an improved ELISA with recombinant B. gibsoni thrombospondin-related adhesive protein (BgTRAP). A total of 1206 dogs from 27 prefectures were examined and 128 (10.6%) tested positive. In the eastern part of Japan, 39 dogs out of the 559 (7.0%) examined were positive, while 89 dogs out of 647 (13.8%) tested positive in the western part of Japan. Although the percentage of dogs that tested positive was significantly (p=0.0001) lower in the eastern part compared to the western part of Japan, overall these results indicate that B. gibsoni infection of dogs has a widespread geographic distribution throughout the country. A history of tick infestation was identified as a significant risk factor for B. gibsoni infection (p=0.0091), while sex (p=0.9411), age (p=0.0920) and breed (p=0.0549) of dogs were not statistically significant risk factors. These results indicate that tick infestation is the most dominant risk factor for B. gibsoni infection of non-fighting dogs in Japan and suggest that other B. gibsoni transmission routes, such as fighting and transplacental transmission, may be less important.     

Epidemiological Aspects of Babesia equi in Horses in Minas Gerais,Brazil

The prevalence of Babesia equi in two climatic regions of Minas Gerais state was determined using the indirect fluorescent antibody test (IFAT) with blood samples obtained from horses in two slaughterhouses. Of 399 samples, 241 (60.4%) showed a positive reaction. Anti-B. equi antibody was detected in every county studied, the prevalence being 59.7% for horses in the area where the temperature rises above 18°C in winter and 61.4% in the area where it remains below 18°C, indicating that climatic variation has no substantial effect on the prevalence of the infection in Brazil. Blood samples collected from all 95 horses on a ranch in the state of Minas Gerais, Brazil, on which clinical babesiosis had never been reported, were subjected to the IFAT. Anti-B. equi antibodies were detected in horses of all ages, but with a significantly lower prevalence in animals less than 6 months old.     

Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5''-nucleotidase subclass I and purine-specific 5''-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5''-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.     

Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs

A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.     

Identification and phylogenetic analysis of Babesia parasites in domestic dogs in Nigeria

The present study examined the presence of Babesia parasites in 104 domestic dogs in Nigeria. Sequentially, Babesia parasites infecting domestic dogs underwent genetic and phylogenetic analyses. The results of nested PCR based on the Piroplasmida 18S rRNA gene illustrated that 13.5% (14/104) of the samples were positive. The obtained positive samples determined the nucleotide sequences of the 18S rRNA genes. In the genetic and phylogenetic analyses, four of five nucleotide sequences were similar to Babesia canis rossi, and one sample exhibited a close similarity to a Babesia sp. isolated from a raccoon in Hokkaido, Japan. The present study revealed the widespread presence of B. canis rossi among domestic dogs in Nigeria.     

Incidence of canine Babesia gibsoni infection and subclinical infection among Tosa dogs in Aomori Prefecture, Japan

To identify the incidence of Babesia gibsoni (B. gibsoni) in Aomori Prefecture, northeastern Japan, dogs with acute B. gibsoni infection were investigated at the Animal Teaching Hospital, Kitasato University, between April 2002 and March 2003. Eighteen dogs with acute B. gibsoni infection were recognized; they were all male dogs of the fighting dog breed Tosa. Their platelet counts were below normal and their packed cell volumes (PCVs) were at various levels. We collected blood samples from 141 Tosa dogs from Aomori Prefecture and used polymerase chain reaction assay to investigate the incidence of subclinical B. gibsoni infection. We also looked into the serological abnormalities associated with thrombocytopenia or anemia in subclinical infection. Forty-one of 87 dogs (47.1%) with histories of dog fighting, and one dog of 54 without a history of dog fighting were positive for B. gibsoni; that is, 42 of 141 dogs (29.8%) showed a positive result. The mean platelet counts of dogs with subclinical infection were significantly lower and levels of anti-platelet IgG were significantly higher than levels for dogs without infection. Anti-erythrocyte membrane IgG levels were significantly higher in dogs with subclinical infections, although mean PCVs were not significantly different. Tosa dogs from Aomori Prefecture, Japan, were highly infected with B. gibsoni subclinically and this pathogen might be successfully transmitted during dog fighting. Dogs with subclinical infections were at risk of chronic thrombocytopenia, which may be due to autoimmune mechanisms.