Establishment Of Babesia Gibsoni Detection Technique Based On Recombinant Expression Of Tbgtrap And Its Application On Epidemiological Investigation

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.