| The bovine piroplasmosis is a tick-born hemoparasitic disease which mainly caused by Babesia bovis, Babesia bigemina and Theilaria annulata. Many kinds of animals can be infected, especially in ruminants, sometime in man. The common clinical symptoms are fever, anaemia, haemoglobinuria, even death in severe cases. The bovine piroplasmosis can be divided into acute and chronic infections, according to the course of disease, and can also be grouped into clinical, sub-clinic and persistent infections by their infection types. The traditional detection and identification of piroplasms depends mainly on microscope, especially for the acute and clinical infection, but is hard for the sub-clinical and persistent infected cases, because the number of hemoparasites in the peripheral blood is too low to detect and easily to make misdiagnosis. The sub-clinical, chronic and persistent infected cases, even carriers can be detected by serological methods, such as complement fixation test (CFT), indirect hemagglutination test (IHA), latex agglutination test (LAT), indirect fluorescent antibody test (IFAT), and enzyme-linked immunosorbent assay (ELISA), etc. The crude, natural proteins as diagnostic antigens are used in the most of existing serological tests for bovine piroplasmosis which are hard to eliminate the cross reactions among the species of piroplasms. The tests of molecular biology, such as DNA probes, rRNA probe and PCR have highly sensitive and specific tools for diagnosis of bovine piroplasmosis, but still hard to use as routine method, because vitriol conditions, such as irradiation, professional personnel and special laboratory materials and equipment, are unsuitable for use in the field. Therefore, it is an important turning point to improve the specificity of serological tests for detection of specific antibodies against bovine piroplasmas. Recently years, some specific antigens have been found, such as merozoite surface antigen-2c (MSA-2c) in Babesia bovis, heat shock protein20(HSP20) in Babesia bigemina, and merozoite/piroplasm surface antigen1(Tams1) in Theileria annulata, etc. The common features for the antigens are highly conserved and immunogenic. Some scientists postulates that they may be not only the useful diagnostic candidates for detection of bovine piroplasmosis, but also the useful subunit or NDA vaccine candidates for improved control of bovine piroplasmosis. At present, the reports of the same or relative studies have not been found in China.The purpose of this study are:(1) to clone and express the MSA-2c, HSP20and Tams1and to obtain three fusion proteins by the optimal conditions for expression of these genes;(2) to develop the improved indirect enzyme-linked immunosorbent assays (iELISAs) using recombinant proteins as diagnostic antigens;(3) to perform the epidemiologic surveys of bovine piroplasmosis in Xinjiang;(4) to obtain the firsthand information for development of prevent and control program against bovine piroplasmosis in the future. The results from this study are as following:1. Three genes MSA-2c, HSP20and Tams1were cloned from extracted genome DNA in bovine blood samples infected with hemoparasite in Xinjiang by PCR with specific designed primers, respectively, and HSP20exon gene was loned from extracted total RNA in bovine blood samples infected with hemoparasites by RT-PCR with specific designed primers. The sequence analysis was shown:(1) the total length of MSA-2c gene was798kb, and had an intact open reading frame encoding281amino acids. It shared99.90%nucleotide homology with that of type strain, Texas AY052538, and also shared97.7%nucleotide homology with that of stains isolated from other different countries and regions.(2) The total lengths of HSP20gene and HSP20exon gene were699kb and534kb, respectively, and they shared99.43%and99.63%nucleotide homology, respectively, with those of the Mexico type strain.(3) The total length of Tamsl gene was846kb, and had an intact open reading frame encoding281amino acids. It shared99.36%-99.80%nucleotide homology with that published in GenBank. Analysis of phylogenetic tree was shown that it was closely related to others isolated from India, Mauritania, Turkey and Tunisia. Analysis of antigen determinants was shown that it had13putative cross-antigen determinants.The results above mentioned suggested these genes MSA-2c for B. bovis, HSP20/HSP20(exon) for B. bigemina and Tamsl for T. annulata were highly conserved.2. The cloned MSA-2c, HSP20(exon) and Tamsl genes were sub-cloned into expression vector pGEX-4T-2and the resultant constructs were respectively transformed into E. coli cells and induced by IPTG. SDS-PAGE and Western blot analysis were shown that fussion proteins of55kDa for MSA-2c gene,46kDa for HSP20(exon) and56kDa for Tamsl were expressed which could be recognized by bovine antisera against B. bovis, B. bigemina and T. annulata, respectively. These results were indicated the expressed fusion proteins by these genes possessed strong immunogenicity and reactionogenicity, which could be used as recombinant antigens to develop the improved serological tests for detection of specific antibodies against bovine piroplasmosis.3.Three improved indirect enzyme-linked immunosorbent assays (indirect ELISAs) for the detection of specific antibody against bovine piroplasmosis were developed using MSA-2c, HSP20(exon) and Tams1fusion proteins after optimizing its reacting conditions.(1) For indirect MSA-2c ELISA, the optimal concentration of coating recombinant antigen was2.5μg/ml and the optimal dilution of serum sample was1:40in the cross assay. The cutoff was chosen as an OD450>0.347for positive response. The variation coefficient of intra-batch and the inter-batch in the repeating tests was less than10%.(2) For indirect HSP20(exon) ELISA, The optimal concentration of coating recombinant antigen was5μg/ml and the optimal dilution of serum sample was1:40in the cross assay. The cutoff was chosen as an OD450>0.292for positive response. The variation coefficient of intra-batch and the inter-batch in the repeating tests was less than10%.(3) For indirect Tams1ELISA, the optimal concentration of coating antigen was10μg/ml and the optimal dilution of serum sample was1:80in the cross assay. The cutoff was chosen as an OD450≥0.282for positive response. The variation coefficient of intra-batch and the inter-batch in the repeating tests was less than15%.The results of specificity for the three indirect ELISAs were shown that (1) positive serum samples for B. bovis identified by Npcr were still detected positive in the indirect MSA2c ELISA, but not from the sera of B. bigemina, T. annulata and B. orientalis, and negative control sera.(2) Identified positive serum samples for B. bigemina were detected positive in the indirect HSP20(exon) ELISA, but not from the sera of B. bovis, T. annulata and B. orientalis, and negative control sera.(3) Identified positive serum samples for T. annulata were detected positive in the indirect Tamsl ELISA, not for sera of B. bigemina, B. bovis and B. orientalis, and negative control sera. The concordances of identified positive serum samples for bovine piroplasmosis in indirect MSA-2c, HSP20(exon) and Tamsl ELISAs were96%(14/16, n=50),96%(9/11, n=50) and96%(21/23, n=50), respectively, comparing with those by nested-PCR. The results above mentioned were shown three improved indirect ELISAs were highly sensitive and specific for the diagnosis and identification of bovine piroplsmas, which could be useful tools of epidemiological survey for detection of bovine piroplasmosis in large scale. 4. The epidemiologic survey for detection of bovine piroplasmosis in Xinjiang by the three improved indirect ELISAs was performed in this study. The results were shown that (1) Bovine babesiosis caused by B. bovis was presented in Xinjiang which was first time to be confirmed by the serological test. The prevalence for B. bovis from the tested cattle in2006,2007and2008were2.25%(7/278),3.31%(32/532) and5.28%(43/530), respectively. The rate of B. bovis infection was increased to28%(14/50) in some endemic regions where the disease occurred in2008. The number of infected prefectures in Xinjiang was also increased from2in2006to9in2008.(2) Bovine babesiosis caused by B. bigemina was also presented in Xinjiang, which was first time to be confirmed by the serological test. The infection rates for B. bigemina in2006,2007and2008were5.40%(11/278),4.70%(25/532) and7.17%(53/530), respectively. The infection rate was increased to30%(15/50) in some endemic regions where the disease occurred in2008. The number of infected prefectures in Xinjiang was also increased from8in2006to13in2008.(3) Bovine theileriosis caused by T. annulata was still the main hemoparasitic disease in cattle of Xinjiang. The infection rates in2006,2007and2008were11.15%(31/278),11.47%(61/532) and11.51%(61/530), respectively. The infection rate was increased to24%(15/50) in some regions where the disease occurred in2008.(4)280positive serum examples for bovine piroplasmosis were detected from1340sera collected from14prefectures in Xinjiang during2006to2008. The total infection rate reached to20.90%(280/1340). of those, the infection rates of bovine piroplasmosis caused by B. bovis, B. bigemina and T. annulata alone were8.92%(23/280),15.71%(44/280) and43.92%(123/280), respectively, during that time. The rate of mix infections among them was15.35%(43/280) which included some triple infections. The infection rate was unexpectedly increased to74%(37/50) in some endemic regions where the disease occurred in2008. The mixed infections reached to35.15%(13/50) in that areas. These results were indicated the category of bovine piroplasmosis was increased from1kind of hemoparasitic disease caused by T. annulata in past years to3kinds of ones caused by T. annulata, B. bigemina and B. bovis, respectively. The features of bovine piroplasmosis in Xinjiang were that rate of single infection and mixed infections were increased year by year, which made epidemic situations more complicated. It was more dangerous for animal and human health and a question which could be disregarded.This is a first time to perform the epidemiological survey in large-scale for detection the specific antibodies against bovine piroplasmosis in Xinjiang by improved indirect ELISAs using recombinant proteins as diagnostic antigens and the first time that B. bovis and B. bigemina have been detected in Xinjiang. We basically obtain the firsthand informa-tion for development of prevent and control program against bovine piroplasmosis in the future in Xinjiang. |
PDF Full Text Request