Attenuation of Babesia bovis by in vitro cultivation

A virulent strain of Babesia bovis was adapted to grow in erythrocyte culture in the presence of equine serum and in lieu of bovine serum. Four splenectomized calves inoculated with the adapted strain, 429, developed hematologic signs of infection and a low grade fever, but remained free of central nervous system (CNS) signs and recovered. All of six control animals inoculated with a virulent strain reacted severely and five showed CNS signs and died. The calves injected with the attenuated strain were solidly immune when challenged with the virulent strain at 44 or 78 days after vaccination.     

The invasion process of bovine erythrocyte by Babesia divergens: knowledge from an in vitro assay

ABSTRACT: Babesia divergens is a tick-transmitted apicomplexan parasite for which asexual multiplication in its vertebrate hosts is restricted to erythrocytes. Current knowledge of invasion of these target cells is limited. An efficient in vitro invasion assay was set up to gain access to this information. Parasites prepared from infected RBC, lysed by electroporation, and mixed with bovine RBC in a selected synthetic medium (RPMI 1640 supplemented with calcium) were able to establish subsequent cultures with parasitemia ranging from 6 to 14%. Free parasites remaining in the invasion medium could be eliminated by Percoll gradient and culture could be pursued with the freshly invaded erythrocytes. In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion. With this assay we demonstrate that 1) erythrocyte invasion by B. divergens is a rapid process since 70% of the invasion-competent parasites invaded the RBC in less than 45 s; 2) all invasion-competent parasites achieved invasion within 10 min of contact; 3) one erythrocyte could be invaded concomitantly by two merozoites; 4) despite a synchronous start, the parasite population evolved heterogeneously resulting in a progressive loss of synchronisation. Western blot analysis of proteins collected from invasion medium were performed with sera from animals experimentally infected with B. divergens and highlighted several proteins. The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process. Further investigations are required for their characterisation.     

Studies on failure of T strain live Babesia bovis vaccine

SUMMARY Field investigations of the protection afforded by the Australian live Babesia bovis vaccine used in the early 1990s (T strain) revealed inadequate vaccine-induced protection in certain herds. Vaccination/challenge trials using 207 experimental cattle were conducted to evaluate the protection afforded by T strain B bovis against field isolates from these herds. The trials investigated whether isolates that could ‘break-through’ T strain immunity were present in the field, the ability or inability of specific cattle to develop protective immunity after vaccination with T strain and the effect of attenuation and maintenance procedures on the immunogenicity of T strain. The results showed that B bovis parasites present early in the process of attenuation of T strain were more protective than those remaining late in the process. They also showed that cattle from properties experiencing vaccine failures were less protected by T strain vaccination than Bos taurus cattle randomly selected from the general population if vaccinated with highly attenuated T strain. A hypothesis is offered to explain these findings .     

Human serum for in vitro cultivation of Babesia bovis.

Various combinations of human serum (from blood of groups A and Rhesus positive) with bovine serum, i.e. 20% + 20% (Medium I), 30% + 10% (II), 40% + 0% (III) and 0% + 40% (IV) and Medium-199 (60%) were used in the propagation of Babesia bovis. Babesia bovis stabilate revived by inoculation in a bovine calf was used at a level of 6% parasitized erythrocytes (PPE). The medium was replenished every 24 h. The medium changed from bright red to dark-coffee color every 24 h. The increase in PPE was maximal between 24 and 48 h. It was also observed that the increase in PPE was significantly higher in a 1:2 dilution compared with a 1:1 dilution. The increase in PPE was highest in Medium I with a 50% replacement of bovine with human serum. However, the parasites could not be subcultured and maintained continuously. Fifty percent replacement was thus optimal for the human-bovine serum combination in a microaerophilous stationary phase (MASP) system. The results indicate that B. bovis can also multiply in a human/bovine serum MASP culture system at least for a period of 48 h, and this is consistent with the zoonotic nature of Babesia species.     

Babesia bovis: studies of parameters influencing microvascular stasis of infected erythrocytes

Bovine erythrocytes infected with Babesia bovis were analysed for parameter changes known to influence rigidity and deformability of erythrocytes. Marked increases in malonyldialdehyde were detected indicating that lipid peroxidation occurs during infection. Consequently, increases in membrane lipid, methaemoglobin and membrane-bound haemoglobin were detected. Conversely, decreases in the antioxidant vitamin E and decreases in sialic acid were also detected. The cumulative effect of these changes would be to increase erythrocyte rigidity and decrease deformability thus contributing to the microvascular stasis characteristic of acute B bovis infection.     

牛巴贝斯虫PCR检测及体外培养试验

本试验通过PCR方法检测出牛巴贝斯虫单一感染的牛全血样本,并探讨了牛巴贝斯虫病患牛全血体外培养方法及培养条件。试验结果表明,PCR检测结果显示检测方法效果较好,单一感染样本的新鲜血液中虫体在含40%牛血清的完全培养液和37 ℃、5% CO₂的条件下能建立起牛巴贝斯虫的体外培养,虫体可以连续培养14 d,传代13次,最高染虫率可以达到12.5%,平均染虫率为4%。     

Heterologous strain immunity in bovine babesiosis using a culture-derived soluble Babesia bovis immunogen

The cross-protective capacity of culture-derived soluble immunogens against heterologous Babesia bovis strains from different geographical locations of Latin America was examined. Susceptible yearling cattle were either immunized with immunogens derived from Venezuelan or Mexican strains, or were administered a multi-component immunogen containing antigens of the Australian, Mexican and Venezuelan strains. Cattle were challenged with virulent B. bovis organisms of the Argentinian, Colombian, Ecuadorean, Mexican and Venezuelan strains. The major parameters used to evaluate cross-protection were the following: presence, level and duration of parasitemia; maximal PCV reduction; level and duration of fever; determination of fibrinogen and cryofibrinogen; homologous and heterologous antibody levels; and net gains in body weight. Results showed good protection with a Venezuelan B. bovis immunogen after homologous and heterologous challenge exposures. A low degree of cross-immunity was observed when cattle vaccinated with the Mexican immunogen were challenged with each of the heterologous strains.     

光生物素标记DNA探针检测牛巴贝斯虫研究初报

光生物素标记重组质粒DNA(c51A)探针检测牛巴贝斯虫研究的初步试验结果表明,能特异地检出感染牛血中的巴贝斯虫,灵敏度为10pg质粒DNA,相当于约150个感染红细胞的虫体DNA。     

Goat serum, a substitute of bovine serum in cultivation of Babesia bovis.

Babesia bovis (a Mexican isolate) was cultivated in MASP culture system using goat serum in various concentrations as substitute of bovine serum. It was observed that 20% goat serum + 20% bovine serum + 60% Parker's medium 199 supported the growth of the parasite, which was maintained in this medium through 8 subcultures. The soluble exoantigen (vaccine) present in the culture supernatant is to be quantified and tested in vitro. Goat serum from slaughterhouses may be utilized for in vitro cultivation of the parasite and, expectedly, production of vaccine. This study may prove to be useful in reducing the cost of vaccine at least in tropical countries.     

快速检测牛巴贝斯焦虫LAMP方法的建立

为提高牛巴贝斯焦虫(Babesia bovis)病检出率,本研究采用环介导等温扩增技术(LAMP)建立一种快速、灵敏、特异的检测B.bovis的方法。根据GenBank中登录的B.bovis细胞色素b基因(cyt b)序列,设计4条LAMP引物,优化反应体系条件,在Bst DNA聚合酶的作用下,62℃反应60min,加入EvaGreen后,肉眼观测。结果表明,该LAMP检测体系特异性强,与双芽巴贝斯焦虫(B.bigemina)DNA等不发生交叉反应;敏感性高,最小检测值为0.014fg(相当于1.58×10-3虫体的拷贝数),为一般PCR方法的1000倍。该方法具有简单、快速、低成本的特点,可用于B.bovis病的现场快速检测。     

The role of specific immunoglobulins in antibody-dependent cell-mediated cytotoxicity assays during Babesia bovis infection

A stabilate prepared from Babesia bovis-infected Boophilus microplus ticks was used to infect intact adult cattle. Whole sera and immunoglobulin fractions from representative sera were tested by complement fixation (CF), indirect fluorescent antibody (IFA), and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The last test utilized 51Cr-labeled chicken erythrocytes coated with Babesia bovis antigen as targets. Mononuclear cell preparations, obtained from peripheral blood of normal donors and consisting of lymphocytes with 2--6% large monocytes, were used as the source of effector cells. Antibody activity was detected by all tests between 14 and 16 days following infection. Specific IgM and IgG1 were reactive in both CF and IFA tests, although the development of high titers was attributable to IgG, alone. The ADCC activity was restricted to IgG1 fractions and was greater in those sera or fractions with greater CF activity. No activity was demonstrated in IgG2 fractions by any test used.     

Alternative complement pathway in bovine serum: lysis of human erythrocytes.

The hemolysis of unsensitized human erythrocytes by fresh bovine serums was investigated. Lysis occurred in ethylene glycol bis-amino tetraacetate buffers and with serums depleted of Clq. Serums extensively absorbed with packed human erythrocytes at 0 C effectively lysed human erythrocytes, but optimal lytic capacity required target cells "sensitized" with a heat-stable serum factor. Lysis did not occur with serums absorbed with zymosan at 17 C or heat inactivated at 50 C. These results indicate that human erythrocytes can activate the alternative pathway of complement in bovine serums. Lysis can proceed in the apparent absence of antibodies, although their presence may enhance the reaction.     

Serological survey of Babesia bovis and Babesia bigemina in cattle in South Africa

A total of 719 serum samples collected from clinically healthy cattle from eight provinces located in different districts of South Africa were examined by the indirect enzyme-linked immunosorbent assay (ELISA) and the standard indirect fluorescent antibody test (IFAT) to determine the serological prevalence of Babesia bovis and Babesia bigemina. The results showed that 35.3% and 39.7% of cattle were positive for B. bovis and 30% and 36.5% were positive for B. bigemina antibodies on ELISA and IFAT, respectively. Mixed infections were detected in 18.2% and 26.3% of the samples using ELISA and IFAT, respectively. Consequently, the ELISAs with recombinant B. bovis spherical body protein-4 (BbSBP-4) and B. bigemina C-terminal rhoptry-associated protein-1 (BbigRAP-1/CT) were proven to be highly reliable in the serological diagnoses of bovine babesiosis in South African cattle, as evidenced by the significant concordance rates when the results were compared to those of IFAT. Moreover, the serological prevalence was significantly different among the tested provinces, in which the ranges exhibited between 15% and 73% for B. bovis infection and between 13% and 54% for B. bigemina infection. High sero-positive rates were present in Mpumalanga and KwaZulu-Natal provinces, while the lowest rate was in the North West province. Our data provide important information regarding the current seroprevalence of bovine babesiosis in South Africa, which might be beneficial in developing rational strategies for disease control and management.     

Studies on the use of gerbil-derived Babesia divergens antigen for diagnosis of bovine babesiosis

Babesia divergens antigen obtained from bovine and gerbil erythrocytes was compared for the diagnosis of bovine babesiosis by the indirect immunofluorescence antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA). Using a range of sera from the field and from experimental cattle, it was found that antigen from the two sources did not differ significantly in sensitivity or specificity. Antigen from gerbils may, therefore, be used for serological surveys of bovine babesiosis caused by B. divergens. The IFAT and ELISA were also compared and it was concluded that they are equally effective in the detection of positive samples. For small-scale surveys, the IFAT using gerbil-derived antigen is adequate, but for large surveys the ELISA is preferable.     

Calcium ions are involved in egress of Babesia bovis merozoites from bovine erythrocytes

Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host’s erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca²⁺ in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca²⁺ concentration in the cytosol of the parasite cells. The increased intracellular Ca²⁺ concentration following these treatments was confirmed using live cell Ca²⁺ imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca²⁺ signalling pathway in the egress of B. bovis merozoites.     

微嗜氧稳定期体外培养法分离巴贝西虫试验

自然感染的牛巴贝西虫（Babesiaboris）通常用来制作活疫苗和进行寄生虫的生物学研究，上述两个目的需要纯的B．boris，不能混有其他的血液寄生虫，但是B．boris的分布范围和其他的以节肢动物为媒介的血液寄生虫（包括牛泰勒焦虫（Theileria buffeli）、