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.     

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.     

Lyophilised bovine serum as a substitute for frozen serum in the cultivation of Babesia bigemina and B bovis.

Lyophilised serum offers significant advantages over frozen serum when it comes to shipping such material over long distances. Babesia bigemina and B bovis were cultured in medium supplemented by either frozen-thawed or lyophilised-rehydrated serum. There were no significant differences between the two types of medium in the growth of parasites and percentage of infected cells during subcultivation for 18 days.     

Cryopreservation of Babesia bigemina for in vitro cultivation

Babesia bigemina-infected RBC and merozoites were cryopreserved and used to initiate in vitro cultures in normal bovine RBC; the cryoprotectant was a final 10% polyvinylpyrrolidone in Vega y Martinez solution. A cooling rate of 20 C/min until -80 C and then rapid transfer to liquid N2 storage was satisfactory. Samples for culture initiation were rapidly thawed at 37 C, washed in Vega y Martinez solution and resuspended in complete culture media containing 10% normal bovine RBC. The optimum culture conditions to reestablish cultures were a 24-well plate (16 mm ID), 5 mm in depth, and an atmosphere of 2% to 5% O2, 5% CO2, and 93% to 90% N2.     

In vitro cultivation of Babesia bigemina

A strain of Babesia bigemina was isolated from an infected calf and propagated in vitro. Culture conditions included washing of infected and normal bovine erythrocytes in a special solution, and the use of a 5% to 10% (v/v) erythrocyte suspension in medium 199 (with 20% to 50% fresh normal bovine serum) at a depth of 4 mm in a 5% CO2, 2% O2, 93% N2 atmosphere. After 36 days in vitro and 9 subcultures, the cultured organism was inoculated into a susceptible calf. This calf developed clinical signs of disease and recovered when treated with 1% trypan blue solution. The strain was also reisolated from the second calf. The original isolate had been maintained in continuous in vitro cultivation for more than 99 days.     

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%。     

A microplate enzyme immunoassay for detecting and measuring antibodies to Babesia bovis in cattle serum

A microplate enzyme immunoassay (EIA) is described for measuring IgG antibody to Babesia bovis in cattle serum. B. Bovis antibody status (whether positive or negative) and the amount of B. Bovis antibody (EIA score), were measured by comparison with reference serums. The EIA was shown to be specific for B. Bovis, and EIA score correlated well with EIA titre. Comparison of EIA with the Indirect Fluorescent Antibody Test (IFAT) showed more than 95% agreement between the methods and disagreement in only 1.6% of serum samples tested. The remaining 3.2% were positive by EIA and suspected positive by IFAT. The EIA was shown, by titrating positive serums, to be more sensitive than IFAT, which explained its tendency to detect more positive serums than IFAT. EIA detected B. bovis antibody in experimentally infected cattle by day 14 post infection (pi) and for at least 268 days pi. EIA score for B. bovis antibody in immune cattle increased significantly (p less than 0.05) following heterologous strain challenge.     

牛巴贝斯虫巢式PCR诊断方法的建立

根据GenBank发表的XJ-MSA-2c核苷酸序列(登录号:EU328267)设计的2对特异性引物MS-1、MS-2、MS-3以及MS-4,建立牛巴贝斯虫病巢式PCR快速检测方法。在特异性检测试验中,仅从MSA-2c质粒样本中扩增出622、350bp2条目的片段,与预期片段大小相符,而作为对照样本的双芽巴贝斯虫、牛环形泰勒虫、东方巴贝斯虫基因组DNA均无此扩增目的条带出现。第1次和第2次扩增的敏感性分别为1.75、1.75×10-2μg/L。在对46份全血的DNA样本巢式PCR和显微镜检测中,阳性检出率分别为34.8%(16/46)和23.9%(11/46)。结果表明,所建立的巢式PCR方法准确、敏感、特异,作为牛巴贝斯虫病的快速检测和小范围的流行病学调查,具有重要的临床意义。     

In vitro cultivation of a newly recognized Babesia sp. in dogs in North Carolina

A novel large Babesia sp. from an infected dog was cultivated in vitro by microaerophilous stationary phase culture methodology. A primary culture initiated in enriched RPMI-1640 medium supplemented with 40% canine serum and incubated in a 2% oxygen environment supported parasite growth in vitro. Subsequent subcultures into enriched HL-1 medium with 20% fetal bovine serum also supported parasite propagation. Cultures were successfully introduced to 5% carbon dioxide in air atmosphere at passage 4. To date, the parasites have been continuously cultured through 35 passages, although the parasitemias are low, ranging from 0.2 to 0.3%. Parasites cultured in RPMI with canine serum were cryopreserved and successfully recovered from liquid nitrogen storage. The small subunit ribosomal rRNA gene sequence was identical in blood-derived and culture-derived parasites, differing in a single base position from the previously reported sequence for this Babesia sp. The ultrastructure of the parasite was consistent with that of other large Babesia spp., except that the spherical body contained numerous round particles unlike the inclusions previously described in Babesia spp.     

Immune control of Babesia bovis infection

Babesia bovis causes an acute and often fatal infection in adult cattle, which if resolved, leads to a state of persistent infection in otherwise clinically healthy cattle. Persistently infected cattle are generally resistant to reinfection with related parasite strains, and this resistance in the face of infection is termed concomitant immunity. Young animals are generally more resistant than adults to B. bovis infection, which is dependent on the spleen. Despite the discovery of B. bovis over a century ago, there are still no safe and effective vaccines that protect cattle against this most virulent of babesial pathogens. Immunodominant antigens identified by serological reactivity and dominant T-cell antigens have failed to protect cattle against challenge. This review describes the innate and acquired immune mechanisms that define resistance in young calves and correlate with the development of concomitant immunity in older cattle following recovery from clinical disease. The first sections will discuss the innate immune responses by peripheral blood- and spleen-derived macrophages in cattle induced by B. bovis merozoites and their products that limit parasite replication, and comparison of natural killer cell responses in the spleens of young (resistant) and adult (susceptible) cattle. Later sections will describe a proteomic approach to discover novel antigens, especially those recognized by immune CD4+ T lymphocytes. Because immunodominant antigens have failed to stimulate protective immunity, identification of subdominant antigens may prove to be important for effective vaccines. Identification of CD4+ T-cell immunogenic proteins and their epitopes, together with the MHC class II restricting elements, now makes possible the development of MHC class II tetramers and application of this technology to both quantify antigen-specific lymphocytes during infection and discover novel antigenic epitopes. Finally, with the imminent completion of the B. bovis genome-sequencing project, strategies using combined genomic and proteomic approaches to identify novel vaccine candidates will be reviewed. The availability of an annotated B. bovis genome will, for the first time, enable identification of non-immunodominant proteins that may stimulate protective immunity.     

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.     

Experimental Babesia bovis infection in Holstein calves

One intact and two splenectomized Holstein calves were infected intravenously with a Mexican strain of Babesia bovis and killed following the onset of severe clinical disease. A light and electron microscopic study was conducted on selected tissues to examine the relationship between parasitized erythrocytes and microvascular endothelial cells. The pattern and degree of specific organ sequestration of parasitized erythrocytes was assessed and correlated to lesions. Red blood cells infected with Babesia bovis exhibited stellate membrane protrusions. This morphological change appeared to mediate erythrocyte sequestration in the microvascular and capillary beds of the brain, kidney, and adrenal gland by an as yet unknown mechanism(s).     

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）、     

牛巴贝斯虫实时荧光PCR检测方法的建立

为建立牛巴贝斯虫(B.bovis)的TaqMan实时荧光PCR检测方法,本研究根据GenBank中B.bovis的18S rRNA基因保守序列,设计引物和TaqMan探针,通过优化反应体系,建立检测B.bovis的实时荧光PCR方法.试验结果表明:实时荧光PCR对靶基因的最低检测值为1.31×101 copies/μL,比常规PCR的敏感性高1 000倍;而且与牛的其他血液原虫无交叉反应;组内及组间重复性试验的变异系数均小于3％,具有良好的重复性;在23份被检样品中,实时荧光PCR和常规PCR的检出率分别为52.17％和30.43％.该检测方法的建立为B.bovis的检测提供了一种快速、敏感、特异的技术手段.     

Investigations of breakdowns in protection provided by living Babesia bovis vaccine.

Field investigations of protection afforded by live Babesia bovis vaccine in Australia revealed that a ninefold increase in vaccine failures occurred in the period from 1985 to 1990. Laboratory trials using 189 experimental cattle were conducted to evaluate the protection afforded by the Babesia bovis strain used in the commercial vaccine during this time. Four isolates from clinical cases of babesiosis in vaccinated cattle were assessed. The results showed that the strain used in the vaccine during the 5 year period was poorly protective against three isolates while a recently isolated and prepared vaccine strain was strongly protective. Circumstantial evidence is provided that indicates the vaccine failures were due to change in the field populations of Babesia bovis, rather than change in the strain used in the vaccine. Implications of the results for the future of Babesia bovis vaccines are discussed.     

Detection of IgM antibodies against Babesia bovis in cattle

The diagnosis of acute babesiosis by direct examination of blood smears has some limitations and the indirect serological methods currently in use are designed for detection of IgG, which may not be detectable at an early stage of infection. There is a need, therefore, for rapid and reliable procedures to diagnose acute infections. An ELISA system using a crude antigenic preparation of Babesia bovis was standardized for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Serum samples of cattle imported from tick-free areas collected before and during an immunization process were used to validate the tests. The specificity was 94% and sensitivity 100%. Specific IgM antibodies against B. bovis first appeared on the 11th day post-inoculation (p.i.) in animals infested with Boophilus microplus ticks and on the 19th day p.i. in animals which had been inoculated with infected blood. Antibody titers decreased after Day 33; however, all animals remained positive until the end of the experiment (124 days).     

A specific DNA probe which identifies Babesia bovis in whole blood.

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.     

Continuous in vitro cultivation of a recently identified Babesia that infects small ruminants in China

Babesia sp. Xinjiang was isolated from a splenectomised sheep infested by Rhipicephalus sanguineus and Hylomma anatolicum anatolicum, collected from sheep and cattle in Xinjiang province. It was considered to be a novel ovine Babesia species on the basis of its morphology, pathogenicity, vector tick species and alignments of 18S ribosomal RNA (18S rRNA) and internal transcribed spacers (ITS) gene sequences. Continuous in vitro cultures of the ovine parasite were established using infected sheep blood. In RPMI 1640 medium with 7.5% sheep red blood cells (RBCs) maintained in an incubator at 37 °C and 5% CO(2), the percentage of parasitized erythrocytes (PPE) peaked at 10% in 24- and 6-well plates. It increased to 20-50% with the same culture medium but with 2.5% RBC in 75 cm(2) flasks. Two clonal lines of Babesia sp. Xinjiang were screened using the limiting dilution method. Growth characteristics of these lines in vitro were measured by a microtiter-based spectrophotometric method and from the PPE. The generation time in sheep erythrocytes was between 15.20 h and 16.27 h. Furthermore, the host range of parasite was identified with in vitro culture and in vivo infection. Erythrocytes of sheep, cattle, sika deer and humans could be invaded into by lines in vitro, but the parasites could not propagate in human erythrocytes. The parasites could not enter erythrocytes from goats in vitro. However, in vivo, only sheep could be infected by lines. Finally, a Babesia sp. Xinjiang-like parasite (which shared 99.5% identity with the original strain of Babesia sp. Xinjiang) was isolated using this in vitro culture system from 1 of 19 sheep blood samples collected from western Gansu province, China.