Cloning of in vitro propagated Babesia bigemina

The original Babesia bigemina culture conditions were modified with regard to infected bovine erythrocyte concentration and atmospheric environment. A procedure was designed which would yield a homogeneous parasite population, beginning with a single infected erythrocyte. Calculated dilutions were made in 96-well tissue culture plates to approach one infected erythrocyte per four wells. Growth of parasites in wells was detected between 16 and 28 days after cultures were initiated. Clones were transferred to 24-well tissue culture plates for regular maintenance. Three primary clones were selected for additional recloning. The probability that the parasites detected in one well are the progeny of a single infected erythrocyte approaches 0.99 for tertiary clones.     

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.     

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.     

The detection of IgM and IgG antibodies against Babesia bigemina in bovine sera using semi-defined antigens in enzyme immunoassays

Soluble extracts prepared from Babesia bigemina merozoites were tested for antigenicity in class-specific enzyme immunoassays currently being evaluated for the differential serodiagnosis of bovine babesiosis. Intact merozoites were harvested from erythrocytes from an experimentally-infected calf by controlled hypotonic lysis and differential ultra-centrifugation. The merozoites were disrupted by ultrasonication and a crude soluble extract obtained by ultracentrifugation. Fractionation of the crude extract on calibrated Sephadex G-200 columns consistently produced 4 fractions with molecular weights of 600, 40, 15 and 5 k (k = 10(3) daltons). Only the 600 and 15 k fractions proved to be antigenic when reacted against bovine immune sera. These fractions were incorporated into IgM- and IgG-specific enzyme immunoassays and used to determine the kinetics of the host-antibody responses to infection. The use of semi-defined antigens allowed assay standardization and good reproducibility of the results. A calf infected with a cryopreserved stabilate of B. bigemina originating from adult Boophilus microplus ticks developed a mild transient fever from 6-4 days post-infection (d.p.i.) and low parasitaemia levels from 7-16 d.p.i. IgG-antibodies first appeared at 7 d.p.i., peaked in intensity at 12 d.p.i. and then persisted at these levels until the end of the test period at 49 d.p.i. IgM-antibodies appeared at 7 d.p.i., peaked in intensity from 12-22 d.p.i., but then declined to low levels by 28 d.p.i. The importance of this transitory IgM-antibody response in the serodiagnosis of acute B. bigemina infections remains to be determined in clinical and field situations.     

双芽巴贝斯虫在长角血蜱肠内发育的形态学研究

以耳袋法将长角血蜱(Haemaphysalis longicornis)幼虫饲于实验感染双芽巴贝斯虫(Babesia bigemina)牛,幼虫饱血后24h内,其肠管内容物中红细胞内、外见有单梨子型(3.5～4.5μm×1.2～2.6μm)和双梨子型(4.1～4.8μm×1.8～3.0μm)两种裂殖体.饱血后24～48h,随着裂殖体细胞膜及核变性而发生形态变化.48～72h,绝大多数裂殖体出现溶解.72h后,这些裂殖体从肠道消失.其后,在蜱肠上皮及血淋巴中也未能找到双芽巴贝斯虫体.本实验从形态学上证明双芽巴贝斯虫在长角血蜱若虫肠道内不能发育.     

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.     

Overcoming constraints to meeting increased demand for Babesia bigemina vaccine in Australia

Demand for live trivalent tick fever vaccine containing Babesia bovis, Babesia bigemina and Anaplasma centrale produced by the Department of Primary Industries, Queensland, has increased from less than 10,000 doses in 1988 to 500,000 doses in 2001. This paper describes a series of trials aimed at overcoming certain constraints to obtain B. bigemina parasitised erythrocytes (PEs) on a large enough scale from infected splenectomised calves to meet the demand. Passage through a series of splenectomised calves failed to increase the yield per calf but we showed that the dose rate of infected cells could be reduced from the long-time standard of 1x10(7) to 2.5x10(6) without affecting immunogenicity and still leaving a safety margin of at least 50-fold for infectivity. This change quadrupled the potential yield of doses per calf and allowed the DPI to meet the increased demand for B bigemina in vaccine. Due to the high cost and limited availability of suitable, health tested donors, calves previously infected with B. bovis or A. centrale were used to provide B. bigemina organisms but the practice resulted in red cell agglutination in some batches of prepared vaccine. A trial is described where B. bigemina-infected red cells were washed by centrifugation to remove agglutinating antibodies. Washing had no effect on parasite viability and this method is now in routine use in the production of trivalent vaccine.     

PCR-based detection of the transovarial transmission of Uruguayan Babesia bovis and Babesia bigemina vaccine strains

Bovine babesiosis is responsible for serious economic losses in Uruguay. Haemovaccines play an important role in disease prevention, but concern has been raised about their use. It is feared that the attenuated Babesia bovis and Babesia bigemina vaccine strains may be transmitted by the local tick vector Boophilus microplus, and that reversion to virulence could occur. We therefore investigated the possibility that these strains could be transmitted via the transovarial route in ticks using a Babesia species-specific polymerase chain reaction (PCR) assay. DNA was extracted from the developmental stages of the tick vector that had fed on calves immunized with the haemovaccine. It was possible to detect Babesia DNA not only in adult ticks, but also in their eggs and larvae. In addition, it was shown that calves infested with larvae derived from eggs laid by ticks fed on acutely infected calves, were positive for Babesia using PCR. Caution should therefore be shown with the distribution of the haemovaccine in marginal areas. It is still advisable that suitable tick control measures be used to prevent transovarial transmission and the potential risk of attenuated Babesia reverting to virulence.     

Patterns of bovine T cell-mediated immune responses to bovine herpesvirus 1

Lymphocyte proliferation was used to evaluate T cell-mediated immune responses to different isolates of Bovine Herpesvirus 1 (BHV-1). Groups of high, moderate, and low responses were observed when lymphocytes from three breeds of dairy cattle were stimulated with each of the BHV-1 isolates. Proliferation of cells in the low responding group could be augmented by exogenous IL-2. The mechanism of unresponsiveness by cells from one individual whose response was not altered by IL-2 supplementation was further investigated. The patterns of response by limiting dilution frequency analysis eliminated the possibility that this individual lacked responsive cells but suggested the presence of regulatory cell interactions which resulted in the observed low proliferative response. These results show that animals exposed to the same environment can vary greatly in their ability to respond to BHV-1. At least two mechanisms may be responsible for low proliferative responses in vitro: inadequate levels of IL-2 and the presence of suppressor cells.     

Immunity to Babesia in mice. III. The effects of corticosteroids and anti-thymocyte serum on mice immune to Babesia rodhaini

BALB/c mice, immunized against Babesia rodhaini by an amicarbalide controlled infection, were exposed to selective immunosuppressive treatment with corticosteroids and anti-thymocyte serum (ATS) respectively. Hydrocortisone acetate, 100 mg/kg, given i.p. six times during the three weeks after challenge inoculation caused a rising parasitaemia and high mortality (6/7). Dexamethasone in the drinking water at 20 mg/l or 10 mg/l for 22 days had a similar suppressive effect on the protection against B. rodhaini. Mortality, 100% at the dose rate of 20 mg/l and 50% at 10 mg/l, occurred both in challenged and in carrier animals after the reappearance of parasites in the bloodstream. All the ATS-treated immune mice demonstrated parasitaemia after challenge, although at a lower level than did the corticosteroid treated mice. Seven out of 9 animals died. Corticosteroid-sensitive macrophages together with T-lymphocytes are considered to play an important role in protection against B. rodhaini in specifically induced immunity in mice.