Immunization with recombinant surface antigens p26 with Freund's adjuvants against Babesia rodhaini infection

The surface proteins of Babesia rodhaini have previously been shown to induce a high degree of protective immunity. In the present study, one of those proteins, B. rodhaini antigen p26 was expressed in Escherichia coli and in insect cells infected with a recombinant baculovirus. These proteins were recognized by immune serum from a drug-cured BALB/c mouse. While BALB/c mice immunized with both recombinant antigens and Freund's adjuvants showed 40-100% survival rate against challenge infection with B. rodhaini, saponin failed to induce protection, although significant levels of B. rodhaini-specific antibodies were produced in both immunized mice (1:1,000-2,000 by indirect immunofluorescent antibody test). The immunization of IFN-gamma-deficient mice with the recombinant proteins was not protective against B. rodhaini infection, indicating that IFN-gamma is one of the important factors for the survival against lethal B. rodhaini infection.     

Changes of splenic lymphocyte subpopulation in mice inoculated with Babesia microti and Babesia rodhaini.

Changes of splenic lymphocyte subpopulation after Babesia microti and Babesia rodhaini inoculation in mice were examined by flow cytometric analysis. The B. microti inoculated mice showed a longer period of time from inoculation to the onset of increase or decrease parasitaemia (%), packed cell volume, total spleen cell numbers and surface immunoglobulin positive splenic cell numbers than respective periods in B. rodhaini inoculated mice. The Thy-1 positive cell numbers in B. microti inoculated mice and B. rodhaini inoculated mice pre-immunized with homologous parasites were significantly higher than that of B. rodhaini inoculated mice. The ratio of L3T4 positive cell/Lyt-2 positive cell after inoculation with B. microti was quite similar to that in B. rodhaini mice pre-immunized. However, the ratio in B. rodhaini inoculated mice revealed a lack of an increasing phase. These results suggested that the T-cell dependent early immune response, especially suppressor activity, was closely related to the difference in the course of infection between the non-lethal B. microti and the lethal B. rodhaini infection in mice.     

Therapeutic effect of clindamycin and tetracycline on Babesia rodhaini infection in mouse model

In order to identify the alternative effective chemotherapeutic agents for murine babesiosis, some selected drugs were examined for their efficacy against protozoan infection in the mouse-Babesia rodhaini (B. rodhaini) model. Clindamycin was not completely effective for elimination of parasites in a dose of 50 mg or 100 mg/kg BW/day b.i.d. but effective to prolong the life span of hosts, while it completely cured B. rodhaini infections in a dose of 200 mg. On the other hand, a double therapy consisting of 2 treatments with 100 mg clindamycin and 100 mg clindamycin and with 100 mg clindamycin and 100 mg tetracycline; respectively, and a single therapy with 100 mg tetracycline or 200 mg clindamycin, had a possibility to clear away B. rodhaini organisms from hosts. However, almost all the treatment groups, had a relapse of the infection within 10 days post treatment or re-treatment. Cured mice by treatment with clindamycin and clindamycin, or clindamycin and tetracycline showed complete resistance against challenge with B. rodhaini, while mice cured by administration of clindamycin at 200 mg or tetracycline at 100 mg showed incomplete resistance to challenge infection. The present data suggest that the two former chemotherapies can induce effective protective immunity (premunization), but the latter two chemotherapies induce incomplete premunization.     

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.     

Glucose uptake activity in murine red blood cells infected with Babesia microti and Babesia rodhaini

The glucose uptake activity in Babesia rodhaini and B. microti - infected red blood cell (IRBC) was investigated in mice using 2-deoxy-D-glucose (2DOG) and L-glucose (L-Glc), a non-metabolizable analogue of D-glucose and non-incorporative glucose to non-infected RBC (NRBC), respectively. The uptake activities of both DOG and L-Glc were higher in IRBCs than those in NRBC. The concentration dependent uptake of 2DOG and L-Glc in both IRBC revealed a linear curve, indicating non-transporter mediated uptake. In addition, B. microti IRBC showed higher 2DOG uptake than B. rodhaini IRBC, whereas no difference was observed in L-Glc uptake. These results indicated that some new glucose uptake system, at least two systems, developed in both IRBC. The new systems were sodium independent, non-competitive to L-Glc, and sensitive to temperature. One of two systems had no kinetical difference between B. rodhaini and B. microti IRBC, however another one might have higher uptake activity in B. microti IRBC compared to that in B. rodhaini IRBC.     

Immunity to Babesia in mice. I. Adoptive transfer of immunity to Babesia rodhaini with immune spleen cells and the effect of irradiation on the protection of immune mice

Immunisation of Balb/c mice against Babesia rodhaini by an amicarbalide-controlled infection resulted in a solid immunity which lasted for 216 days. With spleen cells of immune mice protection could be transferred both to naive mice pretreated with cyclophosphamide. Treatment of naive mice with cyclophosphamide (300 mg/kg) five days before a lethal B. rodhaini inoculation resulted in over 50% survival. This protective effect of cyclophosphamide is explained by its inhibiting effect on suppressor T-cells. The protection against B. rodhaini challenge infection afforded to immune Balb/c mice was completely resistant to a sublethal irradiation of 400 rad. Since B-lymphocyte function in antibody production is suppressed by this dose, the role of antibodies in the effector phase of the immunity appears to be of minor if any importance. A considerable degree of protection was still preserved after irradiation of immune animals with 875 rad. Sensitivity to this irradiation dose of all immunocompetent cells except macrophages and a small fraction of T-lymphocytes indicates the involvement of these cell types in the effector phase of the specific immunity. Highly radioresistant macrophages are therefore considered to play the major role but T-lymphocytes are also required for complete protection.     

The immunization of cattle against Babesia divergens with fractions of parasitized erythrocytes

A lysate of erythrocytes infected with Babesia divergens was subjected to isoelectric focusing within a pH range of 3.5-9.5. The focused proteins were split into 2 fractions depending on pH (1) those from 3.5-6.5 and (2) those from 6.5-9.5. The acidic fraction was used as an immunogen for groups of 5 adult cows using 2 different adjuvants, saponin and a mixture of muramyldipeptide (MDP) and incomplete Freund's adjuvant (IFA). Two other groups of 5 cows were simultaneously treated with the alkaline fraction and a sample of the complete lysate with the MDP/IFA adjuvant. The treated cows plus a control group were infected with 2.5 X 10(8) erythrocytes infected with a homologous strain of B. divergens. When compared with the controls all pre-treated groups were protected to some degree. There were significant differences between the treated groups with the group which received MDP/IFA combined with the acidic fraction showing most resistance to the infection. The experiment also showed that the MDP/IFA adjuvant produced more protection than the saponin.     

Immunological changes in Babesia rodhaini infected BALB/c mice after treated with anti-babesial drug; diminazene diaceturate.

A model system capable of investigating immunological changes was first established in Babesia rodhaini infected mice with an aid of a drug, diminazene diaceturate (DD). Intraperitoneal (ip) inoculation with B. rodhaini resulted in acute death in euthymic (nu/+) and athymic (nu/nu) BALB/c mice. Treatment with DD at an early stage of infection saved both mice from acute death. Parasitemia recurred in some of them but resulted in death only in nu/nu mice. A re-challenge with 10(5) parasitized erythrocytes (PE) on the surviving mice on day 28 post infection revealed resistance in nu/+ but not in nu/nu mice. The results suggested a participation of the thymus in the protective mechanisms. Immunological changes were then observed on nu/+ and nu/nu mice which were inoculated ip with 10(4)PE and treated with the drug, and then challenged with 10(5)PE ip on day 28. An antibody response was measured with immediate reaction by footpad injection of a soluble antigen of B. rodhaini and by ELISA of serum antibody using the antigen and protein A, on day 10 and later, and further a pronounced response was detected after re-challenge in nu/+ mice. No response was detected by ELISA in nu/nu mice. Delayed footpad reaction was seen in nu/+ mice by day 14 and later but it was suppressed after the re-challenge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycophorin A-knockout mice, which lost sialoglycoproteins from the red blood cell membrane, are resistant to lethal infection of Babesia rodhaini

Recent in vitro-based studies using several Babesia spp. have suggested that sialic acids and/or sialoglycoproteins on host red blood cells (RBCs) play an important role in their invasion of RBCs. In the present study, we analyzed the RBC characteristics of glycophorin A (GPA)-knockout mice and studied their in vivo susceptibility to lethal infection of Babesia rodhaini for the first time. In immunoblot and lectin blot analyses, glycoproteins containing O-linked oligosaccharides terminated with alpha2-3-linked sialic acids disappeared from the RBCs of GPA homozygous ((-/-)) mice. Flow cytometric analysis showed a remarkable reduction of Maackia amurensis lectin II binding to the surface of GPA(-/-) RBCs relative to control RBCs, indicating an appreciable loss of alpha2-3-linked sialic acids on the RBC surface of GPA(-/-) mice. Importantly, while B. rodhaini caused lethal infection in wild-type mice, the infected GPA(-/-) mice showed inhibition of parasite growth and eventually survived. These results indicate that RBC sialoglycoproteins lost in GPA(-/-) mice are involved in the in vivo growth of B. rodhaini, probably functioning as essential molecule(s) for the parasite invasion of host RBCs in the blood circulation.     

不同佐剂GnRH疫苗主动免疫对公猪睾丸发育和血清睾酮的影响

为探讨铝胶佐剂与白油Span佐剂的GnRH疫苗对公猪睾丸发育和血清睾酮的影响,12头初生公猪随机分为GnRH疫苗铝胶佐剂免疫组和GnRH疫苗白油Span佐剂免疫组(下简称铝胶组和白油组).9周龄初免,17周龄加强免疫,25周龄屠宰.游标卡尺测量猪阴囊直径和睾丸大小,石蜡切片观察睾丸组织发育,ELISA法和放射免疫分析(RIA)法分别检测GnRH抗体水平和血清睾酮水平.结果表明,铝胶组与白油组GnRH抗体效价差异不显著(P0.05).铝胶组血清睾酮水平于17,21周龄(P＜0.05)和25周龄(P＜0.01)显著地低于白油组,阴囊直径,睾丸直径、横径、周长及睾丸血量均显著地低于白油组(P＜0.05).铝胶组与白油组猪睾丸曲精小管均出现不同程度的空泡化,白油组零星分布少量精子,铝胶组无精子产生.这表明在公猪免疫去势的效果上,辅以铝胶佐剂的GnRH疫苗比辅以白油Span佐剂的GnRH疫苗好.     

Viability and virulence of Babesia rodhaini eight years after cryogenic preservation with dimethyl sulfoxide

Mouse blood infected with Babesia rodhaini and containing an equal volume of 4 M dimethyl sulfoxide was infective after storage at -196 degrees C for 8 years. The Babesia organisms were still able to cause lethal infections after prolonged low temperature storage.