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.     

Influence of zinc deficiency to the mice infected with Babesia microti

Zinc (Zn) is an essential trace element for DNA synthesis and for cell growth and differentiation. The deficiency induces a wide range of disorders including immunodeficiency. In this study, the influence of Zn deficiency to the mice infected with Babesia microti was examined, and was compared with the influence in the rats infected with B. rodhaini previously reported. Experiments of B. microti infection were conducted using Zn-deficient (ZD; allowed to eat ad libitum on the ZD diet), Zn-adequate (ZA; allowed to eat ad libitum on the ZA diet), and diet-restricted (DR; supplied 2 g/day on the ZA diet) mice. It was suggested that the Zn deficiency exacerbated the infection dynamics of the mice with B. microti by the growth retardation, the reduction of immunity and the decrease in PCV. The results in the mice supported the consequences in the rats previously reported.     

Isolation of a human erythrocyte-adapted substrain of Babesia rodhaini and analysis of the merozoite surface protein gene sequences

Babesia rodhaini is a rodent hemoparasite closely related to B. microti, the major causative agent of human babesiosis. We tested the infectivity of B. rodhaini for human erythrocytes by using the SCID mouse model in which the circulating erythrocytes were replaced with those of humans. Initially, parasites grew very poorly in the mouse model, but a variant capable of propagating in human erythrocytes emerged after an adaptation period of three weeks. In an attempt to identify parasite proteins involved in the alteration of host cell preference, an expression cDNA library of B. rodhaini was constructed and screened with immune mouse sera. Although we were able to obtain three merozoite surface protein (MSP) genes, sequences of these genes from both the parental strain and human erythrocyte-adapted substrain were identical. Our results suggest that B. rodhaini has potential ability to infect human erythrocytes, but development of this ability may not be brought about by an amino acid change in MSPs.     

Detection of a new pathogenic Babesia microti-like species in dogs

Small babesiae in dogs are generally considered to belong to Babesia gibsoni. Here we describe the genotypic characterisation of small piroplasms found in the blood of a dog which suffered from clinical babesiosis. Pairwise identities as well as distance, parsimony and maximum likelihood analyses of the 18S rDNA clearly demonstrated that this isolate was only distantly related to the other canine piroplasms characterised genetically so far, including B. gibsoni. It was more closely related to B. microti, B. rodhaini, and Theileria equi. It is concluded that the small canine piroplasms described in this study represent a hitherto unknown species and that the fauna of piroplasms occurring in dogs is more diverse than assumed so far.     

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.     

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.     

Suppression of Babesia microti infection in mice concurrently infected with Schistosoma mansoni

Blood cell panasitaemias of Babesai microti and associated haematological changes were studied in mice concurrently infected with Schistosoma mansoni and in controls with no schistosome infections. In comparison to the controls B. microti parasit-aemias were suppressed markedly in mice harbouring 6 and 8 weeks old patent S. mansoni infections at the time of infection with B. microti. The suppression was accompanied by an alleviation/annulment of the B. microti induced reductions in haemoglobin and haematocrit levels and in its erythropoetic stimulation. The suppressive effect of patent S. mansoni infections on B. microti is suggested to be at least partly attributable to non-specific immunological factors although the altered erythrocyte kinetic state induced by the schistosoma infection in combination with the preference of B. microti for mature (old) erythrocytes might also play a role in the suppression. In mice harbouring 2 weeks old prepatent S. mansoni infections B. microti was not suppressed.     

Molecular studies on Babesia,Theileria and Hepatozoon in southern Europe. Part II. Phylogenetic analysis and evolutionary history

Following a study on molecular epizootiology of Hepatozoon canis and piroplasmids (Babesia spp. and Theileria spp.) in southern Europe, newly obtained sequences of 18s rRNA gene were used for phylogenetic analysis. Partial sequences were analysed in isolates showing high degree of homology (>99%) with previous GenBank entries: H. canis, B. canis vogeli, B. equi (two isolates, Spain1 and Spain2), T. annulata and Theileria sp. The complete gene sequences were used for B. ovis and B. bovis, that showed lower homology (<95%) with rapport to previously reported species or isolates. A first set of phylogenetic trees constructed with partial 18s rRNA sequences showed that most European isolates clustered unambiguously with previously described species, so that minor sequence dissimilarities found are due probably to strain variations.The second set of phylogenetic trees was made using the complete 18s rRNA sequences of 44 species from GenBank and the newly sequenced B. ovis and B. bovis. The analysis revealed for the first time a division of piroplasmids in five clades: (1) B. microti group, with B. rodhaini, B. felis, B. leo, B. microti and T. annae (proposed name for the group, without taxonomic value: Archaeopiroplasmids), (2) Western USA Theilerid-like group (proposed name: Prototheilerids), (3) Theileria group, containing all Theileria species from Bovinae (proposed name: Theilerids), (4) A first group of Babesia species including B. canis and B. gibsoni from canids together with B. divergens and B. odocoilei (proposed name: Babesids), (5) A second group composed mainly by Babesia species from ungulates: B. caballi, B. bigemina, B. ovis, B. bovis and Babesia sp. from cow (proposed name: Ungulibabesids). The bootstrap support obtained with several analytical procedures for this new dicotomy of Babesiidae was always very high. Taking into account the present phylogenetic analysis and additional paleogeographic, parasitological and zoological evidences, two hypothesis on the origin and evolution of piroplasmids groups are presented.     

Development of a Babesia rodhaini mouse assay for quality control of the serum component of a bovine babesiosis vaccine

The rodent babesia, Babesia rodhaini, survived equally well in basal medium containing either 10 per cent rat serum or 10 per cent bovine serum. As a result, a B rodhaini mouse assay is now performed routinely to determine the suitability of batches of bovine serum for use in a commercial Babesia bovis vaccine issued in Australia. Heat inactivation of bovine serum at 56 degrees C for two hours did not affect the survival of either B bovis or B rodhaini.     

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.     

Comparison and phylogenetic analysis of the heat shock protein 70 gene of Babesia parasites from dogs

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.     

Suppression of Babesia microti infection in mice concurrently infected with Fasciola hepatica

Blood cell parasitaemia of Babesia microti and the associated haematological changes were examined in mice harbouring patent Fasciola hepatica infections and in fluke-free control mice. B. microti parasitaemia was markedly suppressed in mice harbouring primary 7-week F. hepatica infections, as reflected in a reduction in the percentage of erythrocytes parasitised and in the incidence of multiple B. microti infections in the red cells. This suppression was accompanied by an annulment of B. microti induced reductions in haemoglobin and haematocrit levels in F. hepatica infected mice. In naive recipient mice, inoculated with blood from mice concurrently infected with F. hepatica and B. microti, the course of B. microti infection was characterised by a prolonged pre-parasitaemia period, a reduced peak parasitaemia and a delayed fall in haematocrit levels as compared to those inoculated with blood from mice infected with B. microti only. This feature may presumably be dose-related. The present study does not reveal the actual mechanism(s) involved in the suppression of the blood protozoan by F. hepatica. However, since B. microti has a preference for mature erythrocytes, the suppression may be a result of the altered erythrocyte kinetic state induced by the removal of erythrocytes by the blood-sucking fluke resulting in high levels of reticulocytes.     

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.     

Detection of Babesia microti-like parasite in filter paper-absorbed blood of wild rodents

The first case of human babesiosis was reported in Japan. The epidemiology of this disease in Japanese nature remains unclear. In this study, 97 common field mice captured in Hokkaido, Japan, were examined. Blood specimens absorbed onto filter papers were eluted and tested by nested PCR using specific primers for the B. microti nuclear small subunit rRNA genome. Twenty-three percent (11/47) of Apodemus speciosus and four percent (2/50) of Clethrionomys rufocanus were positive. The 159-bp primary sequences of PCR products tested exhibited 97.5% and 96.8% homology with those of the human isolate in Japan and of U.S. strains of B. microti, respectively.     

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.     

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)     

Studies on route of immunization with a mixture of killed parasites and adjuvants against Babesia rodhaini infection in mice

A series of experiments were undertaken to determine the most effective route of immunization with a mixture of killed Babesia rodhaini antigen (S antigen) and formalin-fixed Corynebacterium parvum (Propionibacterium acnes) bacterin (CPB) against challenge infection with B. rodhaini 3 weeks later. The mice pretreated with S antigen and CPB mixture intraperitoneally, but not intramuscularly, were significantly resistant to intraperitoneal (IP) or intravenous (IV) challenge with 10(6) organisms. The survival rates were 70.0 (IP challenge) and 60.0% (IV challenge) respectively. Fairly protective activities were equally produced in mice intravenously pretreated with S antigen and CPB with survival rates of 60.0% against IV challenge, but 30% against IP. These results indicated that the IP injection of S antigen and CPB mixture is desirable route for immunization against subsequent IP or IV challenge with B. rodhaini. On the other hand, lower protective effect was reconfirmed in the mice treated with S antigen and Freund's Complete adjuvant, regardless of immunization routes in the additional experiment. The survival rates were 33.3, 14.3 and 11.8% in the intraperitoneally, intramuscularly and subcutaneously-treated mice respectively against IP challenge with 10(6) organisms.     

Babesia microti-like rodent parasites isolated from Ixodes persulcatus (Acari: Ixodidae) in Heilongjiang Province, China

A Babesia microti-like rodent parasite was isolated from the tick, Ixodes persulcatus, collected from the northern forest area of Heilongjiang province, China. The collected I. persulcatus were allowed to feed on specific pathogen-free SCID mice and red blood cells from the mice were used to isolate Babesia spp. with the microareophilous stationary-phase culture technique. Paired and tetrad forms of merozoites were observed by light microscope in red blood cells of SCID mice. In vitro growth of the parasites was also achieved in mice erythrocytes, which indicated the presence of Babesia spp. in I. persulactus. To further identify the Babesia species, polymerase chain reaction screening and subsequent sequencing of nuclear small subunit ribosomal RNA (nss-rRNA) was employed. The results indicate that the observed parasites might be an isolate strain responsible for human babesiosis -B. microti - which has 99.3% identity with that of B. microti isolate RcM5201 (AB112050) from Mishan in Heilongjiang and Kobe isolates from Japan. In addition, the infection rate of B. microti in I. persulcatus ticks in the region was 3.6-4.0% in adult females and no infection in males. Though the infection rate is low, the high attack frequency of tick species on local residents indicates the risk of human babesiosis in the region and the necessity of precautionary measures.     

High susceptibility of Djungarian hamsters (Phodopus sungorus) to the infection with Babesia microti supported by hemodynamics

As the comparative study was carried out on the susceptibility by the pursuit of parasitemia among the Djungarian, Syrian, and Chinese hamsters as well as BALB/c mice infected with the Syrian hamster-adapted Babesia microti strain, and Djungarian hamsters showed the highest parasitemia among them. Then, the other hematological parameters were pursued in the Djungarian hamsters infected with the hamster-adapted B. microti strain. Remarkable symptoms observed were hemoglobinuria clinically, anemia hematologically, and splenomegaly macroscopically during all over the observation period for 24 weeks post infection (PI). Parasitemia began to rise at 2 weeks and peaked at 4 weeks PI. After that, parasitemia decreased gradually but was maintained with a level of about 10% on average until 24 weeks PI at the end of the experiment. A decrease in the RBC count, Hb, and PCV, and an increase in the reticulocyte and WBC counts due to the development of immature neutrophils, lymphocytes and monocytes were recognized together with a rise of parasitemia. The hamsters had macrocytic hypochromic anemia due to the increase of MCV and the decrease of MCHC in the growth phase of the parasite. It was considered that the Djungarian hamsters will be useful for the infection examination, isolation, maintenance, and passage of B. microti in laboratory.