Attempts to infect T lymphocyte-deficient mice with Babesia species of cattle.

Nu/nu, nu/+, splenectomised nu/nu and Lasat mice were inoculated with freshly collected bovine blood infected with Babesia divergens and B major. There was no evidence that either parasite became established in mice but B divergens persisted in mice up to 10 days whereas B major lasted only one day. B divergens infection generally persisted longer in splenectomised mice but absence of thymus made no apparent difference to persistence of infection. B divergens underwent morphological changes in mice to vacuolated and ring forms.     

Piroplasms of ruminants in Switzerland and zoonotic significance of Babesia

Piroplasms are tick-transmitted blood parasites belonging to the genera Babesia and Theileria. In western and southern Switzerland, B. divergens, a small Babesia species, has been known for a long time as a parasite of cattle. Recent investigations have revealed the autochthonous occurrence of this parasite also in central and eastern Switzerland. On the occasion of an outbreak of anaplasmosis in the canton of Grisons, however, B. bigemina, a large Babesia species, and Theileria of the buffeli/sergenti/orientalis species complex were for the first time identified; the epidemiology of these two piroplasms in Switzerland remains unknown until now. The recent identification by genetic analyses of B. divergens in wild ruminants contradicts the hitherto postulated strict host specificity of this Babesia species for cattle. B. divergens as well as the closely related Babesia spp. genotype EU1 have in single cases also been identified in splenectomized humans.The rodent babesia B. microti which causes a human infection that is considered an "emerging tick-borne disease" in the U.S.A., is widespread in rodent populations in Switzerland, but seems to be of minor relevance as zoonotic pathogen here. Reasons for this could be differences in virulence of the parasites or in the transmission by the respective tick-vectors on the two continents.     

Indirect fluorescent antibody levels in experimental Babesia divergens infections of cattle.

In splenectomised calves infected with Babesia divergens IFA titres appeared after peak parasitaemia and maximum titres were observed 35 days after infection: the IFA response of adult non-splenectomised cattle was similar. It was concluded that the absence of a spleen did not markedly inhibit the IFA response of cattle to B divergens infection.     

Babesia divergens (Phylum Apicomplexa) in vitro growth in the presence of calf serum

Resistance to severe babesiosis in young calves has frequently been ascribed to an unknown serum factor(s) which inhibits growth of Babesia bovis in vitro. Our experiments show that young calf sera are as suitable as adult bovine sera for the in vitro culture of Babesia divergens, indicating that in this species at least inverse age resistance is due to alternative mechanisms. The suitability of commercial foetal calf sera for B. divergens cultures seems highly variable.     

Induction of protective immunity to Babesia divergens in Mongolian gerbils, Meriones unguiculatus, using culture-derived immunogens

Immunogens derived from microaerophilous stationary phase (MASP) cultures of Babesia divergens grown in bovine erythrocytes were used to inoculate the laboratory host of B. divergens, the Mongolian gerbil, Meriones unguiculatus. Animals inoculated subcutaneously twice with preparations of freeze-thawed merozoites in complete Freund's adjuvant were fully protected against homologous challenge, as were gerbils immunised with a non-viable preparation of parasite-enriched lysed infected bovine erythrocytes. Animals which had been infected with small numbers of parasitised erythrocytes from cultures cooled to 4 degrees C, allowed to recover, then challenged, also survived. All three groups had high antibody titres which dropped immediately after challenge and then rose again. Gerbils given culture supernatants containing soluble merozoite protein coat antigens were partially protected only after receiving a third inoculation. Non-immunised animals all died 4 days after challenge.     

Evaluation of the indirect fluorescent antibody test for Babesia major and Theileria mutans in Britain.

Use of the indirect fluorescent antibody (IFA) tests is described to detect antibodies to Theileria mutans and Babesia major in the sera of infected cattle. When antisera against T mutans and B major were tested against homologous antigens high antibody titres were recorded: when they were tested against each other or against Babesia divergens antigen insignificant titres (1/40 or less) were recorded. Thus the test was found to be species specific. Animals recovered from T mutans and B major infections retained significant levels of IFA titres for 22 and 11 months respectively. It is suggested that the IFA test could be used for field survey of the piroplasms of cattle in Britain.     

Inverse age resistance to experimental Babesia divergens infection in cattle

Two groups of calves, 1.5-2 and 7-11 months old respectively, and dairy cows were inoculated i.v. with 3 x 10(7) erythrocytes infected with Babesia divergens. High parasitaemia, fever and other clinical signs of babesiosis occurred among adult animals. A very low parasitaemia and a slightly increased body temperature but no other symptoms occurred in calves. these findings substantiate the conclusion that there exists an inverse age resistance against Babesia divergens. The kinetics of B. divergens IgG antibody formation were similar in all age groups. Consequently this antibody response was not the factor determining the development of the primary parasitaemia and thus the inverse age resistance phenomenon. However, age is not necessarily the only factor involved in the clinical expression of babesiosis. The kinetics of antibody formation was not associated with the intensity of the parasitaemia. In fact only about half the animals had a demonstrable parasitaemia although the antibody responses were similar in all age groups.     

Molecular cloning and genetic polymorphism of Babesia capreoli gene Bcp37/41, an ortholog of Babesia divergens merozoite surface antigen Bd37

Babesia divergens and Babesia capreoli are closely related species with distinct host ranges, a zoonotic feature being described only for B. divergens. The two species are 99.8% similar in the 18S rDNA gene sequence and indistinguishable by morphological or serological means, leading to confusion as to their species status. The phylogenetic relatedness between the two species, and the frequent involvement of surface components in serological cross-reactions led us to postulate that an ortholog of Bd37, the merozoite surface antigen described for B. divergens, could also exist in B. capreoli. We were able to amplify a single partial PCR product from B. capreoli genomic DNA using primers specific for the B. divergens merozoite surface protein coding gene Bd37, and sequencing confirmed the presence of a Bd37 ortholog in B. capreoli, named Bcp37/41. The full sequences of the Bcp37/41 genes and their intron-exon structures were obtained for two cloned lines of B. capreoli. They suggest functional homologies between Bd37 and Bcp37/41 such as their surface localization, their role in immune escape mechanism and in the initial non-specific attachment to the erythrocyte. Restriction fragment length polymorphism (RFLP) analysis of the amplicons and partial sequencing revealed an extreme polymorphism within B. capreoli, greater than the one observed for its ortholog Bd37. Such a marker could thus be useful in epidemiological as well as phylogenetic studies.     

Genetic basis for GPI-anchor merozoite surface antigen polymorphism of Babesia and resulting antigenic diversity

Glycosyl-phosphatidylinositol anchor merozoite surface antigens (GPI-anchor MSA) are proposed to act in the invasion process of infective merozoites of Babesia into host erythrocytes. Because of their essential function in the survival of Babesia parasites, they constitute good candidates for the development of vaccines against babesiosis and they have been extensively analyzed. These include Babesia bovis variable MSA (VMSA) and Babesia bigemina gp45/gp55 proteins of the agents of bovine babesiosis from tropical and subtropical countries, and the Babesia divergens Bd37 and Babesia canis Bc28 proteins of the main agents of bovine and canine babesiosis in Europe, respectively. However, these are very polymorphic antigens and Babesia parasites have evolved molecular mechanisms that enable these antigens to evade the host immune system as a survival strategy. This review focuses on the genetic basis of GPI-anchor MSA polymorphism and the antigenic diversity of B-cell epitopes that might be generated in each of these Babesia species. The picture is incomplete and no Babesia genome sequence is yet available. However, the available sequences suggest that two distinct, non cross-reactive GPI-anchor MSA (i.e., with unique B-cell epitopes) may be required by all Babesia species for invasion, and that these two distinct GPI-anchor MSA would be encoded by a multigene family. Furthermore, the data are consistent with the ability of biological clones from Babesia to use these multigene families for the expression of GPI-anchor MSA, either conserved (B. canis and B. bovis) or polymorphic (B. divergens and B. bigemina) in their amino acid sequence. Moreover, as a consequence for successful parasitism, the data suggest that both conserved and polymorphic GPI-anchor MSA would present unique B-cell epitopes.     

Antigenic diversity in Babesia divergens: preliminary results with three monoclonal antibodies to the rat-adapted strain

Three murine monoclonal antibodies were raised against the rat-adapted strain of Babesia divergens. In the indirect fluorescent antibody test (IFAT) the monoclonals did not react with B microti or B bovis. The monoclonals in IFAT gave high titres with the rat-adapted strain of B divergens, but showed variable reactivity with field isolates of the parasite indicating antigenic diversity in this parasite. Two of the monoclonals, as ascitic fluid, were protective against the rat-adapted strain in splenectomised rats.     

Sheep as a new experimental host for Babesia divergens

Babesia divergens was cultivated in sheep erythrocytes in RPMI 1640 supplemented with 10% Fetal Calf Serum (FCS) or sheep serum. In vitro cultures in sheep red blood cells were initiated with human erythrocytes infected in vitro with B. divergens Rouen 1987 or with gerbil blood infected with several isolates from bovine origin. After the first subcultures on sheep erythrocytes, a ten-fold multiplication of the parasites was obtained within 48 h. Erythrocytes from three splenectomized sheep were infected in vitro with B. divergens; when parasitaemia reached 10%, the animals were inoculated with homologous parasitized erythrocytes. All sheep expressed hyperthermia with a peak between the 6th and the 9th day post-infection (p-i) and a transitory parasitaemia 10 days p-i. In vitro primary cultures were performed on two of these sheep, demonstrating the parasite persistence at very low parasitaemia in the infected animals. Splenectomized sheep can be used as a new model for B. divergens chronic infection.     

Detection of Theileria and Babesia species in ticks collected from cattle

The present study was carried out to detect tick species that infest cattle, and Theileria and Babesia species transmitted by these ticks in Kayseri province (Turkey). A total of 300 cattle were examined for tick infestations. Of the 300 cattle, 117 (39%) were infested with ticks. A total of 1160 ticks belonging to 11 Ixodid genera were collected from the infested animals and their shelters. The most prevalent tick species was Boophilus annulatus 26.37% (306/1160) followed by Hyalomma marginatum marginatum 21.12% (245/1160) and Rhipicephalus turanicus 18.7% (217/1160). The collected ticks were separated into 43 tick pools, according to their species. These pools were examined for bovine Theileria and Babesia species (Theileria sp., Babesia sp., Theileria annulata, T. buffeli/orientalis, Babesia bigemina, B. bovis and B. divergens) by using the reverse line blotting method (RLB). Of the 43 tick pools examined, 6 (14%) were infected with B. bigemina, 4 (9.3%) with T. annulata, and 1 (2.3%) with Babesia sp., whereas 1 (2.3%) displayed mixed infection with T. annulata + B. bigemina. The sequence and phylogenetic analyses of Babesia sp., which could not be identified to the species level by RLB, were performed. In the phylogenetic tree, Babesia sp. (Kayseri 1) grouped with Babesia sp. (Kashi 2), Babesia sp. (Kashi 1), Babesia sp. (Xinjiang) and B. orientalis with 96.8-100% identity.     

Comparisons of serological tests for Babesia in British cattle.

A comparison was made between the microplate enzyme linked immunosorbent assay (ELISA) and the indirect fluorescent antibody (IFA) and complement fixation (CF) tests for the detection of antibodies in the serum of cattle experimentally infected with Babesia divergens and B major. Antibodies were detected using all three tests but they were detected earlier using the CF test. However CF titres were consistently lower than those obtained using the other tests. Although there was little to choose between the IFA and ELISA tests, it was suggested that the ELISA may be preferable since it is less subject to operator error and operator stress, and can be adapted readily to field use.     

Babesia divergens: cloning of a Ran binding protein 1 homologue

Babesia divergens is an Apicomplexa transmitted to bovines by its acarian vector, the tick I. ricinus. Babesia divergens merozoites have an intraerythrocytic development in the blood of infected mammals. The nucleocytoplasmic transport system in this parasite is not yet characterized and no protein involvement in such transport has been described. In this report, we describe the cloning of a protein that shares important homologies with Ran binding protein 1. This protein in Eukaryote belongs to the nucleocytoplasmic transport system.     

Inhibition of Babesia divergens in cattle by oxytetracycline

The effects of continuous oxytetracycline administration on the development of parasitaemia of Babesia divergens during both natural and artificial infections were studied. During natural exposure on grazing heavily infested with Ixodes ricinus, seven out of 42 cattle with no previous exposure to tick-borne diseases were injected every four days with a long acting preparation of oxytetracycline at a dose rate of 20 mg/kg. During the six week grazing period 21 untreated cattle developed a patent parasitaemia of B divergens and all became seropositive by the fluorescent antibody test. In contrast, no parasites were observed in treated cattle and antibody titres remained low. Artificial infections were studied with different dose levels of oxytetracycline and their effects on antibody stimulation noted. First, four groups of cows were infected with 10(8) erythrocytes infected with B divergens, three groups being injected every four days with the long acting oxytetracycline formulation at dose levels of 20, 10 and 5 mg/kg, respectively. The highest level completely inhibited parasite replication and antibody formation; the same was observed in one animal dosed at 10 mg/kg but the remainder, plus those treated at 5 mg/kg, developed both low parasitaemia and high antibody titres. The untreated cows developed severe babesiosis. A further untreated control group was added and three weeks after cessation of oxytetracycline treatment all were infected with 10(9) erythrocytes infected with a homologous isolate of B divergens. The controls, plus those in which the previous infection had been completely inhibited, developed severe clinical babesiosis but the remainder were refractory to parasite development.     

Detection of Babesia and Theileria species infection in cattle from Portugal using a reverse line blotting method

Babesiosis and Theileriosis are tick-borne diseases widespread in tropical and sub-tropical regions with high economic impact worldwide. In Portugal there are at least 4 tick vectors known to be competent for the transmission of Babesia and Theileria sp. identified: Rhipicephalus bursa, Rhipicephalus (Boophilus) annulatus, Ixodes ricinus and Haemaphysalis punctata. All these potential Babesia and Theileria tick vectors are widely distributed in Portugal, although they are predominant in the Southern region. In this study, 1104 cattle blood samples were randomly collected from Central and Southern regions of Portugal and analyzed by PCR-reverse line blotting (RLB) for the detection of Babesia and Theileria sp. Testing indicated that 74.7% of the bovines tested were positive for either Babesia and/or Theileria sp. In addition, five different apicomplexan species, namely, Theileria buffeli, Theileria annulata, Babesia divergens, Babesia bovis, and Babesia bigemina were detected by RLB among the bovines tested. T. buffeli was the most frequently found species, being present in 69.9% of the positive samples either as single infections (52.4%), or as mixed infections (17.5%). The Babesia specie most frequently found was B. divergens, detected in 4.2% of the infected bovines. Overall, infected bovines were found in all regions tested; however the highest number of infected bovines was observed in évora district (96.2%) and in cattle from Limousin breeds (81.7%). The results indicate widespread Babesia and Theileria infections in Portuguese bovines, suggesting the need for improved control of ticks and tick-borne diseases.     

Isolation of Babesia divergens from carrier cattle blood using in vitro culture

Babesia divergens, the main causative agent of bovine babesiosis in Western Europe, was isolated from naturally infected cattle. Ninety-six blood samples were examined by means of an in vitro culture technique in sheep erythrocytes: 19 of them were collected from animals in the acute phase of the disease with visible parasitemia on blood smears, while the 77 remaining animals showed no microscopically detectable parasites. B. divergens was cultured from the 19 first blood samples as well as from 31 samples collected from asymptomatic animals. The time period before parasites could be detected in the culture varied in the latter samples from 6 to 20 days. The effects of sampling condition (anticoagulant used) and storage length were tested. A good correlation was obtained between immunofluorescent antibody test and culture, with identical results (positive or negative) for 89.6% of the samples collected from asymptomatic animals. The sensitivity of the in vitro culture method was determined and was about 10 parasites/mL of whole blood from three independent experiments performed with three different isolates, confirming its suitability to detect and culture diverse B. divergens isolates from carrier cattle. The parasites could indeed be isolated 9 months after the acute babesiosis phase in the blood of naturally infected animals. The 50 isolates collected in this study were successfully subcultured, cryopreserved and resuscitated using the same culture medium. The in vitro isolation of B. divergens from asymptomatic carrier cattle was achieved and will allow the analysis of parasite diversity within cattle herds.     

A Vegetation Index qualifying pasture edges is related to Ixodes ricinus density and to Babesia divergens seroprevalence in dairy cattle herds

Babesia divergens, transmitted by the tick Ixodes ricinus, is the main agent of bovine piroplasmosis in France. This Apicomplexa often is present in asymptomatic carriers; however, clinical cases are rare. While numerous factors are known to influence tick density, no risk factor of contact with B. divergens has been identified for cattle. Our study aimed to explore whether a Vegetation Index could serve as an indirect indicator of within-herd B. divergens seroprevalence. In February 2007, blood samples were taken from all of the cows in 19 dairy cattle herds in Western France and IFAT serology was performed individually to measure B. divergens seroprevalence. The following spring, I. ricinus nymphs were collected by drag sampling along transects on the vegetation of each farm's pasture perimeters. Tick density was related significantly to a Vegetation Index (V.I., ranging from 1 to 5) that took into account the abundance of trees and bushes on the edge of pastures: most ticks (57%) were found in transects with the highest V.I. (covering 15% of the explored surface in the study area). At the farm level, the proportion of transects presenting I. ricinus nymphs was significantly related to B. divergens seroprevalence: the farms with more than 15% of transects with I. ricinus had a significantly higher risk of high seroprevalence. The proportion of pasture perimeters where the V.I.=5 also was significantly related to B. divergens seroprevalence: the farms where more than 20% of transects had a V.I.=5 had a significantly higher risk of high seroprevalence. Given that the Vegetation Index is a steady indicator of the potential I. ricinus density in the biotope, we recommend that the risk of high B. divergens seroprevalence in cows be evaluated using this tool rather than drag samplings.     

Effects of rapid passage in the gerbil (Meriones unguiculatus) on the course of infection of the bovine piroplasm Babesia divergens in splenectomised calves

The antigenicity and virulence of the cattle piroplasm Babesia divergens was compared in splenectomised calves before and after adaptation to the gerbil. It was apparent that adaptation of B divergens to the gerbil did not result in attenuation of the parasite in cattle. The gerbil-adapted parasite had a shorter prepatent period, produced higher parasitaemias, and more severe haematological and biochemical changes than the initial bovine strain. The antigenicity of the parasite was unchanged by gerbil passage. The changes in the virulence of the gerbil-adapted parasite were attributed to selection of a rapidly replicating parasite.     

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.