Babesia Divergens Research Articles

Development of Culture-Based Serological Assays to DiagnoseBabesia divergensInfections

Babesioses are hematic tick-borne diseases that induce malaria-like disorders in domestic, wild animals, and humans. Although indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) commercial kits are available to test the presence of antibodies against most Babesia species, no kit exists to serologically diagnose the infections due to Babesia divergens, one of the most important zoonotic species. To fill this gap and to develop assays to detect animal and human infections, in vitro cultures (microaerophilous stationary phase system) of B. divergens were organized. Infected erythrocytes were adsorbed as corpuscular antigen (CA) on IFAT slides and ELISA microwells. The supernatant medium of the cultures (metabolic antigen, MA) was collected and employed in ELISA and western blot (WB) assays. B. divergens was also used to produce positive sera in Meriones unguiculatus and to infect a calf. Serological tests were set up with sera from experimentally/naturally infected animals, and possible cross-reactions were evaluated using heterologous sera from cattle positive to other piroplasms. Sera from clinically healthy people at risk of infection were also tested. As expected, assays based on the purified MAs from in vitro cultures proved more sensitive and specific than CA-IFAT and CA-ELISA. In fact, MA-ELISA provided satisfactory performances (even if 8.4%-15.7% cross-reactions were evidenced), and the WB developed proved totally sensitive and specific. WB indicated as immunodominant antigens two major protein bands at 33 and 37 kDa, which were also evidenced in 2.2% of the human sera tested, proving the parasite transmission to humans also in Italy.

Longitudinal field study on bovine Babesia spp. and Anaplasma phagocytophilum infections during a grazing season in Belgium

Anaplasmosis and babesiosis are major tick-borne diseases with a high economic impact but are also a public health concern. Blood samples collected in the spring, summer, and autumn of 2010 from 65 cows in seven different farms in Belgium were monitored with an indirect immunofluorescence antibody test to assess seroprevalence against these pathogens. Seroprevalences to Babesia spp. were measured as 10.7%, 20%, and 12.3% in spring, summer, and autumn, respectively, whereas seroprevalences to Anaplasma phagocytophilum were 30.8%, 77%, and 56.9%, respectively. A total of 805 Ixodes ricinus ticks were collected at the same time from both cattle (feeding ticks) and grazed pastures (questing ticks). The infection level of ticks, assessed by PCR assay, for Babesia spp. DNA was 14.6% and 7.9% in feeding and questing ticks, respectively, whereas 21.7% and 3% of feeding and questing ticks were found be positive for A. phagocytophilum cDNA. Fifty-five PCR-positive samples were identified by sequencing as Babesia sp. EU1, of which five from feeding ticks were positive for both A. phagocytophilum and Babesia sp. EU1. The high density of wild cervids in the study area could explain these observations, as deer are considered to be the main hosts for adults of I. ricinus. However, the absence of Babesia divergens both in feeding and questing ticks is surprising, as the study area is known to be endemic for cattle babesiosis. Increasing cervid populations and comorbidity could play an import role in the epidemiology of these tick-borne diseases.

Transport of Babesia venatorum-infected Ixodes ricinus to Norway by northward migrating passerine birds

BackgroundBovine babesiosis is regarded as a limited health problem for Norwegian cows, and the incidence has decreased markedly since the 1930s. Rare cases of babesiosis in splenectomised humans from infection with Babesia divergens and B.venatorum have been described. The objective of this study was to determine whether birds can introduce Babesia-infected ticks. There are between 30 and 85 million passerine birds that migrate to Norway every spring.MethodsPasserine birds were examined for ticks at four bird observatories along the southern Norwegian coast during the spring migrations of 2003, 2004 and 2005. The presence of Babesia was detected in the nymphs of Ixodes ricinus by real-time PCR. Positive samples were confirmed using PCR, cloning and phylogenetic analyses.ResultsOf 512 ticks examined, real-time PCR revealed five to be positive (1.0%). Of these, four generated products that indicated the presence of Babesia spp.; each of these were confirmed to be from Babesia venatorum (EU1). Two of the four B. venatorum-positive ticks were caught from birds having an eastern migratory route (P< 0.001).ConclusionsBirds transport millions of ticks across the North Sea, the Skagerrak and the Kattegat every year. Thus, even with the low prevalence of Babesia-infected ticks, a substantial number of infected ticks will be transported into Norway each year. Therefore, there is a continuous risk for introduction of new Babesia spp. into areas where I. ricinus can survive.

Fosmidomycin Uptake into Plasmodium and Babesia-Infected Erythrocytes Is Facilitated by Parasite-Induced New Permeability Pathways

BackgroundHighly charged compounds typically suffer from low membrane permeability and thus are generally regarded as sub-optimal drug candidates. Nonetheless, the highly charged drug fosmidomycin and its more active methyl-derivative FR900098 have proven parasiticidal activity against erythrocytic stages of the malaria parasite Plasmodium falciparum. Both compounds target the isoprenoid biosynthesis pathway present in bacteria and plastid-bearing organisms, like apicomplexan parasites. Surprisingly, the compounds are inactive against a range of apicomplexans replicating in nucleated cells, including Toxoplasma gondii.Methodology/Principal FindingsSince non-infected erythrocytes are impermeable for FR90098, we hypothesized that these drugs are taken up only by erythrocytes infected with Plasmodium. We provide evidence that radiolabeled FR900098 accumulates in theses cells as a consequence of parasite-induced new properties of the host cell, which coincide with an increased permeability of the erythrocyte membrane. Babesia divergens, a related parasite that also infects human erythrocytes and is also known to induce an increase in membrane permeability, displays a similar susceptibility and uptake behavior with regard to the drug. In contrast, Toxoplasma gondii-infected cells do apparently not take up the compounds, and the drugs are inactive against the liver stages of Plasmodium berghei, a mouse malaria parasite.Conclusions/SignificanceOur findings provide an explanation for the observed differences in activity of fosmidomycin and FR900098 against different Apicomplexa. These results have important implications for future screens aimed at finding new and safe molecular entities active against P. falciparum and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery.

Novel Diamidines with Activity against Babesia divergens In Vitro and Babesia microti In Vivo

Dicationic diamidines, such as diminazene and pentamidine, are well-studied chemotherapeutic agents with significant activity against parasitic diseases. The in vitro activities of novel diamidine compounds against the Babesia divergens strains 1903B and 4201 were investigated. The most potent compound, a diphenyl furan, had a 50% inhibitory concentration (IC(50)) of 1.5 ng/ml. In a murine model, several test compounds were effective enough to cure mice infected with Babesia microti at a dose of 12.5 and/or 25 mg/kg of body weight given by the subcutaneous route for 4 days. The best antibabesial properties were exhibited by terphenyls, benzimidazoles, diphenyl furans, pentamidine, and pentamidine analogues.

Babesiasp. EU1 Infection in a Forest Reindeer, the Netherlands

<i>Babesia</i>sp. EU1 Infection in a Forest Reindeer, the Netherlands

Detection of Babesia divergens using molecular methods in anemic patients in Shandong Province, China

Babesiosis (piroplasmosis) is a zoonotic disease caused by an intraerythrocytic protozoan transmitted by Ixodes ticks. The aim of this study was to detect Babesia spp. infection using molecular methods in 377 blood samples from anemic patients. Sequence analysis showed that the 18S rRNA gene was 439 bases long by polymerase chain reaction (PCR) amplification and that the PCR products from the samples had an identical sequence (named Taian China, HM355854). BLAST search showed that the sequence was identical to the 18S rRNA sequences of Babesia divergens. The 18S rRNA sequence for Toxoplasma gondii was included as the outlier for phylogenetic analysis by using the program MEGA4.0 software. The results showed that the 18S rRNA gene sequences obtained from the present study was most closely related to B. divergens Switzerland (DQ312439) with 98.4% similarity (differing only by seven bases). The phylogenetic analysis also revealed that this sequence closely resembled B. divergens strains from other countries and belonged to the same clade. This is the first report of a human being infected by B. divergens in China.

Prevalence of Three ZoonoticBabesiaSpecies inIxodes ricinus(Linné, 1758) Nymphs in a Suburban Forest in Switzerland

The tick Ixodes ricinus (Linné, 1758) is known as the vector of various Babesia spp. pathogenic for humans. In Switzerland, three of them, Babesia divergens, Babesia venatorum (also known as Babesia EU1), and Babesia microti, have been reported in I. ricinus ticks from various areas. The aim here was to determine how frequently these species infect I. ricinus nymphs in a suburban forest and to determine their prevalence over 3 years along a pathway delimited in four different sections. Babesia spp. was detected and identified in 44/2568 (1.7%) I. ricinus nymphs using Reverse Line Blot. B. venatorum was infecting 1.1% (27/2568) of nymphs, B. divergens 0.2% (4/2568), and B. microti 0.7% (13/1908). Tick infection rates by these three Babesia species between years were not different except for B. microti, which was significantly less frequent in ticks in 2008 than in 2006 and 2007 according to a test using trusted intervals of percentages. B. microti was displaying the greater difference of prevalence among sampling sections, ranging from 1.6% in section 1 to 0% in section 4. The presence of these three Babesia species that are of medical relevance in a suburban forest where I. ricinus tick density is high requires attention from physicians, particularly for patients presenting unspecific symptoms and for patients who are immunocompromised, and who have history of contact with tick biotopes.

Detection and Characterization ofBabesiaSpecies inIxodesTicks in Estonia

The presence of Babesia spp. was studied in 2603 Ixodes ricinus and Ixodes persulcatus ticks collected at seven sites in Estonia. By reverse line blot screening, Babesia spp. was detected in 36 (1.4%) ticks, among them 18 (0.7%) were further recognized by a Babesia microti probe, 3 (0.1%) by a Babesia divergens probe, and the other 15 (0.6%) were recognized only by the universal Babesia spp. "catch all" probe. Sequence analyses of 6 of these 15 samples revealed that all of them belonged to Babesia sp. EU1. B. microti was detected in both tick species I. ricinus and I. persulcatus at the seven sites, whereas B. divergens-like and Babesia sp. EU1 were found only in I. persulcatus and I. ricinus, respectively. Genetic characterization based on partial 18S rRNA showed that the Estonian sequences of B. microti, B. divergens-like, and Babesia sp. EU1 share a high rate of similarity and are closely related to sequences from other European countries, Siberia, and United States. The present study demonstrated for the first time the existence and distribution of Babesia spp. in I. persulcatus and I. ricinus ticks in Estonia.

The invasion process of bovine erythrocyte by Babesia divergens: knowledge from an in vitro assay

Babesia divergens is a tick-transmitted apicomplexan parasite for which asexual multiplication in its vertebrate hosts is restricted to erythrocytes. Current knowledge of invasion of these target cells is limited. An efficient in vitro invasion assay was set up to gain access to this information. Parasites prepared from infected RBC, lysed by electroporation, and mixed with bovine RBC in a selected synthetic medium (RPMI 1640 supplemented with calcium) were able to establish subsequent cultures with parasitemia ranging from 6 to 14%. Free parasites remaining in the invasion medium could be eliminated by Percoll gradient and culture could be pursued with the freshly invaded erythrocytes. In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion. With this assay we demonstrate that 1) erythrocyte invasion by B. divergens is a rapid process since 70% of the invasion-competent parasites invaded the RBC in less than 45 s; 2) all invasion-competent parasites achieved invasion within 10 min of contact; 3) one erythrocyte could be invaded concomitantly by two merozoites; 4) despite a synchronous start, the parasite population evolved heterogeneously resulting in a progressive loss of synchronisation. Western blot analysis of proteins collected from invasion medium were performed with sera from animals experimentally infected with B. divergens and highlighted several proteins. The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process. Further investigations are required for their characterisation.

Babesiosis in Immunocompetent Patients, Europe

We report 2 cases of babesiosis in immunocompetent patients in France. A severe influenza-like disease developed in both patients 2 weeks after they had been bitten by ticks. Diagnosis was obtained from blood smears, and Babesia divergens was identified by PCR in 1 case. Babesiosis in Europe occurs in healthy patients, not only in splenectomized patients.

Novel Diamidines with Activity against Babesia divergens In Vitro and Babesia microti In Vivo

Novel Diamidines with Activity against Babesia divergens In Vitro and Babesia microti In Vivo

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.

Detection of Babesia divergens in southern Norway by using an immunofluorescence antibody test in cow sera

BackgroundThe incidence of bovine babesiosis, caused by Babesia divergens (Apicomplexa: Piroplasmida) has decreased markedly since the 1930 s, but may re-emerge as a consequence of climate change and changes in legislation and pasturing practices. This is a potentially serious disease, with both economical and animal welfare consequences. Therefore, there is a need to survey the distribution of B. divergens.MethodsWe tested sera from 306 healthy pastured cows from 24 farms along the southern Norwegian coast by using an indirect immunofluorescence IgG antibody test (IFAT). Fractions of seropositive cows were compared by calculating 95% CI.ResultsThe results of this test showed that 27% of the sera were positive for B. divergens antibodies. The fraction of antibody-positive sera that we detected showed a two-humped distribution, with a high fraction of positives being found in municipalities in the western and eastern parts of the study area, while the municipalities between these areas had few or no positive serum samples.ConclusionsNeither the farmers' observations nor the Norwegian Dairy Herd Recording System give an adequate picture of the distribution of bovine babesiosis. Serological testing of cows by using IFAT is a convenient way of screening for the presence of B. divergens in an area.

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.

Identification of three CCp genes in Babesia divergens: Novel markers for sexual stages parasites

Babesia divergens, a tick-borne protozoan parasite of red blood cells, is the main agent of bovine and human babesiosis in Europe. Very few data are available concerning its life cycle and sexual reproduction inside the tick vector, Ixodes ricinus. The aim of this study was to define some markers of the B.divergens sexual stage. An in silico post-genomic approach was used to analyze genomic, transcriptomic and proteomic data and to select specific sexual stage proteins of the related apicomplexan genus Plasmodium. Three proteins, based on sequence identity between the available genomes of Plasmodium and Babesia spp., were chosen, as members of a highly conserved and apicomplexan sexual stages specific protein family (CCp) potentially involved in adhesive functions. Degenerate primers were used to amplify and clone three B.divergens orthologs (bdccp1, bdccp2, and bdccp3) corresponding to newly identified genes in this parasite. The opportunities offered by such markers to study parasite development in its vector are discussed.

Fatal Babesiosis in Man, Finland, 2004

We report an unusual case of human babesiosis in Finland in a 53-year-old man with no history of splenectomy. He had a rudimentary spleen, coexisting Lyme borreliosis, exceptional dark streaks on his extremities, and subsequent disseminated aspergillosis. He was infected with Babesia divergens, which usually causes bovine babesiosis in Finland.

Molecular Survey of Babesia microti in Wild Rodents in Central Croatia

Babesia divergens and B. divergens-like organisms are the main causative agents of human babesiosis in Europe. Recently, the first case of human infection with Babesia microti was confirmed in Germany, implicating the presence of zoonotic isolates. To estimate the presence of zoonotic B. microti in Croatia we analyzed 120 small wild mammals that serve as its reservoir by polymerase chain reaction. Yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) were both found to be infected with prevalence of 16.2%. Sequence analysis of the portion of 18S rDNA gene demonstrated that six polymerase chain reaction-positive samples, detected in both rodent species, were identical to that of the human Jena/Germany strain (EF413181). The other two isolates were identical to the nonzoonotic Munich strain (AB071177). The results of this study indicate the presence of zoonotic B. microti in A. flavicollis and M. glareolus in Croatia and a potential risk for human health.

Coexistence ofBorreliaspp. andBabesiaspp. inIxodes ricinusTicks in Middle Germany

Altogether, 430 nymphs and 570 adult Ixodes ricinus ticks were collected in 2006 (n = 506) and 2007 (n = 494) from a forest area in Middle Germany (Thuringia). Single ticks were investigated by polymerase chain reaction and restriction fragment length polymorphism or sequencing for Borrelia spp. (ospA gene) and Babesia spp. (18S rRNA gene). Overall, 27.0% (270/1000) were infected with Borrelia species. Out of these, Borrelia garinii was detected most frequently (133/270)-especially, OspA serotype 6 (51/270), followed by Bo. burgdorferi (70/270), Bo. afzelii (42/270), Bo. valaisiana subgroup I (28/270), not typable Borrelia spp. (5/270), Bo. spielmanii (3/270), Bo. valaisiana subgroup II (2/270), and Bo. lusitaniae (1/270). In 1.4% of investigated ticks mixed infections with several Borrelia spp. occurred. Babesia spp.-specific DNA was detected in 5.0% of ticks. Babesia microti was slightly more prevalent (28/50) than Babesia divergens (20/50). Moreover, 5.9% (16/270) of Borrelia spp.-infected ticks were coinfected with Babesia spp. Knowledge on the degree of heterogeneity of Borrelia species and OspA types is prerequisite not only for local risk assessment, but also for diagnostic test and vaccine development.

One Health approach to identify research needs in bovine and human babesioses: workshop report

BackgroundBabesia are emerging health threats to humans and animals in the United States. A collaborative effort of multiple disciplines to attain optimal health for people, animals and our environment, otherwise known as the One Health concept, was taken during a research workshop held in April 2009 to identify gaps in scientific knowledge regarding babesioses. The impetus for this analysis was the increased risk for outbreaks of bovine babesiosis, also known as Texas cattle fever, associated with the re-infestation of the U.S. by cattle fever ticks.ResultsThe involvement of wildlife in the ecology of cattle fever ticks jeopardizes the ability of state and federal agencies to keep the national herd free of Texas cattle fever. Similarly, there has been a progressive increase in the number of cases of human babesiosis over the past 25 years due to an increase in the white-tailed deer population. Human babesiosis due to cattle-associated Babesia divergens and Babesia divergens-like organisms have begun to appear in residents of the United States. Research needs for human and bovine babesioses were identified and are presented herein.ConclusionsThe translation of this research is expected to provide veterinary and public health systems with the tools to mitigate the impact of bovine and human babesioses. However, economic, political, and social commitments are urgently required, including increased national funding for animal and human Babesia research, to prevent the re-establishment of cattle fever ticks and the increasing problem of human babesiosis in the United States.