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RNA-seq technology has been widely used for the characterization of the transcriptome profile induced by several diseases in both humans and animals. In the present study, RNA-seq was used to identify the differential expression of genes associated with the immune response in cattle infected with two different strains of Babesia bigemina , both derived from the same Mexican field isolate, which exhibit distinct phenotypic characteristics: the virulent strain, capable of producing acute clinical signs, and the attenuated strain, capable of stimulating a protective immune response when used as an immunogen with an efficacy greater than 80%. The differential gene expression analysis performed revealed a total of 620 differentially expressed genes (DEGs). However, the intersection of the edgeR and DESeq2 programs used in the bioinformatics analysis only identified 247 DEGs, of which 108 genes were enriched to be closely correlated with the bovine immune response based on gene ontology terms; most of the DEGs obtained encode proteins associated with the major histocompatibility complex, immunoglobulins, and T-cell surface receptors. The infection caused by the attenuated strain induced higher transcription of immune response genes compared to the infection caused by the virulent strain; nonetheless, in both infections, a greater down-regulation than up-regulation was observed. Different immunoglobulin-associated genes were found to be up-regulated in the group inoculated with the attenuated strain, whereas these were down-regulated in the virulent strain-inoculated group. In addition, an up-regulation of the HSPA6, CD163, and SLC11a1 genes was observed in the group inoculated with the virulent strain, previously reported in other Apicomplexan infections. The findings provide relevant information that could contribute to clarifying the immune response associated with an acute bovine babesiosis infection by B. bigemina .

Babesia microti is a protozoan that mainly parasitizes rodent and human erythrocytes. B. microti infection can result in changes in the expression levels of various proteins in the host serum. To explore the mechanism underlying the regulation of serum proteins by the host during B. microti infection, this study used a data-independent acquisition (DIA) quantitative proteomic approach to perform comprehensive quantitative proteomic analysis on the serum of B. microti-infected mice. We identified and analysed 333 serum proteins during the infectious stage and recovery stage within 30 days of infection by B. microti in mice. Through quantitative analysis, we found 57 proteins differentially expressed in the infection stage and 69 proteins differentially expressed in the recovery stage. Bioinformatics analysis revealed that these differentially expressed proteins were mainly concentrated in organelles, cell parts, and extracellular regions that are mainly involved in immune system, metabolic, and cellular processes. Additionally, the differentially expressed proteins mainly had catalytic activity. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis showed that many of the differentially expressed proteins participate in the complement and coagulation cascade reaction, including complement C3, complement FP, and coagulation factor XII. The results of this study can provide more information for the selection of biomarkers for the early clinical monitoring of babesiosis and help in the treatment of babesiosis.

Bovine babesiosis is a tick-borne disease caused by intraerythrocytic protozoa and leads to substantial economic losses for the livestock industry throughout the world. Babesia bovis is considered the most pathogenic species, which causes bovine babesiosis in Brazil. Genomic data could be used to evaluate the viability of improving resistance against B. bovis infection level (IB) through genomic selection, and, for that, knowledge of genetic parameters is needed. Furthermore, genome-wide association studies (GWAS) could be conducted to provide a better understanding of the genetic basis of the host response to B. bovis infection. No previous work in quantitative genetics of B. bovis infection was found. Thus, the objective of this study was to estimate the genetic correlation between IB and tick count (TC), evaluate predictive ability and applicability of genomic selection, and perform GWAS in Hereford and Braford cattle. The single-step genomic best linear unbiased prediction method was used, which allows the estimation of both breeding values and marker effects. Standard phenotyping was conducted for both traits. IB quantifications from the blood of 1,858 animals were carried using quantitative PCR assays. For TC, one to three subsequent tick counts were performed by manually counting adult female ticks on one side of each animal's body that was naturally exposed to ticks. Animals were genotyped using the Illumina BovineSNP50 panel. The posterior mean of IB heritability, estimated by the Bayesian animal model in a bivariate analysis, was low (0.10), and the estimations of genetic correlation between IB and TC were also low (0.15). The cross-validation genomic prediction accuracy for IB ranged from 0.18 to 0.35 and from 0.29 to 0.32 using k-means and random clustering, respectively, suggesting that genomic predictions could be used as a tool to improve genetics for IB, especially if a larger training population is developed. The top 10 single nucleotide polymorphisms from the GWAS explained 5.04% of total genetic variance for IB, which were located on chromosomes 1, 2, 5, 6, 12, 17, 18, 16, 24, and 26. Some candidate genes participate in immunity system pathways indicating that those genes are involved in resistance to B. bovis in cattle. Although the genetic correlation between IB and TC was weak, some candidate genes for IB were also reported in tick infestation studies, and they were also involved in biological resistance processes. This study contributes to improving genetic knowledge regarding infection by B. bovis in cattle., (Copyright © 2020 Cavani, Braz, Giglioti, Okino, Gulias-Gomes, Caetano, Oliveira, Cardoso and Oliveira.)

Background: Babesiosis, a tick-borne disease caused by protozoans of the genus Babesia, is widespread in subtropical and tropical countries. Mitochondria are essential organelles that are responsible for energy transduction and metabolism, calcium homeostasis and cell signaling. Mitochondrial genomes could provide new insights to help elucidate and investigate the biological features, genetic evolution and classification of the protozoans. Nevertheless, there are limited data on the mitochondrial genomes of ovine Babesia spp. in China., Methods: Herein, we sequenced, assembled and annotated the mitochondrial genomes of six ovine Babesia isolates; analyzed the genome size, gene content, genome structure and cytochrome b (cytb) amino acid sequences and performed comparative mitochondrial genomics and phylogenomic analyses among apicomplexan parasites., Results: The mitochondrial genomes range from 5767 to 5946 bp in length with a linear form and contain three protein-encoding genes, cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 3 (cox3) and cytb, six large subunit rRNA genes (LSU) and two terminal inverted repeats (TIR) on both ends. The cytb gene sequence analysis indicated the binding site of anti-Babesia drugs that targeted the cytochrome bc1 complex. Babesia microti and Babesia rodhaini have a dual flip-flop inversion of 184-1082 bp, whereas other Babesia spp. and Theileria spp. have one pair of TIRs, 25-1563 bp. Phylogenetic analysis indicated that the six ovine Babesia isolates were divided into two clades, Babesia sp. and Babesia motasi. Babesia motasi isolates were further separated into two small clades (B. motasi Hebei/Ningxian and B. motasi Tianzhu/Lintan)., Conclusions: The data provided new insights into the taxonomic relationships and drug targets of apicomplexan parasites.

Canine babesiosis is a serious disease among tick-borne haemoprotozoan diseases, globally. The present study was envisaged for carrying out thorough investigation of the disease among working dogs of organised kennels situated in different agro-climatic zones of India as comprehensive understanding of the disease from this country was pertinently lacking. During the study period of three years (2012-2014), 330 dogs suspected for babesiosis were examined for clinicopathology by their physical examination, haematological and biochemical parameters estimation, while the detection of apicomplexan parasites was confirmed by using various diagnostic techniques i.e. by conventional microscopy, by two different Babesia specific 18S rRNA based PCR protocols (conventional/simple PCR and nested PCR assays) followed by sequencing of obtained PCR amplicons for Babsesia spp. identification. Out of 330 clinical cases screened 5.15% (17/330), 9.09% (30/330) and 15.45% (51/330) were found to be positive in microscopic examination, simple- and nested- PCR assay, respectively. Comparative statistical analyses of these diagnostic assay results revealed that significant difference exists among the three diagnostic methodologies and thus it is recommended that the nested PCR technique be relied upon as a screening molecular assay and also for epidemiological studies of the disease in this country. Phylogenetic analysis based on 18S rRNA depicted the monophyletic nature and clonal expansion among all the B. gibsoni, under study. Sequencing results of PCR amplicons revealed that B. gibsoni has predominantly established itself over B. vogeli as former was incriminated in 47 cases while latter was confirmed in only four animals. Based on the clinical severity, these 51 affected animals were classified into three main groups' of 17 animals each viz., apparently healthy-, simple or uncomplicated babesiosis- and atypical or complicated babesiosis- group. Haematological and biochemical profiling of these dogs confirmed the characteristics findings of infection by both the Babesia spp. It was observed that the infection by small form of Babesia (B. gibsoni) is posing a significant therapeutic challenge and chemosterilization by commonly prescribed anti-protozoal drugs was not achieved as clinical relapses were often observed. The clinical signs, sequence based confirmation and severity of the infection suggested that there is a positive selection of B. gibsoni (smaller form) over B. vogeli (larger form) in this country and raises serious concerns as prognosis in former is considered to be poor compared to latter. Thus, these findings have opened new paradigms for planning of pragmatic control strategies against this emerging canine health problem., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bovine babesiosis caused by protozoan parasites Babesia bovis and B. bigemina is one of the most important causes of losses for the livestock industry in tropical and subtropical regions of the world. Therefore, highly sensitive and specific tools for these hemoparasites detection and monitoring are required, especially in carrier animals, in which low parasite levels were usually present. In this context, qPCR assays have been successfully and fairly used in last years. Aiming to improve the performance of Babesia levels monitoring by qPCR, some of main aspects of this methodology that may influence results were tested: DNA extraction kits, whole blood EDTA pre-treatment, blood source (tip of tail or jugular vein), erythrocytes isolation, FTA card interference and qPCR system of detection. Under our experimental conditions, both EDTA pre-treatment and FTA card application have no influence on the sensitivity of detection, and two DNA extraction kits provided higher sensitivity compared to others. As expected, blood samples collected from the tip of tail vessels presented higher levels of B. bovis DNA compared to those obtained from the jugular vein, and erythrocytes processed isolated has also improved the sensitivity compared to whole blood. Moreover, both qPCR assays here developed using hydrolysis probes for B. bovis and B. bigemina detection, presented enhanced reproducibility compared to qPCR assays using intercalating dye system. Even, qPCR for B. bigemina using hydrolysis probe here developed presented higher sensitivity compared to intercalating dye system. This study has contributed to the improvement of molecular diagnosis of bovine babesiosis, which may improve epidemiological studies related to these pathogens.

Phosphorylation is the most commonly studied protein post-translational modification (PTM) in biological systems due to its importance in controlling cell division, survival, growth, etc. Despite the thorough research in phosphoproteomics of cells and tissues there is little information on circulating phosphoproteins. We compared serum from 10 healthy dogs and 10 dogs affected by B. canis-caused babesiosis with no organ dysfunctions by employing gel-free LC-MS/MS analysis of individual samples and tandem mass tag (TMT) label-based quantitative analyses of pools, both supported by phosphopeptide enrichment. Results showed a moderate number of phosphorylated proteins (50-55), with 89 phosphorylation sites not previously published for dogs although a number of them matched phosphorylation sites found in mammalian orthologs. Three phosphopeptides showed significant variation in babesiosis-affected dog sera compared to controls: Serum amyloid A (SAA) phosphorylated at serine 101 (up-regulation), kininogen 1 phosphorylated at threonine 326, and fibrinogen α phosphorylated at both threonine 20 and serine 22 (down-regulation). 71.9% of the detected phosphorylated sites were phosphoserine, 16.8% phosphothreonine and only 11.2% phosphotyrosine residues. TMT label-based quantitative analysis showed α-2-HS-glycoprotein / Fetuin A to be the most abundant phosphoprotein (50-70% of all phosphoproteins) followed by kininogen-1 (10-20%). The alterations of phosphorylated proteins observed in canine babesiosis caused by Babesia canis suggest new insights into the largely neglected role of extracellular protein phosphorylation in health and disease, encouraging urgent further research on this area. To the best of our knowledge the present study represents the first attempt to characterize canine serum phosphoproteome., Competing Interests: The authors have declared that no competing interests exist.

A 67-year-old woman presented with 5 days of myalgias and fevers on completion of a 21-day course of amoxicillin for Lyme disease ( Borrelia burgdorferi infection). She was found to have profound thrombocytopenia, as well as new anaemia and leucopenia. Workup revealed Babesia microti as the causative agent of her symptoms. The patient quickly improved after appropriate antimicrobial therapy directed against babesiosis was started. This case illustrates the importance of basic microbiology, including epidemiology and common vectors, when creating a differential diagnosis. Because the Ixodes scapularis tick can harbour and transmit multiple parasites simultaneously, the possibility of coinfection should be considered in any patient not responding to appropriate initial medical therapy., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2018. No commercial re-use. See rights and permissions. Published by BMJ.)

The aim of the paper was to study the epizootic situation of babesiosis in the cattle population in eastern Poland and possibly to determine what species of protozoa infects Polish cattle. Blood samples for molecular analysis (real time PCR) were collected from 192 dairy cows from various farms located in eastern Poland. The infection was detected in 10.4% of the samples. All animals were infected with Babesia occultans which sequence of the 18S RNA gene fragment showed a 93.1%, homology with the sequence of B. occultans EU 376017. This is the first report about the detection of B. occultans DNA in asymptomatic cattle in eastern Poland., (Copyright© by the Polish Academy of Sciences.)

Infections of dogs with virulent strains of Babesia canis are characterized by rapid onset and high mortality, comparable to complicated human malaria. As in other apicomplexan parasites, most Babesia virulence factors responsible for survival and pathogenicity are secreted to the host cell surface and beyond where they remodel and biochemically modify the infected cell interacting with host proteins in a very specific manner. Here, we investigated factors secreted by B. canis during acute infections in dogs and report on in silico predictions and experimental analysis of the parasite's exportome. As a backdrop, we generated a fully annotated B. canis genome sequence of a virulent Hungarian field isolate (strain BcH-CHIPZ) underpinned by extensive genome-wide RNA-seq analysis. We find evidence for conserved factors in apicomplexan hemoparasites involved in immune-evasion (e.g. VESA-protein family), proteins secreted across the iRBC membrane into the host bloodstream (e.g. SA- and Bc28 protein families), potential moonlighting proteins (e.g. profilin and histones), and uncharacterized antigens present during acute crisis in dogs. The combined data provides a first predicted and partially validated set of potential virulence factors exported during fatal infections, which can be exploited for urgently needed innovative intervention strategies aimed at facilitating diagnosis and management of canine babesiosis.

Babesia microti, a tick-transmitted, intraerythrocytic protozoan parasite circulating mainly among small mammals, is the primary cause of human babesiosis. While most cases are transmitted by Ixodes ticks, the disease may also be transmitted through blood transfusion and perinatally. A comprehensive analysis of genome composition, genetic diversity, and gene expression profiling of seven B. microti isolates revealed that genetic variation in isolates from the Northeast United States is almost exclusively associated with genes encoding the surface proteome and secretome of the parasite. Furthermore, we found that polymorphism is restricted to a small number of genes, which are highly expressed during infection. In order to identify pathogen-encoded factors involved in host-parasite interactions, we screened a proteome array comprised of 174 B. microti proteins, including several predicted members of the parasite secretome. Using this immuno-proteomic approach we identified several novel antigens that trigger strong host immune responses during the onset of infection. The genomic and immunological data presented herein provide the first insights into the determinants of B. microti interaction with its mammalian hosts and their relevance for understanding the selective pressures acting on parasite evolution.

Human babesiosis is a tick-borne multisystem disease caused by Babesia species of the apicomplexan phylum. Most clinical cases and fatalities of babesiosis are caused by Babesia microti Current treatment for human babesiosis consists of two drug combinations, atovaquone + azithromycin or quinine + clindamycin. These treatments are associated with adverse side effects and a significant rate of drug failure. Here, we provide evidence for radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone (ELQ) prodrug and atovaquone. In vivo efficacy studies in mice using ELQ-271, ELQ-316, and the ELQ-316 prodrug, ELQ-334, demonstrated excellent growth inhibitory activity against the parasite, with potency equal to that of orally administered atovaquone at 10 mg/kg. Analysis of recrudescent parasites after ELQ or atovaquone monotherapy identified genetic substitutions in the Qi or Qo sites, respectively, of the cytochrome bc1 complex. Impressively, a combination of ELQ-334 and atovaquone, at doses as low as 5.0 mg/kg each, resulted in complete clearance of the parasite with no recrudescence up to 122 d after discontinuation of therapy. These results will set the stage for future clinical evaluation of ELQ and atovaquone combination therapy for treatment of human babesiosis., (© 2016 Lawres et al.)

Background: Almost all of the reported US tick-borne and transfusion-associated Babesia cases have been caused by Babesia microti, which is endemic in the Northeast and upper Midwest. We investigated a case caused by B. duncani (formerly, the WA1-type parasite), in a 59-year-old California resident with sickle cell disease (HbSS) whose only risk factor for infection was receipt of red blood cell transfusions., Case Report: The patient's case was diagnosed in September 2008: intraerythrocytic parasites were noted on a blood smear, after a several-month history of increasing transfusion requirements. Molecular and indirect fluorescent antibody (IFA) analyses were negative for B. microti but were positive for B. duncani (IFA titer, 1:1024). The complete 18S ribosomal RNA gene of the parasite was amplified from a blood specimen; the DNA sequence was identical to the sequence for the index WA1 parasite isolated in 1991. The patient's case prompted a transfusion investigation: 34 of 38 pertinent blood donors were evaluated, none of whom tested positive by B. microti IFA. The implicated donor-a 67-year-old California resident-had a B. duncani titer of 1:4096; B. duncani also was isolated by inoculating jirds (Mongolian gerbils) with a blood specimen from March 2009, more than 10 months after his index donation in April 2008. The patient's case was diagnosed more than 4 months after the implicated transfusion in May 2008., Conclusions: This patient had the third documented transfusion case caused by B. duncani. His case underscores the fact that babesiosis can be caused by agents not detected by molecular or serologic analyses for B. microti., (© 2011 American Association of Blood Banks.)

Endemic stability is a widely used term in the epidemiology of ticks and tick-borne diseases. It is generally accepted to refer to a state of a host-tick-pathogen interaction in which there is a high level of challenge of calves by infected ticks, absence of clinical disease in calves despite infection, and a high level of immunity in adult cattle with consequent low incidence of clinical disease. Although endemic stability is a valid epidemiological concept, the modelling studies that underpinned subsequent studies on the epidemiology of tick-borne diseases were specific to a single host-tick-pathogen system, and values derived from these models should not be applied in other regions or host-tick-pathogen systems., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: The aim of this work was to study the prevalence and genetic diversity of Babesia in Ixodes persulcatus ticks and small mammals from Ural and Siberia in Russia., Methods: In total, 481 small mammals and 922 questing adult I. persulcatus from North Ural (Sverdlovsk region) and West Siberia (Novosibirsk region) were examined for the presence of Babesia by nested PCR based on the 18S rRNA gene., Results: Babesia microti of the 'Munich'-type was found in 36.2% of blood samples of the small mammals from the Sverdlovsk region and B. microti of the 'US'-type in 5.3% of the animals from the Novosibirsk region. Babesia DNA was not detected in 133 analysed I. persulcatus from the Sverdlovsk region; however, it was found in 24 of 789 ticks from the Novosibirsk region. Three distinct Babesia species were detected in I. persulcatus. B. microti 'US'-type was identified in 10 ticks, Babesia closely related to B. divergens/B. capreoli in 2 ticks, and Babesia closely related to B. venatorum (EU1) in 12 ticks., Conclusion: To our knowledge, this is the first detection of Babesia sensu stricto in I. persulcatus ticks and of B. microti in I. persulcatus in the Asian part of Russia.

The genetic diversity of Babesia gibsoni strains worldwide is currently poorly defined. The aim of the present study was to characterize B. gibsoni strains in naturally infected dogs in Taiwan using a combination of polymerase chain reaction (PCR) and sequence analysis of both 18S rDNA and the gene encoding thrombospondin-related adhesive protein (TRAP). Genomic DNA was extracted from 29 parasitemic dogs, and the target genes were separately amplified, sequenced and aligned with corresponding sequences available in GenBank. All 18S rDNA sequences (1,262 bp) amplified from the Taiwanese isolates were identical to each other and had very high similarity (99.9-100%) with previously reported B. gibsoni sequences. These results provide the first molecular evidence showing infection of dogs with B. gibsoni from Taiwan. On the other hand, a phylogenetic analysis based on the deduced amino acid sequence of the TRAP gene demonstrated that the Taiwanese isolates were closely related to strains previously identified from Okinawa Island, Japan, but genetically distinct from strains found on Honshu in Japan and Jeju Island in South Korea. The divergence of TRAP among the geographically dispersed strains examined in this study and others supports the conclusion that this gene is useful for molecular genotyping of B. gibsoni strains.

The Babesia species "BQ1 (Lintan)" is infective to sheep and goats. The species was isolated from Haemaphysalis qinghaiensis collected in the Gannan Tibet Autonomous Region, China in April 2000. In this study, an in vitro culture system was developed for the propagation of Babesia sp. BQ1 (Lintan). Continuous cultivation and 5.0% parasitemia was obtained in vitro in RPMI 1640 medium with sheep red blood cells (RBC) (7.5%) supplemented with Fetal Bovine Serum (FBS) (20%), Amphotericin B (0.5 microg/ml) and Gentamicin (50 microg/ml) in an incubator at 37 degrees C and 6% CO(2) in 24-well and 6-well plates. Parasitemia could attain 10% in 75 cm(2) flasks with the same culture medium but with 2.5% RBC. A clonal line of Babesia sp. BQ1 (Lintan) was screened using the limiting dilution method and designated G7. Growth of Babesia sp. BQ1 (Lintan) in vitro was measured by microtitre-based spectrophotometric method and from parasitemia counts. The generation time was between 20.57 h (based the A(405) of the culture supernatant) and 26.41 h (based on parasitemia). Three French sheep were successfully infected with the culture and the infectivity of the clonal line G7 was determined. Finally, this in vitro culture system was used to compare the susceptibility (capacity to sustain Babesia sp. growth in vitro) of RBC from French sheep (Vendéen breed) and Chinese sheep (Tan mutton breed) for Babesia sp. BQ1 (Lintan) and B. divergens. The lower susceptibility to B. divergens and Babesia sp. BQ1 (Lintan) of RBC from French sheep, compared to Chinese sheep, is discussed., ((c) 2010 Elsevier B.V. All rights reserved.)

The Duffy antigen is the only receptor for Plasmodium vivax, a hemoparasite of the phylum Apicomplexa and the cause of vivax malaria in humans. Resistance to this parasite in the majority of black African individuals and their descendents is due to a mutation in the gene promoter region, which blocks its transcription on erythrocytes. Regarding bovine babesiosis, it is known that taurine breeds are more susceptible to parasite infection than zebuine breeds. In order to verify whether the same human resistance occurs in bovine, the 5' flanking region of the DARC gene was isolated and characterized in Bos indicus and Bos taurus. Four single nucleotide polymorphisms were identified and genotyped (SNP1: EF_647729.1:g.91C>T; SNP2: EF_647729.1:g.405C>T; SNP3: EF_647729.1: g.433A>G and SNP4: EF_647729.1:g.588A>G), which showed significant frequency differences among 99 bovines of each species (n=198). Characterization of the isolated region revealed the presence of 6 putative haplotypes, 14 genotypes, which are formed by haplotypes, and numerous putative transcription factor binding sites. Only the thymine presence on SNPs 1 and 2, more common in B. indicus, was observed to alter some of the sites in this region. Despite this fact, analyses through real-time PCR on bovines that present the most common homozygote genotypes of each species, which contrast for all the polymorphism, revealed no difference on the DARC gene transcription. Thus, in principle, it was concluded that the polymorphisms identified would not be useful as molecular markers in an improvement program for resistance to babesiosis.

A multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of canine blood parasites, Ehrlichia canis, Babesia spp and Hepatozoon canis, from blood samples in a single reaction. The multiplex PCR primers were specific to E. canis VirB9, Babesia spp 16S rRNA and H. canis 16S rRNA genes. Specificity of the amplicons was confirmed by DNA sequencing. The assay was evaluated using normal canine and infected blood samples, which were detected by microscopic examination. This multiplex PCR offers scope for simultaneous detection of three important canine blood parasites and should be valuable in monitoring parasite infections in dogs and ticks.

Bovine babesiosis is a tick-borne disease caused by Babesia spp. haemoprotozoans. The disease is of great importance at tick enzootic unstable areas and hampers cattle production in several developing countries. The available immunisation alternatives are pre-immunition and attenuated vaccines. Despite being efficient and protective, they are unsafe as they use cattle blood cells as inoculum and may potentially spread other diseases. Another alternative to help in babesiosis control would be the identification of genetically resistant cattle to Babesia bovis infection. The objective of this work was to phenotype cattle based on primary response against B. bovis infection. Two-hundred and forty half-sib Hereford and Aberdeen Angus heifers (120 animals from each breed), 12-18-month-old naïve cattle, originated from a tick-free area in Southern Brazil, were used in the experiment. Animals were monitored following an inoculation with 1x10(7)B. bovis parasitised erythrocytes. Results showed three different phenotypes: 1-'susceptible', animals with babesiosis clinical signs that received treatment to avoid death; 2-'intermediate', animals with clinical signs: parasitaemia, >or=21.5% reduction in packed cell volume (PCV) and increase in body temperature when compared to their pre-challenge physiological parameters, no specific treatment was needed as animals self recovered from the disease, and 3-'resistant', animals without clinical signs that showed B. bovis presence in blood smears, <21.5% PCV reductions, with little or no increase in body temperature and no need for babesiosis treatment. The frequencies of each phenotype were: 45.4, 26.7, and 27.9%, respectively, demonstrating the existence of phenotypic variation for B. bovis in Bos taurus cattle.

A 42-year-old female patient with acute myeloid leukemia presented with fever and heavy chest pain after her first cycle of specific chemotherapy. Acute myocardial infarction was excluded, but surprisingly, parasitic inclusions in erythrocytes became obvious in Pappenheim and Giemsa-stained peripheral blood smears. The patient did not remember a tick bite but acknowledged having received several blood transfusions in her recent medical history. Suspicion of malaria was ruled out by use of a dip-stick test. The diagnosis of Babesia microti infection was finally established by specific polymerase chain reaction (PCR). Six weeks after initiation of specific treatment, PCR turned negative and a positive immunoflourescence assay (IFA) with an IgG titer of 1:128 indicated seroconversion. Subsequent screening of donors involved in the transfusion of blood products to the patient demonstrated borderline reactivity for Babesia microti (IgG-titer 1:32) in 1 out of 44 individuals. Neither the patient nor the positively tested blood donor had travelled to North America or Asia. Therefore, this is the first confirmed autochthonous human infection in Europe.

Members of the Babesiarap-1 gene family are expressed during multiple parasite stages, and are regulated by both transcriptional and post-transcriptional mechanisms. In all Babesia species, tandemly arranged rap-1 gene copies are separated by an intergenic (IG) region that is hypothesized to regulate gene expression. In this study, we tested that hypothesis by determining whether the Babesia bovisrap-1 IG region could promote extra-chromosomal expression of exogenous genes introduced into merozoites by transfection, and whether a tandem arrangement of IG regions similar to the rap-1 locus enhances exogenous gene expression. Initially, electroporation conditions of B. bovis parasites were determined using expression of the reporter luciferase gene. Both B. bovis transfected by electroporation and Escherichia coli transformed with plasmid p40-15-luc containing the luciferase gene under the control of the B. bovisrap-1 IG and 3' flanking regions were able to express luciferase, indicating that the rap-1 IG region contains a functional promoter. The chromosomal organization of the B. bovisrap-1 locus includes two identical rap-1 open reading frames and IG regions in a head to tail orientation. To determine whether this orientation enhanced expression of exogenous genes, plasmid constructs containing two rap-1-IG regions controlling expression of the luc and human dihydrofolate reductase (hdhfr) genes, and oriented either in head to head (pLuc-H-13) or head to tail (pLuc-H-18) arrangement, were compared. The head to tail orientation of the gene cassettes resulted in a significant increase in the level of luciferase as compared to either head to head orientation or a single IG region construct (p40-15-luc). Thus, an organization that mimics the native structure of the rap-1 locus results in enhanced luciferase expression. These results are the first to demonstrate exogenous gene expression in B. bovis after transfection, and to confirm that the B. bovisrap-1 IG region can promote extra-chromosomal gene expression in vivo.

Our previous report demonstrated that small wild rodents in Japan harbored two types of novel Babesia microti-like parasites (Kobe and Hobetsu types), but not the type widely distributed throughout the temperate zones of North American and Eurasian Continents (U.S. type). In this study, we surveyed small wild mammals collected at various places in the northern part of Japan, seeking for U.S.-type B. microti. A total of 197 small mammals comprising 10 species, Apodemus speciosus, A. argenteus, Clethrionomys rufocanus, C. rutilus, Eothenomys andersoni, Microtus montebelli, Tamias sibiricus, Sorex unguiculatus, S. caecutiens, and Urotrichus talpoides, were examined. Babesia parasites were detected in A. speciosus, C. rufocanus, C. rutilus, M. montebelli, S. unguiculatus, and S. caecutiens by microscopy of blood smears and by PCR targeting babesial nuclear small-subunit rRNA (rDNA) and beta-tubulin genes. Inoculation of their bloods into experimental animals gave rise to 23 parasite isolates, which included 16 from A. speciosus, 4 from C. rufocanus, and 1 each from C. rutilus, M. montebelli and S. unguiculatus. Sequencing analyses of their rDNA and beta-tubulin genes revealed that, of the 23 isolates, 20 and 3 were of Hobetsu and U.S. types, respectively. The U.S.-type B. microti strains isolated in Japan, however, were distinguishable from the isolates in the United States when their beta-tubulin gene sequences and antigen profiles in Western blots were compared. We conclude that U.S.-type B. microti exists in Japan although it has been genetically and antigenically diversified from that distributed in the United States. The results also suggest that not only rodents, but also some insectivores may serve as reservoirs for the agent of human babesiosis.

Background: Although infection by the protozoan Babesia microti is rarely symptomatic in immunocompetent young people, healthy individuals aged >50 years may experience life-threatening disease. To determine the basis for this age relationship, we developed a mouse model of babesiosis using a novel clinical isolate of B. microti., Methods: Mice were infected at 2, 6, 12, or 18 months. Parasitemia was monitored on Giemsa-stained blood smears or by flow cytometry., Results: In DBA/2 mice, early and persistent parasitemias increased with age at infection. BALB/c and C57BL/6 mice were resistant, regardless of age, which indicates that allelic variation determines resistance to B. microti. Unlike immunocompetent mice, SCID mice, which retain an innate immune system but lack the lymphocytes needed for adaptive immunity, developed high and persistent levels of parasitemia that were markedly reduced by transfer of naive BALB/c or DBA/2 splenocytes. BALB/c cells reduced the persistent parasitemia to a greater extent than did age-matched DBA/2 cells. Of importance, there was an age-associated loss of protection by cells of both strains., Conclusion: The resistance to B. microti infection conferred by the adaptive immune system is genetically determined and associated with age. We postulate that there are age-related differences in the expression of alleles critical for adaptive immunity to B. microti.

Recent advances in genomic technology have focused many veterinary researchers on the possibility of producing one multivalent recombinant vaccine against all the haemoparasites that infect cattle in the tropics. Before such a vaccine is developed it is essential to define target cattle populations as well as the range of anti-pathogen vaccines required in order to control disease. To further this objective, we have evaluated a reverse line blot (RLB) assay, which simultaneously detects the principal tick transmitted protozoan and rickettsial cattle pathogens, in different epidemiological scenarios in Uganda. A critical question is the sensitivity, particularly in relation to detecting carrier animals. As Theileria parva is considered to be the most important pathogen in the region, we assessed the sensitivity of the RLB assay for T. parva and showed that 1-2 x 10(3) parasites per ml of blood could be detected-a level comparable with previously developed PCR methods and well below conventional microscopic detection. We applied the RLB assay to evaluate the differences in pathogen profiles between crossbred and indigenous cattle and show that there were different profiles, with a low prevalence of T. parva and Theileria taurotragi in the indigenous cattle compared to a high prevalence in the crossbred cattle. In contrast, we show higher prevalences of Theileria mutans and Theileria velifera in the indigenous compared to the crossbred cattle. Interestingly Anaplasma marginale, Babesia bovis and Babesia bigemina were of low prevalence but a high prevalence of Ehrlichia bovis was seen, raising the question of whether this rickettsial species could be pathogenic in cattle. Analysis of animals with clinical symptoms of East Coast Fever showed that, while T. parva is a major cause of these symptoms, T. mutans and possibly T. taurotragi and T. velifera, may also cause clinical disease. Overall, the results presented here highlight the complexity of tick-borne pathogen infections in cattle in Uganda.

The use of microscopy, infection of golden hamsters and the polymerase chain reaction made it possible to find out that about 30% of common red-backed voles (Clethrionomys glareolus), inhabiting the taiga forests of the southern part of the Western Urals (the Chusovskoi district of the Perm region), were infected with Babesia microti and simultaneously (a third of them) with Ehrlichia (Cytoecetes) phagocytophila, the causative agent of granulocytic ehrlichiosis. The sequencing of 18S rDNA of strain "Mys", isolated in Russia, revealed its identity to American B. microti strain GI, pathogenic for humans. The main vector supporting the circulation of B. microti in the natural foci in the region where these investigations were conducted was, seemingly, the tick Ixodes trianguliceps, Thus, for the first time the data proving the presence of reservoir hosts infected with B. microti and granulocytic E. phagocytophila, pathogenic for humans, in Russia were presented.

Infection of severe combined immunodeficient mice with Babesia sp. strain WA1 was studied to assess the contributions of innate and adaptive immunity in resistance to acute babesiosis. The scid mutation showed little effect in genetically susceptible C3H mice and did not decrease the inherent resistance of C57BL/6 mice to the infection, suggesting that innate immunity plays a central role in determining the course of Babesia infection in these strains. In contrast, the scid mutation dramatically impaired resistance in moderately susceptible BALB/c mice, suggesting that acquired immunity may play an important secondary role. In comparison to their female counterparts, male mice of different genetic backgrounds showed increased resistance to the infection, indicating that the gender of the host may influence protection against babesiosis.

Babesia bovis is an intraerythrocytic protozoan that causes bovine babesiosis. Agarose gel electrophoresis of nucleic acids extracted from two isolates of B. bovis reveals, besides bulk DNA, an ethidium bromide-stainable band at about 5.5 kb. Further characterization of the latter with DNase I, RNase and mung bean nuclease suggested it to be a double-stranded RNA. Sonicated parasites were fractionated in a CsCl buoyant density gradient. A sample containing the 5.5-kb RNA was analysed under an electron microscope and a virus-like particle was observed.

The effect of infection by Babesia microti, a tick-borne piroplasm endemic to the northeastern United States, on the temporal pattern of micronucleated erythrocyte frequencies in peripheral blood was investigated in male Syrian golden hamsters. Significantly greater frequencies of micronucleated erythrocytes occurred in the blood of infected hamsters from 26 to 46 days after injection with B. microti, the magnitude of which within individual hamsters correlated highly with the percentage of polychromatic erythrocytes and the extent of parasitization. These data suggest that parasitic infection and other factors which alter the rate of erythropoiesis should be considered when the micronucleus assay is used in environmental or laboratory studies of genetic toxicity.

Babesia canis infection was diagnosed in a litter of seven 3-week-old Mastiff pups kept in a north Florida kennel. The pups were evaluated because of poor weight gain; the smallest pup also was markedly lethargic. Six of the pups were anemic and thrombocytopenic. A positive linear correlation between PCV and absolute reticulocyte count suggested that the variation in PCV may have been related more to the ability of a pup to increase erythrocyte production than to a difference in magnitude of erythrocyte destruction. All pups recovered from clinical signs and hematologic abnormalities attributable to babesiosis within 2.5 weeks after treatment with diminazene aceturate. Transient neurologic signs observed in 1 pup 3 days after treatment were believed to represent an adverse drug reaction. The dam of the litter had a serum titer of 1:640 for B canis, but appeared healthy, as did approximately 30 other adult dog in the kennel. The strain of B canis infecting dogs in the kennel caused severe illness and death in some pups, but clinically inapparent disease in adult dogs.

Striking differences in the susceptibility to P. chabaudi and B. microti infections among different strains of mice are described. In most of the strains, both parasites induce a moderate, transient parasitaemia, followed by recovery. However, in the A strain P. chabaudi is rapidly lethal and B. microti induces a parasitaemia which persists for life in most animals. In contrast B10.A mice, which have the same H-2q haplotype as the A strain, recover from both infections. In first crosses (A X B10.A)F1 mice are uniformly resistant. Among other correlates, NK cells activity may be relevant. This activity is known to be high in strain of mice which are resistant to both of the parasites studied, such as C57B1 and CBA mice, and low in the susceptible A strain. We have examined the spleen weight, total cell number and NK activity at different times after infection. In the resistant strains there is a rapid increase in size and cell number which is still more marked during recovery. NK activity increases greatly during the infection; this is especially remarkable considering the dilution of nucleated cells with erythrocytes precursors. In contrast, in A mice cell numbers of NK activity remain almost unchanged. Thus, marked activation of NK cells occur, in resistant strains but not in susceptible ones. Possible mechanisms of activation of these cells and their effect in haemoprotozoan infections are discussed.

Two groups of Droughtmaster cattle, one group previously infected, were challenged with Babesia argentina infected erythrocytes. All animals were typed for haemoglobin and the course of infection followed by regular parasite counts. No conclusive relationship between the frequencies of the HbA and HbB genes and the severity of parasitaemia in the different groups was found. The presence of HbF was not associated with any limitations on the severity of infection in 1 1/2 - 2 1/2 months calves.