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Background Babesia bovis, an intra-erythrocytic apicomplexan parasite, is one of the causative agents of bovine babesiosis, the most important tick-borne disease of cattle in tropical and subtropical regions. Babesia bovis has a complex life-cycle that includes sexual development within the tick vector. The development of a transmission blocking vaccine to control bovine babesiosis requires the identification of antigens displayed on the surface of the parasite during its development within tick vectors. Four B. bovis cysteine-rich GCC2/GCC3 domain protein (BboGDP) family members were previously identified and are differentially expressed as discrete pairs by either blood stages or kinetes. In this study we focused on two family members, BboGDP1 and -3, that are expressed by Babesia parasites during tick infection. Methods and results Transcription analysis using quantitative PCR demonstrated that BboGDP1 and -3 were upregulated in in vitro-induced sexual stage parasites and during parasite development in the tick midgut. Moreover, protein expression analysis of BboGDP1 and -3 during the development of sexual stages in in vitro culture was consistent with their transcription profile. Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of BboGDP1 and -3 on in vitro-induced sexual stage parasites. In addition, fixed immunofluorescence analysis showed reactivity of anti-BboGDP1 and -3 polyclonal antibodies to kinetes. Conclusions The collective data indicate that BboGDP1 and -3 are expressed by kinetes and on the surface of sexual stages of the parasites. The identified parasite surface membrane proteins BboGDP1 and -3 are potential candidates for the development of a B. bovis transmission blocking vaccine. Graphical Abstract

Bovine babesiosis caused by Babesia bigemina and Babesia bovis is an economically important disease that affects cattle worldwide. Both B. bigemina and B. bovis are transovarially transmitted by Rhipicephalus ticks. However, little is known regarding parasite gene expression during infection of the tick vector or mammalian host, which has limited the development of effective control strategies to alleviate the losses to the cattle industry. To understand Babesia gene regulation during tick and mammalian host infection, we performed high throughput RNA-sequencing using samples collected from calves and Rhipicephalus microplus ticks infected with B. bigemina. We evaluated gene expression between B. bigemina blood-stages and kinetes and compared them with previous B. bovis RNA-seq data. The results revealed similar patterns of gene regulation between these two tick-borne transovarially transmitted Babesia parasites. Like B. bovis, the transcription of several B. bigemina genes in kinetes exceeded a 1,000-fold change while a few of these genes had a >20,000-fold increase. To identify genes that may have important roles in B. bigemina and B. bovis transovarial transmission, we searched for genes upregulated in B. bigemina kinetes in the genomic datasets of B. bovis and non-transovarially transmitted parasites, Theileria spp. and Babesia microti. Using this approach, we identify genes that may be potential markers for transovarial transmission by B. bigemina and B. bovis. The findings presented herein demonstrate common Babesia genes linked to infection of the vector or mammalian host and may contribute to elucidating strategies used by the parasite to complete their life cycle.

The apicomplexan tickborne parasites Babesia bovis and B. bigemina are the major causative agents of bovine babesiosis, a disease that negatively affects the cattle industry and food safety around the world. The absence of correlates of protection represents one major impediment for the development of effective and sustainable vaccines against bovine babesiosis. Herein we superinfected cattle with attenuated and virulent strains of B. bovis to investigate immune correlates of protection against acute bovine babesiosis. Three 6-month-old Holstein calves were infected intravenously (IV) with the in vitro culture attenuated Att-S74-T3Bo B. bovis strain (106 infected bovine red blood cells (iRBC)/calf) while three age-matched Holstein calves were inoculated IV with normal RBC as controls (106 RBC/calf). All Att-S74-T3Bo-infected calves showed a significant increase in temperature early after inoculation but recovered without treatment. Att-S74-T3Bo-infected calves also developed: (a) monocytosis, neutropenia, and CD4+ lymphopenia in peripheral blood on days 3 to 7 post-inoculation; (b) significant levels of TNFα, CXCL10, IFNγ, IL-4, and IL-10 in sera at day 6 after infection; and (c) IgM and IgG against B. bovis antigens, starting at days 10 and 30 post-inoculation, respectively. At 46 days post-Att-S74-T3Bo inoculation, all experimental calves were infected IV with the homologous virulent B. bovis strain Vir-S74-T3Bo (107 iRBC/calf). All Att-S74-T3Bo-infected calves survived superinfection with Vir-S74-T3Bo without displaying signs of acute babesiosis. In contrast, control animals showed signs of acute disease, starting at day 10 post-Vir-S74-T3Bo infection, and two of them were humanely euthanized at days 13 and 14 after inoculation due to the severity of their symptoms. Also, control calves showed higher (P

Bovine babesiosis caused by

The tick-transmitted disease bovine babesiosis causes significant economic losses in many countries around the world. Current control methods include modified live-attenuated vaccines that have limited efficacy. Recombinant proteins could provide effective, safe, and low-cost alternative vaccines. We compared the expression of the

The apicomplexan tickborne parasites

Tick midgut is the primary infection site required by tick-borne pathogens to initiate their development for transmission. Despite the biological significance of this organ, cell cultures derived exclusively from tick midgut tissues are unavailable and protocols for generating primary midgut cell cultures have not been described. To study the mechanism of Anaplasma marginale-tick cell interactions, we successfully developed an in vitro Dermacentor andersoni primary midgut cell culture system. Midgut cells were maintained for up to 120 days. We demonstrated the infection of in vitro midgut cells by using an A. marginale omp10::himar1 mutant with continued replication for up to 10 days post-infection. Anaplasma marginale infection of midgut cells regulated the differential expression of tick α-(1,3)-fucosyltransferases A1 and A2. Silencing of α-(1,3)-fucosyltransferase A2 in uninfected midgut cells reduced the display of fucosylated glycans and significantly lowered the susceptibility of midgut cells to A. marginale infection, suggesting that the pathogen utilized core α-(1,3)-fucose of N-glycans to infect tick midgut cells. This is the first report using in vitro primary D. andersoni midgut cells to study A. marginale-tick cell interactions at the molecular level. The primary midgut cell culture system will further facilitate the investigation of tick-pathogen interactions, leading to the development of novel intervention strategies for tick-borne diseases.

Bovine babesiosis is caused by apicomplexan pathogens of the genus

Bovine babesiosis is a tick-transmitted disease caused by intraerythrocytic protozoan parasites of the genus Babesia. Its main causative agents in the Americas are Babesia bigemina and Babesia bovis, while Babesia ovata affects cattle in Asia. All Babesia species secrete proteins stored in organelles of the apical complex, which are involved in all steps of the invasion process of vertebrate host cells. Unlike other apicomplexans, which have dense granules, babesia parasites instead have large, round intracellular organelles called spherical bodies. Evidence suggests that proteins from these organelles are released during the process of invading red blood cells, where spherical body proteins (SBPs) play an important role in cytoskeleton reorganization. In this study, we characterized the gene that encodes SBP4 in B. bigemina. This gene is transcribed and expressed in the erythrocytic stages of B. bigemina. The sbp4 gene consists of 834 nucleotides without introns that encode a protein of 277 amino acids. In silico analysis predicted a signal peptide that is cleaved at residue 20, producing a 28.88-kDa protein. The presence of a signal peptide and the absence of transmembrane domains suggest that this protein is secreted. Importantly, when cattle were immunized with recombinant B. bigemina SBP4, antibodies identified B. bigemina and B. ovata merozoites according to confocal microscopy observations and were able to neutralize parasite multiplication in vitro for both species. Four peptides with predicted B-cell epitopes were identified to be conserved in 17 different isolates from six countries. Compared with the pre-immunization sera, antibodies against these conserved peptides reduced parasite invasion in vitro by 57%, 44%, 42%, and 38% for peptides 1, 2, 3, and 4, respectively (p < 0.05). Moreover, sera from cattle infected with B. bigemina cattle contained antibodies that recognized the individual peptides. All these results support the concept of spb4 as a new gene in B. bigemina that should be considered a candidate for a vaccine to control bovine babesiosis.

Babesia bovis natural field strains are composed of several geno-phenotypically distinct subpopulations. This feature, together with possible epigenetic modifications, may facilitate adaptation to variable environmental conditions. In this study we compare geno-phenotypical features among long-term (more than 12 years) (LTCP) and short-term cultured B. bovis parasites (STCP) derived from the B. bovis S74-T3Bo strain. LTCPs intraerythrocytic forms are smaller in size than STCPs and have faster in vitro growth rate. In contrast to its parental strain, the LTCP lack expression of the sexual stage specific 6cysA and 6cysB proteins and are unable to develop sexual forms upon in vitro sexual stage induction. Consistently, in contrast to its parental strain, LTCPs have reduced virulence and are not transmissible to cattle by vector competent Rhipicephalus microplus (R. microplus). Similar to previous comparisons among attenuated and virulent B. bovis strains, the LTCP line has decreased genomic diversity compared to the STCP line. Thus, LTCP may contribute to our understanding of adaptive mechanisms used by the parasites in response to environmental changes, protective immunity, virulence, and transmission by ticks. In addition, LTCPs may be considered as candidates for a non-tick transmissible vaccine against bovine babesiosis.

Control of Brucella ovis infection in sheep flocks in the United States depends on early detection of B. ovis antibodies via serologic testing. We used 2,276 sheep sera and various cutoff values to compare seroprevalence and agreement between 2 ELISAs: the National Veterinary Services Laboratories (NVSL) B. ovis indirect ELISA and the IDEXX B. ovis ELISA kit. A subset of 295 sera was used to compare agreement and evaluate relative sensitivity and specificity of the 2 ELISAs with an agar gel immunodiffusion (AGID) test kit. There was no significant difference in B. ovis seroprevalence between the ELISAs; however, there was poor agreement between them. When the AGID test was used as the reference test, the IDEXX ELISA with a moderate cutoff value (S/P ratio = 45%) had the highest relative sensitivity of 38.1% and specificity of 92.0%. The NVSL ELISA with a lax cutoff value (S/P ratio = 0.75) had relative sensitivity of 19.1% and specificity of 94.6%. Receiver operating characteristic analysis revealed that optimal cutoff values for the NVSL and IDEXX ELISAs were 0.091 and 16.5%, respectively. This results in sensitivity and specificity of 85.7% and 31.8% for the NVSL ELISA, and sensitivity and specificity of 81.0% and 53.6% for the IDEXX ELISA, respectively.

Equine piroplasmosis (EP) is an infectious, tick-borne disease caused by the hemoprotozoan parasites, Theileria equi, Babesia caballi, and a recently reported new species, T. haneyi. Infections by these apicomplexan parasites limit performance and cause economic losses for the horse industry. Equine piroplasmosis is widespread in the northern regions of Nigeria, where an increasing portion of the animal population is composed of horses. This disease has remained epidemiologically challenging, especially as the movement of horses increases across Nigeria. In this study, blood samples from 300 horses were collected in three states of northwestern Nigeria. The presence of piroplasms was screened by nested PCR targeting 18S rDNA and positive samples were analyzed using species-specific-nested PCR-targeting genes including ema1 (T. equi), rap1 (B. caballi), and a gene coding a protein of unknown function (T. haneyi). Species-specific-nPCR results demonstrated that the prevalence of T. equi was 13.0% (39/300), B. caballi was 3.3% (10/300) and T. haneyi was 2.7% (8/300). Mixed infections with T. equi and B. caballi was 2.7% (8/300) while T. equi, B. caballi, and T. haneyi multiple infection prevalence was 0.6% (2/300). We used 18S rDNA sequences to determine close relationships between T. equi by phylogenetic analysis and demonstrated that among 57 sequences of Theileria parasites, 28 samples belonged to clade A (49%), 13 samples were found to be clade C (22%), and 16 were clade D (28%). These results demonstrate the genetic diversity of T. equi circulating in horses from Nigeria.

Background Vector-borne diseases pose an increasing threat to global food security. Vaccines, diagnostic tests, and therapeutics are urgently needed for tick-borne diseases that affect livestock. However, the inability to obtain significant quantities of pathogen stages derived from ticks has hindered research. In vitro methods to isolate pathogens from infected tick vectors are paramount to advance transcriptomic, proteomic, and biochemical characterizations of tick-borne pathogens. Methods Nymphs of Rhipicephalus appendiculatus were infected with Theileria parva by feeding on a calf during an acute infection. Isolation of sporozoites was accomplished by feeding infected adult ticks on an in vitro tick feeding system. Sporozoite viability was tested using in vitro bovine lymphocytes. Results We isolated infectious T. parva sporozoites secreted into an in vitro tick feeding system. Infected adult R. appendiculatus ticks attached to and successfully fed on silicone membranes in the in vitro tick feeding system. Bovine blood in the receptacle was replaced with cell-free medium and the ticks were allowed to feed for 3 h to collect secreted T. parva sporozoites. Secreted sporozoites infected in vitro bovine lymphocytes, demonstrating that isolated sporozoites remained viable and infectious. Conclusions This work is the first to report the isolation of mature infectious T. parva sporozoites using an in vitro tick feeding system, which represents a significant step towards the development of a more efficient control strategy for T. parva. Isolation of infectious tick-stage parasites will facilitate the examination of the vector-pathogen interface, thereby accelerating the development of next-generation vaccines and treatment interventions for tick-borne pathogens. Graphical Abstract

Arthropod-borne apicomplexan pathogens remain a great concern and challenge for disease control in animals and humans. In order to prevent Babesia infection, the discovery of antigens that elicit protective immunity is essential to establish approaches to stop disease dissemination. In this study, we determined that poly-N-acetylglucosamine (PNAG) is conserved among tick-borne pathogens including B. bovis, B. bigemina, B. divergens, B. microti, and Babesia WA1. Calves immunized with synthetic ß-(1→6)-linked glucosamine oligosaccharides conjugated to tetanus toxoid (5GlcNH2-TT) developed antibodies with in vitro opsonophagocytic activity against Staphylococcus aureus. Sera from immunized calves reacted to B. bovis. These results suggest strong immune responses against PNAG. However, 5GlcNH2-TT-immunized bovines challenged with B. bovis developed acute babesiosis with the cytoadhesion of infected erythrocytes to brain capillary vessels. While this antigen elicited antibodies that did not prevent disease, we are continuing to explore other antigens that may mitigate these vector-borne diseases for the cattle industry.

The tick-transmitted disease bovine babesiosis causes significant economic losses in many countries around the world. Current control methods include modified live-attenuated vaccines that have limited efficacy. Recombinant proteins could provide effective, safe, and low-cost alternative vaccines. We compared the expression of the Babesia bovis thrombospondin-related anonymous protein (TRAP) family from parasites in bovine blood, in vitro induced sexual stages, and kinetes from tick hemolymph. Quantitative PCR showed that in blood and sexual stages, TRAP3 was highly transcribed as compared to the other TRAPs. In contrast, the TRAP1 gene was highly transcribed in kinetes as compared to the other TRAPs. Fixed immunofluorescence assays showed that TRAP2, 3, and 4 proteins were expressed by both blood and sexual stages. Conversely, TRAP1 protein, undetected on blood and induced sexual stages, was the only family member expressed by kinetes. Live IFA revealed that TRAP2, 3, and 4 proteins were expressed on the surface of both B. bovis blood and sexual stages. Modeling of B. bovis TRAP1 and TRAP4 tertiary structure demonstrated both proteins folded the metal-ion-dependent adhesion site (MIDAS) domain structure of Plasmodium TRAP. In conclusion, TRAP proteins may serve as potential vaccine targets to prevent infection of bovine and ticks with B. bovis essential for controlling the spread of bovine babesiosis.

Equine piroplasmosis is a disease of horses, mules and donkeys, caused by the hemoprotozoans Babesia caballi and Theileria equi and transmitted by ticks of tropical and subtropical regions. Because the clinical signs are not specific, the diagnosis of equine piroplasmosis is difficult. In Mexico, where the environmental factors are conducive to the persistence of these pathogens, there is a lack of molecular studies to evaluate the occurrence of both parasites in horses. In the present study, matching serum and whole blood samples were obtained from 269 horses residing in 24 locations with tropical or subtropical climate and the presence of ticks. Testing of serum samples by ELISA demonstrated 55.7% seroprevalence of B. caballi and 68.4% prevalence of antibodies to T. equi. Blood samples analyzed with nPCR test were 7.8% positive to B. caballi and 78.8% positive to T. equi, while a duplex qPCR showed 15.24% positive samples to B. caballi and 59.11% to T. equi. From these results, 27 samples were sequenced for T. equi and 13 for B. caballi, confirming the presence of both horse parasites that cause equine piroplasmosis and suggesting that they are widespread in Mexico. This is the first study confirming the presence of B. caballi and T. equi in Mexico using both serological and molecular diagnostic methods. This study shows a high incidence of exposure to the etiological agents of equine piroplasmosis in horses in the studied areas.

Background Babesia bovis is one of the most significant tick-transmitted pathogens of cattle worldwide. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. Each life stage has developmental forms that differ in morphology and metabolism. Differentiation between these forms is highly regulated in response to changes in the parasite’s environment. Understanding the mechanisms by which Babesia parasites respond to environmental changes and the transmission cycle through the biological vector is critically important for developing bovine babesiosis control strategies. Results In this study, we induced B. bovis sexual stages in vitro using xanthurenic acid and documented changes in morphology and gene expression. In vitro induced B. bovis sexual stages displayed distinctive protrusive structures and surface ruffles. We also demonstrated the upregulation of B. bovis calcium-dependent protein kinase 4 (cdpk4), tubulin-tyrosine ligase (ttl), and methyltransferase (mt) genes by in vitro induced sexual stages and during parasite development within tick midguts. Conclusions Similar to other apicomplexan parasites, it is likely that B. bovis upregulated genes play a vital role in sexual reproduction and parasite transmission. Herein, we document the upregulation of cdpk4, ttl, and mt genes by both B. bovis in vitro induced sexual stages and parasites developing in the tick vector. Understanding the parasite's biology and identifying target genes essential for sexual reproduction will enable the production of non-transmissible live vaccines to control bovine babesiosis. Graphical abstract

Bovine babesiosis is the most important protozoan disease transmitted by ticks. In Plasmodium falciparum, another Apicomplexa protozoan, the interaction of rhoptry neck protein 2 (RON2) with apical membrane antigen-1 (AMA-1) has been described to have a key role in the invasion process. To date, RON2 has not been described in Babesia bigemina, the causal agent of bovine babesiosis in the Americas. In this work, we found a ron2 gene in the B. bigemina genome. RON2 encodes a protein that is 1351 amino acids long, has an identity of 64% (98% coverage) with RON2 of B. bovis and contains the CLAG domain, a conserved domain in Apicomplexa. B. bigemina ron2 is a single copy gene and it is transcribed and expressed in blood stages as determined by RT-PCR, Western blot, and confocal microscopy. Serum samples from B. bigemina-infected bovines were screened for the presence of RON2-specific antibodies, showing the recognition of conserved B-cell epitopes. Importantly, in vitro neutralization assays showed an inhibitory effect of RON2-specific antibodies on the red blood cell invasion by B. bigemina. Therefore, RON2 is a novel antigen in B. bigemina and contains conserved B-cell epitopes, which induce antibodies that inhibit merozoite invasion.

The apicomplexan parasite Theileria haneyi is one of two known causative agents of equine theileriosis. It causes milder clinical disease than its more virulent counterpart, Theileria equi, in experimentally infected horses, and can superinfect T. equi-positive horses. The current equi merozoite antigen 1 (EMA1)-based competitive enzyme-linked immunosorbent assay (ELISA)used in the U.S. to detect equine theileriosis detects T. equi but not T. haneyi, and the complexity of molecular assays precludes widespread use for epidemiologic studies. In order to facilitate urgently needed studies on the prevalence of T. haneyi, the goal of this study was to develop a sensitive and specific serologic assay for the diagnosis of T. haneyi based on the equi merozoite antigen 11 (ThEMA11). To achieve this objective, ThEMA11 was recombinantly expressed in eukaryotic cells and its antigenicity assessed using sera from T. haneyi-experimentally infected horses. Confirmation of sera reactivity enabled design and optimization of an indirect ELISA. Specificity of the ELISA for T. haneyi was assessed using a cohort of sera from horses experimentally infected and confirmed PCR-positive for either T. equi or T. haneyi. Data from field samples further demonstrate that the ThEMA11 ELISA is capable of identifying T. haneyi antibodies in horses from multiple continents around the world.

Background Theileria orientalis is a tick-borne hemoparasite that causes anemia, ill thrift, and death in cattle globally. The Ikeda strain of T.orientalis is more virulent than other strains, leading to severe clinical signs and death of up to 5% of affected animals. Within the Asia–Pacific region, where it affects 25% of Australian cattle, T.orientalis Ikeda has a significant economic impact on the cattle industry. In 2017, T.orientalis Ikeda was detected in a cattle herd in Albermarle County, Virginia, United States. Months earlier, the U.S. was alerted to the invasion of the Asian longhorned tick, Haemaphysalis longicornis, throughout the eastern U.S. Abundant H.longicornis ticks were identified on cattle in the T.orientalis-affected herd in VA, and a subset of ticks from the environment were PCR-positive for T.orientalis Ikeda. A strain of T.orientalis from a previous U.S. outbreak was not transmissible by H.longicornis; however, H.longicornis is the primary tick vector of T.orientalis Ikeda in other regions of the world. Thus, the objective of this study was to determine whether invasive H.longicornis ticks in the U.S. are competent vectors of T.orientalis Ikeda. Methods Nymphal H.longicornis ticks were fed on a splenectomized calf infected with the VA-U.S.-T.orientalis Ikeda strain. After molting, a subset of adult ticks from this cohort were dissected, and salivary glands assayed for T.orientalis Ikeda via qPCR. The remaining adult ticks from the group were allowed to feed on three calves. Calves were subsequently monitored for T.orientalis Ikeda infection via blood smear cytology and PCR. Results After acquisition feeding on a VA-U.S.-T.orientalis Ikeda-infected calf as nymphs, a subset of molted adult tick salivary glands tested positive by qPCR for T.orientalis Ikeda. Adult ticks from the same cohort successfully transmitted T.orientalis Ikeda to 3/3 naïve calves, each of which developed parasitemia reaching 0.4–0.9%. Conclusions Our findings demonstrate that U.S. H.longicornis ticks are competent vectors of the VA-U.S.-T.orientalis Ikeda strain. This data provides important information for the U.S. cattle industry regarding the potential spread of this parasite and the necessity of enhanced surveillance and control measures. Graphical Abstract

Control of Brucella ovis infection in sheep flocks in the United States depends on early detection of B. ovis antibodies via serologic testing. We used 2,276 sheep sera and various cutoff values to compare seroprevalence and agreement between 2 ELISAs: the National Veterinary Services Laboratories (NVSL) B. ovis indirect ELISA and the IDEXX B. ovis ELISA kit. A subset of 295 sera was used to compare agreement and evaluate relative sensitivity and specificity of the 2 ELISAs with an agar gel immunodiffusion (AGID) test kit. There was no significant difference in B. ovis seroprevalence between the ELISAs; however, there was poor agreement between them. When the AGID test was used as the reference test, the IDEXX ELISA with a moderate cutoff value (S/P ratio = 45%) had the highest relative sensitivity of 38.1% and specificity of 92.0%. The NVSL ELISA with a lax cutoff value (S/P ratio = 0.75) had relative sensitivity of 19.1% and specificity of 94.6%. Receiver operating characteristic analysis revealed that optimal cutoff values for the NVSL and IDEXX ELISAs were 0.091 and 16.5%, respectively. This results in sensitivity and specificity of 85.7% and 31.8% for the NVSL ELISA, and sensitivity and specificity of 81.0% and 53.6% for the IDEXX ELISA, respectively.

Bovine babesiosis is a tick-borne disease caused by apicomplexan parasites of the Babesia genus that represents a major constraint to livestock production worldwide. Currently available vaccines are based on live parasites which have archetypal limitations. Our goal is to identify candidate antigens so that new and effective vaccines against Babesia may be developed. The perforin-like protein (PLP) family has been identified as a key player in cell traversal and egress in related apicomplexans and it was also identified in Babesia, but its function in this parasite remains unknown. The aim of this work was to define the PLP family in Babesia and functionally characterize PLP1, a representative member of the family in Babesia bovis. Bioinformatic analyses demonstrate a variable number of plp genes (four to eight) in the genomes of six different Babesia spp. and conservation of the family members at the secondary and tertiary structure levels. We demonstrate here that Babesia PLPs contain the critical domains present in other apicomplexan PLPs to display the lytic capacity. We then focused on the functional characterization of PLP1 of B. bovis, both in vitro and in vivo. PLP1 is expressed and exposed to the host immune system during infection and has high hemolytic capacity under a wide range of conditions in vitro. A B. bovis plp1 knockout line displayed a decreased growth rate in vitro compared with the wild type strain and a peculiar phenotype consisting of multiple parasites within a single red blood cell, although at low frequency. This phenotype suggests that the lack of PLP1 has a negative impact on the mechanism of egression of the parasite and, therefore, on its capacity to proliferate. It is possible that PLP1 is associated with other proteins in the processes of invasion and egress, which were found to have redundant mechanisms in related apicomplexans. Future work will be focused on unravelling the network of proteins involved in these essential parasite functions.

In this study, we report Babesia bigemina proliferation in culture medium free of components of animal origin supplemented with a lipid mixture. Babesia bigemina continuously proliferated in VP-SFM with a higher percent parasitized erythrocyte as compare to using other animal component-free culture media. Compared with Advanced DMEM/F12 (ADMEM/F12), VP-SFM had a similar percent parasitized erythrocyte (PPE). Supplementation of VP-SF with a lipid acid mixture improved B. bigemina proliferation in vitro culture, with a maximum PPE of 11.3%. Growth of B. bigemina in a perfusion bioreactor using VP-SFM medium supplemented with lipid mixture resulted in a PPE above 28%. In conclusion, we demonstrated that B. bigemina proliferated in an animal component-free medium supplemented with the fatty acid mixture. This innovation to B. bigemina in vitro culture method presented herein is an important source of biological material for live vaccine production and understanding the mechanisms and molecules involved in parasite attachment and invasion of bovine erythrocytes.

A novel apicomplexan parasite was serendipitously discovered in horses at the United States – Mexico border. Phylogenetic analysis based on 18S rDNA showed the erythrocyte-infective parasite to be related to, but distinct from, Theileria spp. in Africa, the most similar taxa being Theileria spp. from waterbuck and mountain zebra. The degree of sequence variability observed at the 18S rDNA locus also suggests the likely existence of additional cryptic species. Among described species, the genome of this novel equid Theileria parasite is most similar to that of Theileria equi, also a pathogen of horses. The estimated divergence time between the new Theileria sp. and T. equi, based on genomic sequence data, is greater than 33 million years. Average protein sequence divergence between them, at 23%, is greater than that of Theileria parva and Theileria annulata proteins, which is 18%. The latter two represent highly virulent Theileria spp. of domestic cattle, as well as of African and Asian wild buffalo, respectively, which differ markedly in pathology, host cell tropism, tick vector and geographical distribution. The extent of genome-wide sequence divergence, as well as significant morphological differences, relative to T. equi justify the classification of Theileria sp. as a new taxon. Despite the overall genomic divergence, the nine member equi merozoite antigen (EMA) superfamily, previously found as a multigene family only in T. equi, is also present in the novel parasite. Practically, significant sequence divergence in antigenic loci resulted in this undescribed Theileria sp. not being detectable using currently available diagnostic tests. Discovery of this novel species infective to equids highlights exceptional diversity within the genus Theileria, a finding with serious implications for apicomplexan parasite surveillance.

Tropical theileriosis is a serious animal disease transmitted by tick vectors. The agents of theileriosis are obligate intracellular parasites that cause mild to severe disease in the mammalian host. Tropical theileriosis has been recognized as a burden to the development of the dairy industry in Sudan and causes major economic losses. However, knowledge about the distribution of Theileria spp. in Sudan and the extent of sequence variation within the 18S rRNA gene is currently unknown. The aim of this study was to determine the diversity of Theileria spp. using 18S rRNA-based PCR to detect parasites in cattle followed by cloning and sequencing. We observed an overall prevalence rate of 63% hemoparasite infection in cattle from Sennar state. A subset of samples was used for cloning and sequencing of the 18S rRNA gene. Nineteen of 44 animals were co-infected with more than one species of Theilera. Phylogenetic analysis revealed three Theileria spp. that were predominant in cattle including pathogenic T. annulata and apathogenic T. velifera and T. mutans. The present study provides information regarding the prevalence of theileriosis in Sudan and will help to design strategies to control it. Additionally, more study is needed to determine tick vector competence and degree of coinfection with multiple Theileria spp. in Sudan. This represents the first molecular phylogeny report to identify Theileria spp. in cattle from Sudan.

Cattle fever ticks, Rhipicephalus microplus and R. annulatus have been eradicated from the United States and inspectors from the U.S. Department of Agriculture (USDA), Animal Plant Health Inspection Service (APHIS), Cattle Fever Tick Eradication Program (CFTEP) monitor the quarantine zone along the Texas border to prevent the introduction of livestock carrying cattle fever ticks from Mexico. Stray livestock apprehended by CFTEP in the zone are checked for ticks and tested for infectious disease-causing pathogens but are not evaluated for evidence of infection with tick-borne pathogens. We tested blood samples collected from stray cattle by CFTEP inspectors for evidence of infection with tick-borne pathogens. As a comparison group representing U.S. resident cattle, we tested blood samples that had been sent to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) for unrelated testing. Both sets of blood samples were evaluated using the same specific and broad-spectrum PCR assays. For the border cattle the overall prevalence of infection with one or more tick-borne pathogen was 58.5 % (79/135) with many co-infections, including 30 cattle positive for Babesia bovis and/or Babesia bigemina (22.2 %) and 77 cattle positive for Anaplasma marginale (57 %), three of these animals were also positive for Borrelia theileri. No resident cattle represented by the TVMDL samples were infected with either of the Babesia spp., or with Borrelia theileri, but three were positive for Theileria orientalis and 7.3 % (7/96) were positive for A. marginale. These data show that cattle originating in Mexico have a higher prevalence of infection with tick-borne pathogens relative to resident U.S. cattle and specifically, a proportion are infected with bovine Babesia, which is absent from U.S. cattle populations. Consequently, these stray cattle may be a reservoir of tick-borne pathogens and if populations of Boophilus ticks become reestablished in areas where they had previously been eradicated, could pose a significant risk to the U.S. Cattle industry.

Background Rhipicephalus microplus is an efficient biological vector of Babesia bovis, a causative agent of bovine babesiosis. Babesia bovis is passed transovarially to the next generation of ticks, which then transmit the parasite to naïve animals. Due to the importance of the R. microplus ovary for tick reproduction and transmission of B. bovis, we investigated the hypothesis that silencing vitellogenin receptor gene expression in the ovary during tick feeding on B. bovis-infected cattle would affect parasite transmission to the next generation of ticks. Results Silencing expression of the vitellogenin receptor in the ovary by RNA interference, resulted in reduced tick fertility. We observed reduced egg production (i.e. reduced weight of eggs), a lower rate of embryonic development, and a reduction in hatching. Analysis of individual larvae by PCR confirmed that RNAi mediated downregulation of the R. microplus vitellogenin receptor and also interfered with transovarial transmission of B. bovis. None of the larvae (0/58) from the RmVgR dsRNA-injected group were PCR-positive, whereas 12% (7/58) and 17% (10/58) of larvae from the non-injected and buffer-injected control groups, respectively, were infected with B. bovis. Conclusions The combined effects of reduced fecundity and reduced infection in surviving larvae resulting from silencing indicate that vitellogenin receptor is essential for tick reproduction and may play a vital role in B. bovis transmission.

Theileria equi infection, exotic to the United States has reemerged through intravenous (iatrogenic) and tick-borne transmission. Surveillance at the US-Mexico border identified a new species, Theileria haneyi, (T. haneyiEP) (EP = Eagle Pass, Texas) which warranted additional investigation due to inability to detect by PCR targeting of T. equi ema-1 and EMA-1-cELISA validated for T. equi. Infection dynamics of T. haneyiEP were evaluated, including ability to superinfect in the presence of T. equi-Texas (T. equiTX), the isolate responsible for the reemergence of T. equi in the U S. Experimental infection with T. equiTX or T. haneyiEP revealed minimal clinical disease however, T. equiTX infection led to significantly greater neutropenia. Comparison of time to antibody detection following inoculation revealed significantly greater time to detectable anti-T. haneyiEP antibody (26.67 days post-inoculation (DPI)) than T. equiTX (11.67 DPI). Regardless of initial infection with either T. equiTX or T. haneyiEP, superinfection was established. Comparative analysis of antibody responses from a splenectomized horse infected with T. haneyiEP to that of a spleen intact horse infected with T. equiFL revealed a different antibody binding profile to T. haneyiEP, T. equiTX and T. equiFL merozoite antigen and limited shared antigen/cross-reactive antibody(s). Affinity purified T. equi EMA-1 and EMA-2 from T. equiFL were shown as targets for horse antibodies against T. haneyi. Data presented here show (1) T. haneyiEP can superinfect in the presence of T. equiTX infection and co-persists for minimally 25 months, (2) intravenous challenge with T. haneyi is subclinical, and (3) limited cross-reactive antibody between T. haneyiEP and T. equi includes reactivity to EMA-1 and EMA-2.

N-glycosylation has remained mostly unexplored in Piroplasmida, an order of tick-transmitted pathogens of veterinary and medical relevance. Analysis of 11 piroplasmid genomes revealed three distinct scenarios regarding N-glycosylation: Babesia sensu stricto (s.s.) species add one or two N-acetylglucosamine (NAcGlc) molecules to proteins, Theileria equi and Cytauxzoon felis add (NAcGlc)2-mannose, while B. microti and Theileria s.s. synthesize dolichol-P-P-NAcGlc and dolichol-P-P-(NAcGlc)2 without subsequent transfer to proteins. All piroplasmids possess the gene complement needed for the synthesis of the N-glycosylation substrates, dolichol-P and sugar nucleotides. The oligosaccharyl transferase of Babesia species, T. equi and C. felis, is predicted to be composed of only two subunits, STT3 and Ost1. Occurrence of short N-glycans in B. bovis merozoites was experimentally demonstrated by fluorescence microscopy using a NAcGlc-specific lectin. In vitro growth of B. bovis was significantly impaired by tunicamycin, an inhibitor of N-glycosylation, indicating a relevant role for N-glycosylation in this pathogen. Finally, genes coding for N-glycosylation enzymes and substrate biosynthesis are transcribed in B. bovis blood and tick stages, suggesting that this pathway is biologically relevant throughout the parasite life cycle. Elucidation of the role/s exerted by N-glycans will increase our understanding of these successful parasites, for which improved control measures are needed.

The CD4 + T-cell response is central for the control of Anaplasma marginale infection in cattle. However, the infection induces a functional exhaustion of antigen-specific CD4 + T cells in cattle immunized with A. marginale outer membrane proteins or purified outer membranes (OMs), which presumably facilitates the persistence of this rickettsia. In the present study, we hypothesize that T-cell exhaustion following infection is induced by the upregulation of immunoinhibitory receptors on T cells, such as programmed death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3). OM-specific T-cell responses and the kinetics of PD-1-positive (PD-1 + ) LAG-3 + exhausted T cells were monitored in A. marginale -challenged cattle previously immunized with OMs. Consistent with data from previous studies, OM-specific proliferation of peripheral blood mononuclear cells (PBMCs) and interferon gamma (IFN-γ) production were significantly suppressed in challenged animals by 5 weeks postinfection (wpi). In addition, bacteremia and anemia also peaked in these animals at 5 wpi. Flow cytometric analysis revealed that the percentage of PD-1 + LAG-3 + T cells in the CD4 + , CD8 + , and γδ T-cell populations gradually increased and also peaked at 5 wpi. A large increase in the percentage of LAG-3 + γδ T cells was also observed. Importantly, in vitro , the combined blockade of the PD-1 and LAG-3 pathways partially restored OM-specific PBMC proliferation and IFN-γ production at 5 wpi. Taken together, these results indicate that coexpression of PD-1 and LAG-3 on T cells contributes to the rapid exhaustion of A. marginale -specific T cells following infection and that these immunoinhibitory receptors regulate T-cell responses during bovine anaplasmosis.

The factors involved in gain or loss of virulence in Babesia bovis are unknown. Spherical body protein 2 truncated copy 11 (sbp2t11) transcripts in B. bovis were recently reported to be a marker of attenuation for B. bovis strains. Increased cytoadhesion of B. bovis-infected red blood cells (iRBC) to vascular endothelial cells is associated with severe disease outcomes and an indicator of parasite virulence. Here, we created a stable B. bovis transfected line over-expressing sbp2t11 to determine whether up-regulation of sbp2t11 is associated with changes in cytoadhesion. This line was designated sbp2t11up and five B. bovis clonal lines were derived from the sbp2t11up line by limiting dilution for characterisation. We compared the ability of iRBCs from the sbp2t11up line and its five derivative clonal lines to adhere to bovine brain endothelial cells, using an in vitro cytoadhesion assay. The same lines were selected for in vitro cytoadhesion and the levels of sbp2t11 transcripts in each selected line were quantified. Our results demonstrate that up-regulation of sbp2t11 is accompanied by a statistically significant reduction in cytoadhesion. Confirmed up-regulation of sbp2t11 in B. bovis concomitant with the reduction of iRBC in vitro cytoadhesion to bovine brain endothelial cell is consistent with our previous finding that up-regulation of sbp2t11 is an attenuation marker in B. bovis and suggests the involvement of sbp2t11 transcription in B. bovis virulence.

Bovine anaplasmosis is an infectious, non-contagious disease caused by the rickettsial pathogen Anaplasma marginale (A. marginale). The organism has a global distribution and infects erythrocytes, resulting in anemia, jaundice, fever, abortions and death. Once infected, animals remain carriers for life. The carrier status provides immunity to clinical disease, but is problematic if infected and naive cattle are comingled. Knowledge of infection prevalence and spatial distribution is important in disease management. The objective of this study was to assess A. marginale infection in-herd prevalence in Texas cattle using both molecular and serological methods. Blood samples from 11 cattle herds within Texas were collected and analyzed by reverse transcription quantitative real-time PCR (RT-qPCR) and a commercial competitive enzyme-linked immunosorbent assay (cELISA). Samples from experimentally infected animals were also analyzed and RT-qPCR detected A. marginale infection up to 15 days before cELISA, providing empirical data to support the interpretation of herd prevalence results. Herds with high prevalence were located in the north Texas Rolling Plains and west Trans-Pecos Desert, with RT-qPCR prevalence as high as 82% and cELISA prevalence as high as 88%. Overall prevalence was significantly higher in cattle in north and west Texas compared to cattle in east Texas (p

Equine piroplasmosis is a disease of Equidae, including horses, donkeys, mules, and zebras, caused by either of two protozoan parasites, Theileria equi or Babesia caballi. These parasites are biologically transmitted between hosts via tick vectors, and although they have inherent differences they are categorized together because they cause similar pathology and have similar morphologies, life cycles, and vector relationships. To complete their life cycle, these parasites must undergo a complex series of developmental events, including sexual-stage development in their tick vectors. Consequently, ticks are the definitive hosts as well as vectors for these parasites, and the vector relationship is restricted to a few competent tick species. Because the vector relationship is critical to the epidemiology of these parasites, we highlight current knowledge of the vector ecology of these tick-borne equine pathogens, emphasizing tick transmissibility and potential control strategies to prevent their spread.

Dermacentor andersoni, known as the Rocky Mountain wood tick, is found in the western United States and transmits pathogens that cause diseases of veterinary and public health importance including Rocky Mountain spotted fever, tularemia, Colorado tick fever and bovine anaplasmosis. Tick saliva is known to modulate both innate and acquired immune responses, enabling ticks to feed for several days without detection. During feeding ticks subvert host defences such as hemostasis and inflammation, which would otherwise result in coagulation, wound repair and rejection of the tick. Molecular characterization of the proteins and pharmacological molecules secreted in tick saliva offers an opportunity to develop tick vaccines as an alternative to the use of acaricides, as well as new anti-inflammatory drugs. We performed proteomics informed by transcriptomics to identify D. andersoni saliva proteins that are secreted during feeding. The transcript data generated a database of 21,797 consensus sequences, which we used to identify 677 proteins secreted in the saliva of D. andersoni ticks fed for 2 and 5days, following proteomic investigations of whole saliva using mass spectrometry. Salivary gland transcript levels of unfed ticks were compared with 2 and 5day fed ticks to identify genes upregulated early during tick feeding. We cross-referenced the proteomic data with the transcriptomic data to identify 157 proteins of interest for immunomodulation and blood feeding. Proteins of unknown function as well as known immunomodulators were identified.

Arthropod-borne protozoan pathogens have a complex life cycle that includes asexual reproduction of haploid stages in mammalian hosts and the development of diploid stages in invertebrate hosts. The ability of pathogens to invade, survive, and replicate within distinct cell types is required to maintain their life cycle. In this study, we describe a comparative proteomic analysis of a cattle pathogen, Babesia bovis, during its development within the mammalian and tick hosts with the goal of identifying cell-surface proteins expressed by B. bovis kinetes as potential targets for the development of a transmission blocking vaccine. To determine parasite tick-stage-specific cell-surface proteins, CyDye labeling was performed with B. bovis blood stages from the bovine host and kinetes from the tick vector. Cell-surface kinete-stage-specific proteins were identified using 2D difference in gel electrophoresis and analyzed by mass spectrometry. Ten proteins were identified as kinete-stage-specific, with orthologs found in closely related Apicomplexan pathogens. Transcriptional analysis revealed two genes were highly expressed by kinetes as compared with blood stages. Immunofluorescence using antibodies against the two proteins confirmed kinete-stage-specific expression. The identified cell-surface kinete proteins are potential candidates for the development of a B. bovis transmission blocking vaccine.

Rhipicephalus microplus is an important biological vector of Anaplasma marginale, the etiological agent of bovine anaplasmosis. The knowledge of tick immune responses to control bacterial infections remains limited. In this study, we demonstrate that transcription factor Relish from the IMD signaling pathway has an important role in the control of A. marginale infection in ticks. We found that RNA-mediated silencing of Relish caused a significant increase in the number of A. marginale in the midgut and salivary glands of R. microplus. In addition, the IMD pathway regulates the expression of the gene that encodes the antimicrobial peptide (AMP) microplusin. Moreover, microplusin expression was up-regulated in the midgut (2×) and salivary glands (8×) of A. marginale infected R. microplus. Therefore, it is plausible to hypothesize that microplusin may be involved in the A. marginale control. This study provides the first evidence of IMD signaling pathway participation on the A. marginale control in R. microplus.

Within the protective outer membrane (OM) fraction of Anaplasma marginale , several vaccine candidates have emerged, including a family of OM proteins (OMPs) 7 to 9, which share sequence identity with each other and with the single protein OMP7 in the vaccine strain A. marginale subsp. centrale. A. marginale OMPs 7 to 9 are logical vaccine candidates because they are surface exposed, present in the OM immunogen and protective cross-linked OM proteins, recognized by immune serum IgG2 and T cells in cattle immunized with OM, and recognized by immune serum IgG2 from cattle immunized with the A. centrale vaccine strain. We report the identification of a globally conserved 9-amino-acid T-cell epitope FLLVDDAI/VV shared between A. centrale vaccine strain OMP7 and the related A. marginale OMPs 7 to 9, where position 8 of the peptide can be isoleucine or valine. The epitope is conserved in American A. marginale strains, in the Australia Gypsy Plains strain, and in multiple field isolates from Ghana. This epitope, together with additional T-cell epitopes that are present within these proteins, should be considered for inclusion in a multivalent vaccine for A. marginale that can provide protection against disease caused by globally distributed bacterial strains.

Serial blood passage of virulent Babesia bovis in splenectomized cattle results in attenuated derivatives that do not cause neurologic disease. Tick transmissibility can be lost with attenuation, but when retained, attenuated B. bovis can revert to virulence following tick passage. This study provides data showing that tick passage of the partially attenuated B. bovis T2Bo derivative strain further decreased virulence compared with intravenous inoculation of the same strain in infected animals. Ticks that acquired virulent or attenuated parasites by feeding on infected cattle were transmission fed on naive, splenectomized animals. While there was no significant difference between groups in the number of parasites in the midgut, hemolymph, or eggs of replete female ticks after acquisition feeding, animals infected with the attenuated parasites after tick transmission showed no clinical signs of babesiosis, unlike those receiving intravenous challenge with the same attenuated strain prior to tick passage. Additionally, there were significantly fewer parasites in blood and tissues of animals infected with tick-passaged attenuated parasites. Sequencing analysis of select B. bovis genes before and after tick passage showed significant differences in parasite genotypes in both peripheral blood and cerebral samples. These results provide evidence that not only is tick transmissibility retained by the attenuated T2Bo strain, but also it results in enhanced attenuation and is accompanied by expansion of parasite subpopulations during tick passage that may be associated with the change in disease phenotype.

The current study tested the hypothesis that removal of maltose binding protein (MBP) from recombinant antigen used for plate coating would improve the specificity of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay (cELISA). The number of 358 sera with significant MBP antibody binding (≥30%I) in Anaplasma-negative herds was 139 (38.8%) when tested using the recombinant major surface protein 5 (rMSP5)-MBP cELISA without MBP adsorption. All but 8 of the MBP binders were rendered negative (

Theileria equi, one of the causative agents of equine piroplasmosis, is endemic in many regions of the world but is considered a ‘foreign’ animal disease in the USA. In an effort to prevent the importation of T. equi, stringent serological screening of horses is practiced prior to entry to the USA. Current regulatory options available where horses are found to be infected include permanent quarantine with or without chemotherapy, repatriation, or euthanasia. Chemotherapeutics that eliminate infection and subsequently transmission risk are critical in the management of infected horses. In this study, the efficacy of the drug imidocarb dipropionate against experimental T. equi infection was assessed. Of nine horses experimentally inoculated with T. equi isolated from an animal previously imported from Peru, six were treated with imidocarb dipropionate after the resolution of the acute phase of the disease. Elimination of the parasite was demonstrated in 5/6 by nested PCR, blood transfusions to naive horses, and reversion to seronegative status. The findings support the use of this drug as a potential treatment option in controlling outbreaks of T. equi, and also suggest that ‘combination testing’ using both serological and PCR detection methods are necessary to demonstrate clearance of infection.

Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the causative agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) where their orthologues in Plasmodium parasite have been shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while genes A, B and E were also transcribed in the subsequent B. bovis kinete stage. Transcription of gene C was found exclusively in the kinete. In contrast, transcription of genes D, F and G in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore, they are likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis within the tick vector.

Superinfection occurs when a second, genetically distinct pathogen strain infects a host that has already mounted an immune response to a primary strain. For antigenically variant pathogens, the primary strain itself expresses a broad diversity of variants over time. Thus, successful superinfection would require that the secondary strain express a unique set of variants. We tested this hypothesis under conditions of natural transmission in both temperate and tropical regions where, respectively, single-strain infections and strain superinfections of the tick-borne pathogen Anaplasma marginale predominate. Our conclusion that strain superinfection is associated with a significant increase in variant diversity is supported by progressive analysis of variant composition: (i) animals with naturally acquired superinfection had a statistically significantly greater number of unique variant sequences than animals either experimentally infected with single strains or infected with a single strain naturally, (ii) the greater number of unique sequences reflected a statistically significant increase in primary structural diversity in the superinfected animals, and (iii) the increase in primary structural diversity reflected increased combinations of the newly identified hypervariable microdomains. The role of population immunity in establishing temporal and spatial patterns of infection and disease has been well established. The results of the present study, which examined strain structure under conditions of natural transmission and population immunity, support that high levels of endemicity also drive pathogen divergence toward greater strain diversity.

Transmission of tick-borne pathogens requires transition between distinct host environments with infection and replication in host-specific cell types. Anaplasma marginale illustrates this transition: in the mammalian host, the bacterium infects and replicates in mature (nonnucleated) erythrocytes, while in the tick vector, replication occurs in nucleated epithelial cells. We hypothesized that proteins containing ankyrin motifs would be expressed by A. marginale only in tick cells and would traffic to the infected host cell nucleus. A. marginale encodes three proteins containing ankyrin motifs, an AnkA orthologue (the AM705 protein), AnkB (the AM926 protein), and AnkC (the AM638 protein). All three A. marginale Anks were confirmed to be expressed during intracellular infection: AnkA is expressed at significantly higher levels in erythrocytes, AnkB is expressed equally by both infected erythrocytes and tick cells, and AnkC is expressed exclusively in tick cells. There was no evidence of any of the Ank proteins trafficking to the nucleus. Thus, the hypothesis that ankyrin-containing motifs were predictive of cell type expression and nuclear localization was rejected. In contrast, AnkA orthologues in the closely related A. phagocytophilum and Ehrlichia chaffeensis have been shown to localize to the host cell nucleus. This difference, together with the lack of a nuclear localization signal in any of the AnkA orthologues, suggests that trafficking may be mediated by a separate transporter rather than by endogenous signals. Selection for divergence in Ank function among Anaplasma and Ehrlichia spp. is supported by both locus and allelic analyses of genes encoding orthologous proteins and their ankyrin motif compositions.

A novel Babesia bovis gene family encoding proteins with similarities to the Plasmodium 6cys protein family was identified by TBLASTN searches of the B. bovis genome using the sequence of the P. falciparum PFS230 protein as query, and was termed Bbo-6cys gene family. The Bbo-cys6 gene family contains six genes termed Bbo-6cys-A, B, C, D, E and F encoding for proteins containing an arrangement of 6 cysteine residues. The Bbo-6cys genes A, B, C, D, and E are tandemly arranged as a cluster of Chromosome 2 in the B. bovis genome, whereas gene F is located in a distal region in the same chromosome. The Bbo-6cys-E gene, with higher homology to PFS230, was selected for further examination. Immunoblot analysis using recombinant Bbo-6cys-E protein and B. bovis-positive bovine serum demonstrated expression by the parasite and immunogenicity during B. bovis infection. Immunofluorescence analysis using anti-Bbo-6cys-E antibodies confirmed expression of Bbo-6cys-E in in vitro blood stages of B. bovis. In addition, polyclonal antisera against both recombinant Bbo-6cys-E and specific synthetic peptides containing predicted B-cell epitopes of Bbo-6cys-E, significantly inhibited erythrocyte invasion by B. bovis in in vitro neutralization assays, suggesting an important functional role for this protein. Identification of this new gene family in B. bovis and further investigation on its biological significance may aid our understanding of the bovine, tick and parasite relationships and the development of improved control methods against B. bovis infection in cattle.

We tested the stability and tick transmission phenotype of transformed Anaplasma marginale through a complete in vivo infection cycle. Similar to the wild type, the gfp -transformed A. marginale strain established infection in cattle, a natural reservoir host, and persisted in immune competent animals. The tick infection rates for the transformed A. marginale and the wild type were the same. However, there were significantly lower levels of the transformed A. marginale than of the wild type in the tick. Despite the lower levels of replication, ticks transmitted the transformant. Transformants can serve as valuable tools to dissect the molecular requirements of tick colonization and pathogen transmission.

Completion of the Babesia bovis (T2Bo strain) genome provides detailed data concerning the predicted proteome of this parasite, and allows for a bioinformatics approach to gene discovery. Comparative genomics of the hemoprotozoan parasites B. bovis and Theileria parva revealed a highly conserved syntenic block of genes flanking the p67 gene of T. parva, a sporozoite stage-specific vaccine candidate against East Coast fever (ECF). The syntenic gene in B. bovis, designated bov57, encodes a protein of limited amino acid sequence identity (11.8%) to p67. Monoclonal antibodies were produced against recombinant BOV57 and were used to demonstrate expression of BOV57 in merozoite and kinete stages of the T2Bo strain of B. bovis. Transcript levels of bov57 in kinetes were increased 100-fold in comparison to msa-1, a previously identified gene encoding an erythrocyte stage surface protein. Amino acid sequence comparisons between the T2Bo strain and two attenuated and virulent strains from Argentina and Australia revealed a high degree of sequence conservation in BOV57 among these geographically and pathogenically divergent isolates (97% amino acid sequence identity). Additional genomic comparisons show that the bov57 gene locus is also conserved in Babesia bigemina and Babesia equi. While not identifiable through amino acid or nucleotide sequence similarity, the conserved gene order within this locus in multiple piroplasms may suggest a critical function adapted for each species' unique host and life-cycle.

Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne bacterium that causes significant economic losses for cattle industries and is increasingly being detected in other animal species. Rhipicephalus microplus is the main vector of this bacterium and may be found parasitizing small ruminants. In northeastern Brazil, multispecies grazing is a common family subsistence practice on smallholder farms possibly facilitating interspecies transmission of pathogens. Considering that A. marginale infection has been previously molecularly described in sheep, this study has aimed to estimate the prevalence of A. marginale and factors associated with the infection in goats from northeastern Brazil. A total of 403 goat blood samples were included in the study. An epidemiological questionnaire was applied to each farm owner addressing age, gender, presence of ticks and multispecies grazing. All samples were screened for A. marginale- and A. ovis-infection using primers targeting the Anaplasma spp. msp4 gene. The identity of A. marginale in the blood was confirmed by PCR amplification of msp5 followed by sequencing. Anaplasma spp. were differentiated by sequencing of the repeat region of the msp1α gene. For the statistical analysis the Chi-square or the Fisher’s exact test was used to verify association of the individual factors (age, gender, presence of ticks, and multispecies grazing) with Anaplasma spp. infection. We report the first molecular detection of A. marginale in goats from northeastern Brazil, based on msp1α, msp4 and msp5 gene sequencing analysis. Sequencing of the detected A. marginale msp1α gene revealed the F repeat. Amblyomma parvum and R. microplus were found feeding on animals. Fil: Da Silva, Nayara B.. Universidade Federal do Paraná; Brasil Fil: Taus, Naomi S.. United States Department of Agriculture. Agriculture Research Service; Estados Unidos. Washington State University; Estados Unidos Fil: Johnson, Wendell C.. United States Department of Agriculture. Agriculture Research Service; Estados Unidos Fil: Mira, Anabela. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Valente, Jessica D. M.. Universidade Federal do Paraná; Brasil Fil: Vidotto, Odilon. Universidade Estadual de Londrina; Brasil Fil: Masterson, Hayley E.. Washington State University; Estados Unidos Fil: Vieira, Thállitha S. W. J.. Universidade Federal do Paraná; Brasil Fil: Ueti, Massaro W.. United States Department of Agriculture. Agriculture Research Service; Estados Unidos. Washington State University; Estados Unidos Fil: Vieira, Rafael F. C.. Universidade Federal do Paraná; Brasil

Babesia bigemina is one of the most prevalent species causing bovine babesiosis around the world. Antigens involved in host cell invasion are vaccine targets for this disease but are largely unknown in this species. The invasion process of Babesia spp. into erythrocytes involves membrane proteins from the apical complex. A protein stored in the micronemes, called Micronemal Protein 1 (MIC-1), contains a sialic acid binding domain that participates in the invasion process of host cells and is a vaccine candidate in other apicomplexan parasites. It is not known if there is a homologous gene for mic-1 in B. bigemina. Therefore, the aim of this study was to characterize the mic-1 gene homologue in Babesia bigemina. A gene was found with a microneme adhesive repeat (MAR) domain in the predicted amino acid sequence. Transcription was determined by reverse transcription polymerase chain reaction (RT-PCR). Subsequently, antibodies against peptides containing conserved B-cell epitopes were used to confirm the expression of MIC-1 in intraerythrocytic merozoites. The presence of anti MIC-1 antibodies in cattle naturally infected with B. bigemina was determined and up to 97.4% of the cattle sera (113 out of 116) identified MIC-1 using enzyme-linked immunosorbent assay (ELISA) methods. Finally, antibodies against MIC-1 were able to block 70% merozoite invasion in-vitro.