Immunization of cattle with a Rhipicephalus microplus chitinase peptide containing predicted B-cell epitopes reduces tick biological fitness.

Rhipicephalus microplus, the cattle fever tick, is the most important ectoparasite impacting the livestock industry worldwide. Overreliance on chemical treatments for tick control has led to the emergence of acaricide-resistant ticks and environmental contamination. An immunological strategy based on vaccines offers an alternative approach to tick control. To develop novel tick vaccines, it is crucial to identify and evaluate antigens capable of generating protection in cattle. Chitinases are enzymes that degrade older chitin at the time of moulting, therefore allowing interstadial metamorphosis. In this study, 1 R. microplus chitinase was identified and its capacity to reduce fitness in ticks fed on immunized cattle was evaluated. First, the predicted amino acid sequence was determined in 4 isolates and their similarity was analysed by bioinformatics. Four peptides containing predicted B-cell epitopes were designed. The immunogenicity of each peptide was assessed by inoculating 2 cattle, 4 times at 21 days intervals, and the antibody response was verified by indirect ELISA. A challenge experiment was conducted with those peptides that were immunogenic. The chitinase gene was successfully amplified and sequenced, enabling comparison with reference strains. Notably, a 99.32% identity and 99.84% similarity were ascertained among the sequences. Furthermore, native protein recognition was demonstrated through western blot assays. Chitinase peptide 3 reduced the weight and oviposition of engorged ticks, as well as larvae viability, exhibiting a 71% efficacy. Therefore, chitinase 3 emerges as a viable vaccine candidate, holding promise for its integration into a multiantigenic vaccine against R. microplus.

Expression of sex-specific molecular markers by Babesia bovis gametes.

BACKGROUND: Bovine babesiosis caused by Babesia bovis is one of the most important tick-borne diseases of cattle in tropical and subtropical regions. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. To date, little is known about genes and proteins expressed by male gametes. METHODS AND RESULTS: We developed a method to separate male gametes from in vitro induced B. bovis culture. Separation enabled the validation of sex-specific markers. Collected male gametocytes were observed by Giemsa-stained smear and live-cell fluorescence microscopy. Babesia male gametes were used to confirm sex-specific markers by quantitative real-time PCR. Some genes were found to be male gamete specific genes including pka, hap2, α-tubulin II and znfp2. However, α-tubulin I and ABC transporter, trap2-4 and ccp1-3 genes were found to be upregulated in culture depleted of male gametes (female-enriched). Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of HAP2 by male and TRAP2-4 by female gametes. These results revealed strong markers to distinguish between B. bovis male and female gametes. CONCLUSIONS: Herein, we describe the identification of sex-specific molecular markers essential for B. bovis sexual reproduction. These tools will enhance our understanding of the biology of sexual stages and, consequently, the development of additional strategies to control bovine babesiosis.

Vaccination with an in vitro culture attenuated Babesia bovis strain safely protects highly susceptible adult cattle against acute bovine babesiosis.

Introduction: Live in vivo attenuated Babesia bovis vaccines produced by sequential passages in splenectomized calves have historically been used to control acute bovine babesiosis in endemic areas worldwide. However, several constraints prevent the widespread use of these vaccines, including the need for several splenectomized calves to produce vaccine batches, and potential inconsistent parasite attenuation, which contraindicates their use for highly Babesia-susceptible adult cattle. Thus, the use of vaccines based on well-defined in vitro culture attenuated B. bovis strains emerges as a more sustainable and efficient alternative. Previous work demonstrated that the culture attenuated strain Att-S74-T3Bo is non-tick transmissible and able to safely protect calves against needle challenge with a B. bovis virulent strain. Methods and results: Herein we evaluated safety and efficacy of Att-S74-T3Bo in preventing acute babesiosis in adult (>1.5 year of age) cattle. Results demonstrated that Att-S74-T3Bo vaccination of adult animals (n=5) induced self-limiting signs of acute infection and protected the vaccinated animals against challenge with the homologous virulent B. bovis strain Vir-S74-T3Bo. Att-S74-T3Bo-vaccinated adult cattle developed significant (P<0.05) monocytosis, with concomitant neutropenia and CD4+ leukopenia, in peripheral blood early after vaccination. Also, vaccinated animals developed a specific signature of pro- and anti-inflammatory cytokine expression in peripheral blood and significant levels of IgM, total IgG, IgG1, and IgG2 against the B. bovis immunodominant antigen RAP-1 CT. Strikingly, none of the vaccinated animals showed any signs of acute babesiosis after challenge with Vir-S74-T3Bo. In contrast, control adult cattle (n=5) showed pathognomonic symptoms of acute babesiosis, and significant decrease (P<0.05) in lymphocytes, monocytes, and neutrophils, starting on day 7 post-challenge. All control animals developed severe acute disease and were euthanized on days 10 through 12 days post-challenge. Discussion and conclusion: Evidence from this study indicates that Att-S74-T3Bo safely protects highly susceptible adult cattle against challenge with a homologous virulent strain of B. bovis. In conclusion, Att-S74-T3Bo may be considered as a potential efficient and sustainable attenuated candidate vaccine strain to control acute bovine babesiosis in highly susceptible adult cattle. Future studies should focus on increasing the number of animals vaccinated, duration of immunity, and efficacy of this attenuated strain against heterologous virulent parasite strains.

Spherical Body Protein 4 from Babesia bigemina: A Novel Gene That Contains Conserved B-Cell Epitopes and Induces Cross-Reactive Neutralizing Antibodies in Babesia ovata.

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.

Differential paired stage-specific expression of Babesia bovis cysteine-rich GCC2/GCC3 domain family proteins (BboGDP) during development within Rhipicephalus microplus.

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.

Identification of novel immune correlates of protection against acute bovine babesiosis by superinfecting cattle with in vitro culture attenuated and virulent Babesia bovis strains.

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<0.05) parasite load in peripheral blood compared to animals previously exposed to Att-S74-T3Bo. No significant alterations in the profile of leukocytes and cytokines were observed in Att-S74-T3Bo-inoculated after Vir-S74-T3Bo infection. In conclusion, data demonstrate novel changes in the profile of blood immune cells and cytokine expression in peripheral blood that are associated with protection against acute bovine babesiosis. These identified immune correlates of protection may be useful for designing effective and sustainable vaccines against babesiosis in cattle.

Thrombospondin-Related Anonymous Protein (TRAP) Family Expression by Babesia bovis Life Stages within the Mammalian Host and Tick Vector.

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.

A Transfected Babesia bovis Parasite Line Expressing eGFP Is Able to Complete the Full Life Cycle of the Parasite in Mammalian and Tick Hosts.

Bovine babesiosis is caused by apicomplexan pathogens of the genus Babesia, including B. bovis. This protozoan parasite has a complex life cycle involving dynamic changes to its transcriptome during the transition between the invertebrate and vertebrate hosts. Studying the role of genes upregulated by tick stage parasites has been hindered by the lack of appropriate tools to study parasite gene products in the invertebrate host. Herein, we present tfBbo5480, a transfected B. bovis cell line, constitutively expressing enhanced green fluorescent protein (eGFP) created by a whole gene replacement transfection strategy, that was capable of completing the parasite's entire life cycle in both the vertebrate and invertebrate hosts. tfBbo5480 was demonstrated to respond to in vitro sexual stage induction and upon acquisition by the female tick vector, Rhipicephalus microplus, the tick specific kinete stage of tfBbo5480 was detected in tick hemolymph. Larvae from tfBbo5480 exposed R. microplus female ticks successfully transmitted the transfected parasite to a naïve calf. The development of the whole gene replacement strategy will permit a deeper understanding of the biology of parasite-host-vector triad interactions and facilitate the evaluation of upregulated genes during the parasite's journey through the tick vector leading to new intervention strategies for the control of bovine babesiosis.

Anaplasma marginale Infection of Dermacentor andersoni Primary Midgut Cell Culture Is Dependent on Fucosylated Glycans.

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.

Serological and molecular detection of Babesia caballi and Theileria equi in Mexico: A prospective study.

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.

A Culture-Adapted Strain of Babesia bovis Has Reduced Subpopulation Complexity and Is Unable to Complete Its Natural Life Cycle in Ticks.

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.

Comparison of high throughput RNA sequences between Babesia bigemina and Babesia bovis revealed consistent differential gene expression that is required for the Babesia life cycle in the vertebrate and invertebrate hosts.

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.

Immunization against a Conserved Surface Polysaccharide Stimulates Bovine Antibodies with Opsonic Killing Activity but Does Not Protect against Babesia bovis Challenge.

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.

Isolation of infectious Theileria parva sporozoites secreted by infected Rhipicephalus appendiculatus ticks into an in vitro tick feeding system.

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.

Differential expression of calcium-dependent protein kinase 4, tubulin tyrosine ligase, and methyltransferase by xanthurenic acid-induced Babesia bovis sexual stages.

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.

Establishment of Babesia bovis In Vitro Culture Using Medium Free of Animal Products.

Babesia bovis, an etiological agent of bovine babesiosis, causes a significant burden to the cattle industry worldwide. The most efficient method to mitigate bovine babesiosis is a live vaccine produced by serial passage in splenectomized cattle. However, there are several concerns regarding live vaccine production, including variation between batches and the use of many animals. In this study, we report a B. bovis-SF strain continuously cultured in a medium free of components of animal origin enriched with a chemically defined lipid mixture (CD lipid mixture) and the use of a perfusion bioreactor to harvest a large amount of B. bovis. Six culture media were compared, including VP-SFM, CD-CHO, CD-Hydrolyzed, CD-CHO, SFM, and ADMEM/F12. We found that the VP-SFM medium performed the best for B. bovis growth, with a maximum percentage of parasitized erythrocytes (PPE) of 8.6%. The effect of six dilutions of a commercial mixture of CD lipids added to VP-SFM showed that the CD lipid mixture at a dilution of 1:100 had the best B. bovis growth curve, with a maximum PPE of 13.9%. Propagation of the in vitro B. bovis culture was scaled up in a perfusion bioreactor using VP-SFM with a CD lipid mixture, and the PPE reached over 32%. The continuous in vitro B. bovis culture in a medium free of animal origin components could potentially reduce and replace the use of animals to produce a reagent for diagnostics and live vaccines to control bovine babesiosis.

Stray Mexico origin cattle captured crossing into Southern Texas carry Babesia bovis and other tick-borne pathogens.

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.

Molecular and Serological Detection of Piroplasms in Horses from Nigeria.

Equine piroplasmosis, an economically important disease of equids caused by the hemoprotozoan parasites Theileria equi, T. haneyi, and Babesia caballi, has a worldwide distribution. These parasites are transmitted by ixodid ticks. To improve the detection of horses in Nigeria exposed to piroplasm parasites, 72 horses with variable clinical signs of piroplasmosis were sampled from Northwest and Northcentral Nigeria and tested by nPCR and cELISA. Blood and serum samples were collected from each horse via jugular venesection. Individually, nPCR or cELISA failed to identify all horses exposed to piroplasms. A combination of species-specific nPCR and the OIE-approved T. equi and B. caballi cELISAs enhanced the detection of horses exposed to parasites. The results also demonstrated horses showing abnormal hematology were positive for only T. equi, except for one sample that was coinfected with T. equi and T. haneyi. We also identified ticks collected from some of the horses, with Rhipicephalus evertsi evertsi being the most prevalent. This study shows that a larger proportion of horses in the sample set were exposed to T. equi than B. caballi or T. haneyi. Additionally, ticks that have been previously reported as potential vectors for these parasites were found to have infested sampled horses. Further studies are needed to investigate which tick species are competent vectors for Theileria spp. and Babesia caballi in Nigeria.

Assessment of Babesia bovis 6cys A and 6cys B as components of transmission blocking vaccines for babesiosis.

BACKGROUND: Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied. METHODS: The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed. RESULTS: Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro. CONCLUSIONS: These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission.

Development of an Indirect ELISA to Detect Equine Antibodies to Theileria haneyi.

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.