Nilgai antelope display no signs of infection upon experimental challenge with a virulent Babesia bovis strain.

BACKGROUND: Bovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection. METHODS: Nilgai were needle inoculated with ≈108 B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite. RESULTS: Nilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer. CONCLUSIONS: Nilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances.

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.

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.

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.

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.

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.

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.

Comparative analysis of gene expression between Babesia bovis blood stages and kinetes allowed by improved genome annotation.

Throughout their life cycle, Babesia parasites alternate between a mammalian host, where they cause babesiosis, and the tick vector. Transition between hosts results in distinct environmental signals that influence patterns of gene expression, consistent with the morphological and functional changes operating in the parasites during their life stages. In addition, comparing differential patterns of gene expression among mammalian and tick parasite stages can provide clues for developing improved methods of control. Hereby, we upgraded the genome assembly of Babesia bovis, a bovine hemoparasite, closing a 139 kbp gap, and used RNA-Seq datasets derived from mammalian blood and tick kinete stages to update the genome annotation. Of the originally annotated genes, 1,254 required structural changes, and 326 new genes were identified, leading to a different predicted proteome compared to the original annotation. Next, the RNA-Seq data was used to identify B. bovis genes that were differentially expressed in the vertebrate and arthropod hosts. In blood stages, 28% of the genes were upregulated up to 300 fold, whereas 26% of the genes in kinetes, a tick stage, were upregulated up to >19,000 fold. We thus discovered differentially expressed genes that may play key biological roles and serve as suitable targets for the development of vaccines to control bovine babesiosis.

Transcriptome dataset of Babesia bovis life stages within vertebrate and invertebrate hosts.

Babesia bovis is a hemoprotozoan parasite of cattle that has a complex life cycle within vertebrate and invertebrate hosts. In the mammalian host, B. bovis undergoes asexual reproduction while in the tick midgut, gametes are induced, fuse, and form zygotes. The zygote infects tick gut epithelial cells and transform into kinetes that are released into the hemolymph and invade other tick tissues such as the ovaries, resulting in transovarial transmission to tick offspring. To compare gene regulation between different B. bovis life stages, we collected parasites infecting bovine erythrocytes and tick hemolymph. Total RNA samples were isolated, and multiplexed libraries sequenced using paired-end 100 cycle reads of a HiSeq 2500. The data was normalized using the TMM method and analysed for significant differential expression using the generalized linear model likelihood ratio test (GLM LRT) in edgeR. To validate our datasets, ten genes were selected using NormFinder. Genes that had no significant fold change between the blood and tick stages in the RNA-Seq datasets were tested by quantitative PCR to determine their suitability as "housekeeping" genes. The normalized RNA-Seq data revealed genes upregulated during infection of the mammalian host or tick vector and six upregulated genes were validated by quantitative PCR. These datasets can help identify useful targets for controlling bovine babesiosis.

Identification of proteins expressed by Babesia bigemina kinetes.

BACKGROUND: Babesia bigemina is an apicomplexan parasite transovarially transmitted via Rhipicephalus ticks that infect red blood cells and causes bovine babesiosis, a poorly controlled severe acute disease in cattle. New methods of control are urgently needed, including the development of transmission blocking vaccines (TBV). Babesia bigemina reproduces sexually in the gut of adult female R. microplus upon acquisition following a blood meal. Sexual reproduction results in zygotes that infect gut epithelial cells to transform into kinete stage parasites, which invade tick ovaries and infects the egg mass. The subsequent tick generation transmits B. bigemina upon feeding on bovine hosts. An important limitation for developing novel TBV is that the pattern of protein expression in B. bigemina tick stages, such as the kinete stage, remain essentially uncharacterized. RESULTS: We determined the protein expression profile of three B. bigemina putative tick stage candidates BbiKSP (BBBOND_0206730), CCp2 and CCp3. We found that BbiKSP expression was restricted to B. bigemina kinetes. CCp2 and CCp3, previously shown to be expressed by induced sexual stages, were also expressed by kinetes. Importantly, none of these proteins were expressed by B. bigemina blood stages. CONCLUSIONS: Babesia bigemina kinetes express BbiKSP, CCp2 and CCp3 proteins, therefore, these proteins may play important roles during B. bigemina development within tick hemolymph and may serve as potential candidate targets for the development of TBV.

Silencing expression of the Rhipicephalus microplus vitellogenin receptor gene blocks Babesia bovis transmission and interferes with oocyte maturation.

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.

First report of Anaplasma marginale infection in goats, Brazil.

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.

Identification of a putative methyltransferase gene of Babesia bigemina as a novel molecular biomarker uniquely expressed in parasite tick stages.

BACKGROUND: Bovine babesiosis is caused by apicomplexan pathogens of the genus Babesia such as B. bigemina and B. bovis. These tick-borne pathogens have a complex life-cycle involving asexual multiplication in vertebrate hosts and sexual reproduction in invertebrate vectors. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. Understanding the mechanisms that underlie formation of extracellular Babesia tick stages is an important step towards developing control strategies for preventing tick infection and subsequent parasite transmission. RESULTS: We induced B. bigemina sexual stages in vitro by exposing parasites to Tris 2-carboxyethyl phosphine (TCEP). Subsequently, we identified a novel putative methyltransferase gene (BBBOND_0204030) that is expressed uniquely in all B. bigemina tick stages but not in blood stages. In vitro TCEP-exposed B. bigemina presented diverse morphology including parasites with long projections, round forms and clusters of round forms indicative of sexual stage induction. We confirmed the development of sexual stages by detecting upregulation of previously defined B. bigemina sexual stage marker genes, ccp2 and 3, and their respective protein expression in TCEP-induced B. bigemina cultures. Next, transcription analysis of in vitro TCEP-induced B. bigemina culture based on an in silico derived list of homologs of Plasmodium falciparum gamete-specific genes demonstrated differential expression of the gene BBBOND_0204030 in induced cells. Further examination of ex vivo infected ticks demonstrated that BBBOND_0204030 is transcribed by multiple stages of B. bigemina during parasite development in tick midgut, ovary and hemolymph. Interestingly, ex vivo results confirmed our in vitro observation that blood stages of B. bigemina do not express BBBOND_0204030 and validated the in vitro system of inducing sexual stages. CONCLUSIONS: Herein we describe the identification of a B. bigemina gene transcribed exclusively by parasites infecting ticks using a novel method of inducing B. bigemina sexual stages in vitro. We propose that this gene can be used as a marker for parasite development within the tick vector. Together, these tools will facilitate our understanding of parasite-tick interactions, the identification of novel vaccine targets and, consequently, the development of additional strategies to control bovine babesiosis.

Evaluation of glycoprotein Ov8 as a potential antigen for an OvHV-2-specific diagnostic assay.

Gammaherpesviruses in the genus Macavirus establish clinically unapparent persistent infections in reservoir species. Transmission of some of these viruses, including alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2), to clinically susceptible species in the order Artiodactyla can result in malignant catarrhal fever (MCF), a usually fatal lymphoproliferative disease. Serology can be used to identify MCF virus (MCFV)-infected carrier animals. However, all current serological assays utilize AlHV-1 antigens, thus none is specific for OvHV-2. In situations where sheep and other MCFV carriers are present, such as in zoos and game farms, an OvHV-2-specific assay would determine if OvHV-2 is present in the population. In this study, a recombinant protein containing a truncated OvHV-2 Ov8 glycoprotein was expressed and evaluated as a suitable target antigen to specifically detect OvHV-2 infection using an enzyme linked immunosorbent assay (ELISA). A competitive inhibition (CI)-ELISA that detects an epitope conserved among all MCFVs was used to categorize, as positive or negative, sera from 205 domestic sheep. The Ov8 assay showed 100% diagnostic sensitivity, 98.97% diagnostic specificity, 99.07% positive predictive value, and 100% negative predictive value and very high agreement (kappa = 0.990 and 95% CI = 0.971-1.000) with the CI-ELISA. Sera from animals infected with MCFVs other than OvHV-2 did not cross-react with Ov8 (100% negative predictive value). These data support the use of the Ov8 ELISA as an OvHV-2-specific diagnostic assay.

Molecular detection and characterization of Theileria spp. infecting cattle in Sennar State, Sudan.

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.

Analysis of Stage-Specific Protein Expression during Babesia Bovis Development within Female Rhipicephalus Microplus.

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.

Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion.

Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus in the genus Macavirus that is carried asymptomatically by sheep. Infection of poorly adapted animals with OvHV-2 results in sheep-associated malignant catarrhal fever, a fatal disease characterized by lymphoproliferation and vasculitis. There is no treatment or vaccine for the disease and no cell culture system to propagate the virus. The lack of cell culture has hindered studies of OvHV-2 biology, including its entry mechanism. As an alternative method to study OvHV-2 glycoproteins responsible for membrane fusion as a part of the entry mechanism, we developed a virus-free cell-to-cell membrane fusion assay to identify the minimum required OvHV-2 glycoproteins to induce membrane fusion. OvHV-2 glycoproteins B, H, and L (gB, gH, and gL) were able to induce membrane fusion together but not when expressed individually. Additionally, open reading frame Ov8, unique to OvHV-2, was found to encode a transmembrane glycoprotein that can significantly enhance membrane fusion. Thus, OvHV-2 gB, gH, and gL are sufficient to induce membrane fusion, while glycoprotein Ov8 plays an enhancing role by an unknown mechanism.IMPORTANCE Herpesviruses enter cells via attachment of the virion to the cellular surface and fusion of the viral envelope with cellular membranes. Virus-cell membrane fusion is an important step for a successful viral infection. Elucidating the roles of viral glycoproteins responsible for membrane fusion is critical toward understanding viral entry. Entry of ovine herpesvirus 2 (OvHV-2), the causative agent of sheep associated-malignant catarrhal fever, which is one of the leading causes of death in bison and other ungulates, has not been well studied due to the lack of a cell culture system to propagate the virus. The identification of OvHV-2 glycoproteins that mediate membrane fusion may help identify viral and/or cellular factors involved in OvHV-2 cell tropism and will advance investigation of cellular factors necessary for virus-cell membrane fusion. We found that OvHV-2 glycoproteins B, H, and L are sufficient for, and viral glycoprotein Ov8 can significantly enhance, cell-cell membrane fusion.

Replacement of Glycoprotein B in Alcelaphine Herpesvirus 1 by Its Ovine Herpesvirus 2 Homolog : Implications in Vaccine Development for Sheep-Associated Malignant Catarrhal Fever.

Vaccine development is a top priority in malignant catarrhal fever (MCF) research. In the case of sheep-associated MCF (SA-MCF) caused by ovine herpesvirus 2 (OvHV-2), progress toward this objective has been hindered by the absence of methods to attenuate or modify the virus, since it cannot be propagated in vitro. As an alternative for vaccine development, in this study, we tested the hypothesis that one of the SA-MCF vaccine candidate targets, OvHV-2 glycoprotein B (gB), could be expressed by a nonpathogenic alcelaphine herpesvirus 1 (AlHV-1) and then evaluated the potential of the AlHV-1/OvHV-2 chimera to be used as a vaccine and a diagnostic tool. The construction and characterization of an AlHV-1/OvHV-2 chimeric virus that is nonpathogenic and expresses an OvHV-2 vaccine target are significant steps toward the development of an SA-MCF vaccine and also provide a valuable means to study OvHV-2 biology.