Theileria secretes proteins to subvert its host leukocyte.

Theileria parasites are classified in the phylum Apicomplexa that includes several genera of medical and veterinary importance such as Plasmodium, Babesia, Toxoplasma and Cryptosporidium. These protozoans have evolved subtle ways to reshape their intracellular niche for their own benefit and Theileria is no exception. This tick transmitted microorganism is unique among all eukaryotes in that its intracellular schizont stage is able to transform its mammalian host leukocytes into an immortalised highly disseminating cell that phenocopies tumour cells. Here, we describe what is known about secreted Theileria-encoded host cell manipulators.

Preparation of Monoclonal Antibody Against EMA-1 and Development of Rapid Serological Detection Method for Theileria equi Infection, Xinjiang, China.

The erythrocytic-stage surface protein equi merozoite antigen 1 (EMA-1) of Theileria equi is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In this study, BALB/c mice were immunized with purified recombinant EMA-1 to prepare monoclonal antibody (mAb) against T. equi EMA-1, and 1 mAb 5H2 was obtained that showed good reaction with infected red blood cells (RBC) in the indirect immunofluorescence assay (IFA). To develop a rapid serological detection method for T. equi infection in Xinjiang Uygur Autonomous Region, China, recombinant EMA-1 originating from the local T. equi strain and the mAb to EMA-1 were employed to develop an immunochromatographic test (ICT) to detect antibodies to T. equi in horse sera. The ICT showed high sensitivity and specificity and no cross-reaction with Babesia caballi. Ninety-two horse serum samples collected from Ili, Xinjiang, were tested by ICT and compared with the detection results of a commercial ELISA kit. The results showed that 56 of 92 (61%) serum samples were seropositive according to the ICT assay, and 50 (54%) samples were seropositive according to the ELISA kit. The ICT had a high coincidence (91.3%) but was more sensitive than the reference ELISA kit. To confirm whether the horses were infected by T. equi, 30 blood DNA samples from 92 horses were examined by PCR. The results showed that 14 of 30 (47%) horses were confirmed to be infected with T. equi by PCR, while 16 of 30 (53%) horses were seropositive by ICT. All PCR-positive horses were ICT-positive. The findings indicate that T. equi is endemic in Ili, Xinjiang, and that the ICT is reliable as a serological diagnosis method. The ICT developed in this study could be an efficient diagnostic tool to detect T. equi infection in horses in the Xinjiang area.

Immune parameters to p67C antigen adjuvanted with ISA206VG correlate with protection against East Coast fever.

East Coast fever (ECF) is a lymphoproliferative disease caused by the tick-transmitted protozoan parasite Theileria parva. ECF is one of the most serious cattle tick-borne diseases in Sub-Saharan Africa. We have previously demonstrated that three doses of the C-terminal part of the sporozoite protein p67 (p67C) adjuvanted with ISA206VG confers partial protection against ECF at a herd level. We have tested the efficacy of two doses of this experimental vaccine, as reducing the vaccination regimen would facilitate its deployment in the field. We reconfirm that three antigen doses gave a significant level of protection to severe disease (46%, ECF score < 6) when compared with the control group, while two doses did not (23%). Animals receiving three doses of p67C developed higher antibody titers and CD4+ T-cell proliferation indices, than those which received two doses. A new panel of immune parameters were tested in order to identify factors correlating with protection: CD4+ proliferation index, total IgG, IgG1, IgG2 and IgM half maximal titers and neutralization capacity of the sera with and without complement. We show that some of the cellular and humoral immune responses provide preliminary correlates of protection.

Genotyping of Theileria lestoquardi from sheep and goats in Sudan to support control of Malignant Ovine Theileriosis.

Theileriosis, caused by parasitic protozoa of the genus Theileria parasites, are among the major tick-borne diseases of ruminant livestock. The largest economic losses are attributed in particular to those caused by the leukoproliferative species of Theileria: T. parva, T. annulata and T. lestoquardi. Theileria lestoquardi is transmitted by Hyalomma ticks and causes malignant ovine theileriosis (MOT), a disease that is particularly prevalent in Sudan. The disease is considered of a high economic importance in Sudan, where export of sheep is a major component of the national economy. A live vaccine based on a Sudanese isolate of T. lestoquardi (Atbara strain) was previously developed for the control of MOT in Sudan, but not yet deployed in the field. The present study aims to genetically characterize and compare samples of T. lestoquardi circulating in Sudan as well as the live vaccine isolate in order to understand vaccine breakthroughs and failure that may occur. Sheep and goats blood samples were collected from six regions in Sudan that are known to be endemic for T. lestoquardi infection or have experienced outbreaks of MOT. Blood samples infected with T. lestoquardi were identified by PCR or RLB. Genotyping was carried out by (1) sequencing the homologues of two T. parva CD8+ T cell antigen genes, Tp1 and Tp2, and (2) using a panel of seven micro- and mini-satellite markers. A total of 100 T. lestoquardi positive field samples and the T. lestoquardi (Atbara) vaccine were genotyped. The results showed that all samples had mixed genotypes, with several alleles identified at one or more loci. The gene diversity ranged from 0.7840 (TS8) to 0.2133 (TS12) with mean values of 0.5470. PCA revealed three clusters of the parasite in Sudan; interestingly one independent cluster was clearly seen, corresponding to the vaccine isolate. The T. lestoquardi Tp1 homologue showed higher homology with T. annulata than with T. parva sequences included the defined single CD8+ T cell target epitope region. The result indicates that multiple genotypes are a common feature of T. lestoquardi infection in Sudan. Both genotyping and the sequencing results clearly showed that the vaccine isolate is highly distinct from the field samples. This finding raised the question whether vaccination with the prepared lived vaccine will effectively protect animals against challenges by the field isolates of T. lestoquardi. The results of this work will inform on the best approach for controlling MOT in Sudan.

Identification of Theileria lestoquardi Antigens Recognized by CD8+ T Cells.

As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles.

Mechanical transfer of Theileria orientalis: possible roles of biting arthropods, colostrum and husbandry practices in disease transmission.

BACKGROUND: The intracellular protozoal parasite Theileria orientalis has rapidly spread across South-eastern Australia, substantially impacting local cattle industries since 2006. Haemaphysalis longicornis appears to be a biological vector in the endemic regions. Mechanical transfer of blood by biting arthropods, in colostrum or iatrogenic transmission though husbandry procedures is another possible mode of transmission. This study assesses the risk of these mechanical modes of transmission. METHODS: Blood was collected from a T. orientalis Ikeda positive Angus steer, and was inoculated into the jugular vein of 9 calves in 3 treatment groups, each with 3 animals. Calves in Group 1 received 10 ml of cryopreserved blood, while those in Groups 2 and 3 received 1 ml (fresh blood) and 0.1 ml (cryopreserved), respectively. An additional three animals remained as negative controls and the donor calf was also followed as a positive control. Blood was collected over 3 months, and analysed via qPCR for the presence of the parasite. Samples of the sucking louse Linognathus vituli were collected opportunistically from calves 5 months after inoculation and tested for T. orientalis. For the colostral transmission study, 30 samples of blood and colostrum were collected from cows at calving in an endemic herd. These samples along with blood from their calves were tested by qPCR for T. orientalis and for antibodies to the major piroplasm surface protein (MPSP). RESULTS: Eight of the nine inoculated calves became positive for T. orientalis. The prepatent period of these infections was inversely correlated with inoculation dose. All negative control calves remained negative and the positive control calf remained positive. Samples of L. vituli tested positive for T. orientalis Ikeda, while some samples of colostrum were also shown to be qPCR and anti-MPSP positive. All calves in the colostral study tested qPCR negative although one was antibody-positive. CONCLUSIONS: T. orientalis is capable of being mechanically transferred by intravenous inoculation with small volumes of blood and is detectable up to 5 months post-infection. Animals infected by this means may play a significant role in the transmission of the disease by acting as asymptomatic carriers. Other modes of blood transfer, including biting arthropods and colostral transfer are also possible modes of disease transmission.

Local pulmonary immune responses in domestic cats naturally infected with Cytauxzoon felis.

Cytauxzoonosis is a hemoprotozoal disease of cats and wild felids in the South and Southeastern United States caused by Cytauxzoon felis. Although the causative agent has been recognized since the seventies, no study has examined the local immune response in affected organs, such as the lung, and compared them to the lungs of uninfected domestic cats. Previous studies have suggested that the histopathologic findings in the lungs of C. felis-infected cats are caused by the release of pro-inflammatory mediators, such as cytokines and increased production of inducible nitric oxide synthase (iNOS), by the infected macrophages. Our laboratory had previously found an upregulation of the adhesion molecule CD18, which can stimulate the release of these pro-inflammatory mediators. The objective of this study was to characterize local pulmonary immune responses in cats naturally infected with C. felis. Immunohistochemistry was performed to detect tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, iNOS, and major histocompatibility complex (MHC) II in 19 lungs from affected cats that died between 2005 and 2013. Results showed increased expression of all of these molecules when compared to lungs from uninfected, healthy cats. Furthermore, MHC II is expressed in the endothelium of C. felis naturally infected cats. These results support that there is a marked, local, pro-inflammatory immune response that can contribute to the pathogenesis of cytauxzoonosis in the lungs.

NKp46+ CD3+ cells: a novel nonconventional T cell subset in cattle exhibiting both NK cell and T cell features.

The NKp46 receptor demonstrates a high degree of lineage specificity, being expressed almost exclusively in NK cells. Previous studies have demonstrated NKp46 expression by T cells, but NKp46+ CD3+ cells are rare and almost universally associated with NKp46 acquisition by T cells following stimulation. In this study we demonstrate the existence of a population of NKp46+ CD3+ cells resident in normal bovine PBMCs that includes cells of both the αß TCR+ and γδ TCR+ lineages and is present at a frequency of 0.1-1.7%. NKp46+ CD3+ cells express transcripts for a broad repertoire of both NKRs and TCRs and also the CD3ζ, DAP10, and FcεR1γ but not DAP12 adaptor proteins. In vitro functional analysis of NKp46+ CD3+ cells confirm that NKp46, CD16, and CD3 signaling pathways are all functionally competent and capable of mediating/redirecting cytolysis. However, only CD3 cross-ligation elicits IFN-γ release. NKp46+ CD3+ cells exhibit cytotoxic activity against autologous Theileria parva-infected cells in vitro, and during in vivo challenge with this parasite an expansion of NKp46+ CD3+ cells was observed in some animals, indicating the cells have the potential to act as an anti-pathogen effector population. The results in this study identify and describe a novel nonconventional NKp46+ CD3+ T cell subset that is phenotypically and functionally distinct from conventional NK and T cells. The ability to exploit both NKRs and TCRs suggests these cells may fill a functional niche at the interface of innate and adaptive immune responses.

Seroprevalence rates of antibodies against Theileria equi in team roping horses from central-western region of Paraná / Soroprevalência de anticorpos contra Theileria equi em equinos atletas da região centro-oeste do Paraná

The purpose of this study was to estimate the prevalence of Theileria equi in horses from central western region of Paraná state, Brazil. The presence of antibodies IgG against T. equi was determined in serum samples obtained from 400 team roping horses of the district of Guarapuava by the enzyme-linked immunosorbent assay (ELISA). Results showed that 242 (61%) animals were positive which demonstrates that equine piroplasmosis is widespread and therefore it might be a contributing factor for the irregular performance among athletes horses in the region studied. No association regarding age and sex were observed (p>0.05). To our knowledge, this is the first report describing a serological survey on equine piroplasmosis in the state of Paraná, Brazil.

O objetivo deste estudo foi estimar a prevalência de Theileria equi em equinos da região centro-oeste do Estado do Paraná, Brasil. A presença de anticorpos IgG contra T. equi foi determinada em amostras de soro obtidas a partir de 400 cavalos atletas do distrito de Guarapuava pelo ensaio imunoenzimático (ELISA). Os resultados mostraram que 242 (61%) animais foram positivos, o que demonstra que a piroplasmose equina apresenta ampla distribuição e, portanto, poderá contribuir para a performace irregular de cavalos que participam de eventos desportivos na região. Não foi observada associação com a idade ou sexo dos equinos (p>0,05). Pelo que se sabe, este é o primeiro relato de levantamento sorológico sobre piroplasmose equina no Estado do Paraná, Brasil.

Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.

Attenuation of Theileria lestoquardi infected cells and immunization of sheep against malignant ovine theileriosis.

Malignant ovine theileriosis caused by Theileria lestoquardi is an economically important disease infecting small ruminants in the Sudan. The disease causes massive losses among sheep in many regions of Northern Sudan. The present studies were done to isolate lymphoblastoid cells infected with malignant ovine theileriosis and attenuate them by passage using culture media to develop and produce schizonts candidate vaccine, then test its efficacy and safety by exposing immunized lambs to field challenge in an area endemic with T. lestoquardi. In the present experiments we isolated and established an in vitro culture of T. lestoquardi infected lymphoblast cell line. Long-term culture of T. lestoquardi infected lymphoplastoid cells was shown to result in attenuation of their virulence and lambs inoculated with different doses of such cells at passage 105 exhibited very mild reactions with fever that lasted for 1-5 days and parasitaemia of <0.2%. The experimental lambs immunized with this candidate vaccine were immune and protected when exposed to field challenge in an area endemic of ovine theileriosis, while morbidity and mortality among non-immunized animals reached 76.9% and 46.15%, respectively, and they exhibited the clinical signs of malignant ovine theileriosis that included, high fever, loss of appetite, enlargement of lymph nodes, jaundice, loss of weight and death. The present study demonstrates the efficacy and the safety of this attenuated cell line as a live attenuated candidate vaccine.

Prime-boost immunisation against tropical theileriosis with two parasite surface antigens: evidence for protection and antigen synergy.

Current methods for control of tropical theileriosis in cattle suffer from several disadvantages that could be circumvented by development of an effective sub-unit vaccine. Previous work has utilised two major surface antigens (SPAG-1 and Tams1) and conventional adjuvants to provide partial protection against parasite challenge. In this study we have delivered these antigens using the prime-boost system and analysed whether a combination regime can enhance protection against lethal challenge. Delivery of the boost as recombinant protein or expressed from a recombinant MVA vector was also assessed. The results confirmed that immunisation with Tams1 alone could reduce the severity of several disease parameters compared to non-immunised controls and these effects were more marked when recombinant protein was used for boosting compared to MVA delivery. A similar outcome was obtained by immunisation with SPAG-1 alone. Significantly, delivery of SPAG-1 and Tams1 as a cocktail showed enhanced protection. This was manifest by significant improvement in a large range of clinical and parasitological parameters and, most dramatically, by the survival and recovery of 50% of the immunised animals compared to 0% of the controls. Analysis of the antibody response post-challenge showed that while there was a strong response to Tams1, no response to SPAG-1 was detected. In contrast, lymphoproliferation assays showed a significant enhancement of response at day 7 post-challenge in calves of the SPAG-1 group but a dramatic decrease of the proliferation activity in all three groups receiving Tams1. We conclude that immunisation with a cocktail of SPAG-1 and Tams1 generates a synergistic protective response that significantly improves the efficacy of recombinant vaccination against tropical theileriosis. Potential effector mechanisms that could mediate this response are discussed.

Occurrence of Theileria equi in horses raised in the Jaboticabal microregion, São Paulo State, Brazil / Ocorrência de Theileria equi em equinos criados na microrregião de Jaboticabal, Estado de São Paulo, Brasil

Blood and serum samples from 170 horses raised in the Jaboticabal microregion, São Paulo State, Brazil, were collected and tested by microscopic examination of blood smears, indirect fluorescent antibody test (IFAT) and nested polymerase chain reaction (nPCR) for Theileria equi infections. The association among the test results was verified by the McNemar test. During the examination of thin blood smears, parasites were detected in six (3.52 percent) horses. Anti-T. equi antibodies were detected in 100 percent sera samples, with titers ranging between 1:80 and 1:5120. The nPCR based on the T. equi merozoite antigen gene (EMA-1) allowed the visualization of specie-specific amplified product in 108 (63.53 percent) horses. All six samples judged positive microscopically were also positive for nPCR. Statistical analysis indicated general disagreement (p < 0.0001) between IFAT and nPCR; IFAT and blood smear; and nPCR and blood smear on the detection of parasite carriers. The results of the present study indicate that T. equi is widely spread among horses in the Jaboticabal microregion, Northeast region of São Paulo State, Brazil.

Amostras de sangue e soro de 170 equinos criados na microrregião de Jaboticabal, Estado de São Paulo, Brasil, foram coletadas e avaliadas pelo exame direto em esfregaço sanguíneo, reação de imunofluorescência indireta (RIFI) e nested reação em cadeia da polimerase (nPCR) para a detecção de infecções por Theileria equi. A concordância dos resultados entre os testes de diagnóstico foi verificada pelo teste de McNemar. Durante o exame dos esfregaços sanguíneos, parasitos foram detectados em seis (3,52 por cento) equinos. Anticorpos anti-T. equi foram detectados em 100 por cento das amostras de soros, com títulos variando entre 1:80 e 1:5120. O nPCR, baseado na sequência do gene do antígeno de merozoíto de T. equi (EMA-1), permitiu a visualização de produtos de amplificação espécie-específico em 108 (63,53 por cento) equinos. Houve diferença altamente significativa (p < 0,0001) entre RIFI e nPCR; RIFI e esfregaço sanguíneo; e nPCR e esfregaço na detecção do parasito. O resultado do presente estudo indica que a infecção por T. equi está amplamente distribuída entre os equinos na microrregião de Jaboticabal, região Nordeste do Estado de São Paulo, Brasil.

Generation of IFN-gamma-producing cells that recognize the major piroplasm surface protein in Theileria orientalis-infected bovines.

Theileriosis is a tick-borne protozoan disease caused by Theileria species. The Theileria species are classified into two groups depending on the cell type in which they proliferate and the clinical symptoms. The first group consists of lymphoproliferative Theileria species (T. parva and T. annulata), which mainly proliferate in lymphocytes, causing uncontrolled lymphocyte proliferation. The other group consists of a nonlymphoproliferative Theileria species (T. orientalis, also known as T. sergenti) that proliferates in erythrocytes and causes hemolytic anemia. Based on reports of generation of antigen-specific CD4(+) and CD8(+) T cells in lymphoproliferative theileriosis, we investigated whether T cells specific to the T. orientalis antigen are present in the nonlymphoproliferative form of the disease. In this study, we developed a new assay based on an enzyme-linked immunospot (ELISpot) to detect interferon-gamma (IFN-gamma)- and interleukin-10 (IL-10)-secreting cells in a series of cryogenically preserved bovine peripheral blood mononuclear cells (PBMCs). We first determined that IFN-gamma- and IL-10-secreting T cells were present in PBMCs by stimulating them with phytohemagglutinin L (PHA-L=red kidney bean lectin L, known as T cell stimulator), and then determined whether T. orientalis-specific T cells are present in T. orientalis-infected bovines. Peptides derived from T. orientalis major piroplasm surface protein (MPSP) were used as a T. orientalis-specific stimulator in the ELISpot assay, and peptides from glycoprotein B (gB) of the bovine herpes virus-1 (BHV-1) were used as a BHV-1-specific stimulator as a control for monitoring the immune response. Compared with results obtained using the BHV-1 (gB peptides)-specific IFN-gamma ELISpot assay to assess BHV-1-immunized Holsteins, prominent T. orientalis MPSP peptide-specific IFN-gamma and IL-10 positive spots were detected in T. orientalis-infected Holsteins but weak positive responses were exhibited by T. orientalis-infected Angus and Japanese Black cattle. As far as we are aware, this is the first report to show direct evidence of the presence of T. orientalis-specific T cells in T. orientalis-infected bovines using an antigen-specific ELISpot assay system and that T. orientalis-specific, IFN-gamma- and IL-10-producing T cells are produced in T. orientalis-infected Holsteins.

Seroprevalence of Babesia caballi and Theileria equi in the Swiss horse population.

In Switzerland, the prevalence and incidence of equine piroplasma parasite (EPP) infections are unknown. In order to obtain a first insight into the prevalence, a representative sample of 689 sera of horses from Switzerland was serologically tested for the presence of antibodies directed against T. equi and B. caballi using the Indirect Fluorescence Antibody Test (IFAT). A total of 50 (7.3%) horses were seropositive for EPP: overall, the seroprevalence of T. equi was significantly higher than that of B. caballi (p=0.002). The seropositivities in indigenous horses (animals bred and raised in Switzerland) and in imported horses were 4.8% (11/230) and 8.5% (39/459), respectively. Unlike in indigenous horses, where no significant difference in seroprevalences could be observed between the two parasite species, the seroprevalence of T. equi was significantly higher (p<0.001) than that of B. caballi in imported horses. Horses imported from France, Spain and Portugal exhibited a significantly higher seroprevalence, and horses imported from Germany a significantly lower seroprevalence of EPP compared to indigenous horses. There were no associations between sex, age, weight loss, surgery or blood transfusions with T. equi and B. caballi seroprevalences. The overall seroprevalence of 7.3% clearly shows that infection with EPP is a threat to the health of the horses in Switzerland. With the presumed expansion of permissive tick vectors, EPP infections will potentially increase in importance in the future. Therefore, continuous monitoring is mandatory.

Prevalence of equine Piroplasmosis and its association with tick infestation in the State of São Paulo, Brazil / Prevalência da Piroplasmose equina e sua associação com infestação por carrapatos no Estado de São Paulo

Serum samples were collected from 582 horses from 40 stud farms in the State of São Paulo and tick (Acari: Ixodidae) infestations were evaluated on them. Serum samples were subjected to the complement fixation test (CFT) and a competitive inhibition ELISA (cELISA) for Babesia caballi and Theileria equi. Logistic regression analyses were performed to construct multivariate models that could explain the dependent variable (horses positive for B. caballi or T. equi) as a function of the independent variables (presence or abundance of each one of the tick species found on the farms). A higher overall prevalence of B. caballi (54.1 percent) than of T. equi (21.6 percent) was found by the two tests. The ticks Dermacentor nitens Neumann, 1897, Amblyomma cajennense (Fabricius, 1787) and Rhipicephalus (Boophilus) microplus (Canestrini, 1887) were present on horses on 38 (95 percent), 20 (50 percent), and 4 (10 percent) farms, respectively. Infestations by D. nitens were statistically associated with B. caballi-positive horses on the farms by either the CFT or cELISA. Infestations by A. cajennense were statistically associated with T. equi-positive horses on the farms by either CFT or cELISA.

Amostras de soro sanguineo foram coletadas de 582 equinos de 40 haras no estado de São Paulo, onde as infestações por carrapatos foram avaliadas nos animais. Os soros foram testados por reação de fixação do complemento (RFC) e ELISA competitivo por inibição (cELISA) com antígenos de Babesia caballi e Theileria equi. Análises de regressão logística foram realizadas para construir modelos multivariados que pudessem explicar as variáveis dependentes (equinos positivos para B. caballi ou T. equi) em função de variáveis independentes (presença e abundância de cada uma das espécies de carrapatos encontradas nos equinos dos haras). Em geral, os dois testes sorológicos indicaram uma prevalência maior para B. caballi (54,1 por cento) do que para T. equi (21,6 por cento). Os carrapatos Dermacentor nitens Neumann, 1897, Amblyomma cajennense (Fabricius, 1787) e Rhipicephalus (Boophilus) microplus (Canestrini, 1887) estiveram presentes em equinos de 38 (95 por cento), 20 (50 por cento) e 4 (10 por cento) haras, respectivamente. As infestações por D. nitens estiveram estatisticamente associadas com equinos positivos para B. caballi tanto pela RFC como pelo cELISA. As infestações por A. cajennense estiveram estatisticamente associadas com equinos soropositivos para T. equi, tanto pela RFC como pelo cELISA.

Progress towards understanding the immunobiology of Theileria parasites.

The pathogenic Theileria species Theileria parva and T. annulata infect bovine leukocytes and erythrocytes causing acute, often fatal lymphoproliferative diseases in cattle. The parasites are of interest not only because of their economic importance as pathogens, but also because of their unique ability to transform the leukocytes they infect. The latter property allows parasitized leukocytes to be cultured as continuously growing cell lines in vitro, thus providing an amenable in vitro system to study the parasite/host cell relationship and parasite-specific cellular immune responses. This paper summarizes important advances in knowledge of the immunobiology of these parasites over the last 40 years, focusing particularly on areas of relevance to vaccination.

Estudo comparativo de três métodos de diagnóstico para detecção de anticorpos anti-Theileria equi em eqüinos de áreas endêmicas do estado do Rio de Janeiro / Comparative studies of three methods for detection Theileria equi antibody in endemic areas horses from Rio de Janeiro state

Este trabalho teve o objetivo de avaliar a reação de imunofluorescência indireta (RIFI), ensaio imunoenzimático (ELISA) e a reação de fixação do complemento (RFC) no diagnóstico de Theileria equi em amostras de soro de 79 equinos na Universidade Federal Rural do Rio de Janeiro(UFRRJ), Seropédica, RJ, Brasil. Houve reação positiva para Theileria equi em 74,7, 75,9 e 60,8% das amostras testadas pela RIFI, ELISA eRFC, respectivamente. Observou-se discrepância em 16,45% (n=13) das amostras de soro testadas pelo ELISA indireto e RIFI. Quando comparado a RIFI e a RFC, a discrepância observada entre os soros testados foi de 36,70% (n=29). O teste ELISA indireto e a RFC apresentaram discordância em 37,97% (n=30) das amostras de soros. Os resultados do presente estudo sugerem que a melhor alternativa para o diagnóstico sorológico de T. equi em eqüinos portadores é aassociação dos testes de RIFI e ELISA indireto, especialmente para a realização de estudos soroepidemiológicos

This study was carried out to evaluate indirect fluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and complement fixation test (CFT) of Theileria equi diagnosis in sera samples of 79 horses at Universidade Federal Rural do Rio de Janeiro(UFRRJ), Seropédica, RJ, Brazil were tested. Positive reaction was obtained in 74.7, 75.9 and 60.8% of samples tested by IFAT, indirect ELISA and CFT, respectively. Discrepancy was observed in 16.45%(n=13) of serum samples tested by ELISA and IFAT. While IFAT and CFT were compared, the discrepancy observed among the samples tested were 36.71% (n=29). Indirect ELISA and CFT test presented disagreement in 37.97% (n=30) of serum samples tested. Results of present study suggests that the best alternative for serological diagnosis T. equi in carriers horses is the combined use of IFAT and indirect ELISA test, especially for accomplishment of seroepidemiological studies.

Frequency of seropositive equines for Theileria equi in the Southern Rio Grande do Sul State, Brazil

Foi realizado um levantamento sobre theileriose equina em uma propriedade da região sul do estado do Rio Grande do Sul (RS). Amostras de sangue foram coletadas de 108 éguas de um haras localizado no municipio de Bagé, latitude 31°30' Sul e longitude 54Ú10' Oeste. A sorologia foi realizada com o uso da imunofluorescência indireta (IFAT) e utilizou-se a técnica de Nested Reacão em Cadeia Polimerase (nPCR). Do total de amostras examinadas, 22 por cento (24) foram positivas por IFAT e 15 por cento (16) por nPCR). Na análise por raca, 15.05 por cento (14) dos animais Puro Sangue Inglês (PSI) foram soro-positivos, e dos animais da raça Crioula (RC), 55 por cento (11) foram positivos por IFAT.

A study on equine theileriosis was carried out in the southern region of the Brazilian state of Rio Grande do Sul (RS). Blood samples were collected from 113 mares from an equine breeding farm located in the city ofBagé, latitude 31 "30'S and a longitude of 54 U10' W. The serological testing was carried out with the use of indirect fluorescence test (IFAT) and compared with Nested Polimerase Chain Reaction (nPCR). Among the sera collected from 118 horses, 25 were found positive to Theileria equi by the IFAT, while by nPCR 17 positive for T. equi was observed, corresponding to a frequency of22.1 percent> and 15.0 percent, respectively. The racial analysis showed 15.05 percent (14) thoroughbred and 55 percent (11) Crioulo breed horses to be positive by IFAT.

Study on Theileria lestoquardi antigens as potential vaccine candidates.

Theileria lestoquardi is the causative agent of malignant theileriosis of sheep and goats, causing morbidity and mortality in these animals worldwide. Western blot analysis based on T. lestoquardi schizont antigens was carried out using sera collected from Iranian sheep, which had been immunized with T. lestoquardi schizont-infected cells. The results of Western blot analysis demonstrated that schizont-immunized animals produced antibodies reacting with protein bands at 73, 42, 20, 14, and 12 kDa. Comparison of the results of the current Western blotting test with earlier studies of Theileria spp. revealed two immunogenic schizont proteins with molecular weights of 73 and 42 kDa shared between T. annulata and T. lestoquardi. Two other proteins with molecular weights of 14 and 12 kDa have not been previously found in other Theileria species. Our results suggest that the 73-kDa protein could be a potential vaccine candidate and that the 14- and 12-kDa proteins could be considered as diagnostic antigens.