Comparison of protectiveness of recombinant Babesia ovis apical membrane antigen 1 and B. ovis-infected cell line as vaccines against ovine babesiosis.

Babesiosis is a disease complex caused by unicellular Babesia parasites and among them, malignant ovine babesiosis caused by B. ovis has a devastating economical impact on the small ruminant industry. The control of disease is mainly based on chemotherapy and preventing animals from tick infestation and to date no vaccine is available against ovine babesiosis. The requirement for vaccination against B. ovis infection in endemically unstable regions is necessary for implementation of effective disease control measures. The aim of the present study was to evaluate the effectiveness of different immunisation protocols against disease in sheep experimentally vaccinated with recombinant B. ovis apical membrane antigen-1 (rBoAMA-1) and/or live, a B. ovis-infected cell line. Sheep were divided into four experimental groups, plus a control group. Animals were immunised either with the B. ovis stabilate, or with rBoAMA-1, or with both rBoAMA-1 and the B. ovis stabilate. Western blots and ELISAs indicated that immunisation with rBoAMA-1 resulted in generation of a specific response against the recombinant protein, but the degree of antibody response did not correlate with the level of induced protection against challenge. The strongest immune response was induced in animals co-immunised with the live B. ovis stabilate plus rBoAMA-1. Both the hematological and parasitological findings indicated that this co-immunisation regimen has vaccine potential to limit losses incurred by ovine babesiosis in endemic countries.

Serological detection and risk factors for equine piroplasmosis in the semiarid region of Pernambuco, Northeastern Brazil / Detecção sorológica e fatores de riscos para piroplasmose equina na região semiárida de Pernambuco, Nordeste do Brasil

Abstract Equine piroplasmosis, an economically important disease in horses, has so far not been reported in Pernambuco state, Brazil. This study aimed to evaluate the seroprevalence of anti-Babesia caballi and anti-Theileria equi antibodies based on the detection of these agents in equine blood and in ticks on horses in the municipality of Petrolina, Pernambuco, northeastern Brazil. Blood samples were drawn from 393 horses and sera were examined by ELISA. The presence of tick infestations was evaluated, and 101 ticks were subjected to DNA amplification for the detection of Babesia spp. by polymerase chain reaction (PCR). No parasites were detected in the blood smears. Anti-B. caballi and anti-T. equi antibodies were found in 27.2% (107/393) and 34.8% (137/393) horses, respectively. Infestation by Dermacentor nitens was detected in 4.3% (17/393) of the horses. There was no DNA amplification of the agents in ticks. The risk factors for the presence of anti-T. equi antibodies (P < 0.05) were: purebred (P < 0.001), animals older than 156 months (P = 0.014), and the presence of ticks (P = 0.001). No risk factors for B. caballi were identified. This study confirmed the circulation of agents of equine piroplasmosis in the municipality of Petrolina, state of Pernambuco, Brazil.

Resumo Piroplasmose equina é uma doença economicamente importante em equinos e não possui relatos no Estado de Pernambuco, Brasil. O objetivo deste estudo foi avaliar a soroprevalência de anticorpos anti-B. caballi e anti-T. equi pela detecção destes agentes no sangue e carrapatos de equinos no município de Petrolina, Pernambuco, Nordeste do Brasil. Amostras de sangue de 393 equinos foram coletadas e submetidas ao esfregaço sanguíneo e ELISA. A presença de infestação por carrapatos foi avaliada, e 71 carrapatos foram submetidos à Reação em Cadeia da Polimerase (PCR) para Babesia spp. Nenhum parasito foi detectado na análise de esfregaços de sangue. Anticorpos anti-B. caballi e anti-T. equi foram verificados em 27,2% (107/393) e 34,8% (137/393) dos equinos, respectivamente. A infestação por Dermacentor nitens foi verificada em 4,3% (17/393) dos equinos. Não houve amplificação do DNA dos agentes nos 71 carrapatos submetidos à PCR. Os fatores de risco para presença de anticorpos anti-T. equi (P < 0,05) foram: raça definida (P < 0,001), animais > de 156 meses (P = 0,014) e presença de carrapatos (P = 0,001). Nenhum fator de risco foi identificado para B. caballi. Esse estudo permitiu a confirmação da presença de agentes da piroplasmose equina no município de Petrolina, Pernambuco.

Molecular detection of vector-borne agents in cats in Southern Brazil / Detecção molecular de agentes transmitidos por vetores em felinos domésticos no Sul do Brasil

Abstract This study used serological and molecular methods to investigate the occurrence of vector-borne pathogens (VBP) with zoonotic potential in cats neutered at the University Veterinary Hospital in Canoinhas, Santa Catarina. The combined PCR and serological results revealed that 17 (56.6%) cats were positive for one or more pathogens. The sampled cats had antibodies to Ehrlichia spp. (7/30), Anaplasma phagocytophilum (3/30) and Leishmania infantum (2/30). The PCR assay detected DNA closely related to Ehrlichia canis in 6/30 cats, Mycoplasma haemofelis in 2/30 cats, A. phagocytophilum and Cytauxzoon sp. in one cat each. While Bartonella clarridgeiae and B. henselae were detected in two cats each, and B. koehlerae was detected in one cat.

Resumo Como os felinos podem ser parasitados por diversos patógenos transmitidos por vetores (PTV), alguns com caráter zoonótico, este estudo objetivou detectar por métodos sorológicos e moleculares, patógenos transmitidos por vetores hematófagos, em gatos atendidos em um Hospital Veterinário Universitário em Santa Catarina. Os resultados da PCR e da sorologia combinados, revelaram que 17 (56,6%) gatos foram positivos para um ou mais patógenos. Na sorologia, foram positivos 7/30 gatos para Ehrlichia, 3/30 para Anaplasma phagocytophilum e 2/30 para Leishmania infantum. Na PCR foi detectado DNA filogeneticamente associado a: Ehrlichia canis em 6/30 gatos; Mycoplasma haemofelis, em 2/30 gatos; A. phagocytophilum e Cytauxzoon sp. em 1/30 gatos cada. Enquanto Bartonella clarridgeiae e B. henselae foram detectadas, cada uma, em dois gatos, B. koehlerae foi detectada em um gato.

Babesia divergens glycosylphosphatidylinositols modulate blood coagulation and induce Th2-biased cytokine profiles in antigen presenting cells.

Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs.

A new immunoreactive recombinant protein designated as rBoSA2 from Babesia ovis: Its molecular characterization, subcellular localization and antibody recognition by infected sheep.

Ovine babesiosis, caused by the intra-erythrocytic protozoan parasite Babesia ovis, is an infectious and economically important tick-borne disease of sheep. Diagnostic testing is an essential tool used for the control of the disease. In order to identify and characterize the immunoreactive proteins which are useful in serological diagnosis of the disease, a complementary DNA (cDNA) expression library was constructed from B. ovis merozoite mRNA. A cDNA clone designated as BoSA2 was identified by immunoscreening of a cDNA library using immune sheep serum. The sequence of the BoSA2 cDNA had a partial open reading frame of 1156 nucleotides encoding a polypeptide of 384 amino acid residues. Theoretical molecular mass for the mature protein was 43.5 kDa. The sequence of the BoSA2 was inserted into the expression vector pGEX-4T-1 and then expressed in Escherichia coli DH5α cells as a glutathione S-transferase (GST)-tagged fusion protein. This recombinant fusion protein (rBoSA2) was purified by GST-affinity chromatography. Immunoreactivity of the rBoSA2 was evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using the sera from the animals naturally and experimentally infected with B. ovis. ELISA results demonstrated that this antigen was useful for the diagnosis of ovine babesiosis. The localization of the BoSA2 protein was shown in and on the parasite and in the cytoplasm of the infected erythrocyte by confocal laser microscope. To our knowledge, rBoSA2 is the second immunoreactive recombinant protein of B. ovis until the present.

A member of the HSP90 family from ovine Babesia in China: molecular characterization, phylogenetic analysis and antigenicity.

Heat shock protein 90 (HSP90) is a key component of the molecular chaperone complex essential for activating many signalling proteins involved in the development and progression of pathogenic cellular transformation. A Hsp90 gene (BQHsp90) was cloned and characterized from Babesia sp. BQ1 (Lintan), an ovine Babesia isolate belonging to Babesia motasi-like group, by screening a cDNA expression library and performing rapid amplification of cDNA ends. The full-length cDNA of BQHsp90 is 2399 bp with an open reading frame of 2154 bp encoding a predicted 83 kDa polypeptide with 717 amino acid residues. It shows significant homology and similar structural characteristics to Hsp90 of other apicomplex organisms. Phylogenetic analysis, based on the HSP90 amino acid sequences, showed that the Babesia genus is clearly separated from other apicomplexa genera. Five Chinese ovine Babesia isolates were divided into 2 phylogenetic clusters, namely Babesia sp. Xinjiang (previously designated a new species) cluster and B. motasi-like cluster which could be further divided into 2 subclusters (Babesia sp. BQ1 (Lintan)/Babesia sp. Tianzhu and Babesia sp. BQ1 (Ningxian)/Babesia sp. Hebei). Finally, the antigenicity of rBQHSP90 protein from prokaryotic expression was also evaluated using western blot and enzyme-linked immunosorbent assay (ELISA).

Characterisation of a Babesia orientalis apical membrane antigen, and comparison of its orthologues among selected apicomplexans.

In the present study, we identified and characterised the complete coding sequence of Babesia orientalis apical membrane antigen 1 (designated Bo-ama1); it is 1803bp in length and encodes a polypeptide of 601 amino acids (aa). The Bo-ama-1 gene product (Bo-AMA1) is predicted to be 67kDa in size and contains a signal peptide. Mature Bo-AMA1 is predicted to have one transmembrane region and a short cytoplasmic tail (C-terminal domain). The extracellular part of Bo-AMA1 has three functional domains (DI, DII and DIII) with 14 conserved cysteine residues. A Bo-AMA1 fragment containing all three of these domains (designated Bo-AMA1-DI/II/III) was cloned into the plasmid vector pET-28a and expressed as a recombinant (His-fusion) protein of 53kDa. Antibodies in the serum from a B. orientalis-infected water buffalo specifically recognised this protein in immunoblotting analysis. Rabbit antibodies raised against the recombinant protein were able to detect native Bo-AMA1 (67kDa) from erythrocytes of B. orientalis-infected water buffalo. Bo-AMA1 is a new member of the AMA1 family and might be a good antigen for the specific detection of antibodies produced in B. orientalis infected cattle. This protein is likely to play critical roles during host cell adherence and invasion by B. orientalis, as the AMA1s reported in other organisms such as Plasmodium falciparum and Toxoplasma gondii. Further research is required to explore the biological functions of this protein and to determine whether its immunisation can induce protective effects in water buffalo against B. orientalis infection.

Survey of Ehrlichia canis, Babesia spp. and Hepatozoon spp. in dogs from a semiarid region of Brazil / Pesquisa de Ehrlichia canis, Babesia spp. e Hepatozoon spp. em cães de uma região semiárida do Brasil

This study assessed the occurrence of Ehrlichia spp., Babesia spp. and Hepatozoon spp. infections in 100 tick-harboring dogs from a semiarid region of the State of Paraíba, Northeastern Brazil. Blood samples and ticks were collected from the animals, and a questionnaire was submitted to dog owners to obtain general data. Blood samples were used to perform hemogram, direct blood smear and immunological and molecular hemoparasite detection. The 1,151 ticks collected were identified as Rhipicephalus sanguineus; direct smears revealed E. canis-like morulae in the monocytes of 4% (4/100) of the non-vaccinated female dogs, and 34% and 25% of the dogs tested positive for Ehrlichia canis by indirect immunofluorescence assay (IFA) and polymerase chain reaction (PCR), respectively. Blood smear examination revealed Babesia-suggestive merozoites in the erythrocytes of 2% (2/100) of the animals. Babesia vogeli was detected by PCR in ten animals (10%) and was correlated with young age (p = 0.007) and thrombocytopenia (p = 0.01). None of the animals showed Hepatozoon spp. positivity. These results indicate that E. canis is the main tick-borne canine pathogen in the study area and provide the first report of B. vogeli infection in dogs from Paraiba State.

O presente estudo avaliou a ocorrência de infecção por Ehrlichia spp., Babesia spp. e Hepatozoon spp. em 100 cães, infestados por carrapatos, oriundos de uma região semiárida do Estado da Paraíba, Nordeste do Brasil. Amostras de sangue e de carrapatos foram coletadas dos animais, e um questionário foi submetido aos proprietários dos cães para obter dados gerais. As amostras de sangue foram utilizadas para realização de hemograma, esfregaço sanguíneo e detecção molecular dos hemoparasitos. Os 1.151 carrapatos coletados foram identificados como Rhipicephalus sanguineus; os esfregaços sanguíneos revelaram mórulas sugestivas de E. canis em 4% (4/100) de cães fêmeas não vacinadas, e 34% e 25% dos cães foram positivos para Ehrlichia canis pela imunofluorescência indireta (IFI) e reação em cadeia pela polimerase (PCR), respectivamente. Os esfregaços sanguíneos revelaram merozoítas sugestivas de Babesia em eritrócitos de 2% (2/100) dos animais. Babesia vogeli foi detectada por PCR em dez animais (10%) e foi correlacionada com a idade jovem (p=0,007) e trombocitopenia (p=0,01). Nenhum dos animais apresentou positividade para Hepatozoon spp. Esses resultados indicam que E. canis é o principal patógeno canino transmitido por carrapato, na área estudada, e fornece o primeiro relato de infecção por B. vogeli em cães do Estado da Paraíba.

A serological and molecular survey of Babesia vogeli, Ehrlichia canis and Rickettsia spp. among dogs in the state of Maranhão, northeastern Brazil / Detecção sorológica e molecular de Babesia vogeli, Ehrlichia canis e Rickettsia spp. em cães do Estado do Maranhão, Nordeste do Brasil

This study evaluated exposure and infection by tick-borne agents (Babesia vogeli, Ehrlichia canis and Rickettsia spp.) in 172 dogs in rural areas and 150 dogs in urban areas of the municipality of Chapadinha, state of Maranhão, northeastern Brazil, using molecular and serological methods. Overall, 16.1% of the sampled dogs (52/322) were seroreactive to B. vogeli, with endpoint titers ranging from 40 to 640. For E. canis, 14.6% of the dogs (47/322) were seroreactive, with endpoint titers from 80 to 163,840. Antibodies reactive to at least one of the five species of Rickettsia were detected in 18.9% of the dogs (61/322), with endpoint titers ranging from 64 to 4,096. High endpoint titers were observed for Rickettsia amblyommii. Three (0.9%) and nine (2.8%) canine blood samples were PCR-positive for Babesia spp. and E. canis. The ticks collected from urban dogs were all Rhipicephalus sanguineus sensu lato, whereas the rural dogs were infested by R. sanguineus s.l, Amblyomma cajennense sensu lato and Amblyomma ovale. One A. ovale tick was found to be infected by Rickettsia bellii. This study provides an epidemiological background for controlling and preventing canine tick-borne diseases in a neglected region of Brazil.

Este estudo avaliou por métodos sorológicos e moleculares a exposição e infecção por agentes transmitidos por carrapatos (Babesia vogeli, Ehrlichia canis, and Rickettsia spp.) em 172 cães de áreas rurais e 150 cães de áreas urbanas do município de Chapadinha, Estado do Maranhão, Nordeste do Brasil. No geral, 16,1% dos cães amostrados (52/322) apresentaram soros reagentes para B. vogeli, com títulos finais variando de 40 a 640. Para E. canis, 14,6% cães (47/322) apresentaram soros reagentes com títulos finais de 80 a 163,840. Anticorpos reativos para pelo menos uma das cinco espécies de Rickettsia foram detectados em 18,9% dos cães (61/322), com os títulos que variam de 64 a 4096. Foram observados altos títulos para Rickettsia amblyommii. Três amostras de sangue canino (0,9%) e 9 (2,8%) foram PCR positivas para Babesia spp e E. canis. Os carrapatos coletados de cães urbanos eram todos Rhipicephalus sanguineus sensulato, e os cães rurais estavam infestados por R. sanguineus s.l , Amblyomma cajennense sensu lato e Amblyomma ovale. Um carrapato A. ovale foi encontrado infectado por Rickettsia bellii. Este estudo fornece um conhecimento epidemiológico para o controle e prevenção de doenças transmitidas por carrapatos de cães em uma região negligenciada do Brasil.

Serological cross-reactivity of Trypanosoma cruzi, Ehrlichia canis, Toxoplasma gondii, Neospora caninum and Babesia canis to Leishmania infantum chagasi tests in dogs

Introduction: The aim of this study was to evaluate the serological cross-reactivity between Leishmania sp. and other canine pathogens. Methods: Positive serum samples for Ehrlichia canis, Babesia canis, Toxoplasma gondii, Neospora caninum and Trypanosoma cruzi were tested using three serological methods enzyme linked immunosorbent assay (ELISA), indirect immunofluorescent antibody test (IFAT) and Kalazar Detect™, for canine visceral leishmaniasis. Results: Of the 57 dog samples tested, 24 (42.1%) tested positive using one of the three serological methods: 10/57 (17.5%) for ELISA, 11/57 (19.3%) for IFAT and 3/57 (5.3%) for Kalazar Detect™. Conclusions: Our results demonstrated that the presence of other infectious agents may lead to cross-reactivity on leishmaniasis serological tests. .

Molecular and serological detection of tick-borne pathogens in dogs from an area endemic for Leishmania infantum in Mato Grosso do Sul, Brazil / Detecção sorológica e molecular de patógenos transmitidos por carrapatos em cães de uma área endêmica para Leishmania infantum do estado do Mato Grosso do Sul

Tick-borne pathogens affect a wide range of vertebrate hosts. To identify tick-borne pathogens among dogs from Campo Grande, MS, Brazil testing seropositive for Leishmania infantum (syn. L. chagasi), a serological and molecular study was conducted to detect Ehrlichia canis, Anaplasma platys and Babesia vogeli in 60 serum and spleen samples. A confirmatory diagnosis of L. infantum based on serological and molecular assays was also performed, as was sequence alignment and phylogenetic analysis to assess the identity of the parasite species infecting these animals. IgG antibodies to Ehrlichia spp., B. vogeli and L. infantum were found, respectively, in 39 (65%), 49 (81.6%) and 60 (100%) of the sampled dogs. Twenty-seven (45%), fifty-four (90%), fifty-three (88.3%), two (3.3%) and one (1.6%) dog were positive, respectively, for E. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. and Anaplasma sp. in PCR assays. After sequencing, the amplicons showed 99% of identity with E. canis, B. vogeli, A. platys and Leishmania chagasi isolates. The findings of this study indicate that L. infantum-seropositive dogs from Campo Grande are exposed to multiple tick-borne pathogens, which should therefore be included in the differential diagnosis of dogs with clinical suspicion of leishmaniasis.

Patógenos transmitidos por carrapatos atingem uma variedade de hospedeiros vertebrados. Para identificar os agentes patogênicos transmitidos por carrapatos entre cães soropositivos para Leishmania infantum no município Campo Grande-MS, foi realizado um estudo sorológico e molecular para a detecção de Ehrlichia canis, Anaplasma platys e Babesia vogeli em 60 amostras de soro e baço, respectivamente. Adicionalmente, foi realizado o diagnóstico confirmatório de L. infantum por meio de técnicas sorológicas e moleculares. Também foi realizado o alinhamento e análise filogenética das sequências para indicar a identidade das espécies de parasitas que infectam esses animais. Anticorpos IgG anti-Ehrlichia spp., anti-B. vogeli e anti-L. infantum foram detectados em 39 (65%), 49 (81,6%) e 60 (100%) dos cães amostrados, respectivamente. Vinte e sete (45%), cinquenta e quatro (90%), cinquenta e três (88,3%), dois (3,3%) e um (1,6%) cães mostraram-se positivos na PCR para E. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. e Anaplasma sp., respectivamente. Após o seqüenciamento, os amplicons mostraram 99% de similaridade com isolados de E. canis, B. vogeli e A. platys e Leishmania chagasi. Os resultados deste estudo indicaram que os cães soropositivos para L. infantum de Campo Grande, MS, são expostos a vários agentes transmitidos por carrapatos, e, portanto, devem ser incluídos no diagnóstico diferencial em cães com suspeita clínica de leishmaniose.

Babesiosis and anaplasmosis in dairy cattle in Northeastern Brazil / Babesiose e anaplasmose em bovinos leiteiros no Nordeste do Brasil

The goal of this study was to characterize the epidemiological situation and the factors involved in the prevalence of babesiosis and anaplasmosis in cattle in the dairy basin of Parnaíba, Piauí, Brazil. The study was conducted in 22 farms, and collected blood samples from 202 cattle to study serological, molecular and determination of the packed cell volume (PCV). On the farms were applied surveys involving epidemiological aspects. Seroprevalence rates were: Babesia bigemina 52.5%, B. bovis 68.8%, and Anaplasma marginale 89.1%. Of the samples analyzed, 73.3% were reactive for Babesia spp. and A. marginale, showing co-infection. In PCR, B. bigemina and B. bovis were positive in 52.0% and 33.2% respectively, and A. marginale in 76.2%. Of these, 51.5% amplified DNA of Babesia spp. and A. marginale. The semi-intensive management predominated in 68.0% of the farms studied. The clinical history of babesiosis and anaplasmosis, was reported from 73% of the farms. There was no significant difference (p>0.05) between age groups and for the PCV of positive compared with negative animals. The study indicates that in this region is enzootic instability for babesiosis and enzootic stability for anaplasmosis, reinforcing the fact that in Brazil there are areas of enzootic instability, even in tropical regions of the country. The PCR technique was a valuable tool for the diagnosis of these diseases and may be used to characterize a geographic region.

O objetivo deste estudo foi caracterizar a situação epidemiológica e os fatores envolvidos na prevalência da babesiose e anaplasmose em bovinos da bacia leiteira de Parnaíba, Piauí, Brasil. O estudo foi realizado em 22 propriedades, sendo coletadas amostras de sangue de 202 bovinos para estudos sorológicos, moleculares e determinação do volume globular (VG). Nas propriedades foram aplicadas inquéritos envolvendo aspectos epidemiológicos. As taxas de soroprevalência foram: 52,5% para Babesia bigemina, 68,8% B. bovis, e 89,1% para Anaplasma marginale. Das amostras analisadas, 73,3% foram reagentes para Babesia spp. e A. marginale, demostrando co-infecção. Na PCR, B. bigemina e B. bovis foram positivas em 52,0% e 33,2% respectivamente, e A. marginale em 76,2%. Destes, 51,5% amplificaram DNA de Babesia spp. e A. marginale. O manejo semi-intensivo predominou em 68,0% das propriedades estudadas. O histórico clínico de babesiose e anaplasmose foi relatado em 73% das propriedades. Não houve diferença significativa (p>0,05) entre as faixas etárias e para o VG de animais positivos comparados com os negativos. O estudo indica que nesta região há instabilidade enzoótica para babesiose e estabilidade enzoótica para anaplasmose, reforçando o fato de que, no Brasil, existem áreas de instabilidade enzoótica, mesmo em regiões tropicais do país. A técnica de PCR demonstrou ser uma ferramenta valiosa para o diagnóstico destas doenças e pode ser utilizada para caracterizar uma região geográfica.

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP).

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.

Seroprevalence rates of antibodies against Leishmania infantum and other protozoan and rickettsial parasites in dogs / Soroprevalência de anticorpos contra Leishmania infantum e outras espécies de protozoários e rickettsia em cães

Canine visceral leishmaniasis (CVL) is caused by the protozoan Leishmania infantum, which infects dogs and humans in many regions of Brazil. The present study involved an indirect fluorescent antibody test (IFAT) to analyze L. infantum, Ehrlichia spp., Babesia canis, Toxoplasma gondii and Neospora caninum infection rates in serum samples from 93 dogs in a rural settlement in Ilha Solteira, SP, Brazil. The seroprevalence rates of anti-L. infantum, anti-Ehrlichia, anti-B. canis, anti-T. gondii and anti-N. caninum antibodies were 37.6%, 75.3%, 72%, 47.3% and 6.4%, respectively. In addition to IFAT, direct microscopic examination of popliteal lymph node aspirates revealed 26.9% of CVL positive dogs. Serological tests revealed that 17.2% of the dogs were seropositive for a single parasite, 29% for two parasites, 33% for three, 16.1% for four, and 1.1% for five parasites, while 3.2% were seronegative for five parasites. The presence of antibodies against these parasites in serum samples from dogs confirmed their exposure to these parasites in this rural area. Because of the potential zoonotic risk of these diseases, mainly leishmaniasis, ehrlichiosis and toxoplasmosis, special attention should focus on programs for the improvement of diagnostic assays and control measures against these parasites.

Leishmaniose Visceral Canina (LVC) é causada pelo protozoário Leishmania infantum, podendo infectar cães e humanos em várias regiões do Brasil. O presente estudo teve por objetivo realizar a reação de imunofluorescência indireta (RIFI) para analisar os índices de infecção parasitária para L. infantum, Ehrlichia spp., Babesia canis, Toxoplasma gondii e Neospora caninum, em 93 amostras de soro de cães de um assentamento rural no município de Ilha Solteira, SP, Brasil. A taxa de soroprevalência de cães com anticorpos anti-L. infantum, anti-Ehrlichia, anti-B. canis, anti-T. gondii e anti-N. caninum foi de 37,6%, 75,3%, 72%, 47,3% e 6,4%, respectivamente. Pelo exame microscópico direto dos parasitas nos esfregaços de aspirados de linfonodos poplíteos dos cães, a positividade para LVC foi de 26,9%. Pelos exames sorológicos, 17,2% dos cães estavam positivos com um único parasita, 29% com dois, 33% com três, 16,1% com quatro e 1,1% com cinco parasitas. Além disso, 3,2% eram soronegativos para todos os cinco agentes parasitários. A presença de anticorpos aos parasitos em amostras sorológicas confirmam a exposição dos cães às doenças parasitárias nesse assentamento rural. Devido ao potencial risco zoonótico destas doenças, principalmente leishmaniose, erliquiose e toxoplasmose, atenção especial deve ser dada aos programas que objetivam o aprimoramento de testes diagnósticos e de medidas de controle dessas parasitoses.

Macrophages are critical for cross-protective immunity conferred by Babesia microti against Babesia rodhaini infection in mice.

Although primary infection of mice with Babesia microti has been shown to protect mice against subsequent lethal infection by Babesia rodhaini, the mechanism behind the cross-protection is unknown. To unravel this mechanism, we investigated the influence of primary infection of mice with nonlethal B. microti using different time courses on the outcome of subsequent lethal B. rodhaini infection. Simultaneous infections of mice with these parasites resulted in rapid increases in parasitemia, with 100% mortality in BALB/c mice, as observed with control mice infected with B. rodhaini alone. In contrast, mice with acute, resolving, and chronic-phase B. microti infections were completely protected against B. rodhaini, resulting in low parasitemia and no mortalities. Mice immunized with dead B. microti were not protected from B. rodhaini infection, although high antibody responses were induced. Interestingly, the protected mice had significantly decreased levels of antibody response, cytokines (including gamma interferon [IFN-γ], interleukin-2 [IL-2], IL-8, IL-10, and IL-12), and nitric oxide levels after infection with B. rodhaini. SCID mice and IFN-γ-deficient mice with chronic B. microti infections demonstrated protective responses comparable to those of immunocompetent mice. Likewise, in vivo NK cell depletion did not significantly impair the protective responses. Conversely, macrophage depletion resulted in increased susceptibility to B. rodhaini infection associated with changes in their antibody and cytokines profiles, indicating that macrophages contribute to the protection against this challenge infection. We conclude that future development of vaccines against Babesia should include a strategy that enhances the appropriate activation of macrophages.

Comparison of the Babesia duncani (WA1) IgG detection rates among clinical sera submitted to a reference laboratory for WA1 IgG testing and blood donor specimens from diverse geographic areas of the United States.

All reported cases of WA1 babesiosis have occurred in the Pacific coast region of the United States, suggesting that WA1 is limited to this geographic area. However, we detected WA1 IgG in 27% of clinical sera sent to our laboratory for WA1 IgG testing from across the United States over a 2-year period, suggesting that exposure to WA1 or a closely related organism occurs outside Pacific coast states. We sought to determine if this high WA1 IgG detection rate among clinical specimens merely reflects WA1 seroprevalence outside the Pacific region. WA1 IgG, as well as Babesia microti IgG, was measured in 900 blood donor specimens from 9 states. Overall seroprevalence was 2.0% for WA1 and 0.4% for B. microti; regional seroprevalences ranged from 0 to 4% and 0 to 2%, respectively. Additional studies were performed to determine if WA1 IgG reactivity was attributable to polyclonal B-cell activation associated with acute Epstein-Barr virus (EBV) infection; 40 WA1 IgG-positive clinical sera and the 18 WA1 IgG-positive blood donor specimens were all negative for EBV capsid antigen (EBVCA) IgM (a marker of acute EBV infection), and 40 EBVCA IgM-positive sera were all negative for WA1 IgG. These findings indicate that the high WA1 IgG detection rate among clinical specimens does not simply reflect the national WA1 seroprevalence among blood donors or nonspecific reactivity due to acute EBV infection. Rather, the findings suggest that infection with WA1 or a related organism is more common than indicated by the literature and is not limited to Pacific coast states.

Detection of antibodies against Babesia bovis and Babesia bigemina in calves from the region of Araguaína, State of Tocantins, Brazil / Detecção de anticorpos anti-Babesia bovis e anti-Babesia bigemina em bezerros na região de Araguaína, Estado do Tocantins, Brasil

The aim of this study was to determine the seroprevalence of antibodies against B. bovis and B. bigemina in calves from the region of Araguaína, State of Tocantins, Brazil. In this research we used sera obtained from 506 calves, from both genders and of 8 to 24 months old, to detect antibodies by indirect Enzyme-Linked Immunosorbent Assay (ELISA-test). Statistical analysis of the data was performed using the Chi-square (χ2) test with Yates correction. The seroprevalence obtained was 90.5 and 91.7 percent for B. bigemina and B. bovis, respectively, characterizing the region as an area of enzootic stability for the species analyzed. The seroprevalence to B. bovis showed higher positivity among calves 19-24 months old.

O objetivo desse estudo foi determinar a soroprevalência de anticorpos anti-B. bovis e anti-B. bigemina em bezerros da região de Araguaína, Estado do Tocantins, Brasil. Nesta pesquisa foram coletadas 506 amostras de soros de bezerros com faixa etária entre 8 e 24 meses, fêmeas e machos, as quais foram processadas pelo ensaio imunoenzimático indireto (ELISA-teste). Como análise estatística utilizou-se o Qui-Quadrado (χ2) com correção de Yates. As prevalências obtidas em bezerros foram de 90,5 e 91,7 por cento para B. bigemina e B. bovis, respectivamente, caracterizando esta região como de estabilidade enzoótica para as espécies analisadas. A soroprevalência para B. bovis apresentou maior positividade entre os bezerros com faixa etária de 19-24 meses.

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.

Fatores associados à soropositividade para Babesia, Toxoplasma, Neospora e Leishmania em cães atendidos em nove clínicas veterinárias do município de Lavras, MG / Factors associated the seropositivity for Babesia, Toxoplasma, Neospora e Leishmania in dogs attended at nine veterinary clinics in the municipality of Lavras, MG

O objetivo deste estudo foi determinar a frequência e avaliar a influência da idade, sexo e raça na soropositividade anti-Babesia canis, Toxoplasma gondii, Leishmania (L.) chagasi e Neospora caninum, por meio da reação de imunofluorescência indireta (RIFI), em amostras de soros coletadas de cães atendidos em nove clínicas veterinárias particulares do município de Lavras, MG, no período de agosto de 2000 a abril de 2002. De 300 cães, 73,3% foram soropositivos (RIFI > 1:80) para B. canis, e houve um aumento significativo de reagentes (p < 0,05) nos animais adultos se comparados com os jovens. Apenas um cão (0,3%), proveniente do município de Belo Horizonte, apresentou anticorpos anti-L. (L.) chagasi (RIFI > 1:40). Para T. gondii, de 218 cães, 60,7% foram positivos (RIFI > 1:16). Em 228 amostras de soros, 3,1% foram positivas (RIFI > 1:50) para N. caninum. Infecções por B. canis e T. gondii são endêmicas em cães atendidos em clínicas veterinárias particulares em Lavras. Não há evidências de casos autóctones de leishmaniose visceral canina em Lavras. Além disso, a infecção por N. caninum é pouco comum em cães criados na zona urbana do município.

The aim of the present study was to determine the frequency and evaluate the influence of age, sex and breed in seropositivity anti-Babesia canis, Toxoplasma gondii, Leishmania (L.) chagasi and Neospora caninum, by means of the indirect immunofluorescence antibody test (IFAT), in serum samples collected from dogs attended in nine private veterinary clinics in municipality of Lavras, Minas Gerais, Brazil, from August 2000 to April 2002. Of 300 dogs, 73.3% were seropositive (IFAT > 1:80) to B. canis, and there was a significant increase (p < 0.05) of the reagent in adult animals when compared with young. Only one dog (0.3%) from Belo Horizonte there was antibodies anti-L. (L.) chagasi (IFAT > 1:40). T. gondii, of 218 dogs, 60.7% were positive (IFAT > 1:16). In 228 serum samples, 3.1% were positive (IFAT > 1:50) to N. caninum. Infections to B. canis and T. gondii occur as endemic form in dogs examined at private veterinary clinics in Lavras. There is no evidence that there are autochthonous cases of canine visceral leishmaniosis in Lavras. Besides this the infection by N. caninum is uncommon in dogs breed at the urbane zone of the municipality.

Comparison between a soluble antigen-based ELISA and IFAT in detecting antibodies against Babesia canis in dogs / Comparação entre ELISA feito com antígeno solúvel e imunofluorescência na detecção de anticorpos anti-Babesia canis em cães

An available enzyme-linked immunosorbent assay (ELISA) was studied for the detection of anti-B. canis antibodies in the sera of dogs using, indirect fluorescent antibody test (IFAT) as a reference test. ELISA uses a soluble antigenic preparation of B. canis and the optimal dilutions of the antigen, serum and conjugate were determined by check board titration, using positive and negative reference serum. The soluble antigen preparation of B. canis merozoites was 10 µg.mL-1, with reference sera from positive and negative in a single dilution of 1:100, and conjugated to 1:4.000. A total of 246 serum samples were collected from dogs during the rabies vaccination campaign in Jaboticabal, São Paulo, Brazil and examined for the presence of antibodies against B. canis by ELISA and IFAT. Under these conditions, the average absorbance of negative serum was 0.129 ± 0.025, resulting in a cut-off value of 0.323 (ELISA level 3) and the average absorbance of positive reference serum was 2.156 ± 1.187. The serological positive samples tested for B. canis by ELISA and IFAT were 67.89 percent (n = 167) and 59.35 percent (n = 146), respectively. These results suggest that ELISA described may prove to be an effective serological test to diagnose canine babesiosis.

O presente trabalho estudou um ensaio imunoenzimático (ELISA) indireto para a detecção de anticorpos anti-Babesia canis no soro de cães, tendo a Reação de Imunofluorescência Indireta (RIFI), como teste de referência O antígeno utilizado no ELISA do presente estudo consistiu em uma preparação antigênica solúvel de merozoítas B. canis e as diluições ótimas do antígeno, soros e conjugado foram determinadas por titulação em bloco, utilizando soros de referência positivos e negativos. A preparação antigênica solúvel de B. canis ótima foi de 10 µg.mL-1, com soros de referência positivos e negativos em uma única diluição de 1:100, e conjugado a 1:4.000. Um total de 246 amostras séricas foram colhidas em cães, durante a campanha de vacinação anti-rábica em Jaboticabal, São Paulo, Brasil e a presença de anticorpos anti-B. canis foi avaliada pelo ELISA e RIFI. Nestas condições, a média de absorbância dos soros de referência negativos foi de 0,129 ± 0,025, resultando em um ponto de corte de 0,323 (Nível de ELISA 3) e a média da absorbância dos soros de referência positivos foi de 2,156 ± 1,187. As amostras com sorologia positiva para B. canis por ELISA e RIFI foram 67,89 por cento (n = 167) e 59,35 por cento (n = 146), respectivamente. Os resultados obtidos sugerem que o ELISA descrito revelou-se um teste sorológico eficaz no diagnóstico da babesiose canina.