Differential diagnosis of theileriosis through blood smear examination and polymerase chain reaction in small ruminants from Pakistan.

Background: Ovine and caprine theileriosis is a tick-borne hemoprotozoan disease, caused by Theileria spp., responsible for heavy economic losses in terms of high mortality and morbidity rates. Diagnosis of ovine theileriosis is primarily based on clinical symptoms, microscopic screening of stained blood smears, and lymph node biopsy smears, but the limitations of these detection methods against Theileria spp. infection limits their specificity. Aim: To overcome these limitations, the current study reports the differential diagnosis of theileriosis through a blood smear examination and polymerase chain reaction (PCR) in small ruminants from Pakistan. Methods: The study was conducted on 1,200 apparently healthy small ruminants (737 sheep and 463 goats). First, blood smears were screened for the presence of Theileria piroplasms in red blood cells. Second, PCR amplification based on 18S rRNA gene was performed by using primers specific to Theileria spp. Results: Out of the 1,200 samples of examined blood smears, 100 animals (8.33%) were found positive for Theileria species, which showed intra-erythrocytic bodies in the form of dot and comma shapes. Amplification of the isolated DNA from randomly collected blood samples of 737 sheep and 463 goats showed that an amplicon size of 1,098 bp was positive for Theileria spp. In total, 315 out of the 1,200 small ruminants examined in this study were found positive for Theileria spp. DNA through PCR amplification. Notably, out of the 885 blood samples negative by PCR amplification, only 15 blood samples were found positive by the blood smear test. Conversely, 230 blood samples that tested negative in the smear technique produced a specific band through PCR amplification. Overall, the sensitivity and specificity rates were 26.98% and 98.31% for the blood smear method and 73.01% and 100% for the PCR assay, respectively. Conclusion: Our finding suggests that PCR is the gold standard method compared to the conventional method of smear examination for the diagnosis of ovine and caprine theileriosis in Pakistan.

Natural infection and molecular detection of Cytauxzoon felis in a free-ranging Puma concolor in the state of Goiás, Brazil / Infecção natural e detecção molecular de Cytauxzoon felis em Puma concolor de vida livre no estado de Goiás, Brasil

The puma (Puma concolor Linnaeus, 1771), the most widely distributed felid species in the Americas, can be found in all Brazilian biomes. Nevertheless, few studies have focused on hemoparasites in this species. Cytauxzoon felis, a hemoparasite that can infect domestic cats, has also been described in wild felids in Brazil. To the best of our knowledge, this study is the first to diagnose the natural infection and molecular detection of C. felis in a P. concolor in the state of Goiás. This animal presented non-regenerative anemia and inclusion suggestive of piroplasmids within red blood cells. The amplified 551 bp fragment of partial Piroplasmida 18S rRNA gene sequence was 100% identical to corresponding sequences of C. felis available in GenBank. No specific treatment for cytauxzoonosis was administered, and after rehabilitation, the animal was reintroduced into the wild. This finding provides some evidence that P. concolor may act as a natural host of the parasite. The epidemiology, vector and pathogenicity of this hemoparasite in wild and domestic cats in Brazil deserves further investigation.

O puma (Puma concolor Linnaeus, 1771) tem a maior distribuição entre os felídeos das Américas e é encontrado em todos os biomas do Brasil. No entanto, poucos estudos têm se concentrado nos hemoparasitos nesta espécie. Cytauxzoon felis, um hemoparasito que pode infectar gatos domésticos, também foi descrito em felídeos selvagens no Brasil. A saber, este estudo é o primeiro diagnóstico de infecção natural e detecção molecular de C. felis em um P. concolor do estado de Goiás. Este animal apresentou anemia arregenerativa e inclusão de piroplasmídeos nos glóbulos vermelhos. A amplificação do fragmento de 551 pb da sequência parcial do gene Piroplasmorida 18S rRNA foi 100% idêntica a sequências correspondentes de C. felis disponíveis no GenBank. Nenhum tratamento específico para citauxzoonose foi administrado e, após a reabilitação, o animal foi reintroduzido na natureza. Essa descoberta fornece algumas evidências de que P. concolor pode atuar como um hospedeiro natural do parasito. A epidemiologia, vetor e patogenicidade deste hemoparasito em gatos selvagens e domésticos no Brasil merecem uma investigação mais aprofundada.

Serological and molecular detection of Theileria equi in horses from Sinop, Mato Grosso state, Brazil / Detecção sorológica e molecular de Theileria equi em equinos oriundos de Sinop, Mato Grosso, Brasil

Equine piroplasmosis is the most important tick-borne disease to affect horses in Brazil. Theileria equi is one of the causative agents of equine piroplasmosis. Chronic cases are expected, in which the animals show no apparent signs of infection and remain asymptomatic but constitute a source of the infectious agent that ticks can spread. This study was conducted across 81 ranches located in the municipality of Sinop, State of Mato Grosso, Brazil. A sample calculation was performed to estimate the apparent prevalence of T. equi among horses. A total of 1,853 animals were included in the sampling analysis based on the information available from the Institute of Agricultural and Livestock Defense of Mato Grosso State. The serological analysis of 367 serum samples using an indirect enzyme-linked immunosorbent assay (ELISA) to detect anti-T. equi antibodies revealed that 337 animals were positive, representing a frequency of 90.70%. The molecular analysis to amplify the EMA-1 gene showed positivity in 20 of 89 tested samples. The fragments of four samples were sequenced and analyzed to determine their similarities to sequences from other species, based on sequences deposited at GenBank. All showed 100% similarity with T. equi. Our study represents the first report of T. equi antibodies among the equids in north-central region of Mato Grosso, revealing the widespread distribution of seropositive animals.

A piroplasmose equina é a doença transmitida por carrapatos mais importante em cavalos no Brasil. Theileria equi é um dos agentes causadores da piroplasmose equina. São esperados casos crônicos, nos quais os animais não apresentam sinais aparentes de infecção e permanecem assintomáticos, mas constituem uma fonte de infecção e disseminação por carrapatos. Este estudo foi realizado em 81 fazendas localizadas no município de Sinop, Estado de Mato Grosso, Brasil. Um cálculo amostral foi realizado para estimar a prevalência aparente de T. equi entre cavalos. No total, 1.853 animais foram incluídos na análise amostral com base nas informações disponíveis no Instituto de Defesa Agropecuária do Estado de Mato Grosso. A análise sorológica de 367 amostras de soro por meio de ensaio imunoenzimático indireto (ELISA) para detecção de anticorpos anti-T. equi revelou que 337 animais eram positivos, representando uma frequência de 90,70%. A análise molecular para o gene EMA-1 mostrou positividade em 20 das 89 amostras testadas. Os fragmentos de quatro amostras foram sequenciados e analisados para determinar suas semelhanças com sequências de outras espécies, a partir das sequências depositadas no GenBank. Todos mostraram 100% de similaridade com T. equi. Nosso estudo representa o primeiro relato de anticorpos contra T. equi entre os equídeos na região centro norte de Mato Grosso, revelando a ampla distribuição de animais soropositivos.

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.

Development and evaluation of a chemiluminescence immunoassay for detecting tropical theileriosis.

Tropical theileriosis is a tick-borne lymphoproliferative disease of cattle caused by the apicomplexan parasite Theileria annulata, and leads to substantial economic losses to the livestock industry worldwide. Although various enzyme-linked immunosorbent assays (ELISAs) have been established to detect antibodies against T. annulata infection, a specific, rapid and reliable diagnostic assay is urgently needed for prevention and control of the disease. In the present study, a chemiluminescence immunoassay (CLIA) was developed based on the subtelomeric variable secreted protein (SVSP) of T. annulata as a sero-diagnostic antigen. Following optimization of the CLIA working parameters, the working time of the method was less than 4.5 h. The sensitivity and specificity of the established CLIA was 98.8% and 97.5%, respectively, when the cut-off value of the percent positive (PP) was 26.1% for detecting serum samples (n = 242 T. annulata positive sera, n = 158 T. annulata negative sera). After comparing 180 serum samples from Gansu province, China, the concordance rate between the CLIA and a published rSpm2 ELISA method was 72.8%. In addition, 565 serum samples of cattle collected between 2017 and 2018 from four provinces in China were detected by the CLIA, and the seroprevalence for T. annulata ranged from 53.3% to 67.3% in these regions. Our findings demonstrated that the CLIA has high specificity, sensitivity and reliability, and could be used as a rapid detection assay for epidemiological investigations of T. annulata infection.

Molecular detection and occurrence of equine theileriosis in Arabian horses in Al-Najaf province/Iraq / Detecção molecular e prevalência de teileriose equina em cavalos árabes na província de Al-Najaf/Iraque

This study was designed to detect equine piroplasmosis using the molecular technique in Al-Najaf province during the season that showed an increment in tick activities. Blood samples were collected from 110 horses with more than two signs of piroplasmosis. After DNA extraction, the product was examined by a polymerase chain reaction to amplify 18SrRNA. The results showed that the overall percentage of equine theileriosis was 38.18%. According to gender, the percentage of infection was 43.48% and 29.27% in females and males, respectively. Significant variations appeared between infected horses according to age, and the percentage of infection was 50% and 35.22% in less than 2 years and more than 2 years age, respectively. Moreover, the percentage of infection was 62.5% and 19.35% in animals with and without acariasis, respectively. Significant variations were also seen in equine theileriosis according to geographical areas, and the higher percentage was reported in Hera district (60.87%), while the lowest percentage was in the center of Al-Najaf (21.43%). This difference may be due to different distribution of vector of disease (tick), which may be the availability of the suitable weather that helped in the multiplication of the intermediate vectors. In conclusion, this study proved the variations in the occurrences of equine piroplasmosis according to gender, age, and geographical areas.(AU)

Este estudo foi desenvolvido para detectar piroplasmose equina usando a técnica molecular na província de Al-Najaf durante o período do ano com maior ocorrência de carrapatos. Foram coletadas amostras de sangue de 110 cavalos que apresentaram mais de dois sinais de piroplasmose. Após a extração do DNA, o produto foi examinado por reação em cadeia da polimerase para amplificar o 18SrRNA. Os resultados mostraram que a porcentagem geral de teileriose equina foi de 38, 18%. De acordo com o sexo, o percentual de infecção foi 43,48% e 29,27% no sexo feminino e masculino, respectivamente. Apareceram variações significativas entre os cavalos infectados de acordo com a idade, e a porcentagem de infecção foi 50% e 35,22% em menos de 2 anos e mais de 2 anos, respectivamente. Além disso, as porcentagens de infecção foram 62, 5% e 19, 35% em animais com e sem acariasis, respectivamente. Também foram observadas variações significativas na teileriose dos equídeos, de acordo com as áreas geográficas, e o maior percentual foi relatado no distrito de Hera (60, 87%), enquanto o menor percentual foi no centro de Al-Najaf (21,43%). Essa diferença pode ser devido à distribuição diferente do vetor da doença (carrapato), que pode ser a disponibilidade do clima adequado que ajuda na multiplicação dos vetores intermediários. Em conclusão, este estudo provou as variações nas ocorrências de piroplasmose eqüina de acordo com sexo, idade e áreas geográficas.(AU)

Frequency and factors associated with Theileria equi, Babesia caballi and Trypanosoma evansi in equids from Bahia (Northeast Brazil) / Frequência e fatores associados a Theileria equi, Babesia caballi e Trypanosoma evansi em equídeos da Bahia (Nordeste do Brasil)

Abstract The aim of this study was to determine the frequency and factors associated to Babesia caballi, Theileria equi and Trypanosoma evansi in naturally infected equids from the northeast Brazil. Blood samples from 569 equids (528 horses, 8 mules, and 33 donkeys) were collected and tested for the presence of DNA of each of these protozoan parasites by PCR. Generalized linear models were used to evaluate risk factors associated with the infection. The frequency of T. equi infection was 83.5% (475/569) - 84.3% in horses, and 73.2% in donkeys and mules. The results of the final model indicated that age (senior group) and animal species (mule and donkey group) were protective factors against this pathogen. The frequency of B. caballi infection was 24.3% (138/569) - 23.5% in horses and 34.1% in donkeys and mules. Age (adult and senior group) was considered a protective factor against B. caballi infection whereas animal species (donkey and mule group) were considered a risk factor for the infection. Trypanosoma evansi infection was not detected in any of animals. Our results suggest that equids from the area studied may be infected earlier in life with the etiological agents of equine piroplasmosis and become asymptomatic carriers.

Resumo Este estudo teve como objetivos conhecer a frequência e os fatores associados à infecção por Babesia caballi, Theileria equi e Trypanosoma evansi em equinos naturalmente infectados do nordeste do Brasil. Amostras de sangue de 569 equídeos (528 equinos, 8 muares e 33 asininos) foram coletadas e testadas para a presença do DNA destes parasitos através da PCR. Modelos lineares generalizados foram utilizados na avaliação dos fatores associados às infecções. A frequência de infecção por T. equi foi de 83,5% (475/569) -84,3% (445/528) em eqüinos e 73,2% (30/41) em asininos e muares. Os resultados do modelo final indicam idade (sênior) e espécie (muar e asinina) como possíveis fatores de proteção para este patógeno. A frequência de infecção por B. caballi foi de 24,3% (138/569) - 23,5% (124/528) em eqüinos e 34,1% (14/41) em asininos e muares. As faixas etárias (adulto e sênior) foram identificadas como possíveis fatores de proteção, e a espécie (asinina e muar) como risco para ocorrência de infecção por B. caballi. Infecções por Trypanosoma evansi não foram detectadas. Estes resultados indicam que os equídeos na região estudada se infectam precocemente com agentes da piroplasmose equina tornando-se portadores assintomáticos.

Atypical theileriosis with cutaneous involvement in a cow in India: a case report.

Bovine tropical theileriosis caused by Theileria annulata is an overwhelming haemoprotozoan tick-borne disease in taurine and cross-bred cattle in Punjab, India. However, there seems to be no report from India of cutaneous nodules associated with the disease. This report describes a five-year-old cross-bred cow presented to a university clinic with a history of fever, inappetence and malaise for the past six to seven days. Clinical examination revealed normal vital parameters, pale mucous membranes, mild enlargement of the prescapular lymph nodes and multiple subcutaneous nodular masses (2-4 cm) on the neck and abdomen. Haematology revealed mild anaemia and leucopenia with 48% neutrophils, 48% lymphocytes and 4% eosinophils. Romanowsky-stained smears of fineneedle aspiration biopsy samples from swollen lymph nodes and subcutaneous masses showed an increased number of lymphoid cells, suggesting cutaneous lymphomatosis. However, a critical examination of the smears from subcutaneous nodules showed a large number of Koch's blue bodies in macrophages and lymphoblasts, and several piroplasms were also noticed within the red blood cells in lymph node smears. A peripheral blood smear revealed mild to moderate parasitaemia. Extracted DNA from the parasitologically positive blood sample was subjected to nested polymerase chain reaction (nPCR) using T. annulata speciesspecific primers encoding the 30-kiloDalton major sporozoite surface antigen. The desired 572-base pair amplified product of the nPCR was comparable to the positive control. This seems to be a rare case of T. annulata in an adult cross-bred cow, showing cutaneous nodular involvement.

La theilériose bovine tropicale est une maladie causée par le protozoaire Theileria annulata et transmise par les tiques, affectant massivement les populations de bovins et de bovidés métis au Pendjab (Inde). Il semble toutefois que la présence de nodules cutanés associés à la maladie n'y ait jamais été rapportée jusqu'à présent. Les auteurs décrivent le cas soumis à une clinique vétérinaire universitaire d'une vache métisse âgée de cinq ans qui présentait depuis six à sept jours un tableau fébrile accompagné d'une perte d'appétit et d'un affaiblissement général. À l'examen clinique, les paramètres vitaux étaient normaux mais une pâleur des membranes muqueuses a été observée, ainsi qu'un gonflement modéré des ganglions lymphatiques préscapulaires et de nombreuses masses nodulaires sous-cutanées (de 2 à 4 cm d'épaisseur) au niveau du cou et de l'abdomen. L'hématologie a mis en évidence une anémie modérée et une leucopénie, les leucocytes se répartissant en 48 % de neutrophiles, 48 % de lymphocytes et 4 % d'éosinophiles. Les frottis à coloration de Romanowsky d'une biopsie par aspiration à l'aiguille fine des ganglions lymphatiques enflés et des masses sous-cutanées ont fait apparaître une augmentation du nombre de cellules lymphatiques évocatrice d'une lymphomatose cutanée. Néanmoins, un examen critique des prélèvements de nodules sous-cutanés a permis de constater la présence d'un grand nombre de corps bleus de Koch dans les macrophages et les lymphoblastes ; en outre, de nombreux piroplasmes ont été trouvés dans les globules rouges des frottis de ganglions lymphatiques. Un frottis de sang périphérique a permis de quantifier la parasitémie comme étant de niveau faible à modéré. L'ADN extrait de l'échantillon de sang à parasitologie positive a été soumis à une amplification en chaîne par polymérase nichée (nPCR) utilisant des amorces spécifiques de T. annulata codant pour l'antigène majeur de surface (30 kDa) du sporozoïte. Le produit amplifié par nPCR de la séquence souhaitée de 572 paires de bases était similaire à celui de l'échantillon de contrôle positif. Il s'agit probablement d'un cas rare d'infection à T. annulata chez une vache adulte métisse présentant des manifestations nodulaires cutanées.

La teileriosis tropical bovina causada por Theileria annulata es una devastadora enfermedad hemoprotozoaria transmitida por garrapatas que afecta al ganado taurino e híbrido del Punjab (India). Ahora bien, en la India no parece haber ningún caso descrito de esta enfermedad que se acompañe de la presencia de nódulos cutáneos. Los autores describen el caso de una vaca de cinco años híbrida que fue presentado a una clínica universitaria con un cuadro de fiebre, pérdida de apetito y decaimiento en los seis a siete días anteriores. El examen clínico puso de manifiesto parámetros vitales normales, mucosas pálidas, leve hipertrofia de los ganglios linfáticos prescapulares y múltiples bultos subcutáneos de tipo nodular (2 a 4 cm) en cuello y abdomen. El análisis hematológico reveló una leve anemia y leucocitopenia, con un 48% de neutrófilos, un 48% de linfocitos y un 4% de eosinófilos. Tras proceder a una biopsia de ganglios inflamados y bultos subcutáneos por aspiración con aguja fina, el examen de frotis de estas muestras con tinción de Romanowsky reveló un número excesivo de células linfáticas, lo que parece apuntar a una linfomatosis cutánea. No obstante, al examinar más a fondo los frotis de nódulos subcutáneos se observó que macrófagos y linfoblastos albergaban un gran número de cuerpos azules de Koch. También se observaron varios piroplasmas dentro de los eritrocitos presentes en los frotis de ganglios linfáticos. Un frotis de sangre periférica reveló una parasitemia entre leve y moderada. El ADN extraído de esta muestra de sangre positiva fue sometido a una técnica de reacción en cadena de la polimerasa (PCR) anidada en la que se emplearon cebadores específicos de la especie T. annulata que codifican el antígeno de superficie principal de 30 kDa del esporozoíto. La deseada secuencia de 572 pares de bases amplificada por PCR resultó comparable con la correspondiente secuencia de la muestra positiva de control. Parece tratarse pues de un caso raro de infestación por T. annulata de una vaca adulta híbrida que se acompaña de nódulos cutáneos.

Theileria sp. in water buffaloes from Maranhão State, northeastern Brazil / Theileria sp. em búfalos do Estado do Maranhão, nordeste do Brasil

Abstract Anaplasma marginale and piroplasm species are widespread among Brazilian cattle herds. Both of these tick-borne pathogens hamper livestock production and cause a significant economic impact. Although buffaloes have demonstrated a high level of adaptability, data on tick-borne pathogens are scarcely reported in Brazil. Thus, the aim of this study was to screen water buffaloes from the state of Maranhão for piroplasm and A. marginale occurrence using PCR assays. All samples were negative for A. marginale. One of the 287 (0.35%) water buffaloes tested was positive for Theileria sp. Sequencing of the 18S rDNA fragment (356 bp) showed that the Theileria sp. identified was closely related to the T. buffeli /orientalis group. Future studies on the clinical signs of infection and the main vector in this country are needed.

Resumo Anaplasma marginale e espécies de piroplasma são amplamente distribuídas no rebanho bovino brasileiro. Ambos os patógenos transmitidos por carrapatos dificultam a produção pecuária e causam um impacto econômico significativo. Embora os búfalos tenham demonstrado um alto nível de adaptabilidade, dados sobre patógenos transmitidos por carrapatos são raramente relatados no Brasil. Assim, o objetivo deste estudo foi investigar búfalos do estado do Maranhão para piroplasmas e A. marginale utilizando-se a técnica da PCR. Todas as amostras foram negativas para A. marginale . Um dos 287 (0,35%) búfalos testados foi positivo para Theileria sp. O sequenciamento de um fragmento do gene 18S rDNA (356 pb) demonstrou que Theileria sp. identificado estava relacionada ao grupo T. buffeli/orientalis . Estudos futuros sobre os sinais clínicos de infecção e o principal vetor neste país são necessários.

Molecular survey and genetic diversity of piroplasmids in equids from Midwestern Brazil / Levantamento molecular e diversidade genética de piroplasmídeos em equídeos do Centro-Oeste do Brasil

Abstract We evaluated the distribution of piroplasmids in equids from the Mato Grosso state in Midwestern Brazil using molecular methods and the interspecific genetic diversity. For this, 1,624 blood samples of equids from 973 farms were examined by PCR, using primer pairs that amplify a fragment of the genes rap-1 and ema-1 of Babesia caballi and Theileria equi, respectively. For molecular characterization and phylogenetic studies, 13 and 60 sequences of the rap-1 and ema-1 genes, respectively, were used to build a dendogram using maximum parsimony. B. caballi and T. equi were detected in 4.11% and 28.16% of the farms, respectively, and molecular prevalence was 2.74% for B. caballi and 25.91% for T. equi. The location of the farms and animals raised in the Pantanal ecoregion influence the probability of equids testing positive for B. caballi and T. equi . Moreover, age and herd purpose were variables significantly associated with T . equi infection. The sequences of B. caballi presented 1.95% intraspecific variability, contrasting with 2.99% in T. equi. Dendrograms for both species demonstrated the presence of subgroups with high values of support of branches. However, it is not possible to associate these groups with geographic origin and/or ecoregion.

Resumo Foi avaliada a distribuição de piroplasmídeos em equídeos do Estado de Mato Grosso, no Centro-Oeste do Brasil, utilizando-se métodos moleculares e a diversidade genética interespecífica. Para isso, 1.624 amostras de sangue de equídeos de 973 fazendas foram examinadas pela PCR, usando pares de oligonucleotídeos que amplificam um fragmento dos genes rap-1and ema-1 de Babesia caballi e Theileria equi, respectivamente. Para caracterização molecular e estudos filogenéticos, foram utilizadas 13 e 60 sequências dos genes rap-1 e ema-1, respectivamente, para construção de um dendograma utilizando máxima parcimônia. B. caballi e T . equi foram detectados em 4,11% e 28,16% das fazendas, respectivamente, e a prevalência molecular foi de 2,74% para B. caballi e 25,91% para T. equi. A localização das fazendas e animais criados na ecorregião do Pantanal influenciam a probabilidade de equídeos serem positivos para B. caballi e T. equi. Além disso, idade e propósito do rebanho foram variáveis, significativamente, associadas à infecção por T. equi. As sequências de B . caballi apresentaram variabilidade intraespecífica de 1,95%, contrastando com 2,99% em T. equi. Dendrogramas para ambas as espécies demonstraram a presença de subgrupos com altos valores de sustentação dos ramos. No entanto, não é possível associar esses grupos com origem geográfica e/ou ecorregião.

Severe meningeal fibrinoid vasculitis associated with Theileria taurotragi infection in two short-horned Zebu cattle.

The Authors describe a severe vasculitis with fibrinoid necrosis of the meningeal arteries observed in two brains of indigenous short-horn zebu (Bos indicus) cattle, with bovine cerebral theileriosis (BCT) caused by a tick-transmitted hemoprotozoan, Theileria taurotragi, from Northern Tanzania. In the Author's opinion, the role of T. taurotragi infection in the angiocentric and angiodestructive detected features remains to be evaluated. A possible immunopathologic cancerous mechanism, secondary to the lymphoid deregulation, could be involved. This report suggests further studies to better characterize the lymphoid cell involvement in the pathogenesis of the meningeal vascular lesions by T. taurotragi.

Comparative in vitro toll-like receptor ligand induced cytokine profiles of Toda and Murrah buffaloes-Identification of tumour necrosis factor alpha promoter polymorphism.

The objective of this study was to assess cytokine production upon activation of pattern recognition receptors responsible for sensing bacterial and viral pathogen associated molecular patterns in two genetically diverse buffalo breeds, Toda and Murrah. A very limited molecular-epidemiological analysis showed a higher prevalence of Anaplasma and Theileria in Murrah than Toda buffaloes. Toda buffalo peripheral blood mononuclear cells (PBMC) produced significantly higher levels of IFN γ and/or TNF α mRNAs in response to peptidoglycan, poly I:C, lipopolysaccharide, imiquimod and CpG. Flagellin stimulation did not result in any significant differences in the expression levels of the cytokines tested between these breeds. The levels of ligand induced IFN γ and TNF α mRNA and proteins also correlated except when induced with CpG. The proximal promoter region of TNF α across these two breeds were also sequenced to detect SNPs and promoter assay performed to determine their role in altering the transcriptional activity. Two polymorphisms were identified at -737 (T/A) and -1092 (G/T) positions in Toda buffalo TNF α promoter and promoter assay revealed higher transcription activity in Toda buffalos than in Murrah. This suggests that disease tolerance of these buffalo breeds could be due to the differences in their cytokine transcription levels in response to the respective PAMPs that may be at least in part determined by polymorphisms in the cytokine promoter regions.

Transcriptional profiling of inflammatory cytokine genes in African buffaloes (Syncerus caffer) infected with Theileria parva.

Theileria parva (T. parva) causes East Coast fever (ECF), which is of huge economic importance to Eastern and Southern African countries. In a previous bovine model, inflammatory cytokines were closely associated with disease progression in animals experimentally infected with T. parva. The African Cape buffalo (Syncerus caffer), the natural reservoir for T. parva, is completely resistant to ECF despite a persistently high parasitaemia following infection with T. parva. Characterizing basic immunological interactions in the host is critical to understanding the mechanism underlying disease resistance in the African Cape buffalo. In this study, the expression level of several cytokines was analyzed in T. parva-infected buffaloes. There were no significant differences in the expression profiles of inflammatory cytokines between the infected and uninfected animals despite a remarkably high parasitaemia in the former. However, the expression level of IL-10 was significantly upregulated in the infected animals. These results indicate a correlation between diminished inflammatory cytokines response and disease resistance in the buffalo.

First confirmed report of outbreak of malignant ovine theileriosis among goats in Sudan.

An outbreak of malignant ovine theileriosis among goats was confirmed and documented. In this outbreak, 16 out of 22 (72.7%) goats died within 4 days showing clinical signs of malignant ovine theileriosis as well as in the postmortem findings. The goats were reared in a mixed flock with sheep in Atbara Town, Northern Sudan. The infection was detected microscopically and confirmed serologically by IFA test and molecularly by PCR technique using specific primer for Theileria lestoquardi. Hyalomma anatolicum was the most prevalent (dominant) tick species found in the farm. It is recommended to undertake future research on the role of goats on the epidemiology of malignant ovine theileriosis.

Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immunoassay in horses from São Paulo State, Brazil.

The erythrocytic-stage surface protein, Equi Merozoite Antigen 1 (EMA-1), is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding the entire EMA-1 of Theileria equi Jaboticabal strain was cloned and expressed in Escherichia coli as a histidine-tagged protein (His6-EMA1). The expressed EMA-1 reacted with specific antibodies in Western blot and had an apparent molecular mass of 34 kDa which was largely consistent with its theoretical value. The nucleotide sequence of the EMA-1 gene of Jaboticabal strain was comparatively analyzed with other published sequences. The results indicated a high degree of homology with EMA-1 genes of all other strains isolated from various countries. The recombinant purified His6-EMA1 protein was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies anti-T. equi in horses. The ELISA clearly differentiated T. equi-infected from Babesia caballi-infected horse sera or normal horse sera. Field serum samples collected from horses in the State of São Paulo, Southeastern Brazil, were examined for the diagnosis of T. equi infection by ELISA. Of 170 samples analyzed, 95.88% (163/170) were positive for T. equi infection. These results suggest that the His6-EMA1 protein expressed in E. coli could be a reliable immunodiagnostic antigen for ELISA test and that T. equi infection is a serious concern in the State of São Paulo, Brazil.

Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immuno assay in horses from São Paulo State, Brazil / Produção da proteína recombinante EMA-1 e sua aplicação para o diagnóstico baseadono imuno ensaio enzimático de Theileria equi em equinos do Estado de São Paulo, Brasil

The erythrocytic-stage surface protein, Equi Merozoite Antigen 1 (EMA-1), is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding the entire EMA-1 of Theileria equi Jaboticabal strain was cloned and expressed in Escherichia coli as a histidine-tagged protein (His6-EMA1). The expressed EMA-1 reacted with specific antibodies in Western blot and had an apparent molecular mass of 34 kDa which was largely consistent with its theoretical value. The nucleotide sequence of the EMA-1 gene of Jaboticabal strain was comparatively analyzed with other published sequences. The results indicated a high degree of homology with EMA-1 genes of all other strains isolated from various countries. The recombinant purified His6-EMA1 protein was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies anti-T. equi in horses. The ELISA clearly differentiated T. equi-infected from Babesia caballi-infected horse sera or normal horse sera. Field serum samples collected from horses in the State of São Paulo, Southeastern Brazil, were examined for the diagnosis of T. equi infection by ELISA. Of 170 samples analyzed, 95.88 percent (163/170) were positive for T. equi infection. These results suggest that the His6-EMA1 protein expressed in E. coli could be a reliable immunodiagnostic antigen for ELISA test and that T. equi infection is a serious concern in the State of São Paulo, Brazil.

A proteína de superfície eritrocitária, Antígeno 1 do Merozoíta de Theileria equi (EMA-1), é um potencial candidato para o desenvolvimento de antígenos de valor diagnóstico para a piroplasmose equina. Com o objetivo de estabelecer um método de diagnóstico efetivo e prático, o gene EMA-1 da amostra Jaboticabal - SP de T. equi foi clonado e expresso em Escherichia coli contendo uma cauda de poli-histidina (His6-EMA1). O EMA-1 expresso reagiu com anticorpos específicos no Western blot e apresentou peso molecular aparente de 34 kDa, sendo altamente consistente com seu valor teórico. A sequência nucleotídica do gene EMA-1 da amostra Jaboticabal foi analisado comparativamente com outras sequências públicas, e os resultados indicam elevado grau de homologia com amostras de diversos países. A proteína recombinante purificada His6-EMA1 foi testada no ensaio imunoenzimático (ELISA) para a detecção de anticorpos anti-T. equi em equinos. O teste de ELISA diferenciou-se claramente entre soros de equinos infectados por T. equi, soros de animais infectados por Babesia caballi e soro normal de equino. Amostras de soros coletadas de equinos do Estado de São Paulo, sudeste do Brasil, foram examinadas para o diagnóstico da infecção por T. equi pelo ELISA. Das 170 amostras analisadas, 95,88 por cento (163/170) foram positivas para T. equi. Os resultados sugerem que a proteína His6-EMA1 expressa em E. coli pode ser um antígeno confiável para diagnóstico imunológico pelo teste de ELISA, e que T. equi merece grande atenção no Estado de São Paulo.

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.

Concurrent infection with bovine leukaemia virus and Theileria annulata in a Friesian calf.

Concurrent infection with bovine leukaemia virus (BLV) and Theileria annulata was diagnosed in a Friesian calf about 6 months of age at a dairy farm at the Qassim region of central Saudi Arabia. The disease ended fatally with signs of liver and heart failure. There was anorexia, pyrexia, anaemia, generalized oedema and jaundice. Haematology showed low RBC counts, PCV percentage and haemoglobin concentration and WBC counts. Lymphocyte differential was high. Examination of blood smears stained with Giemsa's stain showed the presence of piroplasms in red blood cells. Autopsy showed enlarged lymph nodes and lymphosarcoma lesions in the omentum and the heart. There was hydroperitoneum, hydropericardium and hydrothorax. The liver was pale yellow and friable. Impression smears from sliced lymph nodes and stained with Giemsa's stain showed presence of Koch's blue bodies in lymphoblasts. Histopathological examination revealed fatty degeneration of hepatocytes and pleomorphic lymphoblasts and giant cells in lymph nodes. Lymphoblasts infiltrated the omentum and heart tissues. Amyloid was found around blood vessels in the liver, kidneys and lymph nodes. BVL infection was diagnosed by demonstrating antibodies against the virus in serum using agar gel immunodiffusion and was confirmed with ELISA.

Purification of macroschizonts of a Sudanese isolate of Theileria lestoquardi (T. lestoquardi [Atbara]).

Research on malignant theileriosis is affected by the limited access to biological materials required for studies aiming at controlling the disease through the establishment of diagnostic tools and vaccines. The main aims of this work were to isolate, establish, and characterize a Theileria lestoquardi-infected cell culture (line) as a source of biological material and to generate a schizont cDNA library for further studies aiming at the identification of antigenic proteins. The T. lestoquardi isolate used originated from a sheep showing typical signs of malignant theileriosis in Atbara town in northern Sudan, and was maintained as an infected cell culture. A high-quality representative schizont cDNA library was established by isolating and purifying the schizonts using a nocodazole/aerolysin protocol followed by Percoll gradient ultracentrifugation. As a parameter to assess the quality of the schizont library, a provisional estimation of the percentage of recombinant phage clones originating from T. lestoquardi (Atbara) was undertaken. Ten clones with inserts ranging in size between 600 and 1200 bp were selected randomly, sequenced, and subjected to BLAST similarity searches. As 6 of the 10 sequenced clones showed similarities to T. parva, T. annulata, and other apicomplexan genes, it was concluded that the majority of the library phage clones originated from the parasite and not from host cell transcripts. The cDNA library will be used for screening of antigenic proteins using sera from infected sheep.