Babesia bovis: expression of adhesion molecules in bovine umbilical endothelial cells stimulated with plasma from infected cattle / Babesia bovis: expressão de moléculas de adesão em células endoteliais de cordão umbilical de bovinos estimuladas com plasma de bovinos infectados

Ten male, 12-month-old Jersey with intact spleens, serologically and parasitologically free from Babesia were housed individually in an arthropod-free isolation system from birth and throughout entire experiment. The animals were randomly divided into two groups. Five animals (group A) were intravenously inoculated with 6.6 X10(7) red blood cells parasitized with pathogenic sample of Babesia bovis (passage 7 BboUFV-1), for the subsequent "ex vivo" determination of the expression of adhesion molecules. Five non-inoculated animals (group B) were used as the negative control. The expression of the adhesion molecules ICAM-1, VCAM, PECAM-1 E-selectin and thrombospondin (TSP) was measured in bovine umbilical vein endothelial cells (BUVECs). The endothelial cells stimulated with a pool of plasma from animals infected with the BboUFV-1 7th passage sample had a much more intense immunostaining of ICAM-1, VCAM, PECAM-1 E-selectin and TSP, compared to the cells which did not received the stimulus. The results suggest that proinflammatory cytokines released in the acute phase of babesiosis may be involved in the expression of adhesion molecules thereby implicating them in the pathophysiology of babesiosis caused by B. bovis.

Dez bezerros machos, da raça Jersey, com 1 ano de idade com baços "in situ", sorológica e parasitologicamente livres de Babesia, foram mantidos em baias individuais no isolamento a prova de artrópodes do Depto de Veterinária desde o nascimento e ao longo de toda a experimentação. Os animais foram divididos aleatoriamente em dois grupos. Cinco animais (grupo A) foram inoculados por via intravenosa com 6,6 x10(7) hemácias parasitados com amostra patogênica de Babesia bovis (BboUFV - 1 7ª passagem) , para a determinação subseqüente "ex vivo" da expressão de moléculas de adesão . Cinco animais não inoculados (Grupo B ) foram utilizados como controlo negativo . A expressão de moléculas de adesão ICAM - 1, VCAM , PECAM - 1, E - selectina e trombospondina ( TSP ) foi medida em células endoteliais da veia umbilical de bovinos (BUVECs). As células endoteliais estimuladas com um pool de plasma proveniente de animais infectados com BboUFV - 1 7ª passagem tinham uma imunocoloração muito mais intensa de ICAM - 1 , VCAM , PECAM - 1 de E - selectina e de TSP , em comparação com as células que não receberam o estímulo . Os resultados sugerem que as citocinas pró-inflamatórias liberados na fase aguda da babesiose pode estar envolvida na expressão de moléculas de adesão , implicando , assim, elas na fisiopatologia da babesiose causada por B. bovis.

Assessing bovine babesiosis in Rhipicephalus (Boophilus) microplus ticks and 3 to 9-month-old cattle in the middle Magdalena region, Colombia

Babesia sp. is a protozoan hemoparasite that affects livestock worldwide. The Colombian Middle Magdalena is an enzootic region for babesiosis, but there is no previous research providing detail on its transmission cycle. This study aims to assess some Babesia sp. infection indicators in cattle and ticks from the area, by using direct microscopic and molecular techniques to detect the infection. In the cattle, 59.9% and 3.4 % positivity values for B. bigemina and mixed infection (B. bovis + B. bigemina) were found respectively. In ticks, the positivity of B. bigemina reached 79.2% and 9.4% for the mixed infection. The degree of infestation in the region was 3.2 ticks per bovine. There was positive correlation between tick control acaricide frequencies and infestation in bovines. This leads us to infer that control periodicity greater than 90 days, in stable zones, is an abiotic factor that benefits the acquisition of protective immunity in calves, the natural control of the infection and eventual disease absence. It is necessary to monitor the disease by applying new entomological and parasitological indicators showing the complexity of this phenomenon.

Molecular and antigenic characterisation of ribosomal phosphoprotein P0 from Babesia bovis

Babesia bovis is a tick-borne pathogen that remains an important constraint for the development of cattle industries in tropical and subtropical regions of the world. Effective control can be achieved by vaccination with live attenuated phenotypes of the parasite. However, these phenotypes have a number of drawbacks, which justifies the search for new, more efficient immunogens based mainly on recombinant protein technology. In the present paper, ribosomal phosphoprotein P0 from a Brazilian isolate of B. bovis was produced and evaluated with regard to conservation and antigenicity. The protein sequence displayed high conservation between different Brazilian isolates of B. bovis and several Apicomplexa parasites such as Theileria, Neospora and Toxoplasma. IgG from cattle experimentally and naturally infected with B. bovisas well as IgG1 and IgG2 from naturally infected cattle reacted with the recombinant protein. IgG from cattle experimentally infected with Babesia bigemina cross-reacted with B. bovis recombinant P0. These characteristics suggest that P0 is a potential antigen for recombinant vaccine preparations against bovine babesiosis.

Cloning and characterization of peroxiredoxin in Babesia bovis.

We have identified the 2-Cys peroxiredoxin (Prx) from a bovine Babesia parasite, B. bovis. Prx is a recently described family of antioxidant enzymes that are highly conserved in eukaryotes and prokaryotes. B. bovis 2-Cys Prx (BbTPx-1) contained two conserved cysteine residues that corresponded to Cys47 and Cys170 of the yeast Prx and the amino acid sequences of two catalytic domains showed significant similarities to those of mammalian typical 2-Cys Prx. The antioxidant activity of the recombinant BbTPx-1 protein expressed in E. coli was demonstrated by a thiol mixed-function oxidation assay. Furthermore, we confirmed that BbTPx-1 was expressed in the cytoplasm of intra-erythrocytic B. bovis merozites. These results suggest that B. bovis likely uses TPx-1 as a way to reduce peroxides as a control of its intracellular redox balance so that it can live and grow in the host cell.

Tristeza parasitária bovina na região sul do Rio Grande do Sul: estudo retrospectivo de 1978-2005 / Tick fever in southern Brazil: a retrospective study of 1978-2005

Foi realizado um estudo retrospectivo dos casos de tristeza parasitária bovina (TPB) ocorridos no sul do Rio Grande do Sul, área de influência do Laboratório Regional de Diagnóstico (LRD) da Faculdade de Veterinária da Universidade Federal de Pelotas entre 1978 e 2005. De um total de 4.884 materiais de bovinos provenientes de necropsias realizadas e órgãos ou sangue enviados ao laboratório 231 (4,7%) tiveram o diagnóstico de TPB. Desses 231 surtos foram resgatados os dados de 221 diagnósticos dos quais 91 (41,1%) foram causados por Babesia bovis, 11 (4,9%) por Babesia bigemina, e 65 (29,41%) por Anaplasma marginale. Em outros 33 (14,93%) surtos de babesiose não foi informada a espécie de Babesia e em 21 (9,5%) surtos foi detectada infecção mista por Babesia sp e A. marginale. Os índices gerais médios de morbidade, mortalidade e letalidade, resgatados em 149 dos 221 surtos da doença, foram de 11,17%, 6,81% e 70,04%, respectivamente. Verificou-se que, na região estudada, a maioria dos surtos ocorre durante os meses de verão e outono, e que os animais com um a três anos de idade são os mais afetados. Os sinais clínicos nos surtos caracterizaram-se por apatia, orelhas caídas, debilidade, febre, anorexia e emagrecimento. Os valores de hematócrito eram baixos. Hemoglobinúria foi frequentemente observada nos casos de babesiose. Sinais neurológicos estavam presentes nos casos de babesiose por B. bovis e se caracterizaram por transtornos da locomoção, tremores musculares, agressividade e quedas com movimentos de pedalagem. As lesões macroscópicas principais relatadas nos casos de babesiose foram esplenomegalia, hepatomegalia, fígado amarelo, hemoglobinúria, icterícia, hemorragias cardíacas e bile espessa. Congestão do córtex cerebral foi relatada nos casos de babesiose por B. bovis. Nesta região, com população de bovinos de aproximadamente 2.630.000 cabeças as perdas anuais por morte de bovinos pela enfermidade podem ser estimadas em 6.220 cabeças por ano...

A retrospective study of tick fever was made, which occurred from 1978-2005 in southern Rio Grande do Sul in the influence area of the Regional Diagnostic Laboratory of the Federal University of Pelotas. From 4,884 cattle specimens, sent by practitioners or which were from necropsies performed at the Diagnostic Laboratory, 231 (4.7%) were diagnosed as tick fever. Data from 221 of those outbreaks were analyzed. Ninety one (41.1%) outbreaks were caused by Babesia bovis, 11 (4.9%) by Babesia bigemina, and 65 (29.41%) by Anaplasma marginale. In other 33 (14.93%) outbreaks of babesiosis there is no information if the disease was caused by B. bovis or B. bigemina, and 21 (9.5%) outbreaks were caused by mixed infection of A. marginale and B. bovis or B. bigemina. Mean morbidity, mortality, and letality rates in 149 outbreaks were 11.17%, 6.81%, and 70.04%, respectively. Most outbreaks occurred during summer (January-March) and autumn (April-June), mainly in 1 to 3-year-old cattle. Clinical signs were depression, weakness, fallen ears, fever, and weight loss. Low packed cell volume values were always found. Hemoglobinury was observed in babesiosis. Neurological signs characterized by gait alterations, muscular tremors, aggressiveness and falling down with tonic and clonic convulsions were observed in babesiosis by B. bovis. The main gross lesions were anemia, jaundice, splenomegaly, hepatomegaly, yellow liver and cardiac hemorrhages. Hemoglobinury was observed in babesiosis, and congestion of the cerebral cortex in babesiosis by B. bovis. It is concluded that B. bovis is the main agent causing thick fever in southern Rio Grande do Sul. In that region with a cattle population of 2,630,000 heads the annual losses due to tick fever can be estimated in 6,220 cattle or US$ 1,623,000.00. Preventive measures to diminish tick fever losses in the region are necessary.

Desenvolvimento de uma prova de imunoadsorção enzimática para detecção de anticorpos contra Babesia bovis / Development of an enzyme-linked immunosorbent assay for the detection of antibodies against Babesia bovis

Uma prova de imunoadsorção enzimática (ELISA) para detecção de anticorpos contra Babesia bovis foi desenvolvida e avaliada em comparação à imunofluorescência indireta (IFI). A sensibilidade e especificidade do ELISA, determinadas pela análise de 100 soros positivos de bovinos infectados experimentalmente com B. bovis e 108 soros negativos colhidos de bovinos livres de infecção por este hemoparasito, foram de 98,0 por cento e 98,1 por cento, respectivamente. Os valores preditivos positivo e negativo foram, respectivamente, 98,0 por cento e 98,1 por cento e a precisão do teste foi de 98,1 por cento. Não foram detectadas reações cruzadas com 80 soros de bezerros experimentalmente inoculados com Babesia bigemina. O ELISA foi comparado à IFI usando 110 soros de rebanhos de área de estabilidade endêmica e 168 soros de rebanhos de áreas de instabilidade endêmica. Em ambos os casos, houve concordância significativa (P=0,631 e 0,4725, respectivamente) entre os resultados demonstrados pelos dois testes. Em um estudo epidemiológico realizado com o ELISA na região do Pantanal de Mato Grosso do Sul, com 1.365 soros de bovinos, 83,9 por cento foram positivos para anticorpos contra B. bovis, caracterizando a região estudada como endemicamente estável.

An enzyme-linked immunosorbent assay (ELISA) for antibodies to Babesia bovis was developed and evaluated in comparison with the indirect fluorescent antibody test (IFAT). The ELISA sensitivity and specificity, estimated with 100 positive sera from cattle experimentally infected with B. bovis and 108 negative sera collected from B. bovis-free herds, were 98.0 percent and 98.1 percent, respectively. Positive and negative predictive values were, respectively, 98.0 percent and 98.1 percent, and precision was 98.1 percent. No cross-reactions were detected with 80 sera from calves experimentally inoculated with Babesia bigemina. The ELISA was compared with IFAT using 110 cattle sera from an enzootically stable area and with 168 cattle sera from an enzootically unstable area. In both cases, there was a significant agreement between results of both tests (P=0.631 and 0.4725, respectively). In an epidemiological study performed with ELISA in the Pantanal region of the State of Mato Grosso do Sul with 1,365 cattle sera, 83.9 percent were positive for antibodies against B. bovis, characterizing this region as enzootically stable.

Hemoparásitos en bovinos, caninos y equinos diagnosticados en el laboratorio de Parasitología de la Facultad de Medicina Veterinaria y Zootecnica de la Universidad Autónoma de Yucatán (1984-1999) / Hemoparasityes in domestic animals, diagnosed in the parasitology laboratory of the Facultad de Medicina Veterinaria y Zootecnia of the universidad Autónoma de Yucatan (1984-1999)

Introducción. Los hemoparásitos son organismos que pueden ser transmitidos a los animales domésticos por vectores mecánicos y biológicos. Su presencia en los animales domésticos produce cuadros hemáticos que afectan la salud animal. Material y métodos. Se revisaron los archivos del laboratorio de Parasitología de la Facultad de Medicina Veterinaria y Zootecnia de la Universidad Autónoma de Yucatán, de marzo de 1984 a diciembre de 1999. Se obtuvo la información de las muestras sanguíneas de bovinos, caninos y equinos que fueron remitidas y procesadas mediante las técnicas de Knott y frotis sanguíneos teñidos con Giemsa al 10 por ciento. Resultados. Se analizaron un total de 3010 muestras sanguíneas, de las cuales 2438 fueron de bovinos, 493 de caninos y 79 de equinos. Los hemoparásitos que se diagnosticaron en las distintas especies animales fueron los siguientes: bovinos: Babesia bovis (2.78 por ciento), Babesia bigemina (1.23 por ciento) y Anaplasma marginale (15.79 por ciento); caninos: Dirofilaria immitis (7.42 por ciento), Dipetalonema reconditum (5.88 por ciento) y Babesia canis (3.92 por ciento), y equinos: Babesia equi (3.79 por ciento) y Babesia caballi (2.53 por ciento). Conclusiones. Se concluye que los bovinos, caninos y equinos del estado de Yucatán se encuentran afectados por hemoparásitos que pueden afectar la salud y/o producción animal.

Transmissäo transovariana de Babesia bovis em Boophilus microplus: obtençäo de cepa de carrapato livre de Babesia spp / Babesia bovis transovarian transmission in Boophilus microplus: obtention of a Babesia free tick strain

O presente trabalho objetivou o estudo de parte do ciclo da Babesia bovis no seu hospedeiro invertebrado, o carrapato Boophilus microplus. Analisou-se a capacidade de infecçäo e transmissäo transovariana de B. bovis em partenóginas de B. microplus, alimentadas em bovinos portadores e enfermos por este protozoário. No 18§ dia após a infestaçäo, coletaram-se partenóginas diretamente do corpo dos bovinos e teleóginas após o desprendimento natural, a partir do 21§ dia. Todos os grupos foram incubados a 27§C e umidade relativa superior a 70 por cento. No 5§ dia após o início da postura, realizou-se o exame de hemolinfa a fim de diagnosticar a infecçäo dos ínstares por B. bovis. A ausência de infecçäo detectada no exame de hemolinfa foi confirmada posteriormente com o teste biológico, revelando que partenóginas näo transmitem B. bovis transovarianamente. Esses resultados oferecem uma técnica simplificada para a obtençäo de cepas de carrapatos livres de B. bovis.

Conservation of merozoite membrane and apical complex B cell epitopes among Babesia bigemina and Babesia bovis strains isolated in Brazil.

Babesia merozoite polypeptides bear surface exposed and neutralization-sensitive B cell epitopes and have been shown to induce partial protection against experimental challenge. Variation in these epitopes has been examined in a limited number of strains. In this study, utilizing strains of Babesia bovis and Babesia begemina from Matto Grosso do Sul in Brazil, we examined the conservation of epitopes bound by monoclonal antibodies developed against Mexico strains of B. bovis and B. bigemina. Apical complex B-cell epitopes, previously shown to be species-specific but common among otherwise antigenically distinct strains, were also conserved between clones of the Mexico strains and the Matto Grosso do Sul strains of each Babesia species. Mexico strain polypeptides bearing these epitopes were recognized by sera from cattle infected with the Matto Grosso do Sul strains. Two distinct epitopes on the B. bovis neutralization-sensitive merozoite surface antigen-1 (MSA-1) were also conserved between the Mexico Mo7 clone and the Matto Grosso do Sul strain, in contrast to previous studies which demonstrated variability among strains. Sera from cattle with B. bovis infections naturally acquired in Matto Grosso do Sul bound Mexico Mo7 MSA-1, demonstrating that conserved MSA-1 epitopes were recognized by the bovine immune system. Similarly, merozoite surface epitopes on the B. bigemina 45 kDa and 55 kDa glycoproteins were conserved on the Matto Grosso do Sul strain of B. bigemina.

Isolation of Babesia bigemina and Babesia bovis merozoites by ammonium chloride lysis of infected erythrocytes

1. We describe the isolation of viable merozoites from erythrocytes infected with Babesia bovis or Babesia bigemina organisms by ammonium chloride lysis. 2. Parasite morphology was examined by both light and transmission electron microscopy. Erythrocyte-free parasites maintain their viability and infectivity, retain their antigenicity and are suitable for use in the indirect fluorescent antibody assay

Isolate-specific parasite antigens of the Babesia bovis-infected erythrocyte surface.

Bovine erythrocytes taken from in vitro cultures of Babesia bovis parasites from Mexico and the United States were assayed for the presence of new epitopes on the erythrocyte surface. New surface-exposed epitopes were detected by means of a whole-cell antigen capture assay. These epitopes were subsequently demonstrated only on infected erythrocytes by immunofluorescence staining of intact, living cells. Parasite-synthesized antigens were identified on each isolate using a surface-specific immunoprecipitation technique to analyze metabolically-labeled infected erythrocytes. In the Mexico isolate these antigens were 120 kDa and 107 kDa, whereas in the United States isolate polypeptides of 135, 120 and 107 kDa were detected. In each of these assays, reaction of immune sera with the infected erythrocyte surface was found to be isolate-specific.