Rapid conglutination test for diagnosis of Babesia bovis antibodies

Neste trabalho objetivou-se desenvolver e avaliar um teste de conglutinação rápida para diagnóstico de anticorpos contra Babesia bovis (TCR-B. bovis), com desempenho similar ao do teste da imunofluorescência indireta. O TCR-B. bovis apresentou sensibilidade de 95,7%, especificidade de 97,6% e precisão de 96,5%. Os mesmos parâmetros para imunofluorescência indireta (IFI) foram respectivamente 99,1%, 99,7% e 99,4%. O TCR demonstrou ter capacidade de determinar imunoglobulinas específicas anti-B. bovis tão cedo quanto a IFI, após infecção experimental com este hemoprotozoário. As reações cruzadas do TCR-B. bovis com soros de animais infectados com Babesia bigemina ocorreram em 4% dos soros. O antígeno para TCR produzido com hemácias não parasitadas apresentou 3,5%, 1,5% e 2,2% de reações positivas com soros de animais infectados experimentalmente com B. bovis, B. bigemina e Anaplasma marginale, respectivamente. Entretanto, não foi observada nenhuma destas reações positivas com soros de áreas endêmicas com esta preparação de antígeno. Considerando-se os resultados acima e ainda que o antígeno TCR-B. bovis se manteve viável após um período de 172 a 245 dias de armazenamento a 4°C, conclui-se que esta prova sorológica poderá vir a ser utilizada em estudos epidemiológicos e na avaliação de medidas preventivas contra esta espécie de Babesia.The objective of this work was to develop and evaluate a rapid conglutination test (RCT) for detection of antibodies against Babesia bovis (B. bovis-RCT), with similar performance as the indirect immunofluorescence test. The B. bovis-RCT had a sensitivity of 95.7%, a specificity of 97.6% and the precision of 96.5%, the same parameters for the IFAT being 99.1%, 99.7% and 99.4% respectively. The RCT was able to detect immunoglobulins anti-B. bovis as early as IFAT after bovine challenge with these hemoprotozoans. B. bovis-cross reactions with sera of Babesia bigemina infected cattle were 4%. The RCT prepared with non-parasitized erythrocytes (negative antigen) showed 3.5%, 1.5% and 2.2% of positive reactions with sera of animals infected with B. bovis, B. bigemina and Anaplasma marginale. None of sera from animals in endemic areas had positive reactions with this antigen. Considering these results and the viability of B. bovis-RCT (172 to 245 days, stored at 4ºC), it can be concluded that this serological test can be used for epidemiological studies and the evaluation of control measures against this species of Babesia

Babesiose cerebral por Babesia bovis (Babés 1888 Starcovici 1893) em bezerros, no Estado de Mato Grosso do Sul

Three cases of death caused by Babesia bovis, One in a 13-day-old Nelore x Fleckvieh female calf, one in a 25-day-old Ibagé male calf and one in a 108-day-old Nelore male calf are described. All were acute cases without the possibility of an in vivo diagnosis or treatment. The diagnosis was based on necropsy, microscopic examination of twain, kidney and heart preparations stained by May-Grünwald-Giemsa and histopathology examination of the various, internal organs stained by Haematoxylin-Eosin. The specific in diagnosis was based on the morphology of the organisms and the characteristical localization of parasitized erythrocytes in the blood capillaries of the internal organs. This is the first notification of the occurrence of clinical cases of babesiosis by S. bovis in Mato Grosso do Sul, Brazil.São descritos três casos de morte de bezerros causadas por B. bovis, sendo uma bezerra cruzamento Nelore x Fleckvieh, de treze dias de idade, um bezerro Ibagé, de 25 dias, e um bezerro Nelore de 108 dias. Todos os casos foram agudos, não havendo possibilidade de diagnóstico in vivo ou tratamento. O diagnóstico foi baseado nos achados de necropsia, exame microscópico de esfregaços de cérebro, rins e coração, corados por May-Grünwald-Giemsa e exame histopatológico de diversos órgãos, corados por hematoxilina-eosina. As características morfológicas e a localização característica dos eritrócitos parasitados nos capilares dos órgãos internos basearam o diagnóstico específico. Esta é a primeira notificação da ocorrência de casos clínicos de babesiose por B. bovis no Mato Grosso do Sul, Brasil

Development of live attenuated strains of Babesia bovis and Babesia bigemina: preliminary test

Foram efetivadas 16 passagens de B. bovis em bezerros esplenectomizados e quatro passagens de B. bigemina em bezerros intactos com o objetivo de desenvolver cepas atenuadas para posterior uso como vacina, de acordo com a tecnologia australiana. O teste preliminar da virulência e antigenicidade destas cepas, realizado a campo, em novilhos Hereford de dois anos de idade revelou que: 1) a cepa de B. bovis apresenta baixa virulência, uma vez que os animais inoculados não apresentaram sinais clínicos da doença durante a reação vacinal; 2) a cepa de B. bigemina apresenta ainda um grau elevado de virulência pois, dos cinco novilhos inoculados, três apresentaram sintomatologia clínica embora tenham se recuperado espontaneamente da infecção; 3) ambas as cepas apresentaram alta antigenicidade pois todos os animais inoculados apresentaram sorologicamente positivos ao teste de imunofluorescência indireta específico e resistiram ao desafio homólogo com as respectivas cepas virulentas, enquanto nos grupos testemunha, todos os novilhos sofreram a doença aguda e, apesar de devidamente medicados, um morreu.With the objective of developing local strains of Babesia bovis and Babesia bigemina attenuated according to the Australian method, sixteen rapid passages of B. bovis in splenectomized calves and four slow passages of B. bigemina in intact calves were made. A preliminary test of the virulence and antigenicity of these strains in two-year old Hereford steers in the field showed that: 1) the B. bovis strain was of low virulence as the inoculated animals did not present clinical signs of the disease during the vaccine reaction; 2) the B. bigemina strain was still too virulent since three of five inoculated steers presented visible clinical signs, although all recovered spontaneously from the infection; 3) both strains were highly antigenic as all inoculated animals were positive for the indirect fluorescent antibody test and resisted the homologous challenge with the respective virulent strain, while all of the non-vaccinated controls became acutely ill and although properly medicated, one died

In Vitro Cultivation of Anaplasma Marginale

72 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.Virulent Anaplasma marginale propagated in culture of bovine erythrocytes showed an increasing rate of replication in a sequence of three experiments. The changes in percentage of parasitized erythrocytes (PPE) were monitored by Giemsa staining and the organism was identified by the direct fluorescent antibody (FA) test and transmission electron microscopy. The PPE increased more than 3 times in the first experiment during a period of 14 days. Regression analysis of the data showed that the trend of growth was significantly (P = 0.05) higher after the third passage of the organism in culture. Serologic and ultrastructural studies demonstrated that the in vitro propagated organism was similar to A. marginale isolated from naturally infected bovine blood. Organisms recovered from 13- and 33-day cultures of the first and second experiments were infective for susceptible cattle.Ovine erythrocytes infected with the attenuated A. marginale were co-cultured with either normal ovine erythrocytes or normal bovine erythrocytes for 42 days. In both systems, the organism showed an initial period of rapid growth followed by a gradual decrease in PPE and accompanied by cyclic peaks. The presence of bovine erythrocytes in infected ovine erythrocytes resulted in a higher degree of parasitization. Transmission of the organism from infected ovine erythrocytes to normal bovine erythrocytes was demonstrated by use of a two-step FA method which allowed for differentiation between cells and identification of the organism. It is concluded that under in vitro conditions A. marginale is directly transmitted from erythrocyte to erythrocyte without any intermediate cell type.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Isolamento de cepas puras de Babesia bovis, Babesia bigemina e Anaplasma marginale em área enzoótica

Pure strains of Babesia bovis and Babesia bigemina were isolated by placing larvae and numphs respectively of the tick Boophilus microplus, on splenectomized calves. Anaplasma marginale was isolated by the inoculation of blood from a bovine carrier into a splenectomized calf, followed by medication with a babesicide. Larvae transmitted B. bovis only when removed from the calf on the fifth day of infestation. However, B. bigemina was transmitted when ticks were removed on the sixth day. Nymphs and adults transmitted only B. bigemina. No transmission of Anaplasma by larvae, nymphs or adults occurred.Foram isoladas cepas puras de Babesia bovis e Babesia bigemina infestando-se bezerros esplenectomizados com larvas e ninfas, respectivamente, do carrapato Boophilus microplus. O Anaplasma marginale foi isolado por inoculacão de sangue de um bovino portador em bezerro esplenectomizado, seguido de medicação específica com babesicida. As larvas transmitiram somente B. bovis quando foram removidas do animal no quinto dia da infestação. Entretanto, quando a remoção foi feita, no sexto dia, houve transmissão de B. bigemina. As ninfas e os adultos transmitiram somente B. bigemina. Não ocorreu transmissão de Anaplasma pelas larvas, ninfas ou adultos

Antibody levels anti-Babesia bigemina and Babesia bovis in calves of Nelore and Ibagé breeds and Nelore crosses

Foram analisados pela técnica de anticorpos fluorescentes, os soros dos bezerros da raça Nelore, Ibagé e cruzamentos de Nelore x Fleckvieh, Nelore x Chianina e Nelore x Charolês, do nascimento ao desmame, com a finalidade de determinar os níveis de imunoglobulinas anti-Babesia bigemina e Babesia bovis. No período de três a quatorze dias de vida, foi observada correlação positiva e significante entre os níveis de imunoglobulinas circulantes das vacas e os anticorpos séricos dos bezerros contra B. bigemina e/ou B. bovis, em algumas raças e cruzamentos. A média do título sorológíco dos grupos experimentais apresentou um decréscimo nos níveis de anticorpos colostrais entre 28 e 56 dias de idade contra B. bigemina e entre 56 e 84 dias anti - B. bovis. A produção ativa de anticorpos contra B. bigemina foi observada aos 84 dias e aos 112 contra B. bovis. Em geral, os níveis de anticorpos contra B. bigemina foram mais elevados que o da B. bovis e houve maior semelhança na curva de anticorpos dos bezerros da raça Nelore e seus cruzamentos que os da raça lbagé. Embora a região seja considerada área de estabilidade enzoótica, conclui-se que existe um período crítico de baixa resistência humoral, no qual podem ocorrer casos clínicos de babesiose.Antibody levels of Babesia bigemina and Babesia bovis were analysed by the indirect fluorescent antibody technique in calves of Nelore and Ibagé breeds, Nelore x Fleckvieh and Nelore x Chianina, and Nelore x Charolais cross up to weaning age. Three and 14 day-old calves of some breeds and crosses showed significant positive correlation with the immunoglobulins anti - B. bigemina and/or B. bovis of the cows. Lower levels of immunoglobulins against B. bigemina were found between 28 and 56 days of age and against B. bovis between 56 and 84 days of age. The active antibody production was observed against B. bigemina at 84 days and at 112 days of age against B. bovis. The antibody level anti B. bigemina was generally higher than anti -B. bovis and the antibody curve was more similar to Nelore breed and its calf crosses than to the lbagé breed. Although the region is considered to be enzootically stable, it was concluded that there is a critical time of low humoral resistance during which the clinical symptoms of babesiosis can occur

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction