4 research outputs found

    Identificación inicial de genes en Babesia bigemina mediante análisis de Etiquetas de Secuencia Expresadas en el estadio intraeritrocítico del parásito

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    In this study, Expressed Sequence Tags (EST) were obtained and analyzed from 2208 randomly selected clones containing plasmids with cDNA inserts derived from a Babesia bigemina library. The obtained sequences were extracted and subject to Blast homology search in the Genbank databases. Sequence homology analysis resulted in the identification of 470 clones (grouped in 267 distinct clusters) which contained EST with no significant sequence identity with Babesia sp genes or other Apicomplexan parasites. Presumably, these EST would correspond either to new, unreported B. bigemina transcribed genes present in the erythrocyte stages of the parasite, or to non-translated sequences of the putative genes. 21 clones were identified which contained EST corresponding to 6 genes coding for B. bigemina antigens already reported in the literature; 1285 clones (grouped in 159 clusters of distinct sequences) had significant sequence identity with genes coding for hypothetical proteins previously identified in the Babesia bovis genome. Moreover, 32 clones had EST corresponding to 16 different Theileria sp. genes; 51 clones (26 distinct sequences) showed EST with sequence similarity to genes of Plasmodium sp., 25 EST had low identity with 13 different Toxoplasma gondii genes; and 4 clones with EST for 4 different Cryptosporidium sp genes. The results obtained, in addition to EST analysis of a larger number of B. bigemina cDNA clones, will allow the characterization and, eventually, the manipulation of gene coding regions, essential for the establishment of improved control strategies for cattle babesiosis caused by B. bigemina.En este estudio se realizó el análisis de Etiquetas de Secuencias Expresadas (EST) obtenidas a partir de 2208 clonas de Escherichia coli, con plásmidos recombinantes conteniendo insertos de cDNA de Babesia bigemina. Las secuencias se analizaron mediante búsqueda de homología en las bases de datos de genes. El análisis de homología en secuencia permitió identificar 470 clonas (agrupadas en 267 clusters) conteniendo EST con similitud de secuencia estadísticamente no significativa con algún gen de Babesia spp o de otro organismo Apicomplexa, sugiriendo la presencia de genes nuevos de B. bigemina; Se identificaron 21 clonas con EST correspondientes a 6 secuencias de genes previamente reportados para B. bigemina; además de 1285 clonas (conformando 159 clusters de genes distintos) de identidad significativa con proteínas hipotéticas o correspondientes a genes ya reportados en el genoma secuenciado de Babesia bovis; 32 clonas con EST homólogas a 16 genes distintos de Theileria spp; 51 clonas (26 genes distintos) con similitud en secuencia a genes de Plasmodium spp; 25 clonas con EST de moderada similitud con 13 genes distintos genes de Toxoplasma gondii; y 4 clonas con EST de mayor identidad con 4 genes diferentes de Cryptosporidium spp. Los resultados obtenidos permiten elaborar una base de datos sobre EST del estadio intraeritrocítico de Babesia bigemina, información básica esencial para la caracterización molecular del parásito, que permite llevar a cabo la identificación y regulación de nuevas regiones génicas codificadoras y, eventualmente el establecimiento de nuevas estrategias de control de la babesiosis bovina causada por B. bigemina

    Innovative Alternatives for Continuous In Vitro Culture of Babesia bigemina in Medium Free of Components of Animal Origin

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    In this study, we report Babesia bigemina proliferation in culture medium free of components of animal origin supplemented with a lipid mixture. Babesia bigemina continuously proliferated in VP-SFM with a higher percent parasitized erythrocyte as compare to using other animal component-free culture media. Compared with Advanced DMEM/F12 (ADMEM/F12), VP-SFM had a similar percent parasitized erythrocyte (PPE). Supplementation of VP-SF with a lipid acid mixture improved B. bigemina proliferation in vitro culture, with a maximum PPE of 11.3%. Growth of B. bigemina in a perfusion bioreactor using VP-SFM medium supplemented with lipid mixture resulted in a PPE above 28%. In conclusion, we demonstrated that B. bigemina proliferated in an animal component-free medium supplemented with the fatty acid mixture. This innovation to B. bigemina in vitro culture method presented herein is an important source of biological material for live vaccine production and understanding the mechanisms and molecules involved in parasite attachment and invasion of bovine erythrocytes

    Activación in vitro de monocitos de bovino con Lactobacillus casei: producción de óxido nítrico

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    Abstract: This research was carried out with the objective of determine the production of nitric oxide (NO) from monocytes of bovine peripheral blood exposed in vitro to L. casei. The NO production was measured every 24 hours during of 4 d in monocyte cultures of bovine peripheral blood of 4 to 6 months of age (MOA) (n = 4), 8 MOA (n = 3), and 12 MOA (n = 8), exposed (E) or not exposed (NE) to Lactobacillus casei. On each of the 24 h periods, a significant production (p < 0.01) of NO was observed on the monocytes exposed in comparison with those not exposed. The difference was more clear on the monocytes of groups 4 and 6 MOA. The use of L. casei can be important in establishing stimulation measures for the innate immunity in bovines against diseases produced by various pathogens.Resumen: La presente investigación se llevó a cabo con el objetivo de determinar la producción de óxido nítrico (ON) a partir de monocitos de sangre periférica de bovino, expuestos in vitro a L. casei. Se midió la producción de ON cada 24 h durante cuatro días, en cultivos de monocitos de sangre periférica de bovinos de 4 a 6 meses de edad (MDE) (n = 4), 8 MDE (n = 3) y 12 MDE (n = 8), expuestos (E) o no (NE) a Lactobacillus casei. En cada uno de los periodos de 24 h se observó una producción significativa (p < 0.01) de ON en los monocitos expuestos en comparación con los no expuestos. La diferencia fue más marcada en los monocitos del grupo 4 a 6 MDE. El uso de L. casei puede ser importante para establecer medidas de estimulación de la inmunidad innata en bovinos contra enfermedades producidas por diversos patógenos

    Molecular Identification of Babesia spp. and Anaplasma marginale in Water Buffaloes in Veracruz and Tabasco, Mexico: A Retrospective Study

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    Two hundred and thirty-three blood samples of water buffalo were collected on four farms in Veracruz state and Tabasco state, Mexico, to detect and confirm the identities of Babesia and Anaplasma spp. sequences. Nested PCR assays were used for the amplification of specific genes encoding B. bovis rhoptry-associated protein (RAP-1), B. bigemina SpeI-AvaI restriction fragment, and Anaplasma marginale major surface protein 5 (MSP5). Using DNA sequencing and BLASTn analysis for DNA homology hemoparasite identification, the identities of the hemoparasites were established by comparing the nucleotide sequences obtained in this study with those available in the GenBank database at the National Center for Biotechnology Information (NCBI). Water buffalo infection with at least one of the hemoparasites under study was detected in 45% (105/233) of the blood samples, while a mixed infection with B. bovis and B. bigemina was detected in 6.4% (15/233) of samples. For this cross-sectional study, mixed infections with the three hemoparasites were not detected. BLASTn analysis revealed that the nucleotide sequences of the water buffalo isolates shared sequence identity values ranging from 88 to 100% with previously published gene sequences of B. bovis, B. bigemina, and A. marginale. The current results confirm that water buffalo, as cattle, are also carriers of hemoparasite infections that are tick-transmitted, and suggest that they probably have an important role in the epidemiology of bovine babesiosis in Mexico
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