Peptidasas en piroplasmas bovinos patógenos: análisis genómico comparativo entre Babesia bovis y Theileria annulata

Las enzimas proteolíticas de los patógenos Apicomplexa juegan un rol sumamente importante en la invasión, supervivencia y egreso de la célula hospedera. La función vital que cumplen para el ciclo de vida del parásito y su estructura altamente conservada, ha hecho que este tipo de enzimas sean consideradas como interesantes candidatos vacunales o blancos para el desarrollo de nuevos fármacos. Se diseñó un algoritmo para predecir mediante herramientas de bioinformática los repertorios putativos de proteasas en los genomas de dos piroplasmas bovinos patógenos: B. bovis y T. annulata.Asociación Parasitológica Argentin

Historia evolutiva y filogenia molecular del género Babesia

Babesia comprende hemoprotozoos transmitidos por garrapatas que infectan mamíferos y aves y que son reconocidos mundialmente por su impacto en la salud de animales domésticos y sus costos económicos asociados. Además, la babesiosis puede ser fatal en animales silvestres si está asociada con prácticas de manejo estresantes, y es también de creciente interés como zoonosis emergente. Debido a la gran diversidad de hospedadores de Babesia hallados, se puede considerar que todos los vertebrados son portadores potenciales, siempre y cuando puedan ser parasitados por las garrapatas vectores. En esta presentación se proveerá una reseña de las especies de Babesia más relevantes, y una discusión sobre criterios taxonómicos clásicos. Se describirá un posible panorama de la historia natural de los piroplásmidos, que agrupa a los géneros cercanamente relacionados Babesia, Cytauxzoon y Theileria, y se discutirán sus implicancias para futuras líneas de investigación. Asimismo, se presentará una clasificación molecular revisada de los piroplásmidos, basada en nuevos árboles filogenéticos generados con todas las secuencias disponibles de los genes 18S rRNA y hsp70. Finalmente, para reconciliar las estimaciones existentes para el origen de los piroplásmidos y las garrapatas (~300 millones de años, Ma, respectivamente) y la radiación de los mamíferos (60 Ma), hipotetizamos que el ciclo de vida dixénico de los piroplásmidos evolucionó con el origen de las garrapatas. Así, el salto observado entre el origen de estos artrópodos y la radiación de los mamíferos indica la existencia de linajes de piroplásmidos previamente desconocidos y/o especies en taxones de vertebrados existentes, incluyendo reptiles y quizás también anfibios. Un muestreo más amplio de taxones que incluya todos los potenciales hospedadores, tanto entre los vertebrados como entre las garrapatas, y la construcción de árboles utilizando múltiples genes permitirá perfeccionar la filogenia y taxonomía del género Babesia.Asociación Parasitológica Argentin

Pursuing effective vaccines against cattle diseases caused by apicomplexan protozoa

Apicomplexan parasites are responsible for important livestock diseases that affect the production of much needed protein resources, and those transmissible to humans pose a public health risk. Vaccines, recognized as a cost-effective and environmentally friendly method for the prevention of infectious diseases in livestock, can avert losses in food production and decrease the exposure of humans to zoonotic pathogens. This review focuses on the need for and advances in vaccine development against the apicomplexan parasites Theileria spp., Babesia spp., Toxoplasma gondii, Neospora caninum, Eimeria spp., Besnoitia spp., Sarcocystis spp., and Cryptosporidium parvum. Together, the effect of these parasites on the cattle industry worldwide causes an enormous burden, yet they remain poorly controlled and very few effective and practical vaccines against them are available.Vaccine development is hampered by our scarce and limited knowledge of the biology and mechanisms of pathogenesis of these microorganisms, and the absence of correlates of host immune protection. More studies focused on these aspects as well as on the identification of parasite vulnerabilities that can be exploited for vaccine design are needed. Novel “omics” and gene editing approaches in understanding complex parasite biology together with advances in vaccinology will facilitate the development of effective, sustainable, and practical vaccines against cattle diseases caused by apicomplexan parasites. Such vaccines will help prevent animal and human diseases and allow production of enough animal protein to feed the growing human population in the twenty-first century and beyond

Development of fluorogenic probe-based PCR assays for the detection and quantification of bovine piroplasmids.

This paper reports two new quantitative PCR (qPCR) assays, developed in an attempt to improve the detection of bovine piroplasmids. The first of these techniques is a duplex TaqMan assay for the simultaneous diagnosis of Babesia bovis and B. bigemina. This technique is ideal for use in South America where bovids harbour no theilerids. The second technique, which is suitable for the diagnosis of both babesiosis and theileriosis worldwide, involves fluorescence resonance energy transfer (FRET) probes. In FRET assays, Babesia bovis, B. divergens, Babesia sp. (B. major or B. bigemina), Theileria annae and Theileria sp. were all identifiable based on the melting temperatures of their amplified fragments. Both techniques provided linear calibration curves over the 0.1fg/microl to 0.01ng/microl DNA range. The assays showed good sensitivity and specificity. To assess their performance, both procedures were compared in two separate studies: the first was intended to monitor the experimental infection of calves with B. bovis and the second was a survey where 200 bovid/equine DNA samples from different countries were screened for piroplasmids. Comparative studies showed that duplex TaqMan qPCR was more sensitive than FRET qPCR in the detection of babesids

Expresión heteróloga y localización subcelular del candidato vacunal GPI4 de babesia bovis en el ciliado tetrahymena thermophila

Los glicosilfosfatidilinositoles (GPIs) se encuentran en abundancia en la superficie de protozoos parásitos como glicolípidos libres y como anclas de proteínas de superficie a la membrana plasmática. Se ha demostrado que las proteínas ancladas por GPIs están involucradas en el mecanismo de invasión a la célula huésped, por lo que representan blancos moleculares muy interesantes para el diseño de nuevas estrategias vacunales contra protozoos patógenos de importancia veterinaria. Además, los GPIs libres actúan como potentes moduladores de la respuesta inmunológica. Tetrahymena thermophila es un protozoo ciliado de vida libre que expresa en la superficie abundantes proteínas ancladas por GPI, y ha sido utilizado como plataforma biotecnológica para la expresión de antígenos recombinantes.Instituto de PatobiologíaFil: Montes, M. G. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Flores, Daniela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Rodriguez, Anabel Elisa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: González Maglio, D. H. Universidad de Buenos Aires. Instituto de Estudios de la Inmunidad Humoral (IDEHU); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; ArgentinaFil: Nusblat, Alejandro David. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentin

Unraveling the complexity of the Rhomboid Serine Protease 4 Family of Babesia bovis using bioinformatics and experimental studies.

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host?pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen

Genetic and Chemical Evaluation of Trypanosoma brucei Oleate Desaturase as a Candidate Drug Target

Background: Trypanosomes can synthesize polyunsaturated fatty acids. Previously, we have shown that they possess stearoyl-CoA desaturase (SCD) and oleate desaturase (OD) to convert stearate (C18) into oleate (C18:1) and linoleate (C18:2), respectively. Here we examine if OD is essential to these parasites. Methodology: Cultured procyclic (insect-stage) form (PCF) and bloodstream-form (BSF) Trypanosoma brucei cells were treated with 12- and 13-thiastearic acid (12-TS and 13-TS), inhibitors of OD, and the expression of the enzyme was knocked down by RNA interference. The phenotype of these cells was studied. Principal Findings: Growth of PCF T. brucei was totally inhibited by 100 mM of 12-TS and 13-TS, with EC50 values of 4062 and 3062 mM, respectively. The BSF was more sensitive, with EC50 values of 763 and 261 mM, respectively. This growth phenotype was due to the inhibitory effect of thiastearates on OD and, to a lesser extent, on SCD. The enzyme inhibition caused a drop in total unsaturated fatty-acid level of the cells, with a slight increase in oleate but a drastic decrease in linoleate level, most probably affecting membrane fluidity. After knocking down OD expression in PCF, the linoleate content was notably reduced, whereas that of oleate drastically increased, maintaining the total unsaturated fatty-acid level unchanged. Interestingly, the growth phenotype of the RNAi-induced cells was similar to that found for thiastearate-treated trypanosomes, with the former cells growing twofold slower than the latter ones, indicating that the linoleate content itsel

Trypanosoma cruzi Epimastigotes Are Able to Store and Mobilize High Amounts of Cholesterol in Reservosome Lipid Inclusions

Reservosomes are lysosome-related organelles found in Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells.Using Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones.Taken together, our results suggest that epimastigote forms of T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation

Diagnosis and predictive molecular analysis of non-small cell lung cancer in the Africa-Middle East region : challenges and strategies for improvement

The identification of tumor biomarkers provides information on the prognosis and guides the implementation of appropriate treatment in patients with many different cancer types. In nonesmall cell lung cancer (NSCLC), targeted treatment plans based on biomarker identification have already been used in the clinic. However, such predictive molecular testing is not currently a universally used practice. This is the case, in particular, in developing countries where lung cancer is increasingly prevalent. In September 2012 and November 2013, a committee of 16 lung cancer experts from Africa and the Middle East met to discuss key issues related to diagnosis and biomarker testing in NSCLC and the implementation of personalized medicine in the region. The committee identified current challenges for effective diagnosis and predictive analysis in Africa and the Middle East. Moreover, strategies to encourage the implementation of biomarker testing were discussed. A practical approach for the effective diagnosis and predictive molecular testing of NSCLC in these regions was derived. We present the key issues and recommendations arising from the meetings.Pfizer Inc.http://www.journals.elsevier.com/clinical-lung-cancer/hb201

Microtomography of the Baltic amber tick Ixodes succineus reveals affinities with the modern Asian disease vector Ixodes ovatus

BACKGROUND: Fossil ticks are extremely rare and Ixodes succineus Weidner, 1964 from Eocene (ca. 44–49 Ma) Baltic amber is one of the oldest examples of a living hard tick genus (Ixodida: Ixodidae). Previous work suggested it was most closely related to the modern and widespread European sheep tick Ixodes ricinus (Linneaus, 1758). RESULTS: Restudy using phase contrast synchrotron x-ray tomography yielded images of exceptional quality. These confirm the fossil’s referral to Ixodes Latreille, 1795, but the characters resolved here suggest instead affinities with the Asian subgenus Partipalpiger Hoogstraal et al., 1973 and its single living (and medically significant) species Ixodes ovatus Neumann, 1899. We redescribe the amber fossil here as Ixodes (Partipalpiger) succineus. CONCLUSIONS: Our data suggest that Ixodes ricinus is unlikely to be directly derived from Weidner’s amber species, but instead reveals that the Partipalpiger lineage was originally more widely distributed across the northern hemisphere. The closeness of Ixodes (P.) succineus to a living vector of a wide range of pathogens offers the potential to correlate its spatial and temporal position (northern Europe, nearly 50 million years ago) with the estimated origination dates of various tick-borne diseases