Bovipain-2, the falcipain-2 ortholog, is expressed in intraerythrocytic stages of the tick-transmitted hemoparasite Babesia bovis

<p>Abstract</p> <p>Background</p> <p>Cysteine proteases have been shown to be highly relevant for Apicomplexan parasites. In the case of <it>Babesia bovis</it>, a tick-transmitted hemoparasite of cattle, inhibitors of these enzymes were shown to hamper intraerythrocytic replication of the parasite, underscoring their importance for survival.</p> <p>Results</p> <p>Four papain-like cysteine proteases were found to be encoded by the <it>B. bovis </it>genome using the MEROPS database. One of them, the ortholog of <it>Plasmodium falciparum </it>falcipain-2, here named bovipain-2, was further characterized. Bovipain-2 is encoded in <it>B. bovis </it>chromosome 4 by an ORF of 1.3 kb, has a predicted molecular weight of 42 kDa, and is hydrophilic with the exception of a transmembrane region. It has orthologs in several other apicomplexans, and its predicted amino acid sequence shows a high degree of conservation among several <it>B. bovis </it>isolates from North and South America. Synteny studies demonstrated that the <it>bovipain-2 </it>gene has expanded in the genomes of two related piroplasmids, <it>Theileria parva </it>and <it>T. annulata</it>, into families of 6 and 7 clustered genes respectively. The <it>bovipain-2 g</it>ene is transcribed in <it>in vitro </it>cultured intra-erythrocyte forms of a virulent and an attenuated <it>B. bovis </it>strain from Argentina, and has no introns, as shown by RT-PCR followed by sequencing. Antibodies against a recombinant form of bovipain-2 recognized two parasite protein bands of 34 and 26 kDa, which coincide with the predicted sizes of the pro-peptidase and mature peptidase, respectively. Immunofluorescence studies showed an intracellular localization of bovipain-2 in the middle-rear region of <it>in vitro </it>cultured merozoites, as well as diffused in the cytoplasm of infected erythrocytes. Anti-bovipain-2 antibodies also reacted with <it>B. bigemina</it>-infected erythrocytes giving a similar pattern, which suggests cross-reactivity among these species. Antibodies in sera of two out of six <it>B. bovis</it>-experimentally infected bovines tested, reacted specifically with recombinant bovipain-2 in immunoblots, thus demonstrating expression and immunogenicity during bovine-infecting stages.</p> <p>Conclusions</p> <p>Overall, we present the characterization of bovipain-2 and demonstrate its <it>in vitro </it>and <it>in vivo </it>expression in virulent and attenuated strains. Given the involvement of apicomplexan cysteine proteases in essential parasite functions, bovipain-2 constitutes a new vaccine candidate and potential drug target for bovine babesiosis.</p

Comparison of DNA extraction methods to improve the molecular diagnosis of Cryptosporidium spp. from fecal samples of calves

Cryptosporidium sp. es un parásito, protozoo que infecta a una gran variedad de hospedadores vertebrados. Entre las más de 30 especies validadas en el género, la especie zoonótica Cryptosporidium parvum es la principal causante de la criptosporidiosis bovina y representa una de las mayores causas de diarrea neonatal bovina. La vía de transmisión esfecal-oral, siendo el ooquiste, eliminado con las heces, el elemento infectante. La extracción de ADN genómico del parásito a partir de materia fecal es esencial para la determinación de la especie o de la subgenotipificación de Cryptosporidium spp. y uno de los desafíos actuales es mejorar la sensibilidad de los métodos moleculares de diagnóstico. En este trabajo evaluamos diferentes combinaciones de métodos de lisis de ooquistes y de extracción de ADN específico con el fin de aumentar la sensibilidad de su detección en aplicaciones downstream como por ejemplo la PCR diagnóstica, basada en la amplificación del gen ARN ribosomal 18S. Tanto la combinación de lisis alcalina y extracción con fenol-cloroformoalcohol isoamílico seguida de un kit comercial, como la aplicación directa del kit comercial -sin pasos previos- resultaron efectivas cuando utilizamos materia fecal como punto de partida. Posteriormente, comparamos la detección de ADN de Cryptosporidium spp. a partir de materia fecal versus ooquistes enriquecidos, resultando esta última más sensible ya que se incrementa el volumen de muestra procesable. Finalmente, a partir de ooquistes enriquecidos de Cryptosporidium spp. comparamos dos métodos para su ruptura y dos de extracción de ADN. Esto incluyó combinaciones de lisis alcalina vs. congelado-descongelado en nitrógeno líquido para la ruptura de ooquistes y la comparación de dos kits comerciales para la extracción de ADN. La combinación de dos pasos de lisis previos a la utilización del kit comercial no mejora la obtención de ADN específico. De esta manera el método más sensible y adecuado consiste en un paso de enriquecimiento de ooquistes yla aplicación directa del kit comercial. En conclusión, este protocolo optimizado logró mejorar la sensibilidad del diagnóstico molecular de Cryptosporidium sp. notablemente, lo cual posibilitará la detección del parásito en muestras con bajo número de ooquistes.Cryptosporidium sp. is a parasitic protozoa that infects a wide range of vertebrates. Among the 30 valid species, the zoonotic species Cryptosporidium parvum is the etiological agent of bovine cryptosporidiosis, representing one of the most important causes of neonatal diarrhea of bovines. The transmission route is fecal-oral and the oocyst, excreted with the feces, is the infective stage. Extraction of genomic DNA from oocysts starting from feces is essential for species determination and/or subgenotipification of Cryptosporidium spp. A current challenge is the improvement of the sensitivity of molecular diagnostic methods. In order to increase the quantity of isolated DNA and the sensitivity of its detection, we evaluated in this study the efficiency of different lysis and DNA extraction methods for posterior detection by diagnostic PCR, which is based on the amplification of the 18S rRNA gene. The combination of alkaline lysis and extraction by phenol-chloroform followed by the use of a commercial kit as well as the exclusive use of a commercial kit resulted effective when applied to fecal samples directly. On the other hand, the addition of a freeze-thaw step after alkaline lysis did not increase the efficiency of parasite DNA detection in this type of sample. Later, we compared the detection of DNA of Cryptosporidium spp. from oocyst-contaminated feces and partially purified oocyst suspensions, showing the latter higher sensitivity as the volume of processable sample is increased. Finally, two protocols for oocyst disruption and two for DNA isolation were compared from purified oocysts. These included combinations of alkaline lysis and freezethaw. The combination of two lysis methods did not improved the extraction of specific DNA. Thus, the most sensitive and adequate method consists of an oocyst enrichment step followed by the direct application of the commercial kit. In summary this optimized protocol significantly improved the sensitivity of C. parvum molecular diagnosis which could allow parasite detection in samples contaminated with low numbers of oocysts.Fil: Toledo, Jonathan. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Lombardelli, Joaquín Andrés. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria. Departamento de Patología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Galarza, Roxana. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Tiranti, Karina Ivana. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria. Departamento de Patología Animal; ArgentinaFil: Garro, Carlos J.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Florin Christensen, Mónica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Schnittger, Leonhard. Universidad de Morón; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; Argentina. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Tomazic, Mariela Luján. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

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

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

Centrality evolution of the charged-particle pseudorapidity density over a broad pseudorapidity range in Pb-Pb collisions at root s(NN)=2.76TeV

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