Kinetic characterization of a novel cysteine peptidase from the protozoan Babesia bovis, a potential target for drug design

C1A cysteine peptidases have been shown to play an important role during apicomplexan invasion and egress of host red blood cells (RBCs) and therefore have been exploited as targets for drug development, in which peptidase specificity is deterministic. Babesia bovis genome is currently available and from the 17 putative cysteine peptidases annotated four belong to the C1A subfamily. In this study, we describe the biochemical characterization of a C1A cysteine peptidase, named here BbCp (B. bovis cysteine peptidase) and evaluate its possible participation in the parasite asexual cycle in host RBCs. The recombinant protein was obtained in bacterial inclusion bodies and after a refolding process, presented typical kinetic features of the cysteine peptidase family, enhanced activity in the presence of a reducing agent, optimum pH between 6.5 and 7.0 and was inhibited by cystatins from R. microplus. Moreover, rBbCp substrate specificity evaluation using a peptide phage display library showed a preference for Val > Leu > Phe. Finally, antibodies anti-rBbCp were able to interfere with B. bovis growth in vitro, which highlights the BbCp as a potential target for drug design.Instituto de PatobiologíaFil: Lu, Stephen. Universidade Federal de São Paulo. Escola Paulista de Medicina. Department of Biochemistry; BrasilFil: Ascencio, Mariano E. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Torquato, Ricardo J.S. Universidade Federal de São Paulo. Escola Paulista de Medicina. Department of Biochemistry; BrasilFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; ArgentinaFil: Florin-Christensen, Monica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Tanaka, Aparecida S. Universidade Federal de São Paulo. Escola Paulista de Medicina. Department of Biochemistry; BrasilFil: Tanaka, Aparecida S. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular; Brasi

Molecular diagnosis of Leishmania spp. in dogs of a subtropical locality of Argentina

Leishmaniosis is a tropical and subtropical vector‐borne disease caused by hemoparasites of the genus Leishmania. The disease can infect humans, as well as domestic and wildlife animals. Dogs are the main reservoir for L. infantum, the aetiological agent of visceral leishmaniosis (VL) in America, and a domestic source of L. braziliensis, the most widespread aetiological agent of American tegumentary leishmaniosis. Infected dogs can develop a clinical syndrome called canine leishmaniosis (CanL), which presents with skin lesions, mild fever; additionally hepatomegaly and splenomegaly can be observed, although asymptomatic infections are frequent. Direct microscopic observation of the parasite in bone marrow, blood, skin scrapings and conjunctival swab samples is the gold standard of diagnosis and is usually complemented with serological tests, and to a lesser extent, molecular detection of the parasite. In Argentina, leishmaniosis is an emerging disease, with a growing number of human and canine clinical cases since 2006. Our study was carried out in Mercedes, a town located in the subtropical north‐eastern area of Argentina, where dogs with positive parasitological test results for Leishmania spp. must be euthanized according to local regulations. We evaluated the presence of Leishmania spp. DNA in the blood of dogs (n = 166) from urban and peri‐urban zones. Genomic DNA was extracted from whole blood using Chelex 100 resin and a conserved 116 bp region of the kinetoplastid DNA was amplified by conventional PCR. Clinical signs, age and gender were recorded. Our results showed that 120 out of 166 surveyed dogs (72%) were positive for Leishmania spp. DNA of which only seven were positive by parasitological and serological tests. No significant correlation between positive cases and gender or age groups was found. This report shows the high prevalence of this disease in Argentina and contributes to improve public health policy with regard to diagnosis, prevention and treatment of infected dogs.Instituto de PatobiologíaFil: Ascencio, Mariano E. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sarmiento, Nestor Fabian. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Morón. Facultad de Ciencias Exactas, Químicas y Naturales; ArgentinaFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Morón. Facultad de Ciencias Exactas, Químicas y Naturales; ArgentinaFil: Rodriguez, Anabel Elisa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Epidemiological situation of bovine cryptosporidiosis in Argentina

La criptosporidiosis en terneros neonatos es causada por protozoos enteroparásitos del género Cryptosporidium. Es una afección caracterizada por cuadros de diarrea y deshidratación, dando lugar a una reducción en la producción y a las subsecuentes pérdidas económicas a productores ganaderos alrededor del mundo. La criptosporidiosis es además considerada una enfermedad zoonótica de relevancia para la salud pública ya que la ingesta de ooquistes puede ser fatal para niños, ancianos y personas inmunocomprometidas. El objetivo de este trabajo de revisión es estudiar la epidemiología de las infecciones producidas por Cryptosporidium spp. en bovinos y los factores de riesgo asociados utilizando la literatura disponible para determinar la situación en Argentina. Se determinó que la prevalencia de la infección para terneros entre 1 a 60 días de edad es variable y ronda entre 16,3 y 25,5 %. Notablemente, en un brote se encontró que 84 % de los animales estaban infectados. Estudios moleculares determinaron que C. parvum fue la única especie encontrada en terneros menores a 60 días en Argentina y se identificaron nueve subtipos diferentes pertenecientes a la familia IIa, incluyendo los subtipos zoonóticos IIaA16G1R1, IIaA17G1R1, IIaA18G1R1 y IIaA19G1R1. Se encontró también el subtipo IIaA24G1R1 como un nuevo subtipo reportado únicamente en Argentina hasta el presente. Estudios realizados en el país determinaron que la edad, la presencia de diarrea y los suelos pobremente drenados son factores de riesgo asociados a la infección por Cryptosporidium spp. Este análisis reúne información que servirá para una mayor comprensión de las características epidemiológicas y factores de riesgo, ayudando a la prevención y desarrollo de estrategias de control para la criptosporidiosis bovina.Cryptosporidiosis in neonatal calves is caused by enteroparasites of the genus Cryptosporidium. This infectious disease is characterized by diarrhea and dehydration, leading to a reduction in production, causing economic losses in meat and dairy farming around the world. In addition, cryptosporidiosis is considered a zoonotic disease of relevance to public health since the ingestion of oocysts can be fatal for children, the elderly and immunocompromised people. The objective of this review is to determine the situation of the epidemiology of C. parvum-infections in cattle and associated risk factors in Argentina, based on the literature of recent studies. The prevalence of infection for calves between 1 to 60 days of age was determined to range between 16.3 and 25.5 %. Noteworthy, in a single outbreak, 84 % of calves were found to be infected. Molecular studies determined that C. parvum was the only species found in calves younger than 60 days of age in Argentina and altogether nine subtypes of the IIa family were identified, including the zoonotic subtypes IIaA16G1R1, IIaA17G1R1, IIaA18G1R1 and IIaA19G1R1. Among them, the IIaA24G1R1 subtype has been exclusively reported in this country so far. Studies carried out in Argentina determined that age, occurrence of diarrhea and poorly drained soils are risk factors associated with infection by Cryptosporidium spp. This analysis gathers information that will aid to understand the epidemiological characteristics and risk factors, facilitating the prevention and development of control strategies for bovine cryptosporidiosis.Fil: de Alba, Paloma. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Patobiología Veterinaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Patobiología Veterinaria; ArgentinaFil: Florin Christensen, Mónica Ofelia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Universidad de Morón; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Patobiología Veterinaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Patobiología Veterinaria; Argentin

Highly sensitive nested PCR and rapid immunochromatographic detection of Babesia bovis and Babesia bigemina infection in a cattle herd with acute clinical and fatal cases in Argentina

Bovine babesiosis is a tick‐transmitted haemoparasitic disease caused by Babesia bovis and B. bigemina affecting cattle of tropical and subtropical regions around the world. Pathogens are transmitted by the tick vector Rhipicephalus microplus displaying a widespread distribution in northeastern Argentina. The disease is characterized by significant animal morbidity and mortality resulting in considerable economic loss. In this study, B. bovis and B. bigemina infection was investigated in a cattle herd of 150 adult bovines of pure Braford breed raised in a tick‐hyperendemic field using molecular and serum antibody tests. A highly sensitive nested polymerase chain reaction (nPCR) assay targeting a species‐specific region of the apocytochrome b gene resulted in direct B. bovis and B. bigemina detection in 27.3% and 54.7% of bovines, respectively. A recently developed immunochromatographic strip test (ICT) based on recombinant forms of spherical body protein 4 and the C‐terminal region of rhoptry‐associated protein 1 showed that 71.3% and 89.3% of bovines were seropositive for B. bovis and B. bigemina, respectively. The mixed infection rate as observed by direct (19.3%) and indirect detection (65.3%) coincided with those expected, respectively. Importantly, four months after sampling, nine bovines of the studied herd showed clinical signs of bovine babesiosis of which six animals eventually died. Microscopic detection of infected erythrocytes in Giemsa‐stained blood smears confirmed B. bovis infection. Our study demonstrates that although animals showed a relatively high and very high rate of immunity against infection with B. bovis (71.3%) and B. bigemina (89.3%) parasites, respectively, clinical cases and fatalities due to the infection with B. bovis were observed. It is proposed that the most adequate control measure in the studied epidemiological situation is to vaccinate animals to prevent losses and/or an outbreak of bovine babesiosis.Instituto de PatobiologíaFil: Ganzinelli Sabrina Belen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Benitez, Daniel Francisco. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Gantuya, Sambuu. Obihiro University of Agriculture and Veterinary Medicine. National Research Center for Protozoan Diseases; JapónFil: Guswanto, Azirwan. Obihiro University of Agriculture and Veterinary Medicine. National Research Center for Protozoan Diseases; JapónFil: Florin-Christensen, Mónica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Igarashi, Ikuo. Obihiro University of Agriculture and Veterinary Medicine. National Research Center for Protozoan Diseases; Japó

In silico identification of immunotherapeutic and diagnostic targets in the glycosylphosphatidylinositol metabolism of the coccidian Sarcocystis aucheniae

Meat of the South American camelids (SACs) llama and alpaca is an important source of animal protein and income for rural families in the Andes, and a product with significant growth potential for local and international markets. However, infestation with macroscopic cysts of the coccidian protozoon Sarcocystis aucheniae, a parasitosis known as SAC sarcocystosis, significantly hampers its commercialization. There are no validated methods to diagnose the presence of S. aucheniae cysts other than carcass examination. Moreover, there are no available drugs or vaccines to cure or prevent SAC sarcocystosis. Identification of relevant molecules that act at the host–pathogen interface can significantly contribute to the control of this disease. It has been shown for other pathogenic protozoa that glycosylphosphatidylinositol (GPI) is a critical molecule implicated in parasite survival and pathogenicity. This study focused on the identification of the enzymes that participate in the S. aucheniae GPI biosynthetic pathway and the repertoire of the parasite GPI‐anchored proteins (GPI‐APs). To this aim, RNA was extracted from parasite cysts and the transcriptome was sequenced and translated into amino acid sequences. The generated database was mined using sequences of well‐characterized GPI biosynthetic enzymes of Saccharomyces cerevisiae and Toxoplasma gondii. Eleven enzymes predicted to participate in the S. aucheniae GPI biosynthetic pathway were identified. On the other hand, the database was searched for proteins carrying an N‐terminal signal peptide and a single C‐terminal transmembrane region containing a GPI anchor signal. Twenty‐four GPI‐anchored peptides were identified, of which nine are likely S. aucheniae‐specific, and 15 are homologous to membrane proteins of other coccidians. Among the latter, 13 belong to the SRS domain superfamily, an extensive group of coccidian GPI‐anchored proteins that mediate parasite interaction with their host. Phylogenetic analysis showed a great degree of intra‐ and inter‐specific divergence among SRS family proteins. In vitro and in vivo experiments are needed to validate S. aucheniae GPI biosynthetic enzymes and GPI‐APs as drug targets and/or as vaccine or diagnostic antigens.Instituto de PatobiologíaFil: Decker Franco, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wieser, Sarah Nathaly. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Soria, Marcelo Abel. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Microbiología Agrícola; ArgentinaFil: De Alba Paloma. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

N-Glycosylation in piroplasmids : Diversity within simplicity

N-glycosylation has remained mostly unexplored in Piroplasmida, an order of tick-transmitted pathogens of veterinary and medical relevance. Analysis of 11 piroplasmid genomes revealed three distinct scenarios regarding N-glycosylation: Babesia sensu stricto (s.s.) species add one or two N-acetylglucosamine (NAcGlc) molecules to proteins; Theileria equi and Cytauxzoon felis add (NAcGlc)2-mannose, while B. microti and Theileria s.s. synthesize dolichol-P-P-NAcGlc and dolichol-P-P-(NAcGlc)2 without subsequent transfer to proteins. All piroplasmids possess the gene complement needed for the synthesis of the N-glycosylation substrates, dolichol-P and sugar nucleotides. The oligosaccharyl transferase of Babesia species, T. equi and C. felis, is predicted to be composed of only two subunits, STT3 and Ost1. Occurrence of short N-glycans in B. bovis merozoites was experimentally demonstrated by fluorescence microscopy using a NAcGlc-specific lectin. In vitro growth of B. bovis was significantly impaired by tunicamycin, an inhibitor of N-glycosylation, indicating a relevant role for N-glycosylation in this pathogen. Finally, genes coding for N-glycosylation enzymes and substrate biosynthesis are transcribed in B. bovis blood and tick stages, suggesting that this pathway is biologically relevant throughout the parasite life cycle. Elucidation of the role/s exerted by N-glycans will increase our understanding of these successful parasites, for which improved control measures are needed.Instituto de PatobiologíaFil: Florin-Christensen, Mónica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Florin-Christensen, Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rodriguez, Anabel Elisa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Rodriguez, Anabel Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Suarez, Carlos Esteban. Washington State University. College of Veterinary Medicine. Department of Veterinary Microbiology and Pathology; Estados UnidosFil: Suarez, Carlos Esteban. United States Department of Agricultural-Agricultural Research Service. Animal Disease Research Unit; Estados UnidosFil: Ueti, Massaro W. Washington State University. College of Veterinary Medicine. Department of Veterinary Microbiology and Pathology; Estados UnidosFil: Ueti, Massaro W. United States Department of Agricultural-Agricultural Research Service. Animal Disease Research Unit; Estados UnidosFil: Delgado, Fernando Oscar. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Delgado, Fernando Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

High genetic diversity and differentiation of the babesia ovis population in Turkey

Babesia ovis is a tick‐transmitted protozoan haemoparasite causing ovine babesiosis in sheep and goats leading to considerable economic loss in Turkey and neighbouring countries. There are no vaccines available, therapeutic drugs leave toxic residues in meat and milk, and tick vector control entails environmental risks. A panel of eight mini‐ and micro‐satellite marker loci was developed and applied to study genetic diversity and substructuring of B. ovis from western, central and eastern Turkey. A high genetic diversity (He = 0.799) was found for the sample of overall B. ovis population (n = 107) analyzed. Principle component analysis (PCoA) revealed the existence of three parasite subpopulations: (a) a small subpopulation of isolates from Aydin, western Turkey; (b) a second cluster predominantly generated by isolates from western Turkey; and (c) a third cluster predominantly formed by isolates from central and eastern Turkey. Two B. ovis isolates from Israel included in the analysis clustered with isolates from central and eastern Turkey. This finding strongly suggests substructuring of a major Turkish population into western versus central–eastern subpopulations, while the additional smaller B. ovis population found in Aydin could have been introduced, more recently, to Turkey. STRUCTURE analysis suggests a limited exchange of parasite strains between the western and the central–eastern regions and vice versa, possibly due to limited trading of sheep. Importantly, evidence for recombinant genotypes was obtained in regionally interchanged parasite isolates. Important climatic differences between the western and the central/eastern region, with average yearly temperatures of 21°C versus 15°C, correspond with the identified geographical substructuring. We hypothesize that the different climatic conditions may result in variation in the activity of subpopulations of Rhipicephalus spp. tick vectors, which, in turn, could selectively maintain and transmit different parasite populations. These findings may have important implications for vaccine development and the spread of drug resistance.Instituto de PatobiologíaFil: Mira, Anabela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Unlu, Ahmet Hakan. Van Yuzuncu Yil University. Vocational School of Gevas; TurquíaFil: Bilgic, Huseyin Bilgin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Bakirci, Serkan. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Hacilarlioglu, Selin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Karagenc, Tulin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Carletti, Tamara. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Weir, William. Universityof Glasgow. College of Medical, Veterinary and Life Sciences; Reino UnidoFil: Shiels, Brian. Universityof Glasgow. College of Medical, Veterinary and Life Sciences; Reino UnidoFil: Shkap, Varda. Kimron Veterinary Institute. Division of Parasitology; IsraelFil: Aktas, Munir. Firat University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

International interlaboratory validation of a nested PCR for molecular detection of Babesia bovis and Babesia bigemina, causative agents of bovine babesiosis

Babesia bovis and B. bigemina are tick-transmitted parasites causing bovine babesiosis, characterized by significant morbidity and mortality leading to economic losses to the livestock industry in tropical and subtropical regions worldwide. Animals that recover from acute infection remain carriers with low parasitemia acting as a source of transmission, and often escape detection. An improved diagnosis of a B. bovis and/or B. bigemina infection of carrier animals is enabled by the availability of detection methods with high sensitivity. To this end, two nested PCR assays targeting the cytochrome b (cytb) genes of B. bovis and B. bigemina (cytb-nPCR), have been recently developed and an increased sensitivity with respect to reference protocols has been shown (Romero-Salas et al., 2016). In this study, the specificity against a panel of hemoparasites that potentially co-occur with B. bovis and B. bigemina was demonstrated to ensure applicability of the cytb-nPCR assays in a wide range of regions where bovine babesiosis is endemic. Furthermore, we compared both reported cytb-nPCR assays with reference nPCR and qPCR protocols for (i) their capability to detect carrier animals in the field, and (ii) their reproducibility when performed in different laboratories by independent operators. We show that, in a panel of bovine field samples (n = 100), the cytb-nPCR assays detected a considerably higher number of 25% B. bovis and 61% B. bigemina-positive animals compared to 7% and 20% B. bovis and 55% and 49% B. bigemina-positive animals when tested by reference nPCR and qPCR protocols, respectively. Cytb-nPCRs were also found superior in the detection of carrier animals when field samples from Africa were analyzed. In addition, both the B. bovis and B. bigemina cytb-nPCR assays were independently validated in a single blinded study in three laboratories. Importantly, no significant differences in the number/percentage of infected animals was observed using cytb-nPCR assays. In summary, the cytb-nPCR assays detected a considerably higher number of chronically infected B. bovis and B. bigemina carrier animals compared to reference nPCR and qPCR protocols, when applied in different epidemiological field situations. Furthermore, a high reproducibility between laboratories could be demonstrated.Instituto de PatobiologíaFil: Ganzinelli Sabrina Belen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria; ArgentinaFil: Ganzinelli Sabrina Belen. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Byaruhanga, Charles. University of Pretoria. Faculty of Veterinary Science. Department of Veterinary Tropical Diseases. Vectors and Vector-borne Diseases Research Programme; SudáfricaFil: Primo, María Evangelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea; ArgentinaFil: Primo, María Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lukanji, Zinathi. University of Pretoria. Faculty of Veterinary Science. Department of Veterinary Tropical Diseases. Vectors and Vector-borne Diseases Research Programme; SudáfricaFil: Sibeko, Kgomotso. University of Pretoria. Faculty of Veterinary Science. Department of Veterinary Tropical Diseases. Vectors and Vector-borne Diseases Research Programme; SudáfricaFil: Matjila, Tshepo. University of Pretoria. Faculty of Veterinary Science. Department of Veterinary Tropical Diseases. Vectors and Vector-borne Diseases Research Programme; SudáfricaFil: Neves, Luis. University of Pretoria. Faculty of Veterinary Science. Department of Veterinary Tropical Diseases. Vectors and Vector-borne Diseases Research Programme; SudáfricaFil: Benitez, Daniel Francisco. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Enkhbaatar, Batmagnai. Obihiro University of Agriculture and Veterinary Medicine. National Research Center for Protozoan Diseases; JapónFil: Enkhbaatar, Batmagnai. Mongolian University of Life Sciences. Institute of Veterinary Medicine. Laboratory of Molecular Genetics; MongoliaFil: Nugraha, Arifin Budiman. Obihiro University of Agriculture and Veterinary Medicine. National Research Center for Protozoan Diseases; JapónFil: Nugraha, Arifin Budiman. IPB University. Faculty of Veterinary Medicine. Department of Animal Infectious Diseases and Veterinary Public Health; IndonesiaFil: Igarashi, Ikuo. Obihiro University of Agriculture and Veterinary Medicine. National Research Center for Protozoan Diseases; JapónFil: Florin-Christensen, Mónica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria; ArgentinaFil: Florin-Christensen, Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología Veterinaria; ArgentinaFil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

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

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