Nematodos intestinales de perros en parques públicos de Yucatán, México / Zoonotic intestinal nematodes in dogs from public parks in Yucatán, México

Resumen Introducción. Los perros representan un potencial riesgo para la salud pública debido a que transmiten infecciones parasitarias al hombre. Objetivo. Estimar la frecuencia y determinar los factores asociados a la presencia de huevos de nematodos intestinales en heces de perros recolectadas en parques públicos de Mérida, Yucatán, México. Materiales y métodos. Se analizaron 100 muestras de heces de perros recolectadas en 20 parques públicos de dos zonas de la ciudad. Las muestras se procesaron mediante las técnicas de flotación centrifugada y de McMaster para confirmar la presencia de huevos de nematodos intestinales y cuantificarlos por gramo de heces. Se determinaron los factores asociados a la presencia de los huevos mediante un análisis univariado de χ2. Resultados. Se encontró una frecuencia de 11 %. Se identificaron huevos de tres especies de parásitos y Ancylostoma caninum fue el más frecuente (10 %), seguido por Toxocara canis (1 %) y Trichuris vulpis (1 %). La mayoría de las muestras positivas presentaba infección con un nematodo intestinal únicamente (10 %) y solo el 1 % resultó positivo para infección mixta por A. caninum y T. vulpis. La presencia de perros sin dueño en los parques públicos fue el factor asociado (p=0,046) con un mayor número de heces positivas para huevos de nematodos intestinales. Conclusiones. En los parques de la ciudad se encontraron heces de perros con huevos de nematodos intestinales con potencial zoonótico; la probabilidad de que las muestras fueran positivas fue mayor en los parques con presencia de perros sin dueño.

Abstract Introduction: Dogs represent a potential public health risk because of the natural transmission of zoonotic parasitic infections. Objective: To estimate the frequency and to determine factors associated with the presence of intestinal nematode eggs in dog feces collected in public parks of Mérida,Yucatán, México. Materials and methods: A total of 100 dog fecal samples collected from 20 public parks in two areas of Mérida were analyzed. Samples were processed by the centrifugation-flotation and the McMaster techniques to confirm the presence and to quantify the excretion of intestinal nematode eggs per gram of feces. The factors associated with the presence of nematode eggs were identified using the chi square univariate analysis. Results: We found an 11% frequency of fecal samples positive for intestinal nematode eggs. Eggs of three species of parasites were identified: Ancylostoma caninum was the most common (10%), followed by Toxocara canis (10%), and Trichuris vulpis (1%). Most positive samples were infected with only one intestinal nematode (10%), and only 1 % was positive for a mixed infection by A. caninum and T. vulpis. The presence of stray dogs in public parks was an associated factor (p=0.046) with a higher number of fecal samples positive for intestinal nematode eggs. Conclusions: The frequency of intestinal nematodes in dog feces with zoonotic potential was high in parks of Mérida, Yucatán, México; samples from parks where there were stray dogs had a higher possibility of being positive.

Cross-disciplinary approaches for measuring parasitic helminth viability and phenotype

Parasitic worms (helminths) within the Phyla Nematoda and Platyhelminthes are responsible for some of the most debilitating and chronic infectious diseases of human and animal populations across the globe. As no subunit vaccine for any parasitic helminth is close to being developed, the frontline strategy for intervention is administration of therapeutic, anthelmintic drugs. Worryingly, and unsurprising due to co-evolutionary mechanisms, many of these worms are developing resistance to the limited compound classes currently being used. This unfortunate reality has led to a renaissance in next generation anthelmintic discovery within both academic and industrial sectors. However, a major bottleneck in this process is the lack of quantitative methods for screening large numbers of small molecules for their effects on the whole organism. Development of methodologies that can objectively and rapidly distinguish helminth viability or phenotype would be an invaluable tool in the anthelmintic discovery pipeline. Towards this end, we describe how several basic techniques currently used to assess single cell eukaryote viability have been successfully applied to parasitic helminths. We additionally demonstrate how some of these methodologies have been adopted for high-throughput use and further modified for assessing worm phenotype. Continued development in this area is aimed at increasing the rate by which novel anthelmintics are identified and subsequently translated into everyday, practical applications.

Vermes parasíticos (helmintos) dos filos Nematoda e Platelmintos são responsáveis por algumas das doenças infecciosas crônicas e mais debilitantes das populações humana e animal em todo o globo. Já que nenhuma vacina está prestes a ser desenvolvida para nenhum parasita helmíntico, a frente estratégica de intervenção é a administração de drogas terapêuticas anti-helmínticas. De maneira preocupante, e não surpreendente devido a mecanismos coevolutivos, muitos destes vermes estão desenvolvendo resistência às limitadas classes de compostos que têm sido usados no momento. Esta infeliz realidade levou a um renascimento na descoberta de uma nova geração de anti-helmínticos tanto no setor acadêmico quanto no industrial. Contudo, um importante gargalo neste processo é a falta de métodos quantitativos para testar um grande número de pequenas moléculas em relação aos efeitos sobre o organismo inteiro. O desenvolvimento de metodologias que possam distinguir objetiva e rapidamente a viabilidade dos helmintos ou o fenótipo seria uma ferramenta valiosa para canalizar a descoberta de anti-helmínticos. Para este fim, descrevemos aqui como muitas técnicas básicas, correntemente usadas para avaliar a viabilidade de células únicas de eucariotos, têm sido aplicadas com sucesso para helmintos parasíticos. Adicionalmente demonstramos como algumas destas metodologias foram adotadas para uso em larga escala e além disso modificadas para avaliar o fenótipo de vermes. O desenvolvimento contínuo nesta área está voltado para aumentar a taxa com que novos anti-helmínticos são identificados e subsequentemente traduzidos em aplicações práticas cotidianas.

Efeitos do pareamento no perfil de expressão gênica do parasita Schistosoma mansoni / Effects of pairing on the gene expression profiles of the parasite Schistosoma mansoni

Esquistossomose é uma doença crônica e debilitante. Schistosoma representa a única classe de trematódeos com vida dióica. Um contínuo pareamento com o macho é essencial para a maturação sexual do sexo feminino. Fêmeas adultas provenientes de infecções uni-sexuadas são subdesenvolvidas, apresentam atrofia do tamanho e um sistema reprodutivo imaturo. Para estudar os mecanismos envolvidos no pareamento de vermes adultos foram utilizadas duas plataformas de microarranjos distintas: uma composta por 4 mil sondas de cDNA dupla fita produzida pelo nosso grupo de pesquisas e outra composta por 44 mil sondas de oligonucleotideos desenhadas pelo nosso grupo e produzida pela empresa Agilent Technologies. Com a plataforma de 4 mil sondas detectamos 113 transcritos diferencialmente expressos em fêmeas adultas mantidas separadas de seus respectivos pares durante 24 horas de cultivo in vitro quando comparadas com fêmeas adultas pareadas; para 10 destes genes obtivemos uma confirmação adicional da expressão diferencial por transcrição reversa fita específica seguida de PCR em Tempo Real. Observamos também os efeitos do pareamento no perfil de expressão gênica de machos adultos mantidos separados de seus respectivos pares durante 24 horas de cultivo in vitro; foram encontrados 152 transcritos diferencialmente expressos. Com a plataforma de 44 mil sondas foi detectada a expressão de 5.798 genes transcricionalmente ativos em verme adulto, em um conjunto de 19.907 genes únicos representados nesta plataforma. A análise do conjunto de genes "no match" mostrou que em 156 genes ocorria expressão senso e anti-senso; para 6 destes transcritos obtivemos uma confirmação adicional da expressão nas duas fitas por transcrição reversa fita específica seguida de PCR em Tempo Real. Adicionalmente foram identificados 2717 transcritos diferencialmente expressos em fêmeas separadas de seus respectivos pares durante 13 dias de cultivo in vitro, quando comparadas com fêmeas mantidas pareadas...

Schistosomiasis is a chronic and debilitating disease. Schistosoma represents the only class of trematodes with a dioecious life. A continuous pairing with the male is essential for female sexual maturation. Adult females from uni-sexual infections are underdeveloped, have body atrophy and an immature reproductive system. To study the mechanisms involved in pairing of adult worms two microarray platforms were used: one comprised by 4000 cDNA probes and printed by our research group and another comprised by 44 000 oligonucleotide probes designed by our group and printed by Agilent Technologies Company. With the 4000-probes platform we detected 113 transcripts differentially expressed in adult females kept separated from their mates during 24 hours in vitro when compared with paired adult females; for 10 of these genes we obtained additional confirmation of differential expression by Real Time RT-PCR. We also observed the effects of pairing on the gene expression profile of adult males kept separate from their mates during 24 hours in vitro, where we found 152 differentially expressed transcripts. With the 44 000-probes platform we detected the expression of 5798 genes in adult worms, out of a set of 19 907 unique genes represented on this platform. Analysis of the "no match" genes showed that 156 have transcription from the sense and anti-sense strands; for 6 of them we obtained additional confirmation of expression by strand specific Real Time RT-PCR. Additionally, we identified 2717 differentially expressed transcripts in females separated from their mates during 13 days in vitro when compared to females that remained paired. In the analysis of males separated for 13 days we found 243 differentially expressed transcripts. Finally, we performed a study aimed at observing genes which might be correlated to physical contact pairing (male and female) and compared to genes that might be regulated by the possible diffusion of secreted proteins and hormones in...

RNA Interference in Infectious Tropical Diseases

Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi.

RNA Interference in Infectious Tropical Diseases

Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi.

Identification of parasite DNA in common bile duct stones by PCR and DNA sequencing

We attempted to identify parasite DNA in the biliary stones of humans via PCR and DNA sequencing. Genomic DNA was isolated from each of 15 common bile duct (CBD) stones and 5 gallbladder (GB) stones. The patients who had the CBD stones suffered from cholangitis, and the patients with GB stones showed acute cholecystitis, respectively. The 28S and 18S rDNA genes were amplified successfully from 3 and/or 1 common bile duct stone samples, and then cloned and sequenced. The 28S and 18S rDNA sequences were highly conserved among isolates. Identity of the obtained 28S D1 rDNA with that of Clonorchis sinensis was higher than 97.6%, and identity of the 18S rDNA with that of other Ascarididae was 97.9%. Almost no intra-specific variations were detected in the 28S and 18S rDNA with the exception of a few nucleotide variations, i.e., substitution and deletion. These findings suggest that C. sinensis and Ascaris lumbricoides may be related with the biliary stone formation and development.