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The indiscriminate administration of synthetic anthelmintics such as ivermectin contributes to the selection of subpopulations capable of resisting the drugs’ effects. To understand the mechanisms of ivermectin resistance in Caenorhabditis elegans, this study attempted to identify molecular targets. C. elegans lineages that were sensitive and resistant to ivermectin were used. Collected nematodes were added to an extraction buffer and macerated in liquid nitrogen for protein extraction. The extracted proteins were separated according to molecular weight by SDS-PAGE to verify their integrity. Subsequently, proteins from both lineages were separated using two-dimensional electrophoresis. The gels were analyzed and the relevant spots were excised and identified by mass spectrometry (NanoESI-Q-TOF and MASCOT®) and subsequently assessed by GO enrichment and STRING® analyses. The increased expression of proteins associated with high metabolic activity, such as ATP-2 and ENOL-1, which are responsible for ATP synthesis, was observed. Furthermore, proteins with involvement in mediating muscular function (MLC-1, ACT-1, and PDI-2), signaling (FAR-1 and FAR-2), and embryo development (VHA-2) were identified. Protein interaction analysis indicated that the majority of the identified proteins in the resistant lineages participated in the same reaction triggered by ivermectin. Resumo A administração indiscriminada de anti-helmínticos sintéticos, como a ivermectina, contribui para a seleção de subpopulações capazes de resistir ao efeito das drogas. Para entender os mecanismos de resistência à ivermectina em Caenorhabditis elegans, este estudo visou identificar alvos moleculares. Portanto, linhagens de C. elegans sensíveis e resistentes à ivermectina foram utilizadas. Os nematóides coletados foram adicionados ao tampão de extração e macerados em nitrogênio líquido para obtenção das proteínas. As proteínas extraídas foram separadas por peso molecular em SDS-PAGE para verificar sua integridade. Posteriormente, as proteínas de ambas as linhagens foram separadas por eletroforese bidimensional. Os géis foram analisados, os spots relevantes foram excisados e identificados por espectrometria de massa (NanoESI-Q-TOF e MASCOT®), em seguida, analisados em seus termos de GO e STRING®. A expressão aumentada de proteínas associadas à alta atividade metabólica, como as proteínas ATP-2 e ENOL-1, responsáveis pela síntese de ATP, foi observada. Além disso, foram identificadas as proteínas responsáveis pelo controle da função muscular (MLC-1, ACT-1 e PDI-2), sinalização (FAR-1 e FAR-2) e desenvolvimento embrionário (VHA-2). A análise das interações proteicas indicou que a maioria das proteínas identificadas na cepa resistente participa da mesma reação desencadeada pela ivermectina.

Considering the great efforts towards formulating a vaccine against hookworms, and the concerns about the spread of drug resistance through hookworm populations, it is justified to study the molecular diversity and population genetic structure of these nematodes. This work had the aim to develop microsatellite markers to investigate the genetic structure and the molecular diversity of Brazilian populations of Ancylostoma caninum. Seven microsatellites markers were successfully used to characterize five Brazilian populations. These findings may contribute to a better comprehension of the ecology, patterns of transmission, drug resistances and development of immunotherapeutic strategies in hookworms.

The tetraprimer ARMS-PCR technique is efficient for SNP detection and can be used to search for polymorphisms associated with drug resistance. However, the establishment of this methodology is not always straightforward because of the constraints on primer design due to the restrictions of the polymorphic regions. Here, we describe the standardization of the tetraprimer ARMS-PCR methodology for the detection of a SNP at codon 198 of the Ancylostoma caninum β-tubulin gene. This SNP is associated with resistance to albendazole in various nematodes. The methodology was used to screen 327 individuals from 6 different locations. No mutation was found in any of the samples. This methodology will be useful for screening for the E198A SNP in the β-tubulin gene of canine hookworms in a broader population to determine whether this SNP is associated with benzimidazole resistance in this species. The method could also be adapted for the analysis of other SNPs in other nematode species.

The mitochondrial cytochrome oxidase I gene was partially sequenced for 164 Ancylostoma caninum individuals, originating from five different localities in Brazil, with the aim of describing the genetic diversity and genetic structure of Brazilian hookworm populations. Allelic and nucleotide diversity were moderate (overall h=0.88 and pi=0.016) and were similar among cities. There was moderate genetic differentiation among the populations sampled (approximately Phi(ST)=0.12) and a weak but nonsignificant correlation between geographical and genetic distance. This genetic structure was similar to that observed among populations of the human hookworm, Necator americanus, but distinct from that typically found in trichostrongylid nematode parasites of livestock. Thus, a pattern of different genetic structures among different groups of nematodes is emerging. We also observed a few individuals that had a highly divergent mtDNA sequence (almost 7% sequence divergence from the other sequences). These results in combination with data from other studies suggest that A. caninum populations worldwide consist of a mix of previously differentiated populations, or perhaps even cryptic species. This study contributes to the knowledge of genetic structure and diversity of hookworms, which in turn will be useful in developing methods for their control.

Species of Ancylostoma infecting dogs and sometimes humans are sympatric in many parts of the world. The establishment of a specific molecular diagnostic tool is important, not only to refine information for epidemiological studies, but also to evaluate the efficacy of vaccine programmes and assist in the development of specific drug treatments. The ITS region from 20 specimens of A. braziliense, collected from three separate geographical areas of Brazil, and from 10 specimens of A. caninum, collected from the same area in Brazil were sequenced and analyzed. Alignment of sequences showed that this gene is highly conserved. The intraspecific polymorphism for both species was less then 1%, whereas the interspecific polymorphism was 6.2, 7.3 and 9.4% between A. ceylanicum and A. braziliense; A. caninum and A. ceylanicum and A. ceylanicum and A. braziliense, respectively. Among the three species it was 12.3%. This revealed the ITS region as highly conserved and consequently a good molecular marker for diagnostic studies. In this work, four restriction enzymes were used in a PCR-RFLP using the ITS region of rDNA, to establish a differential diagnosis which discriminates between three Ancylostoma species, A. braziliense, A. caninum and A. ceylanicum. The best pattern was given by the HinfI enzyme, which produced different fragment sizes for each of the three species. Furthermore, the diagnostic tool differentiates DNA extracted directly from faeces of Ancylostoma-infected dogs.

Recombinant DNA studies have been focused on developing vaccines to different cestodes. But few studies involving Dipylidium caninum molecular biology and genes have been done. Only partial sequences of mitochondrial DNA and ribosomal RNA gene are available in databases. Any molecular work with this parasite, including epidemiology, study of drug-resistant strains, and vaccine development, is hampered by the lack of knowledge of its genome. Thus, the knowledge of specific genes of different developmental stages of D. caninum is crucial to locate potential targets to be used as candidates to develop a vaccine and/or new drugs against this parasite. Here we report, for the first time, the sequencing of a fragment of a D. caninum expressed gene.

In a previous work Ancylostoma caninum sex-specific genes had being identified, however without confirmation, by using an non specific RT-PCR comparing male and female cDNA. In this work more fragments had been identified and a semi-quantitative RT-PCR carried out in order to confirm the sex-specific character of the transcripts obtained. Four fragments were confirmed as sex-specific or as being expressed more in one sex than the other.

While several mechanisms of immunoregulation have been demonstrated for hookworm and other neglected tropical infections, the influence of apoptosis in the immunomodulation of hookworm infection is still poorly understood. In this study, we demonstrate the cytotoxic and pro-apoptotic activity of hookworm antigens in Jurkat T cells, mesenteric lymph nodes lymphocytes of healthy and hookworm-infected hamsters and during human natural infection. Our results showed that in vitrostimulation of Jurkat T cells with antigens induces a significant decrease of cell viability leading to a relevant increase of apoptotic cells. Similar results were also observed in experimental conditions, for both healthy and hookworm-infected hamsters` lymphocytes. Flow cytometric analysis demonstrated that hookworm-infected patients presented a significant increase of CD4+, CD8+, and CD19+lymphocytes in early and/or late apoptosis when compared with non-infected individuals. The downmodulation of TNF receptors, as well as the up-regulation of the pro-apoptotic genes belonging to the BCL-2 and P53 families, suggest that hookworm antigens induced apoptosis by an intrinsic mitochondrial pathway, acting as a sophisticated strategy to safeguard parasite long-term survival in their hosts.

O Ancylostoma caninum (Ercolani, 1859) possui uma ampla distribuição geográfica, representando um risco para a saúde animal e humana. Além disso, representa um importante modelo de estudo para as demais espécies antropofílicas de ancilostomídeos. Presentemente, está sendo realizado um projeto para desenvolver uma vacina anti-ancilostomídeos o qual está sendo executado pelo Sabin Vaccine Institute em conjunto com a George Washington University / EUA e outras instituições. Considerando os grandes esforços e investimentos financeiros que estão sendo empregados na tentativa de se formular uma vacina contra ancilostomídeos, se faz necessário a implementação de projetos para estudar a variabilidade molecular e a diversidade e estrutura genética populacional destes patógenos. Este trabalho teve como objetivo investigar a estrutura genética e a variabilidade molecular de populações brasileiras de A. caninum, utilizando-se marcadores moleculares mitocondriais e nucleares, incluindo um gene codificante de uma proteína importante para estratégias imunoprofiláticas, a Ancylostoma secreted protein 2, Ac-ASP-2. Foram coletadas amostras de A. caninum em centros de controle de zoonoses de cinco localidades brasileiras: Belo Horizonte/MG (BH = 36 indivíduos), Campo Grande/MS (CG = 46 indivíduos), Curitiba/PR (CT = 35 indivíduos), Ribeirão Preto/SP (11 indivíduos) e São Luís/MA (49 indivíduos). Os vermes foram identificados morfologicamente e submetidos individualmente à extração de DNA. Posteriormente, avaliou-se a diversidade e estrutura genética de populações brasileiras de A. caninum utilizando o marcador mitocondrial citocromo c oxidase subunidade I (COI), marcadores nucleares conhecidos como espaços transcritos internos (ITS-1 e ITS-2) e microssatélites de DNA. Os níveis de diversidade molecular foram intermediários e constantes nas populações brasileiras de A. caninum. Os resultados revelaram ainda moderada diferenciação entre as populações avaliadas. Os dados obtidos com os marcadores ribossomais ITS-1 e ITS-2 se mostraram inadequados para o estudo de genética populacional em A. caninum devido ao baixo número de sítios polimórficos encontrados. Alguns fatores foram discutidos para explicar a estrutura genética observada nas populações brasileiras de A. caninum: (i) distância geográfica evitando o evento de panmixia entre as localidades avaliadas; (ii) fluxos gênicos parciais entre subpopulações amostradas, incluindo a influência de movimentos recentes de hospedeiros; (iii) a presença de subpopulações distintas geneticamente melhores adaptadas à diferentes hospedeiros e/ou a presença de espécies crípticas dentro de uma mesma localização geográfica; e (iv) outros eventos genéticos (por exemplo, deriva genética) ocorrendo independentemente em cada subpopulação. Além dos estudos de Biologia Populacional com marcadores neutros, um fragmento genômico do gene Ac-ASP-2 foi também seqüenciado utilizando-se os vermes das cinco localidades brasileiras amostradas. As análises reveleram um considerável polimorfismo nucleotídico. A maior parte da variabilidade foi encontrada nos íntrons devido a menor pressão seletiva destes segmentos. As regiões codificantes também revelaram polimorfismos nucleotídicos e aminoacídicos distribuídos de forma não homogênea entre os éxons avaliados. Estes resultados contribuem para uma melhor compreensão da ecologia, padrões de transmissão, desenvolvimento de resistência à drogas e desenvolvimento de vacinas contra ancilostomídeos.

The hookworm Ancylostoma canium represents a serious health problem, not only for animals but also for humans. These blood-feeding parasites produce various proteolytic enzymes in order to digest the host hemoglobin. The female worm ingests more blood than does the male. It is not known whether this difference is accompanied by expression of sex-specific proteinases. The identification of new genes related either to the developmental process of maturation of each sex or to the proteinases secreted by these worms could provide researchers with new tools to be used in control programs for this important parasite. The differential-display technique was used to compare the gene expression patterns of adult male and female worms in order to find specific genes that could be used as new targets in the control strategies for this parasite.

Hookworm infection is considered one of the most important poverty-promoting neglected tropical diseases, infecting 576 to 740 million people worldwide, especially in the tropics and subtropics. These blood-feeding nematodes have a remarkable ability to downmodulate the host immune response, protecting themselves from elimination and minimizing severe host pathology. While several mechanisms may be involved in the immunomodulation by parasitic infection, experimental evidences have pointed toward the possible involvement of regulatory T cells (Tregs) in downregulating effector T-cell responses upon chronic infection. However, the role of Tregs cells in human hookworm infection is still poorly understood and has not been addressed yet. In the current study we observed an augmentation of circulating CD4+CD25+FOXP3+ regulatory T cells in hookworm-infected individuals compared with healthy non-infected donors. We have also demonstrated that infected individuals present higher levels of circulating Treg cells expressing CTLA-4, GITR, IL-10, TGF-β and IL-17. Moreover, we showed that hookworm crude antigen stimulation reduces the number of CD4+CD25+FOXP3+ T regulatory cells co-expressing IL-17 in infected individuals. Finally, PBMCs from infected individuals pulsed with excreted/secreted products or hookworm crude antigens presented an impaired cellular proliferation, which was partially augmented by the depletion of Treg cells. Our results suggest that Treg cells may play an important role in hookworm-induced immunosuppression, contributing to the longevity of hookworm survival in infected people., Author Summary The hookworm infection is characterized by the long-term survival of the parasite and the concomitant modulation of the host immunity. Among several mechanisms that may account for the suppression of T cell response, we here described the presence and role of T regulatory cells (also known as Tregs) in the human hookworm infection. Tregs are a minor subpopulation of CD4+ T-cells, which also express specific cell markers that allow its further identification (CD25 and FOXP3). Our results showed that hookworm infection induce an augmentation of Tregs in the peripheral blood, followed by the higher levels of circulating Treg cells expressing several markers and cytokines associated with cell regulation (CTLA-4, GITR, IL-10, TGF-β and IL-17). We also demonstrated that in vitro depletion of Tregs partially enhanced the naturally impaired cellular proliferation of lymphocytes from infected individuals after antigenic stimulation. Our results suggest that Treg cells may play an important role in hookworm-induced immunosuppression, contributing to the longevity of hookworm survival in infected people.