Microscopic and molecular evaluation of Strongyloides venezuelensis in an experimental life cycle using Wistar rats. / Evaluación microscópica y molecular de Strongyloides venezuelensis en un ciclo de vida experimental utilizando ratas Wistar

INTRODUCTION: Strongyloides venezuelensis is a nematode whose natural host is rats. It is used as a model for the investigation of human strongyloidiasis caused by S. stercoralis. The latter is a neglected tropical disease in Ecuador where there are no specific plans to mitigate this parasitic illness. OBJECTIVE: To evaluate the stages of S. venezuelensis in an experimental life cycle using Wistar rats. MATERIALS AND METHODS: Male Wistar rats were used to replicate the natural biological cycle of S. venezuelensis and describe its morphometric characteristics, as well as its parasitic development. Furthermore, the production of eggs per gram of feces was quantified using two diagnostic techniques and assessment of parasite load: Kato-Katz and qPCR. RESULTS: Viable larval stages (L1, L2, L3) could be obtained up to 96 hours through fecal culture. Parthenogenetic females were established in the duodenum on the fifth day postinfection. Fertile eggs were observed in the intestinal tissue and fresh feces where the production peak occurred on the 8th. day post-infection. Unlike Kato-Katz, qPCR detected parasitic DNA on days not typically reported. CONCLUSIONS: The larval migration of S. venezuelensis within the murine host in an experimental environment was equivalent to that described in its natural biological cycle. The Kato-Katz quantitative technique showed to be quick and low-cost, but the qPCR had greater diagnostic precision. This experimental life cycle can be used as a tool for the study of strongyloidiasis or other similar nematodiasis.

Introducción. Strongyloides venezuelensis es un nematodo cuyo huésped natural son las ratas. Se utiliza como modelo para la investigación de la estrongiloidiasis humana producida por S. stercoralis. Esta última es una enfermedad tropical desatendida que afecta al Ecuador, donde no existen planes específicos para mitigar esta parasitosis. Objetivo. Evaluar experimentalmente los estadios del ciclo de vida de S. venezuelensis utilizando ratas Wistar. Materiales y métodos. Se emplearon ratas Wistar macho para replicar el ciclo biológico natural de S. venezuelensis y describir sus características morfométricas y su desarrollo parasitario. Además, se cuantificó la producción de huevos por gramo de heces mediante dos técnicas de diagnóstico y valoración de carga parasitaria: Kato-Katz y qPCR. Resultados. Se obtuvieron estadios larvarios viables (L1, L2, L3) hasta las 96 horas del cultivo fecal. En el duodeno se establecieron hembras partenogenéticas a partir del quinto día de la infección. Se observaron huevos fértiles en el tejido intestinal inspeccionado y en las heces frescas, en las que el pico de producción ocurrió al octavo día de la infección. A diferencia del método Kato-Katz, la qPCR detectó ADN parasitario en días que usualmente no se reportan. Conclusiones. La migración larvaria de S. venezuelensis dentro del ratón en un ambiente experimental fue equivalente al descrito en un ciclo biológico natural. El método cuantitativo de Kato-Katz dio resultados inmediatos a más bajo costo, pero la qPCR tuvo mayor precisión diagnóstica. Este ciclo de vida experimental puede usarse como una herramienta para el estudio de la estrongiloidiasis u otras nematodiasis similares.

scFv against HSP60 of Strongyloides sp. and Its Application in the Evaluation of Parasite Frequency in the Elderly.

The present study is aimed at evaluating serological method using scFv anti-Strongyloides sp. and reporting the frequencies of the results with conventional parasitological technique (faeces) in elderly individuals. Among 112 elderly individuals (≥60 years of age), 14.28% were positive for at least one enteroparasite, with one individual positive for S. stercoralis. Sera were evaluated for the presence of anti-Strongyloides sp. antibodies using total or detergent fraction extracts of Strongyloides venezuelensis, which presented positivity rates of 19.64% and 10.71%, respectively. An anti-HSP60 single-chain variable fragment from Strongyloides sp. was used to detect parasite antigens, with 5.36% (6 individuals) of ELISA-positive individuals returning a positive result. While the serological test indicates previous or recent infection and may be limited by antigen purification, the anti-HSP60 method reflects the presence of Strongyloides sp. immune complexes and exhibits greater sensitivity and specificity. Our results demonstrate the variable occurrence of enteroparasites in elderly individuals residing in long-term nursing homes and validate a novel epidemiological tool to describe infection cases by Strongyloides sp.

Gain and Loss of Small RNA Classes-Characterization of Small RNAs in the Parasitic Nematode Family Strongyloididae.

The nematode family Strongyloididae is of particular interest because it contains important parasites of medical and veterinary relevance. In addition, species of this family can form parasitic and free-living generations and it also occupies an interesting phylogenetic position within the nematodes. Nematodes differ in several ways from other taxa with respect to their small noncoding RNAs. Recent comparative studies revealed that there is also considerable variability within the nematodes. However, no Strongyloididae species or close relative was included in these studies. We characterized the small RNAs of two developmental stages of three different Strongyloididae species and compared them with the well-studied free-living nematodes Caenorhabditis elegans and Pristionchus pacificus. Strongyloididae have conserved and taxon-specific microRNAs, many of which are differentially regulated between the two developmental stages. We identified a novel class of around 27-nucleotide-long RNAs starting with 5'G or A, of which a large fraction have the potential to target transposable elements. These RNAs most likely have triphosphates at their 5' ends and are therefore presumably synthesized by RNA-dependent RNA polymerases. In contrast to C. elegans but similarly to some other nematode taxa, Strongyloididae have no Piwi-interacting RNAs, nor do their genomes encode Argonaute proteins of the Piwi family. Finally, we attempted but failed to detect circulating parasite small RNAs in the blood of hosts.

Presence of Parasites in Environmental Waters in Samsun and Its Districts.

OBJECTIVE: The aim of this study was to detect the presence of parasites in environmental waters in Samsun and its districts. METHODS: At the center of Samsun, 13 stations were determined. The research was performed between March 2012 and February 2013, and every month, water samples were collected on the dates stated. The samples were stained with Kinyoun acid-fast, modified trichrome, and trichrome dyes after examining with the direct bond. The preparations were evaluated in terms of parasitologic under a light microscope. RESULTS: Totally, 180 of 228 water samples analyzed were from streams; of these, 48 were drinking water samples. The following were found: 142 Giardia spp., 132 Cryptosporidium spp., 56 Cyclospora spp., 38 microsporidia, 47 Blastocystis spp., 38 Entamoeba coli cysts, 18 Dientamoeba, 9 Chilomastix, 9 Strongyloides spp., and 6 hookworms. CONCLUSION: The widespread use of animal husbandry and agriculture in the region and the use of stream surroundings as a grazing area increase the presence of some determined protozoa during a certain period. Parasitological studies in humans and animals in the region should be conducted, and control programs should be applied.

Strongyloides ratti and S. venezuelensis - rodent models of Strongyloides infection.

Strongyloides spp. are common parasites of vertebrates and two species, S. ratti and S. venezuelensis, parasitize rats; there are no known species that naturally infect mice. Strongyloides ratti and S. venezuelensis overlap in their geographical range and in these regions co-infections appear to be common. These species have been widely used as tractable laboratory systems in rats as well as mice. The core biology of these two species is similar, but there are clear differences in aspects of their within-host biology as well as in their free-living generation. Phylogenetic evidence suggests that S. ratti and S. venezuelensis are the result of two independent evolutionary transitions to parasitism of rats, which therefore presents an ideal opportunity to begin to investigate the basis of host specificity in Strongyloides spp.

Effect of different stages of Schistosoma mansoni infection on the parasite burden and immune response to Strongyloides venezuelensis in co-infected mice.

Multiple schistosome and soil-transmitted nematode infections are frequently reported in human populations living in tropical areas of developing countries. In addition to exposure factors, the host immune response plays an important role in helminth control and morbidity in hosts with multiple infections; however, these aspects are difficult to evaluate in human populations. In the current study, female Swiss mice were simultaneously co-infected with Strongyloides venezuelensis and Schistosoma mansoni or infected with St. venezuelensis at 2, 4, or 14 weeks after Sc. mansoni infection. The simultaneously infected mice showed a similar parasite burden for St. venezuelensis compared with mono-infected mice. In contrast, there was a significant reduction of St. venezuelensis burden (primarily during the migration of the larvae) in mice that were previously infected with Sc. mansoni at the acute or chronic phase. Independent of the stage of Sc. mansoni infection, the St. venezuelensis co-infection was capable of inducing IL-4 production in the small intestine, increasing the IgE concentration in the serum and increasing eosinophilia in the lungs and intestine. This result suggests that the nematode infection stimulates local type 2 immune responses independently of the schistosomiasis stage. Moreover, previous Sc. mansoni infection stimulated early granulocyte infiltration in the lungs and trematode-specific IgM and IgG1 production that recognized antigens from St. venezuelensis infective larvae; these immune responses would act in the early control of St. venezuelensis larvae. Our data suggest that the effect of multiple helminth infections on host susceptibility and morbidity largely depends on the species of parasite and the immune response.

Structural and functional characterization of a novel scFv anti-HSP60 of Strongyloides sp.

Phage display is a powerful technology that selects specific proteins or peptides to a target. We have used Phage Display to select scFv (single-chain variable fragment) clones from a combinatorial library against total proteins of Strongyloides venezuelensis. After scFv characterization, further analysis demonstrated that this recombinant fragment of antibody was able to bind to an S. venezuelensis antigenic fraction of ~65 kDa, present in the body periphery and digestive system of infective larvae (L3), as demonstrated by immunofluorescence. Mass spectrometry results followed by bioinformatics analysis showed that this antigenic fraction was a heat shock protein 60 (HSP60) of Strongyloides sp. The selected scFv was applied in serodiagnosis by immune complexes detection in serum samples from individuals with strongyloidiasis using a sandwich enzyme-linked immunosorbent assay (ELISA), showing sensitivity of 97.5% (86.84-99.94), specificity of 98.81 (93.54-99.97), positive likelihood ratio of 81.60 and an area under the curve of 0.9993 (0.9973-1.000). Our study provided a novel monoclonal scFv antibody fragment which specifically bound to HSP60 of Strongyloides sp. and was applied in the development of an innovative serodiagnosis method for the human strongyloidiasis.

Biodiversity threats from outside to inside: effects of alien grey squirrel (Sciurus carolinensis) on helminth community of native red squirrel (Sciurus vulgaris).

Biological invasions are among the major causes of biodiversity loss worldwide, and parasites carried or acquired by invaders may represent an added threat to native species. We compared gastrointestinal helminth communities of native Eurasian red squirrels (Sciurus vulgaris) in the presence and absence of introduced Eastern grey squirrels (Sciurus carolinensis) to detect alterations induced by the alien species. In particular, we investigated whether spillover of a North American nematode Strongyloides robustus occurs and whether prevalence of a local parasite Trypanoxyuris sciuri in red squirrels is affected by grey squirrel presence. The probability of being infected by both parasites was significantly higher in areas co-inhabited by the alien species, where 61 % of examined red squirrels (n = 49) were infected by S. robustus and 90 % by T. sciuri. Conversely, in red-only areas, the two parasites infected only 5 and 70 % of individuals (n = 60). Overall, our findings support the hypothesis that red squirrels acquire S. robustus via spillover from the alien congener and suggest that invaders' presence may also indirectly affect infection by local parasites through mechanisms diverse than spill-back and linked to the increased competitive pressure to which red squirrels are subjected. These results indicate that the impact of grey squirrel on red squirrels may have been underestimated and highlight the importance of investigating variation in macroparasite communities of native species threatened by alien competitors.

Comparative study of cultivation of feces in vermiculite or charcoal to obtain larvae of Strongyloides venezuelensis.

INTRODUCTION: We compared feces culturing in charcoal or vermiculite to obtain Strongyloides venezuelensis larvae. METHODS: Feces (5 g) from infected rats was mixed with vermiculite (10 g) or coal (10 g) in plastic cups and incubated at 28°C for 48 h. Larvae were recovered using Baermann-Moraes method. RESULTS: Significantly higher number of positive larval cultures were recovered from vermiculite than from charcoal (15/17 and 4/17, respectively; p < 0.001; 990.6 ± 307.5 and 215 ± 78.1 larvae, p = 0.027). CONCLUSIONS: Vermiculite yields more larvae and provides cleaner pellets, improving larvae identification and facilitating their use for other purposes.

Statistical experimental design to assess the influence of enzymes of nematophagous fungi versus helminths.

The present work used Plackett-Burman experimental design to assess the influence of enzymes of nematophagous fungi versus Strongyloides westeri and trichostrongylides larvae and Platynosomum fastosum eggs. The variables studied in the Plackett-Burman design were the proteases and chitinases of AC001 or VC4 as destructive agents of S. westeri and trichostrongylides larvae, and P. fastosum eggs. All tested enzymes had a significant effect (P < 0.05) on the destruction of S. westeri larvae. Furthermore, only VC4 and AC001 proteases showed a significant effect (P < 0.05) on the destruction of trichostrongylides larvae. On the other hand, chitinases of VC4 showed the highest significance (P < 0.05) on the destruction of P. fastosum eggs. It is proposed that statistical planning for the use of enzymes derived from nematophagous fungi is a viable way to elucidate some questions about their mechanism of action.

EFFECT OF Bifidobacterium animalis ON MICE INFECTED WITH Strongyloides venezuelensis / Efeito da administração de Bifidobacterium animalis sobre a infecção por Stongyloides venezuelensis em camundongos

The administration of viable Bifidobacterium animalis was tested to induce resistance against Strongyloides venezuelensis infection in mice. Effects on parasite burden, worm length, egg output, and intestinal mucosal histology were evaluated. The oral administration of B. animalis, strain 04450B, starting 14 days before the inoculation of nematode larvae significantly decreased the worm burden and egg output. In probiotic treated animals, the percent reduction of adult worms in the intestine was of 33% and the reduction of egg production was of 21%, compared with those of the control group. The duodenum villous height and villous/crypt ratio were significantly higher in probiotic-treated mice, indicating that this group could be experiencing less intestinal damage. The present findings revealed that the administration of B. animalis for the amelioration of host response to nematode infections is biologically plausible and could have some potential for impacting public health. Meanwhile, further study is needed to delineate the nature and identity of the factor(s) involved in these beneficial effects.

Os efeitos da administração de Bifidobacterium animalis viáveis sobre a infecção por Strongyloides venezuelensis foram avaliados em camundongos experimentalmente infectados. Os parâmetros analisados incluíram a carga parasitária, o comprimento dos vermes, a quantidade de ovos eliminados e a histologia da mucosa intestinal. A administração oral da cepa 04450B de B. animalis, iniciada 14 dias antes da inoculação de larvas do nematódeo, foi acompanhada de uma redução significativa do número de vermes que se estabeleceu no intestino e do número de ovos eliminados nas fezes. Nos animais tratados com o probiótico, o percentual de redução de vermes adultos no intestino foi de 33% e da produção de ovos foi de 21%, em comparação com os do grupo controle. O comprimento das vilosidades do duodeno e a relação vilus/cripta foram significativamente maiores nos animais tratados, indicando que nestes animais as lesões intestinais foram mais leves. Os resultados do presente trabalho revelaram que a administração de B. animalis com o propósito de modular a resposta do hospedeiro contra infecções por nematódeos é uma possibilidade biologicamente plausível com impacto potencial em saúde pública. No entanto, são ainda necessários mais estudos para esclarecer os mecanismos de ação destes microrganismos e identificar os fatores envolvidos na produção dos efeitos benéficos.

Effect of Bifidobacterium animalis on mice infected with Strongyloides venezuelensis.

The administration of viable Bifidobacterium animalis was tested to induce resistance against Strongyloides venezuelensis infection in mice. Effects on parasite burden, worm length, egg output, and intestinal mucosal histology were evaluated. The oral administration of B. animalis, strain 04450B, starting 14 days before the inoculation of nematode larvae significantly decreased the worm burden and egg output. In probiotic treated animals, the percent reduction of adult worms in the intestine was of 33% and the reduction of egg production was of 21%, compared with those of the control group. The duodenum villous height and villous/crypt ratio were significantly higher in probiotic-treated mice, indicating that this group could be experiencing less intestinal damage. The present findings revealed that the administration of B. animalis for the amelioration of host response to nematode infections is biologically plausible and could have some potential for impacting public health. Meanwhile, further study is needed to delineate the nature and identity of the factor(s) involved in these beneficial effects.

Strongyloides cebus (Nematoda: Strongyloididae) in Lagothrix cana (Primates: Atelidae) from the Brazilian Amazon: aspects of clinical presentation, anatomopathology, treatment, and parasitic biology.

Abstract : Seven cases of parasitism by Strongyloides cebus were identified in Lagothrix cana from Brazil. Aspects of the clinical presentation, treatment, pathology, and parasitic biology of these infections are described. Moderate to severe disease was observed, requiring hospitalization of 3 primates, and diarrhea was the most common clinical sign described. One L. cana individual died, for which ulcerative enteritis was the major finding upon histopathological analysis. The use of ivermectin in these atelids was safe and effective against the parasite. Parallel attempts to experimentally infect gerbils with the parasite failed. Lagothrix cana is presented as a new host for S. cebus. The evidence that Strongyloides infections are common in nonhuman primates under free-living conditions, and even more prevalent in captive animals, likely represents a neglected problem.

Dexamethasone reduces bronchial wall remodeling during pulmonary migration of Strongyloides venezuelensis larvae in rats.

Strongyloidiasis is an intestinal parasitosis with an obligatory pulmonary cycle. A Th2-type immune response is induced and amplifies the cellular response through the secretion of inflammatory mediators. Although this response has been described as being similar to asthma, airway remodeling during pulmonary migration of larvae has not yet been established. The aim of this study was to identify the occurrence of airway remodeling during Strongyloides venezuelensis (S. v.) infection and to determine the ability of dexamethasone treatment to interfere with the mechanisms involved in this process. Rats were inoculated with 9,000 S. v. larvae, treated with dexamethasone (2 mg/kg) and killed at 1, 3, 5, 7, 14 and 21 days. Morphological and morphometric analyzes with routine stains and immunohistochemistry were conducted, and some inflammatory mediators were evaluated using ELISA. Goblet cell hyperplasia and increased bronchiolar thickness, characterized by edema, neovascularization, inflammatory infiltrate, collagen deposition and enlargement of the smooth muscle cell layer were observed. VEGF, IL1-ß and IL-4 levels were elevated throughout the course of the infection. The morphological findings and the immunomodulatory response to the infection were drastically reduced in dexamethasone-treated rats. The pulmonary migration of S. venezuelensis larvae produced a transitory, but significant amount of airway remodeling with a slight residual bronchiolar fibrosis. The exact mechanisms involved in this process require further study.

Callithrix penicillata as a nonhuman primate model for strongyloidiasis.

In order to better understand experimental strongyloidiasis in small New World primates, and to evaluate aspects of reinfection and immunosuppression induced by glucocorticoids, nine specimens of Callithrix penicillata (Primates: Cebidae) were administered (by subcutaneous injection, sc) 3000 infective larvae of a strain of Strongyloides venezuelensis (Rhabditida: Strongyloididae) that had been maintained in successive passages through AKR/J mice since 1987. The mean prepatent period was 5.6 ± 0.7 days post-infection (DPI). The mean patent period of infection among the untreated animals (marmosets 1-7) was 123.4 ± 61.4 DPI. Two animals (marmosets 8 and 9) received dexamethasone (2.5 mg/kg, sc) for five consecutive days starting on the 20th day after infection, but this treatment did not alter the course of the infection, and the patent period for these animals was 100.5 ± 58.7 DPI (59 and 142, respectively). Stool examination showed that the highest quantities of parasite eggs were expelled between the 8th and 19th days after inoculation of the larvae. Thereafter, there was a gradual reduction in the number of parasite eggs in feces of all marmosets. During the chronic phase of the infection, before completely negative parasitological findings were obtained, the parasitological examinations were intermittently positive. Reinfection of three of these animals did not result in new positive examinations. However, given the receptiveness of these animals to initial infection with S. venezuelensis and their similarities to human beings, it is proposed that C. penicillata could be used as a nonhuman primate model for experimental strongyloidiasis.

Contribution of IL-33-activated type II innate lymphoid cells to pulmonary eosinophilia in intestinal nematode-infected mice.

When animals are infected with helminthic parasites, resistant hosts show type II helper T immune responses to expel worms. Recently, natural helper (NH) cells or nuocytes, newly identified type II innate lymphoid cells, are shown to express ST2 (IL-33 receptor) and produce IL-5 and IL-13 when stimulated with IL-33. Here we show the relevant roles of endogenous IL-33 for Strongyloides venezuelensis infection-induced lung eosinophilic inflammation by using Il33(-/-) mice. Alveolar epithelial type II cells (ATII) express IL-33 in their nucleus. Infection with S. venezuelensis or intranasal administration of chitin increases in the number of ATII cells and the level of IL-33. S. venezuelensis infection induces pulmonary accumulation of NH cells, which, after being stimulated with IL-33, proliferate and produce IL-5 and IL-13. Furthermore, S. venezuelensis infected Rag2(-/-) mice increase the number of ATII cells, NH cells, and eosinophils and the expression of IL-33 in their lungs. Finally, IL-33-stimulated NH cells induce lung eosinophilic inflammation and might aid to expel infected worms in the lungs.

Trichuris sp. and Strongyloides sp. infections in a free-ranging baboon colony.

We conducted cross-sectional surveys of parasites infecting a large free-living colony of baboons at the Southwest National Primate Research Center in San Antonio in October 2003 and April 2004, immediately before, and 6 mo after, treatment with ivermectin. Trichuris sp. was the predominant species present, infecting 79 and 69% of individual animals in the 2 surveys, with fecal egg counts (FEC) of up to 60,200 eggs per g (epg) (mean = 1,235 in October 2003 and 1,256 in April 2004). Prevalence remained fairly stable across age groups, and intensity was highest in animals <1 or >15 yr old, in contrast to patterns observed in humans, where school-age children show the heaviest infections. Strongyloides sp. was also identified, but the species identity remains uncertain. Small subunit ribosomal DNA sequences differed from published sequences of Strongyloides fuelleborni at multiple sites, but resided in a monophyletic group with other Strongyloides species with 92% bootstrap support. This may reflect a recent acquisition from a local host, or that the published sequence of S. fuelleborni is incorrect. Widespread infections with 2 nematode genera in a free-ranging baboon colony that are an important source of morbidity in human populations provide a useful model system for work on the epidemiology, control, pathology, and genetics of these parasites in a host species that is physiologically, immunologically, and genetically similar to humans.

Synergistic effects of seasonal rainfall, parasites and demography on fluctuations in springbok body condition.

1. Seasonality of rainfall can exert a strong influence on animal condition and on host-parasite interactions. The body condition of ruminants fluctuates seasonally in response to changes in energy requirements, foraging patterns and resource availability, and seasonal variation in parasite infections may further alter ruminant body condition. 2. This study disentangles the effects of rainfall and gastrointestinal parasite infections on springbok (Antidorcas marsupialis) body condition and determines how these factors vary among demographic groups. 3. Using data from four years and three study areas, we investigated (i) the influence of rainfall variation, demographic factors and parasite interactions on parasite prevalence or infection intensity, (ii) whether parasitism or rainfall is a more important predictor of springbok body condition and (iii) how parasitism and condition vary among study areas along a rainfall gradient. 4. We found that increased parasite intensity is associated with reduced body condition only for adult females. For all other demographic groups, body condition was significantly related to prior rainfall and not to parasitism. Rainfall lagged by two months had a positive effect on body condition. 5. Adult females showed evidence of a 'periparturient rise' in parasite intensity and had higher parasite intensity and lower body condition than adult males after parturition and during early lactation. After juveniles were weaned, adult females had lower parasite intensity than adult males. Sex differences in parasitism and condition may be due to differences between adult females and males in the seasonal timing of reproductive effort and its effects on host immunity, as well as documented sex differences in vulnerability to predation. 6. Our results highlight that parasites and the environment can synergistically affect host populations, but that these interactions might be masked by their interwoven relationships, their differential impacts on demographic groups, and the different time-scales at which they operate.