Efficacy of Simparica Trio™, a novel chewable tablet containing sarolaner, moxidectin and pyrantel, against induced hookworm infections in dogs.

BACKGROUND: Ancylostomatids ('hookworms') are among the most important zoonotic nematode parasites infecting dogs worldwide. Ancylostoma caninum and Uncinaria stenocephala are two of the most common hookworm species that infect dogs. Both immature and adult stages of hookworms are voracious blood feeders and can cause death in young dogs before infection can be detected by routine fecal examination. Hence, treatment of both immature and adult stages of hookworms will decrease the risk of important clinical disease in the dog as well as the environmental contamination caused by egg-laying adults, which should reduce the risk of infection for both dogs and humans. The studies presented here were conducted to evaluate the efficacy of a novel, oral chewable tablet containing sarolaner, moxidectin and pyrantel (Simparica Trio™), against induced larval (L4), immature adult (L5) and adult A. caninum, and adult U. stenocephala infections in dogs. METHODS: Eight negative-controlled, masked, randomized laboratory studies were conducted. Two separate studies were conducted against each of the target parasites and stages. Sixteen or 18 purpose bred dogs, 8 or 9 in each of the two treatment groups, were included in each study. Dogs experimentally infected with the target parasite were dosed once on Day 0 with either placebo tablets or Simparica Trio™ tablets to provide minimum dosages of 1.2 mg/kg sarolaner, 24 µg/kg moxidectin and 5.0 mg/kg pyrantel (as pamoate salt). Timing of dosing relative to parasite inoculation allowed for efficacy to be evaluated primarily against the target parasite stage. Worm counts were conducted 7 or 8 days after treatments during necropsy. Efficacy was based on the number of worms recovered at necropsy compared to placebo control. RESULTS: Based on geometric mean worm counts, efficacy of Simparica Trio™ was ≥ 98.4% against L4 larval stage of A. caninum, ≥ 99.8% against immature adult (L5) A. caninum, and 100% against adult A. caninum and adult U. stenocephala. CONCLUSIONS: These studies confirm the efficacy of a single oral dose of a novel, chewable tablet containing sarolaner, moxidectin and pyrantel (Simparica Trio™) against L4 larval and immature adult (L5) A. caninum, and adult A. caninum and U. stenocephala infections in dogs.

Morphometrical and molecular evidence suggests cryptic diversity among hookworms (Nematoda: Uncinaria) that parasitize pinnipeds from the south-eastern Pacific coasts.

Hookworms of the genus Uncinaria parasitize pinniped pups in various locations worldwide. Four species have been described, two of which parasitize pinniped pups in the southern hemisphere: Uncinaria hamiltoni parasitizes Otaria flavescens and Arctocephalus australis from the South American coast, and Uncinaria sanguinis parasitizes Neophoca cinerea from the Australian coast. However, their geographical ranges and host specificity are unknown. Uncinaria spp. are morphologically similar, but molecular analyses have allowed the recognition of new species in the genus Uncinaria. We used nuclear genetic markers (internal transcribed spacer (ITS) and large subunit (LSU) rDNA) and a mitochondrial genetic marker (cytochrome c oxidase subunit I (COI)) to evaluate the phylogenetic relationships of Uncinaria spp. parasitizing A. australis and O. flavescens from South American coasts (Atlantic and Pacific coasts). We compared our sequences with published Uncinaria sequences. A Generalized Mixed Yule Coalescent (GMYC) analysis was also used to delimit species, and principal component analysis was used to compare morphometry among Uncinaria specimens. Parasites were sampled from A. australis from Peru (12°S), southern Chile (42°S), and the Uruguayan coast, and from O. flavescens from northern Chile (24°S) and the Uruguayan coast. Morphometric differences were observed between Uncinaria specimens from both South American coasts and between Uncinaria specimens from A. australis in Peru and southern Chile. Phylogenetic and GMYC analyses suggest that south-eastern Pacific otariid species harbour U. hamiltoni and an undescribed putative species of Uncinaria. However, more samples from A. australis and O. flavescens are necessary to understand the phylogenetic patterns of Uncinaria spp. across the South Pacific.

Hookworm exposure decreases human papillomavirus uptake and cervical cancer cell migration through systemic regulation of epithelial-mesenchymal transition marker expression.

Persistent infection with human papillomavirus (HPV) is responsible for nearly all new cervical cancer cases worldwide. In low- and middle-income countries (LMIC), infection with helminths has been linked to increased HPV prevalence. As the incidence of cervical cancer rises in helminth endemic regions, it is critical to understand the interaction between exposure to helminths and the progression of cervical cancer. Here we make use of several cervical cancer cell lines to demonstrate that exposure to antigens from the hookworm N. brasiliensis significantly reduces cervical cancer cell migration and global expression of vimentin and N-cadherin. Importantly, N. brasiliensis antigen significantly reduced expression of cell-surface vimentin, while decreasing HPV type 16 (HPV16) pseudovirion internalization. In vivo infection with N. brasiliensis significantly reduced vimentin expression within the female genital tract, confirming the relevance of these in vitro findings. Together, these findings demonstrate that infection with the hookworm-like parasite N. brasiliensis can systemically alter genital tract mesenchymal markers in a way that may impair cervical cancer cell progression. These findings reveal a possible late-stage treatment for reducing cervical cancer progression using helminth antigens.

Complex interactions in soil-transmitted helminth co-infections from a cross-sectional study in Sri Lanka.

Background: Co-infection with multiple soil-transmitted helminth (STH) species is common in communities with a high STH prevalence. The life histories of STH species share important characteristics, particularly in the gut, and there is the potential for interaction, but evidence on whether interactions may be facilitating or antagonistic are limited. Methods: Data from a pretreatment cross-sectional survey of STH egg deposition in a tea plantation community in Sri Lanka were analysed to evaluate patterns of co-infection and changes in egg deposition. Results: There were positive associations between Trichuris trichiura (whipworm) and both Necator americanus (hookworm) and Ascaris lumbricoides (roundworm), but N. americanus and Ascaris were not associated. N. americanus and Ascaris infections had lower egg depositions when they were in single infections than when they were co-infecting. There was no clear evidence of a similar effect of co-infection in Trichuris egg deposition. Conclusions: Associations in prevalence and egg deposition in STH species may vary, possibly indicating that effects of co-infection are species dependent. We suggest that between-species interactions that differ by species could explain these results, but further research in different populations is needed to support this theory.

Soil Contamination Rate, Prevalence, Intensity of Infection of Geohelminths and Associated Risk Factors among Residents in Bazou (West Cameroon).

BACKGROUND: Soil-transmitted helminths (STH) infections remain an important public health problem in the tropics. This study aimed to evaluate the soil contamination rate and prevalence of infestations of geohelminth among residents in Bazou. METHODS: Four hundred (400) soil samples and 182 stool specimens were collected respectively from four sites and from inhabitants aged 1 to 40 years and above in 12 households. A questionnaire was administered to evaluate risk factors of STH-infections. Soil samples were screened using sucrose centrifugal flotation method while stool specimens were analyzed using two techniques. RESULTS: Out of the 400 soil and 182 stool samples examined, 13(3.3%) and 9(4.95%) were positive respectively after analyses. Soil contamination rates were 2%, 1% and 0.3% for Ascaris, Trichuris and hookworm eggs respectively while 4.95% of stool samples were positive to one species nematode (Ascaris lumbricoides). The soil contamination rate observed (5%) in the rainy season was significantly different (p>0.05) from the one observed (1.5%) in the dry season. Irrespectively of the season, the most frequent parasite identified was Ascaris spp. Soils around houses were more contaminated 6(12%) as compared to those collected from markets 5(10%) and roads 2(4%) while those around classrooms were not contaminated. Participants in the age group of 1-10 were more infected. Factors such as no hand washing habit and untrimmed finger nails were found to be important risk factors for STH. CONCLUSION: These results suggest the necessity to promote measures that could help to reduce STH infections.

A novel blood-feeding detoxification pathway in Nippostrongylus brasiliensis L3 reveals a potential checkpoint for arresting hookworm development.

As part of on-going efforts to control hookworm infection, the "human hookworm vaccine initiative" has recognised blood feeding as a feasible therapeutic target for inducing immunity against hookworm infection. To this end, molecular approaches have been used to identify candidate targets, such as Necator americanus (Na) haemoglobinase aspartic protease-1 (APR-1), with immunogenicity profiled in canine and hamster models. We sought to accelerate the immune analysis of these identified therapeutic targets by developing an appropriate mouse model. Here we demonstrate that Nippostrongylus brasiliensis (Nb), a phylogenetically distant strongylid nematode of rodents, begins blood feeding early in its development and that immunisation with Na-APR-1 can block its growth and completion of its life cycle. Furthermore, we identify a new haem detoxification pathway in Nb required for blood feeding that can be blocked by drugs of the quinolone family, reducing both infection burden and the associated anaemia in rodents. Collectively, our findings show that haem metabolism has potential as a checkpoint for interrupting hookworm development in early stages of the hookworm life cycle and that the Nippostrongylus brasiliensis rodent model is relevant for identifying novel therapeutic targets against human hookworm.

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.

Restoration of hookworm egg development after prolonged storage in stool suspension.

Hookworm infection is still prevalent in southern Thailand despite control measures. Hookworm eggs submerged for an extended period under water from rainfall or in latrines may not survive, but they may recover their ability to develop into infective larvae when exposed to atmospheric air. This study examined the survival of the hookworm eggs in stool suspension and the restoration of development capability after prolonged storage. In stool mass, eggs developed normally and yielded infective filariform larvae (FL) in 7 days. On the contrary, in 1:10 stool suspension, hookworm eggs were found to remain at the 4-8 cell stage; degenerated eggs were observed after 15 days of storage, and the number of degenerated eggs reached 80 % on day 30. Aeration of the suspension, or transferring to a Petri dish or agar plate, restored the capacity of eggs stored for up to 15 days to develop into FL; thereafter, the capacity declined sharply. Retardation of egg development under water or in stool suspension may be due to a lack of atmospheric air. Use of "night soil" from latrines as fertilizer may be one factor in maintaining hookworm transmission, as worm eggs can undergo normal development upon exposure to atmospheric air.

Secreted proteomes of different developmental stages of the gastrointestinal nematode Nippostrongylus brasiliensis.

Hookworms infect more than 700 million people worldwide and cause more morbidity than most other human parasitic infections. Nippostrongylus brasiliensis (the rat hookworm) has been used as an experimental model for human hookworm because of its similar life cycle and ease of maintenance in laboratory rodents. Adult N. brasiliensis, like the human hookworm, lives in the intestine of the host and releases excretory/secretory products (ESP), which represent the major host-parasite interface. We performed a comparative proteomic analysis of infective larval (L3) and adult worm stages of N. brasiliensis to gain insights into the molecular bases of host-parasite relationships and determine whether N. brasiliensis could indeed serve as an appropriate model for studying human hookworm infections. Proteomic data were matched to a transcriptomic database assembled from 245,874,892 Illumina reads from different developmental stages (eggs, L3, L4, and adult) of N. brasiliensis yielding∼18,426 unigenes with 39,063 possible isoform transcripts. From this analysis, 313 proteins were identified from ESPs by LC-MS/MS-52 in the L3 and 261 in the adult worm. Most of the proteins identified in the study were stage-specific (only 13 proteins were shared by both stages); in particular, two families of proteins-astacin metalloproteases and CAP-domain containing SCP/TAPS-were highly represented in both L3 and adult ESP. These protein families are present in most nematode groups, and where studied, appear to play roles in larval migration and evasion of the host's immune response. Phylogenetic analyses of defined protein families and global gene similarity analyses showed that N. brasiliensis has a greater degree of conservation with human hookworm than other model nematodes examined. These findings validate the use of N. brasiliensis as a suitable parasite for the study of human hookworm infections in a tractable animal model.

Chronic hookworm-related cutaneous larva migrans.

OBJECTIVES: The purpose of this study is to present the 'chronic' or 'persistent' form of hookworm-related cutaneous larva migrans. METHODS: From 1998 to 2011, 13 patients were seen in our department with clinically typical hookworm-related cutaneous larva migrans that had been present for more than 5 months and that, because of the absence of pruritus, had never been treated. RESULTS: The duration of hookworm-related cutaneous larva migrans ranged from 5 to 14 months (mean 7.8 months) in these 13 patients (10 males and three females, aged 23-55 years). The infestation was acquired in Brazil (three patients), Jamaica (three patients), Mexico (two patients), Tanzania (two patients), Thailand (two patients), and Martinique (one patient). The infestation was located on the feet in 10 patients; one of these patients also presented tracks on the back and another presented tracks on a knee. The chest (two patients) and thigh (two patients) were also involved. All patients presented with clinically typical hookworm-related cutaneous larva migrans: seven patients had one track and six patients had two tracks. Laboratory and instrumental examinations were within the normal range or negative. Histopathological examination revealed edema in the papillary and upper dermis, and a perivascular and perifollicular infiltrate in the upper dermis, consisting mainly of lymphocytes and eosinophils. No larvae were detected. CONCLUSIONS: This can be considered the 'chronic' or 'persistent' form of hookworm-related cutaneous larva migrans. Some pathogenetic hypotheses are suggested.

Transgenic C. elegans dauer larvae expressing hookworm phospho null DAF-16/FoxO exit dauer.

Parasitic hookworms and the free-living model nematode Caenorhabtidis elegans share a developmental arrested stage, called the dauer stage in C. elegans and the infective third-stage larva (L3) in hookworms. One of the key transcription factors that regulate entrance to and exit from developmental arrest is the forkhead transcription factor DAF-16/FoxO. During the dauer stage, DAF-16 is activated and localized in the nucleus. DAF-16 is negatively regulated by phosphorylation by the upstream kinase AKT, which causes DAF-16 to localize out of the nucleus and the worm to exit from dauer. DAF-16 is conserved in hookworms, and hypothesized to control recovery from L3 arrest during infection. Lacking reverse genetic techniques for use in hookworms, we used C. elegans complementation assays to investigate the function of Ancylostoma caninum DAF-16 during entrance and exit from L3 developmental arrest. We performed dauer switching assays and observed the restoration of the dauer phenotype when Ac-DAF-16 was expressed in temperature-sensitive dauer defective C. elegans daf-2(e1370);daf-16(mu86) mutants. AKT phosphorylation site mutants of Ac-DAF-16 were also able to restore the dauer phenotype, but surprisingly allowed dauer exit when temperatures were lowered. We used fluorescence microscopy to localize DAF-16 during dauer and exit from dauer in C. elegans DAF-16 mutant worms expressing Ac-DAF-16, and found that Ac-DAF-16 exited the nucleus during dauer exit. Surprisingly, Ac-DAF-16 with mutated AKT phosphorylation sites also exited the nucleus during dauer exit. Our results suggest that another mechanism may be involved in the regulation DAF-16 nuclear localization during recovery from developmental arrest.

Soil transmitted helminths and schistosoma mansoni infections among school children in Zarima town, northwest Ethiopia.

BACKGROUND: In Ethiopia, because of low quality drinking water supply and latrine coverage, helminths infections are the second most predominant causes of outpatient morbidity. Indeed, there is a scarcity of information on the prevalence of soil transmitted helminths and Schistosomiasis in Ethiopia, special in study area. Therefore, the aim of this study was to determine the prevalence and associated risk factors of soil transmitted helminths and intestinal Schistosomiasis. METHODS: Cross-sectional study was conducted among 319 school children of Zarima town from April 1 to May 25, 2009. A pre-tested structured questionnaire was used to collect socio-demographic data and possible risk factors exposure. Early morning stool samples were collected and a Kato Katz semi concentration technique was used to examine and count parasitic load by compound light microscope. Data entry and analysis was done using SPSS-15 version and p-value < 0.05 considered statistically significant. RESULTS: Out of 319 study subjects, 263 (82.4%) of the study participants infected with one or more parasites. From soil transmitted helminths, Ascaris lumbricoides was the predominant isolate (22%) followed by Hookworms (19%) and Trichuris trichiura (2.5%). Schistosoma mansoni was also isolated in 37.9% of the study participants. Hookworm and S. mansoni infections showed statistically significant associations with shoe wearing and swimming habit of school children, respectively. CONCLUSION: Prevalence of soil transmitted helminths (STH) and S.mansoni was high and the diseases were still major health problem in the study area which alerts public health intervention as soon as possible.

New host and locality records of snake intestinal nematode Kalicephalus spp in Indonesia.

OBJECTIVE: To observe three species of Kalicephalus found in three species of snake (Ophiophagus hannah, Ptyas mucosus, and Naja Sputatrix) during research on Capture Snake for Trading in Java and Snake Biodiversity in Kalimantan Islands. METHODS: Specimens for light microscopy examination were fixed with warm 70% alcohol, cleared and mounted in lactophenol for wet mounting. Drawings were made with the aid of a drawing tube attached to a Nikon compound microscope. Measurements were given in micrometers (µ) as the average of findings, followed by the range in parentheses, unless otherwise stated. RESULTS: Kalicephalus (Costatus) indicus was found from 7 Ptyas mucosus, Kalicephalus bungari from 2 Naja sputatrix and 1 Kalicephalus (Costatus) indicus and Kalicephalus assimilis found from 1 Ophiophagus hannah. The morphology and measurement of three species of Kalicephalus found in this study were close to those described before. CONCLUSIONS: New finding of host of Kalicephalus (Costatus) indicus and Kalicephalus bungari was a snake species of Naja sputatrix. New records of locality were Kalimantan island as the new locality of Kalicephalus assimilis, and Java island was new locality of Kalicephalus (Costatus) indicus.

Hookworm (Necator americanus) larval enzymes disrupt human vascular endothelium.

Knowledge of the molecular mechanisms used by Necator americanus larvae to penetrate the human skin and the vasculature would aid the development of effective vaccines against this important pathogen. In this work, the impact of N. americanus exsheathing fluid (EF) and excretory/secretory products (ES) on the endothelial barrier was examined using human umbilical vein endothelial cells (HUVEC). Cellular responses were assessed by investigating molecular changes at cell-cell junctions and by determining levels of secreted IL-6, IL-8, and vascular endothelial growth factor (VEGF) in the culture medium. It would appear that a repertoire of larval proteases caused a dose-related increase in endothelial permeability as characterized by a decrease in monolayer resistance with increased permeation of tracer-albumin. These barrier changes were associated with disruption of junctional vascular endothelial cadherin (VE-cadherin) and F-actin and an increase in endothelial secretion of IL-6 and IL-8. Our data suggest that larval proteases play an important role in negotiating the endothelium.

The role of rabbits in the biology of some gastrointestinal parasites infecting sheep.

Twelve rabbits (Buscat strain) were classified into 4 Gs (3 rabbits each). The first three groups were experimentally infected with the infective 3rd stage larvae of Bunostomum phlebotomum, Trichostrangylus colubriformis and Cooperia onchophora, respectively. The 4th G was used as a control. The faecal examination until slaughtering time (21 days post infection) of all Gs did not show any nematode eggs. The microscopic examination of the gastrointestinal canal contents and mucosal scrapings of all Gs at slaughtering time did not show any gastrointestinal parasites. The 4th stage larvae (L4) of B. phlebotomum and T. colubriformis were obtained after digestion of epithelium of ileum and duodenum of G.1 & G.2 respectively. The histopathologic examination of gastrointestinal canal, ileum of G.1 showed necrosis of epithelium and eosinophilic infiltration associated with parasitic stages (L4) of B. phlebotomum. The intestine of G.2 showed parasitic stages (L4) loaded in the villi of duodenum associated with eosinophilic infiltration of T. colubriformis. The intestine of G.3 showed necrosis of the villi and eosinophilic infiltration due to C. onchophora. The histopathologic examination of the gastrointestinal canal of control group was normal.

Historic importance of some aspects of research by O. Wilford Olsen on hookworms (Uncinaria lucasi) in northern fur seals (Callorhinus ursinus) and Steller sea lions (Eumatopias jubatus) in 1951 on St. Paul Island, Alaska.

The purpose of this paper is to disseminate, more widely, certain historically important aspects of research by O. Wilford Olsen on hookworms (Uncinaria lucasi) in northern fur seals (Callorhinus ursinus) and Steller sea lions (Eumatopias jubatus) in 1951 on St. Paul Island, Alaska. Highly meaningful facets of the research were recorded only in a report with limited availability by Olsen in 1952. Portions of his research presented here include measurements of hookworm free-living infective third-stage larvae (L3) and adults, photographs of bursae, and the conclusion that the species of hookworms (U. lucasi) is probably the same in both pinniped hosts. This information is especially important because very little has been published on taxonomy of hookworms in E. jubatus.

Human hookworm infection in the 21st century.

The scientific study of human hookworm infection began at the dawn of the twentieth century. In recent years, there have been dramatic improvements in our understanding of many aspects of this globally widespread parasite. This chapter reviews recent advances in our understanding in the biology, immunology, epidemiology, public health significance and control of hookworm, and to look forward to the study of this important parasite in the 21st century. Advances in molecular biology has lead to the identification of a variety of new molecules from hookworms, which have importance either in the molecular pathogenesis of hookworm infection or in the host-parasite relationship; some are also promising vaccine targets. At present, relatively little is known about the immune responses to hookworm infection, although it has recently been speculated that hookworm and other helminths may modulate specific immune responses to other pathogens and vaccines. Our epidemiological understanding of hookworm has improved through the development of mathematical models of transmission dynamics, which coupled with decades of field research across multiple epidemiological settings, have shown that certain population characteristics can now be recognised as common to the epidemiology, population biology and control of hookworm and other helminth species. Recent recognition of the subtle, but significant, impact of hookworm on health and education, together with the simplicity, safety, low cost and efficacy of chemotherapy has spurred international efforts to control the morbidity due to infection. Large-scale treatment programmes are currently underway, ideally supported by health education and integrated with the provision of improved water and sanitation. There are also on-going efforts to develop novel anthelmintic drugs and anti-hookworm vaccines.

Uncinaria hamiltoni (Nematoda: Ancylostomatidae) in South American sea lions, Otaria flavescens, from northern Patagonia, Argentina.

Thirty-one South American sea lion pups (Otaria flavescens) found dead in Punta León, Argentina, during the summer of 2002, were examined for hookworms (Uncinaria hamiltoni). Parasite parameters were analyzed in 2 locations of the rookery, i.e., a traditional, well-structured breeding area and an expanding area with juveniles and a lax social structure. Prevalence of hookworms was 50% in both localities, and no difference was observed in prevalence between pup sexes (P > 0.05). Hookworms were concentrated in the small intestine. Transmammary transmission is assumed because only adult hookworms were found in the pups. The mean intensity of hookworms per pup was 135; the mean intensity in females (92.78) was significantly different (P < 0.05) from that of males (230.25). No difference (P > 0.05) in intensity was found between the 2 breeding areas, although prevalence was higher in the traditional breeding area than in the other area. Location was the only factor affecting hookworm prevalence (P log-linear model: 0.9552; chi2: 1.5629). No apparent trend between body condition and intensity of hookworms was observed.