Anthelmintic Activity of Yeast Particle-Encapsulated Terpenes.

Soil-transmitted nematodes (STN) infect 1-2 billion of the poorest people worldwide. Only benzimidazoles are currently used in mass drug administration, with many instances of reduced activity. Terpenes are a class of compounds with anthelmintic activity. Thymol, a natural monoterpene phenol, was used to help eradicate hookworms in the U.S. South circa 1910. However, the use of terpenes as anthelmintics was discontinued because of adverse side effects associated with high doses and premature stomach absorption. Furthermore, the dose-response activity of specific terpenes against STNs has been understudied. Here we used hollow, porous yeast particles (YPs) to efficiently encapsulate (>95%) high levels of terpenes (52% w/w) and evaluated their anthelmintic activity on hookworms (Ancylostoma ceylanicum), a rodent parasite (Nippostrongylus brasiliensis), and whipworm (Trichuris muris). We identified YP-terpenes that were effective against all three parasites. Further, YP-terpenes overcame albendazole-resistant Caenorhabditis elegans. These results demonstrate that terpenes are broad-acting anthelmintics. Terpenes are predicted to be extremely difficult for parasites to resist, and YP encapsulation provides water-suspendable terpene materials without surfactants and sustained terpene release that could lead to the development of formulations for oral delivery that overcome fast absorption in the stomach, thus reducing dosage and toxic side effects.

Zoonotic Ancylostomiasis: An Update of a Continually Neglected Zoonosis.

Hookworm infections are classified as the most impactful of the human soil-transmitted helminth (STH) infections, causing a disease burden of ∼4 million disability-adjusted life years, with a global prevalence of 406-480 million infections. Until a decade ago, epidemiological surveys largely assumed Necator americanus and Ancylostoma duodenale as the relevant human hookworm species implicated as contributing to iron-deficiency anemia. This assumption was based on the indistinguishable morphology of the Ancylostoma spp. eggs in stool and the absence of awareness of a third zoonotic hookworm species, Ancylostoma ceylanicum. The expanded use of molecular diagnostic assays for differentiating hookworm species infections during STH surveys has now implicated A. ceylanicum, a predominant hookworm of dogs in Asia, as the second most common hookworm species infecting humans in Southeast Asia and the Pacific. Despite this, with the exception of sporadic case reports, there is a paucity of data available on the impact of this emerging zoonosis on human health at a population level. This situation also challenges the current paradigm, necessitating a One Health approach to hookworm control in populations in which this zoonosis is endemic. Here, we have summarized the available research studies and case reports on human A. ceylanicum infections in Southeast Asia and the Pacific after 2013 using a systematic review approach. We summarized eight research articles and five clinical case studies, highlighting the importance of future in-depth investigation of zoonotic A. ceylanicum infections using sensitive and cost-effective diagnostic tools.

Análise da ocorrência de parasitos com potencial zoonótico no solo de escolas municipais infantis em dois municípios do Rio Grande do Sul / Analysis of the occurrence of parasites with zoonotic potential in the soil of municipal nursery schools in two municipalities in Rio Grande do Sul state

Introdução: A contaminação de praças ou ambientes de recreação por ovos de geohelmintos constitui um problema de saúde pública. O solo, com relação aos helmintos parasitas se comporta como um hospedeiro intermediário. Recebe fezes ou água contaminada por parasitas em estágios não infectantes, oferecendo-lhes condições para o desenvolvimento e protege os parasitas em estágios infectantes durante certo tempo para, posteriormente, transmiti-lo ao homem. O objetivo é verificar a ocorrência e a contaminação ambiental por parasitas com potencial zoonótico no solo de escolas infantis em dois municípios do Rio Grande do Sul. Métodos: Trata-se de um estudo delineamento transversal, observacional e descritivo. A coleta foi através de amostras de areia em dez escolas de dois municípios, as coletas do material foram de cinco pontos diferentes, tanto da profundidade quanto da superfície, totalizando-se 100 amostras analisadas. As amostras de areia foram processadas em laboratório através dos métodos de Rugai e Hoffman, Pons e Janer. Resultados: Análise total (N=100) dos dois municípios, totalizando 50 amostras de cada. A contaminação das areias, por ovos de Ancylostoma spp., foi de 54% (n=27) no município I, sendo encontrados 56% (n=28) na cidade II. Conclusão: A ocorrência de ovos de Ancylostoma spp. no ambiente de recreação enfatiza a importância da adoção de medidas restritivas rígidas que impeçam a entrada de animais, como cães e gatos em locais de lazer. Além desses parasitas serem capazes de comprometer a saúde de humanos e infectar outros animais. (AU)

Introduction: Contamination of squares or recreational environments with geohelminth eggs is a public health problem. The soil serves as an intermediate host to helminths. It receives feces or water contaminated with parasites in non-infective stages, providing them with conditions for development, and protects parasites in infective stages for some time and then transmits them to humans. The aim was to investigate the occurrence of environmental contamination with parasites with zoonotic potential in the soil of nursery schools in two municipalities of Rio Grande do Sul state. Methods: A cross-sectional, observational and descriptive study was conducted. Soil samples were collected at ten schools in two municipalities, from five different school sites in terms of both depth and surface. Soil samples were processed at a laboratory using the methods of Rugai and Hoffman, Pons and Janer. Results: In total, 100 samples from the two municipalities were analyzed, 50 samples each. Soil contamination with Ancylostoma spp. eggs was 54% (n=27) in city I and 56% (n=28) in city II. Conclusions: The occurrence of Ancylostoma spp. eggs in recreational environments shows the importance of adopting strict restrictive measures to prevent animals, such as dogs and cats, from entering leisure areas. Also, these parasites may compromise human health and infect other animals. (AU)

The genome and transcriptome of the zoonotic hookworm Ancylostoma ceylanicum identify infection-specific gene families.

Hookworms infect over 400 million people, stunting and impoverishing them. Sequencing hookworm genomes and finding which genes they express during infection should help in devising new drugs or vaccines against hookworms. Unlike other hookworms, Ancylostoma ceylanicum infects both humans and other mammals, providing a laboratory model for hookworm disease. We determined an A. ceylanicum genome sequence of 313 Mb, with transcriptomic data throughout infection showing expression of 30,738 genes. Approximately 900 genes were upregulated during early infection in vivo, including ASPRs, a cryptic subfamily of activation-associated secreted proteins (ASPs). Genes downregulated during early infection included ion channels and G protein-coupled receptors; this downregulation was observed in both parasitic and free-living nematodes. Later, at the onset of heavy blood feeding, C-lectin genes were upregulated along with genes for secreted clade V proteins (SCVPs), encoding a previously undescribed protein family. These findings provide new drug and vaccine targets and should help elucidate hookworm pathogenesis.

Identification of Ancylostoma ceylanicum in children from a tribal community in Tamil Nadu, India using a semi-nested PCR-RFLP tool.

BACKGROUND: It is generally assumed that hookworm infections in humans are caused by Necator americanus and Ancylostoma duodenale. However, previous studies have also reported the presence of the animal hookworm A. ceylanicum in human stools. METHODS: We determined hookworm infections in children in a tribal community in Tamil Nadu, India, using a semi-nested PCR-RFLP approach. RESULTS: The results indicate that human species account for a majority of the hookworm infections (N. americanus 39/41 [95%]; A. duodenale 6/41 [15%]), whereas the animal hookworm A. ceylanicum only accounts for a minority of the infections (5%; 2/41). CONCLUSIONS: The results emphasize the need to consider zoonotic ancylostomiasis while developing strategies to control hookworm infections.

Evaluation of parasitological and immunological aspects of acute infection by Ancylostoma caninum and Ancylostoma braziliense in mixed-breed dogs.

This study compared the course of infection by Ancylostoma caninum and Ancylostoma braziliense in mixed-breed dogs infected with L3 larvae. Dogs infected with A. caninum eliminated more eggs than did those infected with A. braziliense. A total of 38 % of A. caninum and 44 % of A. braziliense larvae were recovered as adult worms. There were no marked clinical abnormalities in dogs with either infection. A. caninum was associated with anemia and an increased number of circulating neutrophils, whereas infection with A. braziliense led to a decrease in the number of leukocytes. The humoral response against excreted and secreted antigens from adult worms was more sensitive and specific than the response induced with the crude antigen. No immune response was observed for either crude or excreted-secreted (ES) antigens from larvae of either species. A nonspecific response against the crude antigen of A. braziliense was found at 0 and 7 days postinfection and maintained throughout the infection period. However, antibody titers against ES antigens were elevated in A. caninum infection at patency and death, showing that this antigen has a higher specificity. The immune response elicited by infection with A. braziliense in dogs has not been described previously. No significant differences were observed in the infection processes of the two Ancylostoma species, except for the higher number of eggs eliminated from dogs infected with A. caninum, which may indicate a better evolutionary adaptation of the parasite to its host in comparison with A. braziliense.

Rapid detection and identification of human hookworm infections through high resolution melting (HRM) analysis.

BACKGROUND: Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. METHODS: Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. CONCLUSION: The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species.

Cure of hookworm infection with a cysteine protease inhibitor.

BACKGROUND: Hookworm disease is a major global health problem and principal among a number of soil-transmitted helminthiases (STHs) for the chronic disability inflicted that impacts both personal and societal productivity. Mass drug administration most often employs single-dose therapy with just two drugs of the same chemical class to which resistance is a growing concern. New chemical entities with the appropriate single-dose efficacy are needed. METHODS AND FINDINGS: Using various life-cycle stages of the hookworm Ancylostoma ceylanicum in vitro and a hamster model of infection, we report the potent, dose-dependent cidal activities of the peptidyl cysteine protease inhibitors (CPIs) K11002 (4-mopholino-carbonyl-phenylalanyl-homophenylalanyl- vinyl sulfone phenyl) and K11777 (N-methylpiperazine-phenylalanyl-homophenylalanyl-vinylsulfone phenyl). The latter is in late pre-clinical testing for submission as an Investigational New Drug (IND) with the US Federal Drug Administration as an anti-chagasic. In vitro, K11002 killed hookworm eggs but was without activity against first-stage larvae. The reverse was true for K11777 with a larvicidal potency equal to that of the current anti-hookworm drug, albendazole (ABZ). Both CPIs produced morbidity in ex vivo adult hookworms with the activity of K11777 again being at least the equivalent of ABZ. Combinations of either CPI with ABZ enhanced morbidity compared to single compounds. Strikingly, oral treatment of infected hamsters with 100 mg/kg K11777 b.i.d. (i.e., a total daily dose of 200 mg/kg) for one day cured infection: a single 100 mg/kg treatment removed >90% of worms. Treatment also reversed the otherwise fatal decrease in blood hemoglobin levels and body weights of hosts. Consistent with its mechanism of action, K11777 decreased by >95% the resident CP activity in parasites harvested from hamsters 8 h post-treatment with a single 100 mg/kg oral dose. CONCLUSION: A new, oral single-dose anthelmintic that is active in an animal model of hookworm infection and that possesses a distinct mechanism of action from current anthelmintics is discovered. The data highlight both the possibility of repurposing the anti-chagasic K11777 as a treatment for hookworm infection and the opportunity to further develop CPIs as a novel anthelmintic class to target hookworms and, possibly, other helminths.

Molecular identification of human hookworm infections in economically disadvantaged communities in Peninsular Malaysia.

Species identification of human hookworm infections among eight communities in rural areas of Peninsular Malaysia was determined during 2009-2011. Fecal samples were examined by microscopy and subsequently, the internal transcribed spacer 2 and 28S ribosomal RNA region of Necator americanus and Ancylostoma spp. were sequenced. Overall, 9.1% (58 of 634) were identified positive by microscopy for hookworm infection, and 47 (81.0%) of 58 were successfully amplified and sequenced. Sequence comparison found that N. americanus (87.2%) was the most predominant hookworm identified, followed by Ancylostoma ceylanicum (23.4%). No A. duodenale infection was detected in this study. Detection of A. ceylanicum in humans highlighted the zoonotic transmission among humans living near dogs. Thus, implementation of effective control measures for hookworm infections in future should seriously consider this zoonotic implication.

Molecular characterization of Ancylostoma braziliense larvae in a patient with hookworm-related cutaneous larva migrans.

We report a case of hookworm-related cutaneous larva migrans diagnosed microscopically. Viable hookworm larvae were found by microscopic examination of a skin scraping from follicular lesions. Amplification and sequencing of the internal transcribed spacer 2 allowed the specific identification of the larvae as Ancylostoma braziliense.

Real-time PCR demonstrates Ancylostoma duodenale is a key factor in the etiology of severe anemia and iron deficiency in Malawian pre-school children.

BACKGROUND: Hookworm infections are an important cause of (severe) anemia and iron deficiency in children in the tropics. Type of hookworm species (Ancylostoma duodenale or Necator americanus) and infection load are considered associated with disease burden, although these parameters are rarely assessed due to limitations of currently used diagnostic methods. Using multiplex real-time PCR, we evaluated hookworm species-specific prevalence, infection load and their contribution towards severe anemia and iron deficiency in pre-school children in Malawi. METHODOLOGY AND FINDINGS: A. duodenale and N. americanus DNA loads were determined in 830 fecal samples of pre-school children participating in a case control study investigating severe anemia. Using multiplex real-time PCR, hookworm infections were found in 34.1% of the severely anemic cases and in 27.0% of the non-severely anemic controls (p<0.05) whereas a 5.6% hookworm prevalence was detected by microscopy. Prevalence of A. duodenale and N. americanus was 26.1% and 4.9% respectively. Moderate and high load A. duodenale infections were positively associated with severe anemia (adjusted odds ratio: 2.49 (95%CI 1.16-5.33) and 9.04 (95%CI 2.52-32.47) respectively). Iron deficiency (assessed through bone marrow examination) was positively associated with intensity of A. duodenale infection (adjusted odds ratio: 3.63 (95%CI 1.18-11.20); 16.98 (95%CI 3.88-74.35) and 44.91 (95%CI 5.23-385.77) for low, moderate and high load respectively). CONCLUSIONS/SIGNIFICANCE: This is the first report assessing the association of hookworm load and species differentiation with severe anemia and bone marrow iron deficiency. By revealing a much higher than expected prevalence of A. duodenale and its significant and load-dependent association with severe anemia and iron deficiency in pre-school children in Malawi, we demonstrated the need for quantitative and species-specific screening of hookworm infections. Multiplex real-time PCR is a powerful diagnostic tool for public health research to combat (severe) anemia and iron deficiency in children living in resource poor settings.

Hookworm SCP/TAPS protein structure--A key to understanding host-parasite interactions and developing new interventions.

SCP/TAPS proteins are a diverse family of molecules in eukaryotes, including parasites. Despite their abundant occurrence in parasite secretomes, very little is known about their functions in parasitic nematodes, including blood-feeding hookworms. Current information indicates that SCP/TAPS proteins (called Ancylostoma-secreted proteins, ASPs) of the canine hookworm, Ancylostoma caninum, represent at least three distinct groups of proteins. This information, combined with comparative modelling, indicates that all known ASPs have an equatorial groove that binds extended structures, such as peptides or glycans. To elucidate structure-function relationships, we explored the three-dimensional crystal structure of an ASP (called Ac-ASP-7), which is highly up-regulated in expression in the transition of A. caninum larvae from a free-living to a parasitic stage. The topology of the N-terminal domain is consistent with pathogenesis-related proteins, and the C-terminal extension that resembles the fold of the Hinge domain. By anomalous diffraction, we identified a new metal binding site in the C-terminal extension of the protein. Ac-ASP-7 is in a monomer-dimer equilibrium, and crystal-packing analysis identified a dimeric structure which might resemble the homo-dimer in solution. The dimer interaction interface includes a novel binding site for divalent metal ions, and is proposed to serve as a binding site for proteins involved in the parasite-host interplay at the molecular level. Understanding this interplay and the integration of structural and functional data could lead to the design of new approaches for the control of parasitic diseases, with biotechnological outcomes.

Efficacy of emodepside plus toltrazuril (Procox(®) oral suspension for dogs) against Toxocara canis, Uncinaria stenocephala and Ancylostoma caninum in dogs.

The efficacy of emodepside plus toltrazuril (Procox® oral suspension for dogs) against different species of gastrointestinal nematodes (Toxocara canis, Ancylostoma caninum, Uncinaria stenocephala) was evaluated in nine randomised,blinded and placebo-controlled laboratory studies in naturally or experimentally infected dogs. The product was used at the proposed minimum dose of 0.45 mg emodepside and 9 mg toltrazuril per kg body weight. Efficacy was calculated based on worm counts after necropsy. Worm burdens in the control dogs ranged between 0 and 409 worms of the respective stage for T. canis and between 4 and 655 worms for hookworms. The studies demonstrated 100 % efficacy of emodepside/toltrazuril suspension against mature adult, ≥ 94.7 %efficacy against immature adult and 99.3 % efficacy against the L4 larval stage of T. canis. The efficacy against mature adult A. caninum was ≥ 99.5 % and the efficacy against mature adult U. stenocephala was 100 %. All differences between treatment and control groups were statistically significant and no gender effect was found. It can be concluded that the emodepside/toltrazuril suspension represents a safe and highly effective product in dogs with nematode (T. canis, hookworms) infection.

Larval migration in PERL chambers as an in vitro model for percutaneous infection stimulates feeding in the canine hookworm Ancylostoma caninum.

BACKGROUND: Ancylostoma caninum third-stage larvae are the non-feeding infective stage of this parasite and are able to infect potential hosts via different infection routes. Since percutaneous infection is one of the most important routes and skin penetration is the first step into parasitic life, an existing in vitro model for percutaneous migration was modified and evaluated. The main parameter used to evaluate migration was the migration ratio (migrated larvae as a percentage of total number of larvae recovered). Additionally, the skin lag was calculated, expressing the percentage of larvae remaining in the skin and therefore not being recovered. Since initiation of feeding is proposed to be an important step in the transition from free-living to parasitic A. caninum larvae, feeding assays were performed with in vitro percutaneously migrated larvae. Additionally, infective larvae of A. caninum were activated via serum-stimulation and feeding behaviour was analysed and compared between percutaneously migrated and serum-stimulated larvae. RESULTS: Maximum skin migration levels of infective larvae were observed at temperatures above 32°C when larvae were placed on the epidermal side of skin for more than 12 hours. The medium beneath the skin had no effect on migration ratio, and no significant difference between the migration ratios through fresh and frozen/thawed skin was observed. Maximum feeding levels of 93.2% were observed for percutaneously migrated larvae after 48 h incubation, whereas serum-stimulated larvae reached the maximum of 91.0% feeding larvae after 24 h. CONCLUSIONS: The PERL chamber system was optimised and standardised as an in vitro model for percutaneous migration. The larvae recovered after percutaneous migration showed characteristic signs of activation similar to that of serum-stimulated larvae. The observed difference in time course of resumption of feeding indicates that percutaneously migrated larvae are not identical to serum-stimulated larvae, which are currently representing the model for early parasitic stages.

Angiogenesis and parasitic helminth-associated neovascularization.

Successful metazoan parasitism, among many other factors, requires a supply of nutrients and the removal of waste products. There is a prerequisite for a parasite-defined vasculature. The angiogenic mechanism(s) involved presumably depend on the characteristics of the tissue- and vascular system-dwelling, parasitic helminths. Simplistically, 2 possibilities or a combination of both have been considered in this review. The multifactorial induction of parasitic helminth-associated neovascularization could arise through, either a host-, a parasite- or a host-/parasite-dependent, angiogenic switch. Most studies appear to support the first and third hypotheses, but evidence exists for the intrahepatic cestode Echinococcus multilocularis, the free-living nematode Caenorhabditis elegans and the intravascular trematode Schistosoma mansoni for the second inference. In contrast, the nematode anti-coagulant protein NAPc2 from adult Ancylostoma caninum is also an anti-angiogenic factor.

The mucosal response of hamsters to a low-intensity superimposed secondary infection with the hookworm Ancylostoma ceylanicum.

An experiment was conducted to assess the mucosal response to low-dose superimposed challenge with Ancylostoma ceylanicum. Hamsters were assigned to five treatment groups (1-5 respectively): naïve controls; primary immunizing infection controls; challenge controls; immunized, anthelmintic-treated, challenged group; immunized, superimposed challenge group. Group 4 hamsters were resistant to challenge, whereas most of the challenge inoculum larvae established in Group 5. Villus height and crypt depth measurements were initially markedly divergent between these two groups but over time post-challenge (pc) values for both parameters drew nearer and by day 31 pc they were indistinguishable. The greatest change was experienced by Group 4 which showed increasing inflammation and gut pathology during the challenge infection. Mitotic activity in crypts and mast cell counts in the mucosa were highest in Group 5 on day 10 pc, but there was little to distinguish between Groups 4 and 5 by day 31 pc. Goblet cell, eosinophil and Paneth cell counts were very similar throughout in both groups but, in the case of Paneth cells, they were consistent with a possible role in protective immunity to challenge. Some adult worms survived throughout the period of intense inflammation, emphasizing their tremendous resilience and resistance to mucosal host protective responses.

Intestinal helminths induce haematological changes in dogs from Jabalpur, India.

The effect of canine intestinal helminths on the haematological profile of 200 dogs, of both sexes and variable age, visiting university veterinary clinics for routine examination was investigated. The dogs were assigned to parasitized (n = 39) and non-parasitized (n = 161) groups of animals. Coprological examination revealed a 19.5% prevalence of different species of the helminths. Of these animals, 10.25% had mixed infections with Ancylostoma caninum, Toxascaris spp. and Dipylidium caninum. The intensity of A. caninum infection was the highest, with mean egg counts of 951.43 (standard error 88.66), followed by Toxascaris 283.33 (standard error 116.81) and D. caninum. The parasitized animals had significantly lower levels of haemoglobin, packed cell volume and total erythrocyte counts than non-parasitized animals (P < 0.01). Values of other parameters, except for lymphocytes and eosinophils, were not different between the two groups. Analyses of the haematological profile revealed normocytic hypochromic anaemia in the parasitized group of animals.