The occurrence of macroparasites was studied from 543 stray cats in four urban cities from the west (Kuala Lumpur), east (Kuantan), north (Georgetown) and south (Malacca) of Peninsular Malaysia from May 2007 to August 2010. Five ectoparasites species were recovered namely, Ctenocephalides felis, Felicola subrostratus, Haemaphysalis bispinosa, Heterodoxus spiniger and Lynxacarus radovskyi. Two cats from Georgetown were infested with the dog louse, H. spiniger and this represented the first host record for this species in Malaysia. Up to nine species of helminths were recovered with overall high prevalences of infection of 83% in Kuantan, followed by 75.1% in Kuala Lumpur, 71.6% in Georgetown and 68% in Malacca. The helminth species comprised five nematodes, Toxocara malaysiensis, Toxocara cati, Ancylostoma braziliensis, Ancylostoma ceylanicum, Physaloptera praeputialis, two cestodes Taenia taeniaeformis, Dipylidium caninum and one trematode, Playtnosomum fastosum. The majority of helminths were present in the four study sites except for the absence of P. praeputialis in Kuala Lumpur. The prevalence and abundance of infections were analysed taking intrinsic (host age and sex) and extrinsic (season) factors into consideration. Levels of infection and infestation were mainly influenced by host age and to a lesser extent sex and season, whereas four nematode species exhibited significant interactions within the intestine of the cat host. The potential for transmission of some macroparasite species from stray cats to the human population in urban areas is discussed.

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles - only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM.

Many regulatory agencies require that the efficacy of veterinary anthelmintic medications be evaluated by enumerating parasites in treated and untreated animals after necropsy. Current ethical considerations, i.e., the 3 Rs of research, call for the replacement of this method with less invasive techniques that would not require animal sacrifice. This study tested standard gastrointestinal endoscopy as an in vivo method of quantifying the intestinal hookworm, Ancylostoma caninum. Worm counts were compared with those from gold standard necropsy. Thirteen dogs inoculated with third-stage A. caninum larvae underwent endoscopy 4-6 weeks post-infection, just prior to necropsy. Two-thirds of the adult hookworms were located in the middle section of the small intestine that could not be reached for endoscopic examination. Not surprisingly, the total worm counts obtained by endoscopy did not correlate with those from necropsy (R(2)=0.05, p=0.464). One method to increase small intestinal access would be to use specialized balloon or spiral endoscopes developed for this purpose in human gastroenterology. Based on the results of this study, standard endoscopy alone is unsuitable for quantification of A. caninum in the small intestine. Parasites in more accessible sites, such as whipworms in the cecum and colon, might be more appropriate targets for endoscopic counting.

A single-strand conformation polymorphism (SSCP) analysis and targeted sequencing approach was used for the genetic characterization of some major pathogens from a cohort of 227 people with histories of gastrointestinal disorders. Genomic DNAs from faecal samples were subjected to PCR-amplification of regions in the glycoprotein (gp60) or triose phosphate isomerase (tpi) gene, or the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2). Cryptosporidium, Giardia and strongylid nematodes were detected in 94, 132 and 12 samples. Cryptosporidium hominis subgenotypes IbA10G2, IdA15G1, IgA17, IgA18 and IfA13G1 were identified in 74.6, 16.9, 5.6, 1.4 and 1.4% of 71 samples, respectively. For C. parvum, subgenotypes IIaA17G2R1 (47.6%) and IIaA18G3R1 (23.8%) were identified in 23 samples. Giardia duodenalis assemblage B (78%) was more common than assemblage A (22%). In addition, DNA of the nematodes Ancylostoma ceylanicum (n = 2), Ancylostoma duodenale (4), Necator americanus (5) and Haemonchus contortus (1) was specifically detected. This is the first report of A. ceylanicum in two persons in Australia and, we provide molecular evidence of H. contortus in a child. This SSCP-based approach should provide a useful diagnostic and analytical tool for a wide range of pathogens.

Geophagic soils from selected communities in Eastern Cape, South Africa were characterised to determine their properties and geohelminth content. The soils were coarse-textured with cation exchange capacity values ranging from 6.35 to 18.94 cmol (+)/kg. Quartz was the dominant mineral in the samples with SiO2, Al2O3, and Fe2O3(t) having the highest concentrations among major element oxides. The soil properties, mineralogical composition, and low amounts of particle binding substances may favour the survival of geohelminth ova in the soils. Seven of the samples contained at least one of the following geohelminths: Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale and Strongyloides stercoralis. The presence of these geohelminths in the soils was attributed to agricultural and sanitary practices inherent in the communities and the soil properties. Communities need to be sensitised on the importance of safe sanitary and animal husbandry practices to reduce the prevalence of helminth infection among geophagists.

The prevalence of fleas and gastrointestinal parasites in free-roaming and domestic cats in central Mexico was evaluated. Three hundred and fifty eight cats captured in the street or brought in by owners to the Animal Control Center Unit, a unit of State Government, from June 2010 to May 2011, were included in the study. All cats were examined for the presence of fleas and gastrointestinal worms. One-hundred and ninety (53%) cats were infested with at least one flea species. Single infestations were observed in 106 (30%) cats and mixed infestations in 84 (23%) cats. Four species of fleas were recovered: Ctenocephalides felis in 53% of the cats, C. canis in 18%, Echidnophaga gallinacea in 7% and Pulex irritans in 1%. One-hundred and sixty three (45%) cats were infected with one or more species of gastrointestinal parasites: 48 (13%) with nematodes, 145 (40%) with cestodes, and one animal presented Moniliformis moniliformis. Prevalences and mean intensity of infection were: Physaloptera praeputialis 7 and 18; T. cati 3 and 2; Ancylostoma tubaeforme 2.5 and 2; Toxascaris leonina 0.5 and 2; Dipylidium caninum 36 and 32; Taenia taeniformis 4 and 3 and Moniliformis moniliformis 0.3 and 106, respectively. There was significant association (P<0.01), between season and ectoparasites load, more fleas were obtained in the summer and autumn than in the winter and spring; however, no statistical difference was observed for endoparasites load (P>0.05). The correlation between the total number of ectoparasites and endoparasites was not significant (r = 0.089, P = 0.094).

BACKGROUND:

The four common soil-transmitted helminth species-Ascaris lumbricoides, Trichuris trichiura, and the two hookworm species Ancylostoma duodenale and Necator americanus-are endemic in South America, but their distribution, infection prevalence, and regional burden are poorly understood. We aimed to estimate the risk and number of people infected with A lumbricoides, T trichiura, and hookworm across South America.

METHODS:

We did a systematic review of reports on the prevalence of soil-transmitted helminth infection in South America published up to May 14, 2012. We extracted and georeferenced relevant survey data and did a meta-analysis of the data to assess the geographical distribution of the infection risk with Bayesian geostatistical models. We used advanced Bayesian variable selection to identify environmental determinants that govern the distribution of soil-transmitted helminth infections.

FINDINGS:

We screened 4085 scientific papers and identified 174 articles containing relevant survey prevalence data. We georeferenced 6948 survey locations and entered the data into the open-access Global Neglected Tropical Diseases database. Survey data were sparse for the south of the continent and for the western coast, and we identified no relevant information for Uruguay and little data for smaller countries such as Suriname, Guyana, French Guiana, and Ecuador. Population-adjusted prevalence of infection with A lumbricoides was 15·6%, with T trichiura was 12·5%, and with hookworm was 11·9% from 2005 onwards. Risks of contracting soil-transmitted helminth infection have substantially reduced since 2005 (odds ratio 0·47 [95% Bayesian credible interval 0·46-0·47] for A lumbricoides, 0·54 [0·54-0·55] for T trichiura, and 0·58 [0·58-0·59] for hookworm infection).

INTERPRETATION:

Our findings offer important baseline support for spatial targeting of soil-transmitted helminthiasis control, and suggest that more information about the prevalence of soil-transmitted helminth infection is needed, especially in countries in which we estimate prevalence of infection to be high but for which current data are scarce. FUNDING: UBS Optimus Foundation and Brazilian Swiss Joint Research Programme (BSJRP 011008).

It is widely recognized that only a handful of drugs are available against soil-transmitted helminthiasis, all of which are characterized by a low efficacy against Trichuris trichiura, when administered as single doses. The re-evaluation of old, forgotten drugs is a promising strategy to identify alternative anthelminthic drug candidates or drug combinations.We studied the activity of the veterinary drug oxantel pamoate against Trichuris muris, Ancylostoma ceylanicum and Necator americanus in vitro and in vivo. In addition, the dose-effect of oxantel pamoate combined with albendazole, mebendazole, levamisole, pyrantel pamoate and ivermectin was studied against T. muris in vitro and additive or synergistic combinations were followed up in vivo.We calculated an ED50 of 4.7 mg/kg for oxantel pamoate against T. muris in mice. Combinations of oxantel pamoate with pyrantel pamoate behaved antagonistically in vitro (combination index (CI) = 2.53). Oxantel pamoate combined with levamisole, albendazole or ivermectin using ratios based on their ED50s revealed antagonistic effects in vivo (CI = 1.27, 1.90 and 1.27, respectively). A highly synergistic effect (CI = 0.15) was observed when oxantel pamoate-mebendazole was administered to T. muris-infected mice. Oxantel pamoate (10 mg/kg) lacked activity against Ancylostoma ceylanicum and Necator americanus in vivo.Our study confirms the excellent trichuricidal properties of oxantel pamoate. Since the drug lacks activity against hookworms it is necessary to combine oxantel pamoate with a partner drug with anti-hookworm properties. Synergistic effects were observed for oxantel pamoate-mebendazole, hence this combination should be studied in more detail. Since, of the standard drugs, albendazole has the highest efficacy against hookworms, additional investigations on the combination effect of oxantel pamoate-albendazole should be launched.

Evidence from human studies and mouse models shows that infection with parasitic helminths has a suppressive effect on the pathogenesis of some inflammatory diseases. Recently, we and others have shown that some of the suppressive effects of hookworms reside in their excretory/secretory (ES) products. Here, we demonstrate that ES products of the hookworm Ancylostoma caninum (AcES) suppress intestinal pathology in a model of chemically induced colitis. This suppression was associated with potent induction of a type 2 cytokine response characterized by coexpression of interleukin-4 (IL-4) and IL-10 by CD4(+) T cells, downregulation of proinflammatory cytokine expression in the draining lymph nodes and the colon, and recruitment of alternatively activated (M2) macrophages and eosinophils to the site of ES administration. Protease digestion and heat denaturation of AcES resulted in impaired induction of CD4(+) IL-4(+) IL-10(+) cell responses and diminished ability to suppress colitis, indicating that protein component(s) are responsible for some of the immunosuppressive effects of AcES. Identification of the specific parasite-derived molecules responsible for reducing pathology during chemically induced colitis could lead to the development of novel therapeutics for the treatment of human inflammatory bowel disease.

Uncinaria stenocephala belongs to Ancylostomatidae family. Members of this family - hookworms - infect millions of people and animals worldwide. U. stenocephala is most pathogenic in dogs and other Canidae, which are the main hosts, and infection causes anemia or even death. So far no effective hookworm vaccine has been developed that is economically viable. Attempts to identify vaccine antigens have led to a group of aspartic proteases, which play a key role in parasite feeding, migration through host tissues and immune evasion. The cDNA of an aspartic protease from U. stenocephala was cloned using the RACE-PCR method. Computational analysis showed that the cDNA encodes a 447 amino acid protein with a molecular mass of 52kDa that shows high homology to aspartic proteases from related hookworms. Analysis identified 1 potential N-glycosylation site, 3 potential disulfide bonds and no O-glycosylation sites. The recombinant protein was expressed in Escherichia coli followed by purification and mouse immunization. Using raised anti-Us-APR-1(2) (Uncinaria stenocephala Aspartic protease-1) serum the presence of Us-APR-1 in the adult stage of U. stenocephala and the expression of homologous protease in L3 and adult stages of A. ceylanicum was confirmed. This analysis is the first phase of work exploring the biological role of Us-APR-1 in parasite-host interactions and raises hope for successful vaccine development against Uncinaria sp. and possibly Ancylostoma sp.