Dirofilaria immitis is endemic in Portugal. Several studies have reported the presence of canine heartworm disease, although no previous studies on feline infections have been published. The aim of this study was to determine the prevalence of D. immitis in cats and dogs from central and northern Portugal. Blood samples from 434 cats were tested for circulating anti-D. immitis and anti-Wolbachia antibodies. Furthermore, 386 dogs were tested for circulating D. immitis antigens. Overall feline seroprevalence was 15%, while canine prevalence was 2.1%. The highest feline seroprevalences of 18.7% and 17.6% were found in Aveiro and Viseu, respectively, while the highest canine prevalences of 8.8% and 6.8% were found in Coimbra and Aveiro, respectively. Cats and dogs showing respiratory signs presented higher prevalences of 24.4% and 17%, respectively, while 50% of cats with gastrointestinal signs were seropositive. The present study confirms the seropositivity of D. immitis in the feline population in central and northern Portugal, and suggests the importance of including heartworm disease in the list of differential diagnoses of cats and dogs showing clinical signs compatible with the disease.

Ethiopian wolves, Canis simensis, are an endangered carnivore endemic to the Ethiopian highlands. Although previous studies have focused on aspects of Ethiopian wolf biology, including diet, territoriality, reproduction and infectious diseases such as rabies, little is known of their helminth parasites. In the current study, faecal samples were collected from 94 wild Ethiopian wolves in the Bale Mountains of southern Ethiopia, between August 2008 and February 2010, and were screened for the presence of helminth eggs using a semi-quantitative volumetric dilution method with microscopy. We found that 66 of the 94 faecal samples (70.2%) contained eggs from at least one group of helminths, including Capillaria, Toxocara, Trichuris, ancylostomatids, Hymenolepis and taeniids. Eggs of Capillaria sp. were found most commonly, followed by Trichuris sp., ancylostomatid species and Toxocara species. Three samples contained Hymenolepis sp. eggs, which were likely artefacts from ingested prey species. Four samples contained taeniid eggs, one of which was copro-polymerase chain reaction (copro-PCR) and sequence positive for Echinococcus granulosus, suggesting a spillover from a domestic parasite cycle into this wildlife species. Associations between presence/absence of Capillaria, Toxocara and Trichuris eggs were found; and egg burdens of Toxocara and ancylostomatids were found to be associated with geographical location and sampling season.

Soil contaminated with helminth eggs and protozoan cysts is a potential source of infection and poses a threat to the public, especially to young children frequenting playgrounds. The present study determines the levels of infection of helminth eggs in soil samples from urban and suburban playgrounds in five states in Peninsular Malaysia and identifies one source of contamination via faecal screening from stray animals. Three hundred soil samples from 60 playgrounds in five states in Peninsular Malaysia were screened using the centrifugal flotation technique to identify and determine egg/cyst counts per gram (EPG) for each parasite. All playgrounds, especially those in Penang, were found to be contaminated with eggs from four nematode genera, with Toxocara eggs (95.7%) the highest, followed by Ascaris (93.3%), Ancylostoma (88.3%) and Trichuris (77.0%). In addition, faeces from animal shelters were found to contain both helminth eggs and protozoan cysts, with overall infection rates being 54% and 57% for feline and canine samples, respectively. The most frequently occurring parasite in feline samples was Toxocara cati (37%; EPG, 42.47 ± 156.08), while in dog faeces it was Ancylostoma sp. (54%; EPG, 197.16 ± 383.28). Infection levels also tended to be influenced by season, type of park/playground and the texture of soil/faeces. The occurrence of Toxocara, Ancylostoma and Trichuris eggs in soil samples highlights the risk of transmission to the human population, especially children, while the presence of Ascaris eggs suggests a human source of contamination and raises the issue of hygiene standards and public health risks at sites under investigation.

Generally, women residing in areas endemic for urinary schistosomiasis may suffer from female genital schistosomiasis which is acquired during childhood. The objective of this cross-sectional study was to estimate the prevalence and intensity of infection of Schistosoma haematobium in women of reproductive age (16–45 years) and to investigate whether S. haematobium had any effect on kidney function. A total of 394 women of known pregnancy status (158 pregnant and 236 non-pregnant) were recruited from five villages (known for their high prevalence of infection of S. haematobium) in Kwale County. Serum samples were analysed to determine levels of urea and creatinine as proxy indicators of kidney function. Data revealed that pregnant women did not, on average, have a higher prevalence or intensity of infection of urinary schistosomiasis than non-pregnant women. During pregnancy, the level of prevalence and intensity of infection of S. haematobium was highest in the first trimester (0–13 weeks), dropped in the second trimester (14–26 weeks) and rose again in the third trimester (27–40 weeks). In addition, 24.8% of women were infected with hookworm, while none were diagnosed with malaria parasites. Of 250 samples analysed for serum urea and creatinine, none had significant levels of pathology, either in pregnant or non-pregnant women. Despite World Health Organization (WHO) recommendations that pregnant women should be treated with praziquantel after the first trimester, in practice this has not been the case in many countries, including Kenya. In view of this, healthcare providers should be informed to consider treatment of pregnant women infected with schistosomiasis during antenatal visits and whenever there is mass drug administration as recommended by the WHO.

Onchocerca volvulus is a tissue-dwelling, vector-borne nematode parasite of humans and is the causative agent of onchocerciasis or river blindness. Natural infections of BALB/c mice with Litomosoides sigmodontis and of cattle with Onchocerca ochengi were used as models to study the immune responses to O. volvulus-derived recombinant proteins (OvALT-2, OvNLT-1, Ov103 and Ov7). The humoral immune response of O. volvulus-infected humans against OvALT-2, OvNLT-1 and Ov7 revealed pronounced immunoglobulin G (IgG) titres which were, however, significantly lower than against the lysate of O. volvulus adult female worms. Sera derived from patients displaying the hyperreactive form of onchocerciasis showed a uniform trend of higher IgG reactivity both to the single proteins and the O. volvulus lysate. Sera derived from L. sigmodontis-infected mice and from calves exposed to O. ochengi transmission in a hyperendemic area also contained IgM and IgG1 specific for O. volvulus-derived recombinant proteins. These results strongly suggest that L. sigmodontis-specific and O. ochengi-specific immunoglobulins elicited during natural infection of mice and cattle cross-reacted with O. volvulus-derived recombinant antigens. Monitoring O. ochengi-infected calves over a 26-month period, provided a comprehensive kinetic of the humoral response to infection that was strictly correlated with parasite load and occurrence of microfilariae.

The helminth fauna associated with Leptodactylus latrans, a large frog living in a disturbed environment of Atlantic rainforest in south-eastern Brazil, was evaluated. We found eight helminth taxa, including five nematode species, Falcaustra mascula, Oswaldocruzia subauricularis, Physaloptera sp., Rhabdias sp. and an unidentified cosmocercid, two trematodes, Gorgoderina parvicava and Haematoloechus fuelleborni, and one larval cestode. The overall prevalence of infection was 63.2% with a mean intensity of 11.3 ± 3.8. The cosmocercid nematode and O. subauricularis showed the highest prevalences, although the trematode G. parvicava was the most abundant and dominant parasite species. Host size positively influenced both the intensity of infection and parasite species richness. Our data suggest that the juvenile individuals of L. latrans are more susceptible to parasitic infection than the adults. The comparison of the similarity of this community component with that found in other studies in South America shows that, as well as the characteristics of the host, the sampling area also influences the parasitic fauna. Therefore, the results of this study agree that the helminth communities of frogs have relatively low species richness and dominance of generalist species.

We evaluated the effect of carbendazim on non-target organisms using the parasite Chordodes nobilii as a test organism. The Gordiida act as a link between freshwater and terrestrial ecosystems; and C. nobilii, a neotropical representative species of this group, has been shown to be sensitive to other contaminants even at environmentally acceptable concentrations. The taxa susceptible to carbendazim, however, may not be adequately represented among the standard aquatic test species used in ecotoxicological risk assessment. Moreover, the autochthonous organisms in this area that could be used as bioindicators still need to be found. The aim of the present work was therefore to assess the susceptibility of the preparasitic stages of C. nobilii to noxious effects by carbendazim. The assay protocol consisted in 96- and 48-h acute exposures of early embryonic stages and larvae, respectively, to concentrations ranging from 10 to 360 μg/l. Embryonic development was not inhibited by carbendazim at any of the evaluated concentrations, but the infectivity of larvae emerging from the exposed eggs was significantly diminished. Larval survival rate was also affected at the lowest concentration assayed. Values of the mean inhibition concentration (IC50) were 7 and 11 μg/l for embryos and larvae, respectively. Compared to other freshwater organisms, C. nobilii can be considered a species moderately to highly susceptible to carbendazim. As the expected environmental concentrations of carbendazim range from 6.25 to 41.3 μg/l, C. nobilii could well be a species in danger when exposed to this fungicide.

The study was conducted to determine the sequence variation in two mitochondrial genes, namely cytochrome c oxidase 1 (pcox1) and NADH dehydrogenase 1 (pnad1) within and among isolates of Toxocara cati, Toxocara canis and Toxascaris leonina. Genomic DNA was extracted from 32 isolates of T. cati, 9 isolates of T. canis and 19 isolates of T. leonina collected from cats and dogs in different geographical areas of Iran. Mitochondrial genes were amplified by polymerase chain reaction (PCR) and sequenced. Sequence data were aligned using the BioEdit software and compared with published sequences in GenBank. Phylogenetic analysis was performed using Bayesian inference and maximum likelihood methods. Based on pairwise comparison, intra-species genetic diversity within Iranian isolates of T. cati, T. canis and T. leonina amounted to 0–2.3%, 0–1.3% and 0–1.0% for pcox1 and 0–2.0%, 0–1.7% and 0–2.6% for pnad1, respectively. Inter-species sequence variation among the three ascaridoid nematodes was significantly higher, being 9.5–16.6% for pcox1 and 11.9–26.7% for pnad1. Sequence and phylogenetic analysis of the pcox1 and pnad1 genes indicated that there is significant genetic diversity within and among isolates of T. cati, T. canis and T. leonina from different areas of Iran, and these genes can be used for studying genetic variation of ascaridoid nematodes.

Taeniid infections in intermediate hosts manifest themselves as extraintestinal larval stages which, in early development, lack species-specific characteristics. The inability to distinguish infections of zoonotic importance such as Echinococcus multilocularis from other taeniid infections that have mainly veterinary significance stimulated the development of species-specific molecular diagnostics. In this study, the prevalence of taeniid infections in potential intermediate hosts was evaluated using both morphological diagnosis and a newly described multiplex polymerase chain reaction (PCR) for species determination. Small mammals (N= 719) were trapped in three different types of habitats in north-east Zealand, Denmark. The sensitivity of the multiplex PCR (90.5%) exceeded that of morphological examination (57.9%) for identifying 95 taeniid infections. The use of the multiplex PCR resulted in higher prevalence rates due to improved detection of immature liver infections with Hydatigera taeniaeformis and Versteria mustelae, but did not affect the observed prevalence rates of peritoneal metacestodes of Taenia polyacantha. The prevalence of taeniid infections showed a significant difference according to habitat type, potentially identifying a ‘sylvatic’ transmission and an ‘urban’ transmission, with marked variation among different taeniid species. Versteria mustelae and T. polyacantha were more prevalent in rural forests, while infections with H. taeniaeformis were dominant in urban parks/forests and in residential and farm gardens. The multiplex PCR facilitated a better utilization of wildlife samples by yielding a higher number of definitive diagnoses of ambiguous taeniid infections in liver lesions, allowing for more accurate epidemiological data and, hence, a more accurate risk assessment.

The giant kidney worm Dioctophymerenale is normally found in wild carnivores and domestic dogs, with aquatic oligochaetes acting as intermediate hosts. In the present study a prevalence of 50% of third-stage larvae of D. renale was recorded in 60 specimens of the freshwater turtle Trachemys dorbigni from southern Brazil. Larvae were encysted in muscles, the coelomic cavity and mesentery, the serous lining of the stomach and on the surfaces of the lung, heart, liver, pancreas, spleen and intestines. There are no previous records of reptiles being part of the life cycle of D. renale, although fish and amphibians normally act as paratenic hosts. This is the first report of third-stage D. renale larvae in the freshwater turtle, T. dorbigni.

Cysticercosis is caused by infections with embryonated eggs of the tapeworm Taenia pisiformis. Knowledge of the genetic characteristics of T. pisiformis could be applied to study the epidemiology and transmission of this parasite. In this study, 61 isolates of intraperitoneal cysticerci from eight geographically distinct regions in Sichuan province, China, were subjected to a molecular analysis in order to determine their intra-regional genetic characteristics. Partial sequences of the mitochondrial cytochrome c oxidase subunit I (cox1, 1427 bp) and NADH dehydrogenase 1 (nad1, 738 bp) were concatenated. Five haplotypes were identified, and 89.04% of total genetic variation was found in collections of T. pisiformis isolates from a single region. According to the phylogenetic reconstruction, the T. pisiformis isolates from eight regions did not form geographical clusters. Our study highlights the genetic characteristics of T.pisiformis with the aim of accelerating the genetic research and control of cysticercosis.

Angiostrongylus vasorum is a parasitic nematode that infects the heart and pulmonary artery and its branches of domestic and wild canids. The parasite can use several species of terrestrial and aquatic molluscs as intermediate hosts, although susceptibility varies. Pomacea canaliculata is a mollusc found in lakes, swamps and rivers in South America. In this study, we evaluated the susceptibility, parasite growth, oviposition and larval development of 282 P. canaliculata infected with 500 A. vasorum first-instar larvae (L1). From day 5 post-infection (pi) to day 30 pi, seven specimens per day were sacrificed to recover the larval instars. We compared 50 egg masses from infected and uninfected molluscs to determine the number of eggs per clutch, the hatching rate and the growth of the molluscs. The percentage of recovered larvae ranged from 39.17% to 67.5%. First-stage larvae (L1) were found until day 19 pi, second-stage larvae (L2) were found from days 11 to 25 pi, and third-stage larvae (L3) were recovered only after day 19 pi. Infected snails exhibited the most eggs during spawning, although the rate of hatching and shell size were lower in the infected snails compared with controls. This is the first report of an experimental infection of P. canaliculata with A. vasorum, and the results confirm the non-specificity of the nematode in relation to the intermediate host and indicate the importance of epidemiological surveys of this parasite and mollusc.

Recently, in the Journal of Helminthology (May 2013), Dao et al. reported that Opisthorchis viverrini-like flukes were found in the bile duct of domestic ducks in Vietnam. They stated that this is the first record of Opisthorchis sp. in birds in Vietnam. However, three Opisthorchis species – O. cheelis, O. longissimus and O. parageminus – in birds in Vietnam were described by Le in 2000. Amongst these, O. parageminus was first reported, by Oshmarin in 1970, as a new Opisthorchis species found in domestic ducks (Anas platyrhynchos) in Vietnam. Morphologially O. viverrini-like flukes described by Dao et al. are much more similar to O. parageminus than to O. viverrini. The phylogenetic trees of internal transcribed spacer 2 (ITS2) and cytochrome oxidase 1 (CO1) gene sequences also showed that the O. viverrini-like liver flukes from domestic ducks were closer to O. lobatus than to O. viverrini. Therefore, O. viverrini-like liver flukes reported by Dao et al. (2013) are most likely to be O. parageminus.

To study possible indirect effects of the infection with intestinal helminths, 12 Rattus norvegicus (Wistar) were each experimentally exposed to 100 metacercariae of Echinostoma caproni, and blood samples were taken weekly up to 4 weeks post-exposure for comparison with control rats. Values of haematocrit (HCT), red blood cells (RBC), platelets (PLT), white blood cells (WBC), haemoglobin (HGB) and haematimatrix indices, and mean corpuscular haemoglobin concentrations (MCHC) were determined. In addition, leucocyte counts, including lymphocytes, neutrophils, monocytes, eosinophils and basophils were analysed. These parameters, including the leucocyte counts, showed no significant differences, except for MCHC at 4 weeks post-exposure. The present results indicate that in rats infected with E. caproni, although eosinophilia did not significantly increase, a significant reduction in MCHC was associated with an increase in the number of RBC.

Nematodes of the genus Trichinella which infect wildlife and domestic animals show a cosmopolitan distribution. These zoonotic parasites are the aetiological agents of a severe human disease, trichinellosis. Twelve taxa are recognized in the Trichinella genus, but they cannot be identified by morphology since they are sibling species/genotypes. For epidemiological studies, it is extremely important to identify each taxon since they have different distribution areas and host ranges. In the present study, polymerase chain reaction (PCR) amplification of the mitochondrial large subunit ribosomal RNA (lsu-RNA) gene coupled with a pyrosequencing technique was developed to distinguish among four Trichinella species: Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis. A PCR method was used to amplify the lsu-RNA of Trichinella sp. larvae in mouse muscles and single larvae collected from infected muscles by digestion. The results show that the four Trichinella species can be distinguished by using 26 nucleotides in the target region and the method is sensitive enough to identify individual larvae. The pyrosequencing provides a simple, rapid and high-throughput tool for the differentiation of Trichinella species.

There is evidence of resistance in horses to anthelmintic treatment using ivermectin and pyrantel. However, little information is available about the parasites, treatment practices or anthelmintic resistance in the horse population in Estonia. In the present study, we examined 41 trotting and riding horses aged < 3 years from four stables in Estonia. Faecal samples were collected, and horses were selected for treatment if the nematode egg count per gram faeces exceeded 200. Horses (n= 32) that shed strongyle-type eggs were treated with pyrantel, whereas Parascaris equorum-positive animals received ivermectin. Up to 78% of horses required anthelmintic treatment and the efficiency of the anthelmintics was evaluated using a faecal egg count reduction test. Resistance of P. equorum was observed in 50% of horses treated with ivermectin and of strongyles in 27% of horses treated with pyrantel. Ivermectin treatment resulted in a mean reduction of 100% for strongyle eggs and an 89% reduction in P. equorum, and pyrantel-treated horses exhibited an 88% reduction in strongyle eggs. These results are considered to be the first indication of resistance to pyrantel, but further studies of ivermectin resistance are required. According to questionnaires completed by the owners of horses, resistance might be explained by a lack of evidence-based strategies, a strong preference for using ivermectin and possibly a subjective evaluation of the body weight of horses.

The genetic variations in internal transcribed spacers (ITS) spanning ITS-1, 5.8S and ITS-2 rDNA of Dicrocoelium dendriticum, isolated from sheep and goats in four geographical regions in Shaanxi province, were examined. The lengths of ITS-1, 5.8S and ITS-2 rDNA sequences for D. dendriticum were 749 bp, 161 bp and 234 bp, respectively. Intra-specific sequence variations of D. dendriticum were 0–0.5% for ITS-1 and 0–1.3% for ITS-2 rDNA, while the inter-specific variations among species in genus Dicrocoelium in ITS-2 rDNA were 3.4–12.3%. Phylogenetic analysis based on sequences of ITS-2 rDNA showed that all D. dendriticum isolates in the present study were grouped with reference D. dendriticum isolates from sheep and goats, and D. dendriticum isolates from cattle and Japanese serow were clustered in a sister clade. However, the phylogenetic tree could not reveal geographically genetic relationships of D. dendriticum isolates in different origins and hosts. These findings provided basic information for further study of molecular epidemiology and control of D. dendriticum infection in Shaanxi province as well as in the world.

An in vivo study in goats evaluated the effect of superimposing a single artificial infection with a benzimidazole (BZ)-susceptible Haemonchus contortus isolate upon established H. contortus populations of known BZ resistance by measuring the phenotypic BZ resistance of eggs collected from faeces before and after re-infection. Two H. contortus isolates, one benzimidazole resistant (BZR) and the other susceptible (BZS), were used to infect worm-free goats. Eight goats were initially infected with 2000 third-stage larvae (L3). In each case the inoculum contained a pre-determined proportion of the two isolates: 100% BZS (one goat), 75% BZS/25% BZR (two goats), 50% BZS/50% BZR (two goats), 25%BZS/75% BZR (two goats) and, finally, 100% BZR (one goat). The phenotypic BZ susceptibility of the H. contortus population formed in each goat after the first infection was determined on days 30 and 36 post-infection using an egg-hatch assay (EHA) that estimated the concentration of thiabendazole (TBZ) required for 95% inhibition of larval hatching (EC95) with a 95% confidence interval (95% CI). On day 49 post-infection, goats were re-infected with 2000 L3 of the BZS isolate alone. A second set of EHA bioassays was performed 28 days and 34 days after re-infection. The first infection protocol produced three populations classified as BZS (EC95 0.055–0.065 μg TBZ/ml) while four were categorized as BZR (EC95 0.122–0.344 μg TBZ/ml). The status of one other population could not be determined. After re-infection with BZS L3, the number of susceptible populations increased to six (EC95 0.043–0.074 μg TBZ/ml) while the remaining two were deemed resistant (EC95 0.114–119 μg TBZ/ml). Re-infection with BZS L3 thereby reduced the resistance status of most H. contortus populations.