The influence of habitat structure on helminth communities of three sigomdontinae rodent species (Akodon cursor, A. montensis and Oligoryzomys nigripes) was investigated in forest fragments within an agricultural landscape in south-eastern Brazil. This is a pionner study correlating the occurrence of helminth species of rodent hosts with microhabitat characteristics. Rodents were collected from 12 fragments and in a continuous conserved area. Up to 13 nematode, three cestode and two trematode species were identified, and habitat fragmentation was found to have more influence on the helminth composition of O. nigripes compared to the other two rodent species. Fragmentation appeared to limit the development of some helminths’ life cycles, e.g. with some species such as Trichofreitasia lenti, Protospirura numidica, Cysticercus fasciolaris and Avellaria sp., occurring mostly in areas with less anthropic impact. However, fragmentation did not seem to affect the life cycles of other dominant helminths, such as the trematode Canaania obesa, the nematodes Stilestrongylus lanfrediae,S. eta and S. aculeata, and the cestode Rodentolepis akodontis. The helminth community structure followed a nested pattern of distribution in A. montensis and O. nigripes. Stilestrongylus lanfrediae seemed to be more associated with dense understorey, C. obesa with open canopy and dense understorey, and Guerrerostrongylus zetta with organic matter on the ground. Their presence in each area may be explained by aspects of their life cycles that take place in the external environment outside the host.

The nervous systems of three Tylodelphys metacercariae (T. mashonense, Tylodelphys spp. 1 and 2) co-occurring in the cranial cavity of the catfish, Clarias gariepinus, were examined by the activity of acetylthiocholine iodide (AcThI), with the aim of better understanding the arrangement of sensillae on the body surface and the nerve trunks and commissures, for taxonomic purposes. Enzyme cytochemistry demonstrated a comparable orthogonal arrangement in the three metacercariae: the central nervous system (CNS) consisting of a pair of cerebral ganglia, from which anterior and posterior neuronal pathways arise and inter-link by cross-connectives and commissures. However, the number of transverse nerves was significantly different in the three diplostomid metacercariae: Tylodelphys sp. 1 (30), Tylodelphys sp. 2 (21) and T. mashonense (15). The observed difference in the nervous system of the three metacercariae clearly separates them into three species. These findings suggest that consistent differences in the transverse nerves of digenean metacercariae could enable the differentiation of metacercariae to the species level in the absence of molecular techniques. This, however, might require further testing on a larger number of species of digenean metacercariae.

This study was undertaken to determine whether a parasite substance produces structural pathology in the mouse spleen. A low-molecular-weight Taenia crassiceps metacestode factor (MF) isolated from the peritoneal fluid of female mice infected with T. crassiceps metacestodes induced pathological and immunological changes in mouse spleen cells in vivo. Electron microscopy and confocal microscopy revealed severe changes in the spleen histoarchitecture of T. crassiceps-infected and MF-treated mice. Apoptotic degenerated spleen cells were observed in the white and red pulps and were more conspicuous in the white pulp of the spleen from the T. crassiceps-infected mice than in that of the MF-treated mice. Flow cytometry analysis revealed that the numbers of spleen CD4+T cells were significantly lower in both experimental groups than in control mice. The ex vivo expression of transforming growth factor (TGF)-β and factor Foxp3 were significantly higher in splenocytes of the experimental mice than the basal expression observed in the control cells. These findings may have potential applications for a better understanding of the host–parasite relationship in human neurocysticercosis.

Pseudanoplocephala crawfordi is an important zoonotic cestode of economic significance and public health concern. In spite of its significance as a pathogen, the systematics, genetics, epidemiology and biology of this parasite remain poorly understood. In the present study, we sequenced and characterized the complete mitochondrial (mt) genome of P. crawfordi, which is 14,192 bp long and encodes 36 genes, including 12 protein-coding genes, 22 transfer RNA genes and two ribosomal RNA genes. Phylogenetic analysis of the concatenated amino acid sequences using the Bayesian inference (BI) method showed that P. crawfordi was closely related to the family Hymenolepididae. Considering that the taxonomic status of P. crawfordi has been controversial when based only on morphological features, the mt genome obtained here will provide novel molecular markers to ascertain the phylogenetic position of this parasite accurately.

In the present study, antigens from parthenogenetic females and eggs of Strongyloides venezuelensis, or anti-parthenogenetic-female and anti-egg antigens were used to detect specific IgG and immune complex responses, respectively. Serum samples from experimentally infected immunocompetent and immunosuppressed rats were analysed on days 5, 8, 13 and 21 post-infection (dpi). An enzyme-linked immunosorbent assay (ELISA) was performed using alkaline parasite extract for specific IgG detection, and anti-parthenogenetic-female or anti-egg antigens for immune complex detection. The data were analysed using analysis of variance (ANOVA), followed by a Bonferroni test. When parthenogenetic female or egg extracts were used as antigens, specific IgGs were not detected in either immunocompetent or immunosuppressed rats. When anti-parthenogenetic-female or anti-S. venezuelensis-eggs were used, immune complexes were detected for the duration of the infection in immunosuppressed animals and were only detected between 5 and 13 dpi in immunocompetent animals. The duration of infection was not significantly different between the immunocompetent and immunosuppressed groups when anti-parthenogenetic-female or anti-S. venezuelensis-eggs were used. Parthenogenetic female extracts yielded significant differences between antibody and immune complex responses in immunocompetent rats from 5 to 13 dpi, but only on day 5 dpi in immunosuppressed rats. Exposure to S. venezuelensis egg extract yielded significant differences in both antibody and immune complex detection between immunocompetent and immunosuppressed rats for the duration of the infection. In conclusion, ELISA using alternative antigens may be a successful strategy for identifying immune complexes in serum samples and diagnosing active strongyloidiasis, particularly under conditions of immunosuppression.

Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ–Linker–VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples.

This study examined sequence variability in internal transcribed spacers (ITS) of nuclear ribosomal DNA among Syphacia obvelata and Aspiculuris tetraptera isolates from laboratory mice from different geographical locations in China. ITS1, 5.8S and ITS2 rDNA were amplified separately from adult S. obvelata and A. tetraptera individuals by polymerase chain reaction (PCR), and the amplicons were subjected to sequencing from both directions. The lengths of the sequences of ITS1, 5.8S and ITS2 rDNA from both nematodes were 314 bp and 456 bp, 157 bp, and 273 bp and 419 bp, respectively. The intraspecific sequence variations in S. obvelata ITS1 were 0–0.3%. For A. tetraptera they were 0–0.7% in ITS1 and 0–1.0% in ITS2. However, the interspecific sequence differences among members of the infraorder Oxyuridomorpha were significantly higher, being 54.0–65.5% for ITS1 and 55.3–64.1% for ITS2. Phylogenetic analysis based on the combined partial sequences of ITS1 and ITS2 using three inference methods – Bayesian inference, maximum likelihood and maximum parsimony – revealed that all the S. obvelata and A. tetraptera samples formed independent monophyletic groups. Syphacia obvelata was closer to Syphacia muris than to A. tetraptera, consistent with morphological classification. These results demonstrate that ITS1 and ITS2 rDNA sequences are useful markers for population genetic studies of oxyurid nematodes.

An immunodominant serine protease of Trichinella spiralis named NBL1 showed encouraging potential in early diagnosis of trichinellosis in pigs and elicited protective immune responses during infection of animals. To further define serological reagents for diagnostic use, the specific epitopes on NBL protein recognized by the antibody responses of different susceptible hosts need to be defined. The present study described comprehensive mapping of immunodominant linear epitopes in the antigenic region (NBL-C, the C-terminal part of the protein) using various serum samples obtained from three kinds of hosts – pig, wild boar and mice. We identified six peptides which were commonly recognized by sera from pigs experimentally infected with Trichinella and pigs immunized with rNBL1-C; five and four peptides were recognized by sera from wild boars and mice infected with Trichinella, respectively. Three peptides (NBL1-6, -7 and -9) were commonly recognized by antisera in all three hosts, which share the sequence PSSGSRPTYP. We also found that one peptide (NBL1-12) was only recognized by antibodies from pigs immunized with rNBL1-C. The identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of subunit vaccines and diagnostic tools for trichinellosis.

Gyrodactylus alviga is a generalist species reported in many Black Sea fish species, but whiting is known to be its main host. It is the only monogenean parasite that has been reported so far on the skin, fins and gills of whiting Merlangius merlangus in the Black Sea. A total of 690 fish from Turkey and 423 fish from Russia were examined to detect parasites. Infection indices of prevalence, mean intensity and mean abundance values were calculated for length classes and sex of fish, as well as for the seasons. There is a gradual increase in infection indices in relation with increasing host length classes and there are higher infection indices on female fish. Furthermore, despite lower prevalence indices in spring and winter, higher parasite intensity values were determined in these seasons. It is concluded that larger fish provided more space and food for G. alviga, and female fish were more parasitized as they were more active in searching for food, which enables the parasite to switch between hosts. In addition, winter and spring were more appropriate for reproduction of G. alviga.

Species of the genus Mizelleus Jain (1957) have always been controversial regarding identification and validity. Members of this group of species differ from each other in the morphology of their hard parts, which can be misleading and subject to differing interpretation among scientists. Therefore, the main objective of present study was to identify Mizelleus worms by morphological methods and molecular analysis on the basis of 18S ribosomal DNA to clarify their phylogenetic status. In this study, specimens were isolated from the gill filaments of Wallago attu (Siluriformes) and studied morphologically. In accordance with morphological characters, the specimens were found to be Mizelleus indicus and Mizelleus longicirrus. Partial sequences of nuclear 18S rDNA of these two species were amplified. The results confirm the phylogenetic relationships and taxonomic validation of M. indicus and M. longicirrus in India.

The relationship between the frequency of loiasis objective symptoms and microfilaraemic or amicrofilaraemic infection was assessed in 1148 exposed patients also infected, or not, with Mansonella perstans. Filarial infections were detected by direct microscopy, leucoconcentration and serology, with prevalence values of 39.5% Loa loa, 5.6% M. perstans and 3.4% co-infection with both filarial species. Amicrofilaraemic or occult loiasis (OL) predominated among L. loa-infected individuals, with a prevalence of 58.2%. Hypermicrofilaraemia (>8000 microfilariae (mf)/ml) was found in 18.4% of L. loa microfilaraemic patients, with 25.7% of them harbouring more than 30,000 mf/ml. Up to 34% of patients with OL showed evidence of Calabar swelling, compared with 26.3% of microfilaraemic patients (P= 0.03). Overall 5.3% of patients presented with adult worm migration across the eye, representing 16.3% of microfilaraemic individuals and 11.4% of amicrofilaraemic patients (P= 0.13). This symptom was similarly found in patients with more than 30,000 mf/ml (22%), those with microfilaraemia between 8 and 30,000 mf/ml (15.4%) and also in individuals with low or without microfilaraemia (16.1%) (P= 0.7). Five (14.3%) hypermicrofilaraemic patients did not present any L. loa-specific objective symptoms, as well as all the patients with single M. perstans infection. The presence of adult eye worm migration as a strong predictor of high microfilaraemia density would obscure the real burden of L. loa hypermicrofilaraemia in exposed individuals. For epidemiological purposes and control strategies, the mapping of L. loa in endemic areas should also take into account the group of patients with occult loiasis.

In this study we present the first approach to exploration of the genetic diversity of Cuban Fasciola hepatica populations using microsatellite markers, coupled with observed prevalence in slaughterhouses. Nine populations of flukes recovered from cows and buffalos were studied in the central-western region of Cuba. The observed infection rates of definitive hosts (bovines) were 70–100% in most cases. An important amount of polymorphism was found in the four loci explored. However, no apparent genetic differences were found between populations from different provinces or bovine species. The absence of deviations from Hardy–Weinberg equilibrium suggests a high rate of cross-fertilization between F. hepatica individuals. This result was confirmed when all multilocus genotypes were tested for clonal reproduction and only four individuals differed statistically (Psex< 0.05). High values of expected heterozygosity coupled with highly probable mixing among strains make the metapopulation genetically diversified but similar in terms of certain alleles (low FST values). These results suggest a close relationship between parasite diversity and cattle management in Cuba. Our findings should be taken into consideration by veterinary authorities to help mitigate fasciolosis transmission.

Trichinellosis is a zoonotic disease caused by nematode species of the genus Trichinella. Anthelmintics targeting the intestinal adults and muscle-dwelling larvae of Trichinella spp. have been tested, with limited success. This study was aimed at determining the efficacy of maslinic acid and fenbendazole on muscle larvae of Trichinella zimbabwensis in laboratory rats. Forty-two Sprague–Dawley rats, with an average weight of 270 g and 180 g for males and females respectively, were infected with T. zimbabwensis larvae. Infected rats were randomly assigned to three groups which were subjected to single treatments with each of maslinic acid, fenbendazole and a combination of both on day 25 post-infection (pi), and three groups which were subjected to double treatments with each of these drugs and a combination on days 25 and 32 pi. The untreated control group received a placebo. In single-treatment groups, the efficacy of each treatment, measured by rate of reduction in muscle larvae, was significant (P< 0.001) for both drugs compared to the untreated control group. There was no apparent synergistic effect on the combination of the two drugs in reducing the muscle larval burden, either in single or double treatments. In all the treatment regimens, the reductions were significant (P< 0.001) when compared to the untreated control and not significant when the single treatments were compared with the double treatments (P>0.05). We conclude that the efficacy of maslinic acid against larval stages of T. zimbabwensis in rats was comparable to that of fenbendazole, with no side-effects observed, making maslinic acid a promising anthelmintic against larval stages of Trichinella species.

The presence of the cyclophyllidean cestode Rodentolepis straminea (Cestoda: Hymenolepididae), was confirmed by molecular DNA analysis from a wood mouse (Apodemussylvaticus) population inhabiting urban woodland in Salford, Greater Manchester (UK) with a prevalence of 27.8%. It would appear that the only previously published record of this species in A. sylvaticus in the British Isles is that from south-west Ireland, where 24% of the wood mice examined were infected with R. straminea. This species has been recorded in studies on A. sylvaticus in continental Europe. The current report represents a new record for R. straminea on mainland Britain and a first study of helminth parasites in an urban wood mouse population.

Opisthorchiasis is a public health problem in South-East Asian countries and Eastern Europe. The infection implicates mainly two species of Opisthorchis, namely O. viverrini and O. felineus, that occur mostly in fish-eating mammals and humans, although there are rare reports of human cases involving two other species, O. noverca and O. guayaquilensis. Opisthorchis noverca has been reported frequently in dogs and pigs from the Indian subcontinent, with rare reports from cattle and human subjects. With a view to supplementing morphology-based identification of this species, the present study aimed to provide molecular characterization of O. noverca, using rDNA internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase I (mt COI) markers so as to determine its genetic correlation with other species of Opisthorchiidae, and also to generate a taxon-specific molecular marker based on the ITS2 region. The phylogenetic relationship between O. noverca and other species of the genus was determined using molecular sequence data. To strengthen the result, secondary structure sequence analyses of ITS2 with hemi-compensatory base changes (hCBCs), and amino acid sequence analyses, were also evaluated. Our results confirm that O. noverca is a distinct and valid species.

Adults of Haplosplanchnus pachysomus (Eysenhardt, 1829) were found in the intestine of Liza engeli (Bleeker) from the coastal waters of Cat Ba Island, Ha Long Bay, northern Vietnam. Additionally, Provitellotrema crenimugilis Pan, 1984 was discovered in Liza haematocheila (Temminck & Schlegel) from Vostok Bay, Gulf of Peter the Great, southern Far-East Russia. Data concerning morphology, 18S rDNA and 28S rDNA of these worms were obtained.The molecular data confirmed the validity of these species and showed that specimens identified as H. pachysomus are closely related to specimens of H. pachysomus found in Spain, and that P. crenimugilis is closely related to Haplosplanchnus purii. Molecular differentiation of P. crenimugilis and H. purii was 0.92% by combined ribosomal gene sequences that confirmed species validity. Molecular differentiation between P. crenimugilis and H. purii, on the one hand, and H. pachysomus, on the other hand, was much higher, suggesting that the sequence for H. purii in GenBank is for a misidentified species of the genus Provitellotrema Pan, 1984.

Three digeneans – Parahemiurus merus (Linton, 1910), Aphanurus stossichii (Monticelli, 1891) and Lecithochirium sp. – and one tetraphyllidean cestode larva were used as biological tags to discriminate the stock of Sardinella aurita (Valenciennes, 1847). In total, 579 fish were examined in five zones off the Tunisian coast, including Bizerte and Kelibia in the north, Mahdia in the east, Gabes and Zarzis in the south. Discriminant analyses used for the separation of S. aurita allowed for the identification of two discrete stocks. Sardinella aurita from Bizerte, Kelibia and Zarzis clumped together as a single stock. Parahemiurus merus and A. stossichii were the most important species in determining the location of sampled fish from these regions. Specimens from Mahdia and Gabes were grouped as one stock characterized by the presence of Lecithochirium sp. and larvae of the Tetraphyllidea. These results were corroborated by comparing the parameters of prevalence and mean abundance of parasites among zones. The separation of S. aurita between localities after pooling specimens from Bizerte, Kelibia and Zarzis and separately pooling those from Mahdia and Gabes also allowed the identification of two discrete stocks, one in offshore waters from Bizerte, Kelibia and Zarzis characterized by the digeneans P. merus and A. stossichii and one in inshore waters from Mahdia and Gabes characterized by Lechithochirium sp. and tetraphyllidean larvae.

The life cycle of Proctoeces maculatus (Looss, 1901) (Digenea, Fellodistomidae) was studied in Bizerte Lagoon (Tunisia). Three sequential hosts appear to be involved: the Mediterranean mussel Mytilus galloprovincialis Lamarck, 1819 (Mytilidae) as the first intermediate host; the polychaete Sabella pavonina Savigny, 1822 (Sabellidae), as the second intermediate host; and fishes (Lithognathus mormyrus (Linnaeus, 1758) (Sparidae), Trachinotus ovatus (Linnaeus, 1758) (Carangidae) and Sparus aurata Linnaeus, 1758 (Sparidae) as the definitive hosts. It should be noted that S. pavonina was recorded as second intermediate host for P. maculatus for the first time. Molecular confirmation of the morphological identification of the life-cycle stages of this digenean was obtained using partial 28S rDNA sequences. Comparative sequences revealed that the sporocysts and the metacercariae are conspecific but they diverged by 0.3% from the adults. The present results raised the possibility of the existence of cryptic species within the different developmental stages. However, all the present isolates differed from material from Archosargus probatocephalus in the Gulf of Mexico identified as P. maculatus.

The aim of this work is to compare the taxonomic diversity of parasite species of the large hairy armadillo Chaetophractus villosus in its native range and in another recently introduced population (Tierra del Fuego island), and to evaluate whether the isolation of the latter determines a decrease in its parasitic diversity. Forty specimens from Buenos Aires and Tierra del Fuego Provinces were collected and examined for helminths. Eleven parasite species were found in the native population, and only one species was present in Tierra del Fuego (Trichohelix tuberculata). This may be explained because isolation and climatic conditions prevent encounters between potential host species and infective forms of parasites. Further sampling will be needed throughout the entire Patagonia steppe to confirm how the characteristic parasitic fauna of C. villosus behaves across the armadillo's southern distribution.

Cacajao calvus ucayalii (Thomas, 1928) (Primates: Pitheciidae), a subspecies endemic to the Peruvian Amazon, occurs in patchy and sometimes isolated populations in north-eastern Peru and is in a vulnerable situation, mainly due to habitat loss and hunting. This rareness and remote distribution means that, until now, parasitical studies have been limited. Based on optical and scanning electron microscopy of specimens of both sexes, we report two new species of Trypanoxyuris pinworms occurring in the large intestine of the Peruvian red uakari, namely Trypanoxyuris (Trypanoxyuris) cacajao and Trypanoxyuris (Trypanoxyuris) ucayalii. Both species showed a distinct morphology of the lips and cephalic structure. Sexual dimorphism in the lateral alae was observed in both male and the female worms, with ventral ornamentation being shown in the oesophageal teeth. The finding of these new pinworm species highlights the possibility of discovering other species.