Detection of Wuchereria bancrofti infection in mosquitoes in areas co-endemic with Brugia malayi in Balasore district, Odisha, India.

Lymphatic filariasis (LF) is a crippling and disfiguring parasitic condition. India accounts for 55% of the world's LF burden. The filarial parasite Wuchereria bancrofti is known to cause 99.4% of the cases while, Brugia malayi accounts for 0.6% of the issue occurring mainly in some pockets of Odisha and Kerala states. The Balasore (Baleswar) district of Odisha has been a known focus of B. malayi transmission. We employed molecular xenomonitoring to detect filarial parasite DNA in vectors. In six selected villages, Gravid traps were used to collect Culex mosquitoes and hand catch method using aspirators was followed for collection of mansonioides. A total of 2903 mosquitoes comprising of Cx. quinquefasciatus (n = 2611; 89.94%), Cx. tritaeniorhynchus (n = 100; 3.44%), Mansonia annuliferea (n = 139; 4.78%) and Mansonia uniformis (n = 53; 1.82%) were collected from six endemic villages. The species wise mosquitoes were made into 118 pools, each with a maximum of 25 mosquitoes, dried and transported to the laboratory at VCRC, Puducherry. The mosquito pools were subjected to parasite DNA extraction, followed by Real-time PCR using LDR and HhaI probes to detect W. bancrofti and B. malayi infections, respectively. Seven pools (6.66%) of Cx. quinquefasciatus, showed infection with only W. bancrofti while none of the pools of other mosquito species showed infection with either W. bancrofti or B. malayi. Although the study area is endemic to B. malayi, none of the vectors of B. malayi was found with parasite infection. This study highlights the ongoing transmission of bancroftian filariasis in the study villages of Balasore district of Odisha and its implications for evaluating LF elimination programme.

Comparison of different molecular protocols for the detection of Uncinaria stenocephala infection in dogs.

The present study aims to assess the performance of different molecular targets using various matrices of samples for the detection of Uncinaria stenocephala (US) in hookworm infected dogs. To this end, the DNA extraction was performed on the following matrices of samples: (i) larvae of US obtained from experimentally infected dogs with US with different larvae counts per microliter (µl); (ii) pure US eggs suspension in distilled water with different egg counts per µl; (iii) spiked dog fecal samples with different US eggs per gram (EPG) of feces; (iv) feces from dogs naturally infected with hookworm eggs; (v) fecal suspension with hookworm eggs recovered from the FLOTAC apparatus. All the samples were tested with four different PCR protocols targeting specific regions for the detection of both hookworms US and AC as follows: Protocol A (ITS1, 5.8 S, ITS2) and Protocol B (18 S) for the detection of both species, Protocol C (ITS1) for the detection of AC and Protocol D (ITS1) for the detection of US. The best results were obtained with DNA extracted from US larvae matrix obtained from experimentally infected dogs, showing a detection limit of 3.5 larvae/ml for the protocols A, B and D. A moderate correlation was found between the FLOTAC technique and PCR protocols B and D with respect to fecal samples from dogs naturally infected with hookworms. Indeed, PCR protocols B (18 S) and D (ITS1) gave the best results for feces and fecal suspension from naturally infected dogs. However, all the PCR protocols used showed lower sensitivity than FLOTAC technique. Perhaps, isolating US eggs in advance could help to obtain better quality and quantity of DNA, avoiding some notable factors such as inhibitors present in faecal samples. However, a further study is needed to evaluate and standardise a protocol for the recovery of parasitic elements, that could be applied prior to DNA extraction. Therefore, this could lead to a better amplification of US eggs DNA. In conclusion, our results showed that the type of sample (sample-matrix) used for the DNA extraction samples is crucial, as this affects the diagnostic sensitivity of the technique.

TREMATODE SPECIES DETECTION AND QUANTIFICATION BY ENVIRONMENTAL DNA-qPCR ASSAY IN LAKE CHANY, RUSSIA.

Environmental DNA (eDNA) surveys promise to be a sensitive and powerful tool for the detection of trematodes. This can contribute to the limited studies on trematode ecology, specifically in aquatic ecosystems. Here, we developed species-specific primer and probe sets for Moliniella anceps, Opisthioglyphe ranae, and Plagiorchis multiglandularis cercariae and applied a novel eDNA qPCR assay to detect larval trematodes quantitatively. We evaluated the effectiveness of the assays using filtered lake water samples collected from different sites of Lake Fadikha and Kargat River Estuary in Lake Chany, Russia, showing high species specificity and sensitivity in all 3 assays. Further, all 3 assays had high efficiencies ranging from 94.9 to 105.8%. Moliniella anceps, O. ranae, and P. multiglandularis were detected in the environmental water samples through real-time PCR. Thus, we anticipate that our approach will be beneficial for biomonitoring, measuring, and managing ecological systems.

Proterodiplostomid no longer: Molecular phylogeny reveals the true position of Proalarioides (Digenea: Diplostomoidea).

Proalarioides Yamaguti, 1933 (Digenea Carus, 1863: Diplostomoidea Poirier, 1886) is a small genus of proterodiplostomids parasitic in the intestines of snakes in Asia. Only two species are considered valid: Proalarioides serpentis Yamaguti, 1933 and Proalarioides tropidonotis Vidyarthi, 1937. Unlike other proterodiplostomids, Proalarioides spp. possess pseudosuckers and lack the paraprostate, otherwise extremely characteristic of the Proterodiplostomidae Dubois, 1936. In the present study, we describe the morphology of progenetic metacercariae of a Proalarioides sp. from bicolored frog, Clinotarsus curtipes (Jerdon), collected in India and provide the first DNA sequences from any member of the genus. These specimens differ from previously described metacercariae and adults of P. serpentis and P. tropidonotis in several ways, including body and organ sizes, sucker ratios, and distribution of vitellarium. The newly generated partial large ribosomal subunit (28S) rRNA gene sequence was used to test the phylogenetic position of the genus among other major lineages of diplostomoideans. Our 28S phylogeny clearly demonstrated Proalarioides sp. to be well-separated from other members of the Proterodiplostomidae. Based on morphological and molecular evidence, we transfer Proalarioides out of the Proterodiplostomidae into the Diplostomidae Poirier, 1886.

Prevalence and molecular characterization of Hysterothylacium species infecting Pandora (Pagellus erythrinus) in the Mediterranean Sea of Egypt.

Species of the genus Hysterothylacium are aquatic roundworms (nematodes) belonging to the family Raphidascarididae. Some species in this family are known to be associated with zoonotic diseases in humans after they consume their parasitic larvae in raw or undercooked fish. The aim of this research was to report the prevalence, morphology, and molecular characteristics of Hysterothylacium species in Pagellus erythrinus. A total of Two hundred fish were purchased from the fish market in Damanhour, Beheira Province, between December 2021 and November 2022 and subjected to examination. For molecular characterization, the internal transcribed spacer (ITS) region of nuclear ribosomal DNA and the mitochondrial cytochrome oxidase subunit 2 (COX-2) gene were used. Hysterothylacium species were morphologically described and identified from the intestine of Pagellus erythrinus in Beheira Province, Egypt. The PCR amplified 1087 bp and 629 bp of the target sequences of the ITS region and COX-2 gene, respectively. Sequence analysis revealed the Hysterothylacium thalassini species. The identified species provided novel biological data for the Hysterothylacium nematode in Pagellus erythrinus. The prevalence of Hysterothylacium species recovered from the intestine was 55%. The highest prevalence of 72% has been reported in summer compared to the lowest prevalence of 38% in the winter. Females had a higher prevalence of 61.8% than males, with 44.2%. The first detection, prevalence, and molecular characterization of H. thalassini in Pagellus erythrinus from Beheira Province, Egypt, was presented in this study.

The vicuna (Vicugna vicugna) as a natural host of Dictyocaulus filaria in Peru.

Lungworm infection, or verminous pneumonia, is a parasitic disease that causes serious problems in small and large ruminants. Despite the fact that nematodes of the genus Dictyocaulus in cattle and sheep are the main cause of this disease, there are few studies on the natural infections of South American camelids. For this reason, this study aims to report the natural infection by Dictyocaulus filaria in vicunas (Vicugna vicugna) for the first time. During a shearing season (chaku) in Cuzco, Peru, two accidentally killed adult vicunas were submitted to the IVITA-Marangani research center in Cuzco for their respective necropsies. The tracheas of both vicunas had numerous nematodes, as seen during the necropsy. The nematodes were collected in 70% ethanol and were morphologically identified as D. filaria. Likewise, the DNA of six nematodes was extracted, and the ITS2 region and the 28S rRNA gene were amplified and sequenced. The nucleotide sequences of both genetic markers were up to 100% identical with previously reported D. filaria DNA sequences found in the goat yearlings from Turkey, sheep from Iran, Turkey, and India, and the argali from Uzbekistan, which confirmed the morphological diagnosis. This finding represents the first molecular confirmation of a natural D. filaria infection in a South American camelid. It will be necessary to carry out future studies to know the current situation of verminous pneumonia in domestic and wild South American camelids and to know the negative effects of the disease on them.

Description of three new species of Gyrodactylus von Nordmann, 1832 (Monogenea: Gyrodactylidae) on bitterling fishes (Acheilognathinae) from China.

Three new species of Gyrodactylus are described from three species of bitterling in Donghu Lake, China: Gyrodactylus ocellorhodei n. sp. from Rhodeus ocellatus; G. sinenorhodei n. sp. from Rhodeus sinensis; and G. acheilorhodei n. sp. from Acheilognathus macropterus. All the three new species showed similar opisthaptor morphology, especially the marginal hooks: all had a slender and perpendicular sickle shaft, and flat sickle base with distinct heel and inner arch which was different from the G. rhodei-group species parasitic on bitterling. Multivariate analyses based on hamulus and marginal hooks suggested that these three new species cannot be completely distinguished, despite some morphology divergence observed in certain less reliable morphometric features, such as hamulus root length, ventral bar total length and process shape. These three new species shared an identical 18S ribosomal RNA gene sequence, while the variation in the Internal Transcribed Spacers (ITS1-ITS2) sequence among them (8.4-11.2%, K2P) far exceeded the 1% ITS sequence difference that had been suggested as a threshold for species delimitation of Gyrodactylus. Phylogenetic analysis based on ITS1-ITS2 showed that all these sequenced Gyrodactylus spp. parasitic on the subfamily Acheilognathinae host formed a monophyletic group. However, a clear differentiation (18.9-20.9%, K2P of ITS1-ITS2) could be found between the subgroup from China (G. ocellorhodei n. sp., G. sinenorhodei n. sp. and G. acheilorhodei n. sp.) and that from Europe (G. rhodei).

Identification of third stage larvae of strongyles and molecular diagnosis of Strongylus vulgaris in the feces of Thoroughbred horses kept in training centers in Rio de Janeiro, Brazil.

The aims of the present study were to identify strongyles in the feces of Thoroughbred horses based on larval morphology; to detect Strongylus vulgaris using molecular diagnosis and compare results to those of feces culture; and to determine the association between the presence of S. vulgaris with corresponding animal information (age range, gender, and anthelmintic use). Feces of horses kept in six Training Centers in Rio de Janeiro State, that showed the presence of ≥500 eggs per gram of feces (EPG) were subjected to strongyle identification. Of the 520 fecal samples collected, 35 had an EPG ≥ 500. After fecal culture for L3 larvae identification, DNA was extracted, subjected to PCR to amplify the ITS2 region DNA fragment of S. vulgaris, and sequenced. A total of 3500 larvae were analyzed. Most were classified as small strong (99.7%), with an emphasis on the type A subfamily of Cyathostominae. Forms of S. vulgaris only corresponded to 0.2%. In all, 25 samples showed amplified S. vulgaris DNA products and 11 showed nucleotide sequences with high sequence identity. Fecal culture and PCR results showed poor agreement (kappa = 0.105) for S. vulgaris diagnosis. Age, gender, anthelmintic use, and anthelmintic administration interval were not statistically significant. The present study showed the presence of S. vulgaris in the feces of horses kept in Rio de Janeiro Training Centers, mainly seen via PCR, which has emerged as the most effective tool for diagnosis. This study made it possible to identify strongyles that infect horses in the region, emphasizing upon the necessity for constant monitoring of the animals.

Estimating Prevalence and Infection Intensity of Soil-Transmitted Helminths Using Quantitative Polymerase Chain Reaction and Kato-Katz in School-Age Children in Angola.

Quantitative polymerase chain reaction (qPCR) is gaining recognition in soil-transmitted helminth (STH) diagnostics, especially for Strongyloides stercoralis and differentiating hookworm species. However, sample preservation and DNA extraction may influence qPCR performance. We estimated STH prevalence and infection intensity by using qPCR in schoolchildren from Huambo, Uige, and Zaire, Angola, and compared its performance with that of the Kato-Katz technique (here termed Kato-Katz). Stool samples from 3,063 children (219 schools) were preserved in 96% ethanol and analyzed by qPCR, of which 2,974 children (215 schools) had corresponding Kato-Katz results. Cluster-adjusted prevalence and infection intensity estimates were calculated by qPCR and Kato-Katz, with cycle threshold values converted to eggs per gram for qPCR. Cohen's kappa statistic evaluated agreement between qPCR and Kato-Katz. DNA extraction and qPCR were repeated on 191 (of 278) samples that were initially qPCR negative but Kato-Katz positive, of which 112 (58.6%) became positive. Similar prevalence for Ascaris lumbricoides (37.5% versus 34.6%) and Trichuris trichiura (6.5% versus 6.1%) were found by qPCR and Kato-Katz, respectively, while qPCR detected a higher hookworm prevalence (11.9% versus 2.9%). The prevalence of moderate- or high-intensity infections was higher by Kato-Katz than by qPCR. Agreement between qPCR and Kato-Katz was very good for A. lumbricoides, moderate for T. trichiura, and fair for hookworm. Strongyloides stercoralis prevalence was 4.7% (municipality range, 0-14.3%), and no Ancylostoma ceylanicum was detected by qPCR. Despite suboptimal performance, presumably due to fixative choice, qPCR was fundamental in detecting S. stercoralis and excluding zoonotic A. ceylanicum. Further evaluations on sample fixatives and DNA extraction methods are needed to optimize and standardize the performance of qPCR.

First molecular identification of Haemonchus contortus (Nematoda: Trichostrongylidae), a blood-sucking gastric nematode of artiodactyles, in the ground beetle Carabus granulatus (Coleoptera: Carabidae).

Among gastrointestinal nematodes, Haemonchus contortus (Rudolphi) Cobb (order Strongylidae; family Trichostrongylidae) is one of pathogenic and economic importance in domestic and wild ruminants, including the European bison, Bison bonasus Linnaeus (order Cetartiodactyla; family Bovidae); a species on the International Union for Conservation of Nature's Red List of Threatened Species. Carabus granulatus Linnaeus (order Coleoptera; family Carabidae) is one of the most prevalent species of ground beetle, inhabiting a wide range of terrestrial ecosystems in Poland. Twenty-six ground beetles of this species inhabiting the Bialowieza Primeval Forest in eastern Poland were screened for the presence of DNA of pathogenic gastrointestinal nematodes of ruminants. Extracted DNA was sequenced and compared to reference sequences. In six insects, the presence of H. contortus DNA was detected. The obtained nucleotide sequences were homologous to each other and to the majority of the published DNA sequences of H. contortus isolates. The sequences were also identical to a sequence of H. contortus isolated from European bison in Poland. The study provides the first molecular evidence of the presence of H. contortus DNA in C. granulatus. The finding suggests that ground beetles may play a role in the transmission dynamics of this parasite.

Lepocreadiidae (Trematoda) associated with gelatinous zooplankton (Cnidaria and Ctenophora) and fishes in Australian and Japanese waters.

We examined gelatinous zooplankton from off eastern Australia for lepocreadiid trematode metacercariae. From 221 specimens of 17 species of cnidarian medusae and 218 specimens of four species of ctenophores, infections were found in seven cnidarian and two ctenophore species. Metacercariae were distinguished using cox1 mtDNA, ITS2 rDNA and morphology. We identified three species of Prodistomum Linton, 1910 [P. keyam Bray & Cribb, 1996, P. orientale (Layman, 1930), and Prodistomum Type 3], two species of Opechona Looss, 1907 [O. kahawai Bray & Cribb, 2003 and O. cf. olssoni], and Cephalolepidapedon saba Yamaguti, 1970. Two species were found in cnidarians and ctenophores, three only in cnidarians, and one only in a ctenophore. Three Australian fishes were identified as definitive hosts; four species were collected from Scomber australasicus and one each from Arripis trutta and Monodactylus argenteus. Transmission of trematodes to these fishes by ingestion of gelatinous zooplankton is plausible given their mid-water feeding habits, although such predation is rarely reported. Combined morphological and molecular analyses of adult trematodes identified two cox1 types for C. saba, three cox1 types and species of Opechona, and six cox1 types and five species of Prodistomum of which only two are identified to species. All three genera are widely distributed geographically and have unresolved taxonomic issues. Levels of distinction between the recognised species varied dramatically for morphology, the three molecular markers, and host distribution. Phylogenetic analysis of 28S rDNA data extends previous findings that species of Opechona and Prodistomum do not form monophyletic clades.

Molecular and morphological evidence suggests the reallocation from Parastrigea brasiliana (Szidat, 1928) Dubois, 1964 to Apharyngostrigea Ciurea, 1927 (Digenea: Strigeidae), a parasite of boat-billed heron (Cochlearius cochlearius) from the Neotropical region.

Parastrigea brasiliana (Szidat, 1928) Dubois, 1964, was described from (Cochlearius cochlearius) in South America. The taxonomy of this species has been unstable due that it was described as a member of Strigea Abildgaard, 1790. However, the same author one year later transferred it to Apharyngostrigea Ciurea, 1927 and since then, it has been alternatively placed in the genus Apharyngostrigea or Parastrigea Szidat, 1928 from Strigeidae. In the current research, specimens identified as P. brasiliana were collected from type host in southeastern Mexico. We sequenced three molecular markers: the internal transcribed spacers ITS1 and ITS2 including the 5.8S gene (ITS region), the D1-D3 domains of the large subunit (LSU) from nuclear DNA and cytochrome c oxidase subunit I (cox 1) from mitochondrial DNA. These sequences were aligned with other sequences available in the GenBank dataset from Strigeidae. Maximum likelihood and Bayesian analyses inferred with three molecular markers consistently showed that P. brasiliana is not closely related to other members of the genus Parastrigea and are placed in a reciprocal monophyletic clade inside Apharyngostrigea, with very low genetic divergence, varying from 0 to 0.09% for the ITS, from 0 to 0.08% for the LSU and from 0.21 to 0.43% for cox 1. Consequently, we proposed to reallocate it to A. brasiliana. The phylogenetic analyses obtained are key and very useful for re-evaluate the morphology of A. brasiliana because this species share morphological characters with the genera Parastrigea (concentration of vitelline follicles distributed in two lateral expansions on the forebody) and Apharyngostrigea (absence of pharynx). Finally, the current record of A. brasiliana expands its distribution range in four countries, namely, the USA, Mexico, Venezuela and Brazil, in the Neotropical region.

Integrative taxonomy of Mesocriconema onoense (Tylenchida: Criconematidae) from Vietnam highly suggests the synonymization of Mesocriconema brevistylus and related species.

The genus Mesocriconema is one of the most diverse genera within the family Criconematidae, known as ring nematodes, with more than 90 species. Although species in this genus usually show distinct morphological characterizations, the identification based only on morphology can lead to misidentification in many studies resulted in a number of synonymizations in the genus over time. In this study, an integrated approach has been applied in characterizing Mesocriconema onoense from Vietnam. The molecular data of 28S rRNA, ITS, 18S rRNA regions were analyzed and discussed to confirm the correct names on GenBank. Besides, phylogenetic analyses of 28S rRNA, ITS, and 18S rRNA regions of Mesocriconema species revealed that Mesocriconema brevistylus should be considered as a junior synonym of M. onoense. Consequently, M. helicus, M. onostris, and M. paronostris should also be considered as the synonyms of M. onoense.

Detection of six soil-transmitted helminths in human stool by qPCR- a systematic workflow.

Soil-transmitted helminths (STH) infect up to one-quarter of the global population, with a significant associated disease burden. The main human STH are: Ancylostoma spp. and Necator americanus (hookworms); Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis. The aim of this study was to establish a scalable system for stool STH multiplex quantitative real-time polymerase chain reactions (qPCR). Stool samples collected in Fiji and preserved in potassium dichromate were transferred to Melbourne at ambient temperature. Samples were washed to remove potassium dichromate and DNA was extracted with the Mini-Beadbeater-24 and a column-based kit. A SYBR green qPCR to detect the vertebrate mitochondrial gene was used as a DNA extraction control. Samples were tested using a probe-based multiplex qPCR targeting A. lumbricoides, T. trichiura and S. stercoralis, and in a second multiplex reaction to detect hookworms to the species level (A. duodenale, A. ceylanicum, N. americanus). An internal amplification control in both multiplex assays was included to prevent false-negative results due to PCR inhibitors. Samples were homogenised for a single cycle of 40 seconds to release STH DNA and washed stool was stored for up to 15 weeks at -30°C without compromising DNA. Our multiplex qPCR detected multiple species of STH without reduced sensitivity compared to singleplex. qPCR data from 40 stools was validated against STH-positive stools determined by microscopy. We have developed and validated an efficient and staged system for detecting six clinically important STH affecting humans that could be easily implemented without advanced automation in any qPCR-capable laboratory.

High-throughput sequencing of Fasciola spp. shows co-infection and intermediate forms in Balochistan, but only Fasciola gigantica in the Punjab province of Pakistan.

Fasciola gigantica and Fasciola hepatica are digenetic trematodes causing fasciolosis in ruminants. The host and geographical distribution of both Fasciola species are influenced by environmental and climatic conditions favouring survival and development of free-living stages and intermediate hosts, and livestock management practices. The aim of the present study was to describe the host distribution of the two Fasciola species in buffalo, cattle, goats, and sheep in the Balochistan and Punjab provinces of Pakistan. 359 flukes were collected from a total of 32 livers from the four livestock species. Deep amplicon sequencing of the internal transcribed spacer region 2 of ribosomal DNA (rDNA ITS-2) and mitochondrial nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND-1) loci confirmed co-infection of F. hepatica and F. gigantica in Balochistan and single species F. gigantica infection in Punjab. In Balochistan, co-infections and hybrids of both Fasciola species were identified in cattle, with more F. hepatica detected than F. gigantica. However, F. hepatica was the only species identified in goats, and F. gigantica was the only species identified in buffalo. In Punjab, all flukes were confirmed as F. gigantica in each of the four livestock species. Overall, the results indicate differences in the host and geographical distribution of F. gigantica and F. hepatica, and provide useful knowledge for the development of control strategies for livestock and humans.

Redescription of Paradiplozoon opsariichthydis (Jiang, Wu et Wang 1984) Jiang, Wu et Wang, 1989 (Monogenea, Diplozoidae).

Paradiplozoon opsariichthydis (Jiang, Wu et Wang, 1984) Jiang, Wu et Wang, 1989 (Platyhelminthes, Monogenea, Diplozoidae) is blood-feeding parasite from the gills of Asian cyprinid fish Opsariichthys bidens Günther, 1873. In this study, we present a morphological redescription of P. opsariichthydis neotype main morphological features e.g. size of body and clamps due to the fact that the type material is missing. We decided to supplement morphological descriptions by the relevant molecular data (internal transcribed spacer - ITS2) related to P. opsariichthydis adult worm isolates and other representatives of genus Paradiplozoon to cross verify our findings. In addition to that, this study also brings an attention to the host identification. Thus, parasite data were complemented by the determinant cytochrome oxidase b (cytb) sequences of its hosts. All novel sequences are deposited in GenBank. This combination of the morphological and molecular data related to both the parasite and its host seems to be the optimal approach to the general process of (re)description of highly host-specific parasitic organisms, which can then lead to a meaningful phylogenetic analysis.

Morphological and molecular data of new species of Characithecium and Diaphorocleidus (Monogenea: Dactylogyridae) from Neotropical characid fishes.

The present study describes three new species of monogenean parasites of characid fishes from the Upper Paraná River basin, Brazil: Characithecium paranapanemense n. sp. on Psalidodon paranae and Psalidodon bockmanni, Diaphorocleidus magnus n. sp. on Astyanax lacustris and Psalidodon fasciatus, and Diaphorocleidus neotropicalis n. sp. on Astyanax lacustris and P. bockmanni. An amendment for Diaphorocleidus is proposed, since additional characters observed in the new species required to extend the generic diagnostic features mainly to include: articulation process connecting the base of the MCO with accessory piece present or absent, and accessory piece with variable shapes (plate-like, pincer-shaped, wrench-shaped, sheath-shaped), divided or not into subunits. Characithecium paranapanemense n. sp. can be distinguished from other congeners by the morphology of its MCO and accessory piece. Diaphorocleidus magnus n. sp. differs from most of its congeners by the morphology of its accessory piece, the presence of articulation process connecting the base of the MCO with accessory piece, and the morphology of the sclerotized structures of the haptor. Diaphorocleidus neotropicalis n. sp. can be easily distinguished from its congeners by the morphology of the accessory piece, the sclerotized structures of the haptor and the morphology of the vagina. Molecular data of the new species (partial 28S rDNA and mitochondrial cytochrome oxidase I) were obtained and the first phylogenetic analysis based on 28S rDNA gene sequences for species of Characithecium and Diaphorocleidus are provided. Although Diaphorocleidus and Characithecium share some morphological similarities, phylogenetic analysis indicates that species of these two genera are not closely related.

Pulmonary hydatidosis genotypes isolates from human clinical surgery based on sequencing of mitochondrial genes in Fars, Iran.

BACKGROUND: Cystic echinococcosis (CE)/hydatidosis is an important neglected parasitic zoonotic disease caused by the metacestode of Echinococcus granulosus s.l. The present study was designed to identify the pulmonary CE species/genotypes in isolated human underwent to surgery in our center in Southern Iran. METHODS: The study population of this study were all patients in Fars province who were admitted to Namazi Hospitals for pulmonary hydatid cyst surgery. Thoracic surgery was performed in the thoracic ward and the cyst/s was removed by open surgery via posterolateral or lateral thoracotomy. DNA was extracted from the germinal layer or the protoscoleces. PCR technique was performed using the cytochrome C oxidase subunit1 (cox1) gene, and the products were sequenced. RESULTS: A total of 32 pulmonary hydatid cyst samples were collected from 9 (28%) female and 23 (72%) male aged from 4 to 74 years old. A total of 18(56%) cyst/s were in the left lobe and 14 (44%) cysts in the right lobe. Sequence analysis of the cysts showed that 24 samples (75%) were E. granulosus s.s (G1-G3) genotype and 8 (25%) were E. canadensis (G6/G7) genotype. CONCLUSION: E.granulosus s.s genotype was the most prevalent genotype followed by E. canadensis (G6/G7) genotype. There was no significant statistical correlation between cysts' size, location, genotype strain, and patients' age and gender.

Systematics of Crepidostomum species from the Russian Far East and northern Japan, with description of a new species and validation of the genus Stephanophiala.

Current article touched upon the issue of the complicated taxonomic status of some species from the genus Crepidostomum collected from the freshwater fish in the rivers of Primorsky region, Sakhalin, and Hokkaido Islands. Primary morphological analyses showed affiliation of the worms to the species C. farionis (Müller, 1784) Lühe, 1909; C. metoecus Braun, 1900b; C. chaenogobii Yamaguti and Matsumura, 1942; C. nemachilus Krotov, 1959. We described the new species Crepidostomum achmerovi sp. nov. that is a sibling species of C. nemachilus. Molecular-genetic investigation have shown that C. nemachilus and C. achmerovi sp. nov. are closely related to C. metoecus in both 28S rDNA and cox1 mtDNA markers. Crepidostomum nemachilus forms a separate branch within the C. metoecus clade on the 28S BI tree with strong statistical support and separate clade in relation to C. metoecus clade on the cox1 BI tree. Values of p-distances between Crepidostomum species were at intergeneric level. Crepidostomum metoecus species complex including five species (C. metoecus, C. nemachilus, C. oschmarini, C. brinkmanni, and C. achmerovi sp. nov.) was reconsidered as independent genus Crepidostomum sensu stricto. Minimum Spanning Network showed that C. nemachilus, C. metoecus and C. achmerovi sp. nov. were separated by large number of mutational events and represent independent phyletic lines. An amended diagnosis is provided for the subfamily Crepidostomatinae, the genera Crepidostomum s. str. and Stephanophiala Nicoll, 1909, along with keys to species of both genera.

A review of molecular identification tools for the opisthorchioidea.

The superfamily Opisthorchioidea encompasses the families Cryptogonimidae, Opisthorchiidae and Heterophyidae. These parasites depend on the aquatic environment and include marine and freshwater species. Some species, such as Clonorchis sinensis and Opisthorchis viverrini, have a high impact on public health with millions of infected people worldwide and have thus been the object of many studies and tool developments. However, for many species, tools for identification and detection are scarce. Although morphological descriptions have been used and are still important, they are often not efficient on the immature stages of these parasites. Thus, during the past few decades, molecular approaches for parasite identification have become commonplace. These approaches are efficient, quick and reliable. Nonetheless, for some parasites of the superfamily Opisthorchioidea, reference genomic data are limited. This study reviews available genetic data and molecular tools for the identification and/or the detection of this superfamily. Molecular data on this superfamily are mostly based on mitochondrial and ribosomal gene sequence analyses, especially on the cytochrome c oxidase subunit 1 gene and internal transcribed spacer regions respectively.