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.

DEVELOPMENT OF A TRIPLEX REAL-TIME PCR ASSAY TO DETECT ECHINOCOCCUS SPECIES IN CANID FECAL SAMPLES.

Echinococcosis is a zoonotic disease with great significance to public health, and appropriate detection and control strategies should be adopted to mitigate its impact. Most cases of echinococcosis are believed to be transmitted by the consumption of food and/or water contaminated with canid stool containing Echinococcus spp. eggs. Studies assessing Echinococcus multilocularis, Echinococcus granulosus sensu stricto, and Echinococcus shiquicus coinfection from contaminated water-derived, soil-derived, and food-borne samples are scarce, which may be due to the lack of optimized laboratory detection methods. The present study aimed to develop and evaluate a novel triplex TaqMan-minor groove binder probe for real-time polymerase chain reaction (rtPCR) to simultaneously detect the 3 Echinococcus spp. mentioned above from canid fecal samples in the Qinghai-Tibetan Plateau area (QTPA). The efficiency and linearity of each signal channel in the triplex rtPCR assay were within acceptable limits for the range of concentrations tested. Furthermore, the method was shown to have good repeatability (standard deviation ≤0.32 cycle threshold), and the limit of detection was estimated to be 10 copies plasmid/µl reaction. In summary, the evaluation of the present method shows that the newly developed triplex rtPCR assay is a highly specific, precise, consistent, and stable method that could be used in epidemiological investigations of echinococcosis.

Molecular Identification of the Human Pathogen Amphimerus sp. in the Freshwater Snail Aroapyrgus sp. in Ecuador.

Here, we report for the first time the snail intermediate host for the Amphimerus liver fluke, a foodborne trematodiasis. In Ecuador, Amphimerus of the Opisthorchiidae family, infects humans, cats, and dogs, in the tropical Pacific-coast region. Opisthorchiidae comprising also Clonorchis sinensis, Opisthorchis sp., and Metorchis sp., have complex life cycles involving a definitive and two intermediate hosts. We identified morphologically and investigated the presence and prevalence of Amphimerus cercaria and DNA in freshwater snails collected in a human-amphimeriasis endemic region in Ecuador, extracted DNA from snail tissue and emerged cercariae, performed real-time polymerase chain reaction (PCR) with the newly developed primers and probe amplifying the Amphimerus ribosomal internal transcribed spacer 2 (ITS2) region, and sequenced the amplified DNA fragment. We collected 2,800 snails, characterized four species Aroapyrgus sp., Melanoides tuberculata, Biomphalaria cousini, and Aplexa marmorata, isolated three cercariae morphotypes. Of the 640 snails analyzed by qPCR, only Aroapyrgus and one of the three cercariae resulted positive, at a 15% infection prevalence. Polymerase chain reaction revealed that the Aroapyrgus snail and cercaria-morphotype-3 corresponded to Amphimerus, but not to C. sinensis, Fasciola hepatica, or Paragonimus mexicanus. The sequence of amplified DNA product matched that of human-isolated Amphimerus. This finding constitutes the first documentation that Aroapyrgus sp. is the first intermediate host for the Amphimerus sp. that infect humans in Ecuador. The ITS2-gene PCR and sequencing analysis demonstrated a high prevalence of snail infection and proved useful for detecting the infection in snails, which findings can help the establishment of suitable control programs against transmission in any endemic region of interest.

MOLECULAR IDENTIFICATION OF JUVENILE NEOECHINORHYNCHUS SPP. (PHYLUM: ACANTHOCEPHALA) INFECTING OSTRACOD AND SNAIL HOSTS PROVIDES INSIGHT INTO ACANTHOCEPHALAN HOST USE.

The role of invertebrates in some acanthocephalan life cycles is unclear because juvenile acanthocephalans are difficult to identify to species using morphology. Most reports suggest acanthocephalans from turtle definitive hosts use ostracods as intermediate hosts and snails as paratenic hosts. However, laboratory studies of the life cycle suggest that ostracods and snails are both required hosts in the life cycle. To elucidate the role of ostracods and snails in acanthocephalan life cycles better, we collected 558 freshwater snails of 2 species, including Planorbella cf. Planorbella trivolvis and Physa acuta, from 23 wetlands in Oklahoma, U.S.A., and examined them for acanthocephalan infections. Additionally, we examined 37,208 ostracods of 4 species, Physocypria sp. (morphotype 1), Cypridopsis sp., Stenocypris sp., and Physocypria sp. (morphotype 2) for juvenile acanthocephalans from 2 wetlands in Oklahoma. Juvenile acanthocephalans were morphologically characterized, and the complete internal transcribed spacer (ITS) region of nuclear rDNA was sequenced from acanthocephalans infecting 11 ostracod and 13 snail hosts. We also sampled 10 red-eared slider turtles, Trachemys scripta elegans, and 1 common map turtle, Graptemys geographica, collected from Oklahoma, Arkansas, and Texas and recovered 1,854 adult acanthocephalans of 4 species. The ITS of 17 adult acanthocephalans of 4 species from turtle hosts were sequenced and compared to juvenile acanthocephalan sequences from ostracod and snail hosts from this study and GenBank to determine conspecificity. Of the 23 locations sampled for snails, 7 (30%) were positive for juvenile acanthocephalans in the genus Neoechinorhynchus. The overall prevalence and mean intensity of acanthocephalans in Planorbella cf. P. trivolvis and P. acuta were 20% and 2 (1-6) and 2% and 1 (1), respectively. In contrast, only 1 of 4 species of ostracods, Physocypria sp. (morphotype 1), was infected with larval/juvenile Neoechinorhynchus spp. with an overall prevalence of 0.1% and a mean intensity of 1 (1-2). Although 4 species of acanthocephalans infected turtle definitive hosts, including Neoechinorhynchus chrysemydis, Neoechinorhynchus emydis, Neoechinorhynchus emyditoides, and Neoechinorhynchus pseudemydis, all the ITS sequences from cystacanths infecting snail hosts were conspecific with N. emydis. In contrast, the ITS sequences from larval/juvenile acanthocephalans from ostracods were conspecific with 2 species of acanthocephalans from turtles (N. emydis and N. pseudemydis) and 1 species of acanthocephalan from fish (Neoechinorhynchus cylindratus). These results indicate that N. emydis infects freshwater snails, whereas other species of Neoechinorhynchus appear not to infect snail hosts. We document new ostracod and snail hosts for Neoechinorhynchus species, including the first report of an ostracod host for N. pseudemydis, and we provide novel molecular barcodes that can be used to determine larva, juvenile, and adult conspecificity of Neoechinorhynchus species.

Evaluation of targets for Strongyloides genus specific molecular diagnosis in experimental strongyloidiasis.

Strongyloides venezuelensis has been used in different experimental studies, such as those aimed at the evaluation of diagnostic techniques for human strongyloidiasis, mainly the molecular diagnosis. In this study, three regions (genus, 18S and 28S targets) of Strongyloides ribosomal DNA were evaluated for the molecular diagnosis of experimental strongyloidiasis. Rats were infected subcutaneously with 400 or 4000 S. venezuelensis infective larvae (400iL3 and 4000iL3), and kept for 35 days. Fecal samples were collected daily to count eggs per gram of feces (EPG) and to perform the polymerase chain reaction (PCR). Egg count started on the 5th day post-infection (pi) and ended on days 33 and 34 pi, in 400iL3 and 4000iL3 groups, respectively. Based in EPG, fecal samples were selected from days 2, 5, 8, 11, 15, 23 and 35 pi for DNA extraction; PCR (genus, 18S and 28S); and sequencing. The PCR-28S products showed higher values of identity (95-100%) in the database with the Strongyloides sequences. Therefore, it is possible to reinforce the application of PCR-28S in the diagnosis of experimental and human strongyloidiasis.

REDESCRIPTION OF CATHARIOTREMA SELACHII (MACCALLUM, 1916) JOHNSTON AND TIEGS, 1922 (MONOGENOIDEA: MONOCOTYLIDAE), EMENDATION OF MONOTYPIC CATHARIOTREMA JOHNSTON AND TIEGS, 1922, AND PROPOSAL OF CATHARIOTREMATINAE N. SUBFAM. BASED ON MORPHOLOGICAL AND NUCLEOTIDE EVIDENCE.

We herein redescribe the enigmatic Cathariotrema selachii (MacCallum, 1916) Johnston and Tiegs, 1922 based on the holotype, paratypes, and newly collected specimens infecting the olfactory organ of 5 shark species from the Gulf of Mexico (all new host records): scalloped hammerhead shark, Sphyrna lewini (Griffith and Smith, 1834) (Carcharhiniformes: Sphyrnidae); great hammerhead shark, Sphyrna mokarran (Rüppell, 1837); blacktip shark, Carcharhinus limbatus (Müller and Henle, 1839) (Carcharhiniformes: Carcharhinidae); spinner shark, Carcharhinus brevipinna (Müller and Henle, 1839); and Atlantic sharpnose shark, Rhizoprionodon terraenovae (Richardson, 1836) (Carcharhinidae). These specimens were morphologically indistinguishable from each other and from MacCallum's holotype and paratypes. Those sequenced had identical first internal transcribed spacer (ITS1) and large subunit ribosomal DNA (28S) nucleotide sequences. As such, C. selachii infects sharks of 2 orders (Carcharhiniformes, Lamniformes) and 3 families (Carcharhinidae, Sphyrnidae, Lamnidae) in the Northwestern Atlantic Ocean (type locality) and Gulf of Mexico (new records herein). This report is the first of new specimens of C. selachii in the Atlantic Ocean Basin in 95 yr and corrects long-standing error cascades and ambiguities concerning the morphology and systematic placement of C. selachii. Considering morphology and nucleotide-based phylogenetic evidence (28S, Bayesian analysis), we herein emend monotypic CathariotremaJohnston and Tiegs, 1922 and propose Cathariotrematinae Bullard n. subfam. for it and 4 other genera (all formerly assigned to Merizocotylinae Johnston and Tiegs, 1922). These genera comprise species infecting only the nose of sharks (monotypic Cathariotrema, SqualotremaKearn and Green, 1983 and SeptitremaKheddam, Chisholm, and Tazerouti, 2020 plus 3 species of TriloculotremaKearn, 1993) and nose of a chimaera (monotypic HolocephalocotyleDerouiche, Neifar, Gey, Justine, and Tazerouti, 2019). Cathariotrematinae differs from Merizocotylinae by having a 3-part attachment organ and by lacking open loculi that symmetrically encircle a cluster of >2 loculi in the center of the haptor. Monophyletic Cathariotrematinae (with sequences representing species of Cathariotrema, Triloculotrema, and Holocephalocotyle only) was sister to monophyletic Merizocotylinae, which together were sister to monophyletic Calicotylinae Monticelli, 1903. These subfamilies comprise a monophyletic group of monocotylids that have a double vagina and infect extrabranchial, enclosed niches (urogenital system, body cavity, olfactory chamber/nose) on their shark, ray, and chimaera hosts (all other monocotylids have a single vagina and infect the gill or body surfaces of rays only). Monocotylinae Taschenberg, 1879 and Decacotylinae Chisholm, Wheeler, and Beverley-Burton, 1995 were recovered as monophyletic. Heterocotylinae Chisholm, Wheeler, and Beverley-Burton, 1995 remained paraphyletic. We accept ParacalicotyleSzidat, 1970.

First report of marine horsehair worms (Nematomorpha: Nectonema) parasitic in isopod crustaceans.

Nectonema, the only horsehair worm (Nematomorpha) genus found in marine environments, was previously known to be parasitic only in decapod crustaceans. We report Nectonema sp. as the first record of a marine nematomorph parasitic in isopod crustaceans. This is also the third record of marine nematomorphs from the North Pacific. Six infected isopods (Natatolana japonensis) collected from 1425 m of depth in the Sea of Japan each contained one to seven (mean 2.33) nematomorphs in the body cavity in the pereon. There was no correlation between the host body length and number of parasites. For Nectonema sp., we describe and illustrate morphological features of the parasitic juvenile stage and present nucleotide sequences for the cytochrome c oxidase subunit I gene (COI or cox1; 451 nt), 18S rRNA gene (1777 nt), and region spanning the internal transcribed spacer 1 (ITS1) and the 28S rRNA gene including the 5.8S rRNA gene and ITS2 (1218 nt in total). In an 18S maximum-likelihood tree that included 24 nematomorph species, Nectonema sp. grouped with N. agile from the northwestern Atlantic; the 18S gene from these two taxa was divergent by 11.8% K2P distance, suggesting that they are different species. Nectonema species may have a broader range of host groups than previously suspected, but may have been previously misidentified as nematode parasites.

Rapid preparation of gastrointestinal nematode eggs from faeces for PCR identification.

Detection of gastrointestinal nematodes (GIN) as both a qualitative and quantitative test is highly desirable. Methods such as multiplex and qPCR are capable of providing such results, but can be laborious and expensive. This paper presents a rapid, low-cost method of preparing GIN egg from faecal samples that produces DNA suitable for PCR analysis. We also describe a set of primers that are suitable for single-tube multiplex PCR.

Morphological and Molecular Data Reveal Two New Species of Viannaia (Nematoda: Viannaiidae), Parasitizing Opossums (Mammalia: Didelphidae) in Mexico.

Two new species of Viannaia from the intestine of the North American opossums, Didelphis virginiana (Virginia opossum), and Philander opossum (gray four-eyed opossum), are described based on morphological and molecular data, through an integrative taxonomic approach. Maximum likelihood and Bayesian inference analyses for each dataset and the concatenated dataset were performed using a mitochondrial cytochrome c oxidase subunit 1 (COI) gene, and the nuclear ribosomal internal transcribed spacer region (ITS1-5.8S-ITS2). The phylogenetic analyses revealed 2 new species that occur in Mexico, one from the western state of Colima and another from the southern state of Chiapas. Our phylogenetic trees for both molecular markers and concatenated datasets yielded similar topologies with high bootstrap values and posterior probabilities. Viannaia is recovered as a monophyletic group, but the family Viannaiidae appears as non-monophyletic, due to the position of Travassostrongylus scheibelorum, similar to previous studies. Finally, the morphology of Viannaia and Hoineffia is discussed.

Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes.

BACKGROUND: The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time. RESULTS: 11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45-11.5 ng/µL, and on Qubit™ ranged between undetectable - 0.962 ng/µL. Median A260/280 ranged between 0.505-3.925, and median A260/230 ranged - 0.005 - 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity. CONCLUSIONS: A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.

Helminth communities of endemic cyprinoids of the Apennine Peninsula, with remarks on ectoparasitic monogeneans, and a description of four new Dactylogyrus Diesing, 1850 species.

The fauna of the Apennine Peninsula is, in comparison to other southern European peninsulas, relatively species-poor regarding the number of endemic cyprinoid species. Nonetheless, the recent introduction of non-native species has significantly increased the total number of freshwater species in this region. Such invasive species may represent a threat to the native fauna, associated among other things with the introduction of non-native parasites with their original hosts.In the present study, we investigated endemic cyprinoid species for the presence of helminth parasites. A total of 36 ectoparasitic monogenean species and five endoparasitic helminth species were collected from ten cyprinoid species in five localities in northern Italy. Out of 20 Dactylogyrus species (gill monogeneans specific to cyprinoids), four were identified as new to science and herein described: Dactylogyrus opertus n. sp. and Dactylogyrus sagittarius n. sp. from Telestes muticellus, Dactylogyrus conchatus n. sp. from T. muticellus and Protochondrostoma genei, and Dactylogyrus globulatus n. sp. from Chondrostoma soetta. All new Dactylogyrus species appear to be endemic to the Apennine Peninsula; however, they share a common evolutionary history with the endemic Dactylogyrus parasitizing cyprinoids of the Balkans. This common origin of cyprinoid-specific parasites supports a historical connection between these two (currently separated) geographical regions.

Rapid parasite detection utilizing a DNA dipstick.

Molecular diagnostics are powerful tools for disease detection but are typically confined to the laboratory environment due to the cumbersome methods required to extract nucleic acids from biological samples. Accurate diagnosis is essential for early detection of parasitic worm infections and for monitoring control programs, particularly during new transmission outbreaks to limit infection spread. We optimized the recently developed DNA dipstick technology to purify Schistosoma japonicum DNA from different life stages in <60 s. We successfully detected DNA from adult worms, eggs and infected snails. The speed and simplicity of this method enables the point-of-care detection of S. japonicum.

Molecular characterization of Cysticercus tenuicollis isolates from sheep in the Nile Delta, Egypt and a review on Taenia hydatigena infections worldwide.

The predator­prey-transmitted cestode Taenia hydatigena infects a wide range of definitive and intermediate hosts all over the world. Domestic and sylvatic cycles of transmission are considered as well. The parasite has considerable economic importance, particularly in sheep. Here, the molecular characters of T. hydatigena cysticerci in sheep from the Nile Delta, Egypt were investigated for the first time. For this purpose, 200 sheep carcasses and their offal were inspected at the municipal abattoir, Dakahlia governorate, Egypt. Cysticerci of T. hydatigena were collected and molecularly characterized employing the mitochondrial 12S rRNA gene. Cysticerci were found in 42 (21%) sheep, mostly attached to the omenti, mesenteries and livers. After molecular confirmation, nine isolates were sequenced displaying six different haplotypes. Analysis of the T. hydatigena 12S rRNA nucleotide sequences deposited in GenBank revealed 55 haplotypes out of 69 isolates, displaying high haplotype (0.797) and low nucleotide (0.00739) diversities. For the Tajima D neutrality index, a negative value (−2.702) was determined, indicating the population expansion of the parasite. Additionally, global data summarized in this study should be useful to set up effective control strategies against this ubiquitous parasite.

Early Detection of Trichinella spiralis DNA in Rat Feces Based on Tracing Phosphate Ions Generated During Loop-Mediated Isothermal Amplification.

Early diagnosis of trichinellosis is still difficult because of the lack of specific symptoms and limited window for serological detection. Here we established an assay based on tracing phosphate ions generated during loop-mediated isothermal amplification (LAMP) to detect Trichinella spiralis DNA in rat feces during its early stage of infection. By targeting a 1.6-kb repetitive element of Tri. spiralis, the assay was able to detect Tri. spiralis DNA in the feces of all infected rats as early as 1 day postinfection (dpi). The positive detection lasted to 7 dpi in the rats infected with 250 muscle larvae, and 21 dpi in the rats infected with 5,000 larvae. The assay was highly sensitive, and could detect 1.7 femtograms (fg) of Tri. spiralis DNA with high specificity, and with no cross reactivity with the DNA from Anisakis pegreffii, Gnathostoma spinigerum, Angiostrongylus cantonensis, Enterobius vermicularis, Schistosoma japonicum, and Trypanosoma evansi. Our present study provided a reliable technique for the early diagnosis of trichinellosis with the advantages of simplicity and speed, as well as high sensitivity and specificity.

Description and Molecular Differentiation of a New Skrjabinoptera (Nematode: Physalopteridae) from Eutropis macularia (Sauria: Scincidae) in North-Central Vietnam.

Skrjabinoptera vietnamensis n. sp. is described from specimens recovered from the stomach of Eutropis macularia in north-central Vietnam. The new species is characterized by the medium-sized male worms (6.7-8.7 mm in length and 154-182 µm in width) relative to known members of the genus, 2 pointed spicules of unequal length (87-112 µm and 56-72 µm in length), and 10 pairs of caudal papillae. Female worms are larger than male worms (10.7-18.4 mm in length and 264-411 µm in width), with the vulva situated in the anterior part, and embryonated, elliptical eggs, 35-46 µm long by 20-24 µm wide. Skrjabinoptera vietnamensis n. sp. represents the ninth species assigned to the genus and the first species recorded from the Oriental region. Partial sequences of the 18S ribosomal RNA gene (rDNA), and cytochrome c oxidase subunit 1 (COI) are provided for the new species. The molecular phylogenetic position of the genus Skrjabinoptera is briefly discussed.

First Record of a Polystome from Alligator Snapping Turtle, Macrochelys temminckii (Cryptodira: Chelydridae) or Mississippi; with Comments on "Neopolystoma orbiculare (Stunkard, 1916)" and Its Junior Subjective Synonyms.

Herein, we describe several newly-collected specimens of Neopolystoma cf. orbiculare from the urinary bladder of 2 alligator snapping turtles, Macrochelys temminckii (Troost in Harland, 1835) (Cryptodira: Chelydridae Gray, 1831) from Comet Lake (30°35'46.94″N, 88°36'3.12″W), Pascagoula River, Mississippi. Our specimens differed from all previous descriptions of N. orbiculare and its junior subjective synonyms by the combination of having intestinal ceca adorned with triangular pockets and that terminate dorsal to the haptor, distinctive hooklets each having a handle and guard of approximately equal length and having a much longer and curved blade, 16 genital coronet spines that each possess 1-2 flanges per spine, pre-testicular vaginal pores, and vaginal ducts that are anterior to the junction of the oviduct and genito-intestinal canal. Some of our specimens were enantiomorphic (4 and 3 had a dextral and sinistral ovary, respectively). Nucleotide sequences (large subunit ribosomal DNA [28S], small subunit ribosomal DNA [18S], and cytochrome oxidase subunit 1 mitochondrial gene [COI]) for our specimens were most similar to GenBank sequences ascribed to N. orbiculare. Single-gene and concatenated phylogenetic analyses confirmed that NeopolystomaPrice, 1939 is polyphyletic and that our isolates share a recent common ancestor with those ascribed to N. orbiculare. This is the first record of a polystomatid from Mississippi, from the Pascagoula River, and from the alligator snapping turtle (and only the second species of Neopolystoma reported from any snapping turtle).

Description and Molecular Differentiation of a New Falcaustra (Nematode: Kathlaniidae) from the Indochinese Water Dragon, Physignathus cocincinus (Squamata: Agamidae) in North-central Vietnam.

Falcaustra vietnamensis n. sp. is described from the small intestine of Physignathus cocincinus from north-central Vietnam. The new species is characterized by the large male worms (20.2-28.8 mm in length and 557-724 µm in width) relative to known members of the genus, 2 sharply pointed alate spicules of equal length (1,128-1,256 µm in length), gubernaculum including 2 separate pieces, 1 ventral with a pointed distal end and 1 dorsal with a blunt distal end (164-192 µm and 155-172 µm in length, respectively), and 12 pairs of caudal papillae. Female worms are larger than male worms (24.2-34.1 mm in length and 532-735 µm in width), with the vulva situated in the posterior half of body, and elliptical eggs, 60-70 µm long by 42-47 µm wide. Falcaustra vietnamensis n. sp. represents the 38th species assigned to the genus and the third species recorded from a lizard host in the Oriental biogeographical region. Partial sequences of the 18S ribosomal RNA gene (rDNA), internal transcribed spacer regions (ITS), and cytochrome c oxidase subunit 1 (COI) are provided for the new species. The molecular phylogenetic position of the genus Falcaustra is briefly discussed.

Investigation of Echinococcus multilocularis in foxes and dogs in Pakistan by detection of copro-DNA.

Alveolar echinococcosis (AE) is a zoonosis caused by Echinococcus multilocularis, a heteroxenous parasite belonging to Cestoda class. AE is currently considered an important public health issue, but epidemiological and notably molecular data from several endemic countries, including Pakistan, are sparse. Here we report the first detection of Echinococcus multilocularis in wildlife from Pakistan after real-time PCR and sequencing confirmation in the faecal samples of three foxes from northern Kaghan and Siran regions. The occurrence is estimated at 4.4% (95% CI 0.9-12.4). In order to go further in the epidemiological investigations on E. multilocularis and due to the potential presence of other Echinococcus species, we suggest the need for further epidemiological surveys targeting E. multilocularis and E. granulosus sensu lato isolates from humans and intermediate hosts as well as definitive hosts from wildlife in Pakistan.

Potential Mosquito Vectors of Dirofilaria immitis and Dirofilaira repens (Spirurida: Onchocercidae) in Aras Valley, Turkey.

Dirofilaria immitis (Leidy, 1856) and Dirofilaria repens (Railliet & Henry, 1911) are mosquito-borne filarial nematodes that primarily affect dogs, causing heartworm disease and subcutaneous dirofilariosis. The canine heartworm is reported in different provinces in Turkey. However, studies about the transmitting mosquito species are limited. Hence, this study aimed to investigate potential vectors of D. immitis and D. repens in Aras Valley, Turkey. In total, 17,995 female mosquitoes were collected from eight villages during three mosquito seasons (2012-2014) in Aras Valley, located in north-eastern Turkey. A total of 1,054 DNA pools (527 abdomen and 527 head-thorax) were tested with Dirofilaria primers by multiplex-polymerase chain reaction (PCR). Aedes caspius was the most abundant species in collection sites with 90%; this was followed by Culex theileri Theobald, 1903 (Diptera: Culicidae) (7.31%), Anopheles maculipennis Meigen 1818 (Diptera: Culicidae) (1.28%), Culex pipiens Linnaeus, 1758 (Diptera: Culicidae) (0.43%), (Anopheles) hyrcanus (Pallas, 1771) (Diptera: Culicidae) (0.37%), Aedes vexans (Meigen, 1830) (Diptera: Culicidae) (0.25%), and Culiseta annulata Schrank, 1776 (Diptera:Culicidae) (0.02%). Dirofilaria immitis and D. repens were detected in mosquito pools from five villages. The total Dirofilaria spp. estimated infection rate was 1.33%. The highest estimated infection rate was found in Ae. vexans (6.66%) and the lowest was in Ae. caspius (1.26%). The results show that An. maculipennis sl, Ae. caspius, Ae. vexans, Cx. theileri and Cx. pipiens are potential vectors of D. immitis and D. repens with DNA in head-thorax pools; An. hyrcanus is also a likely vector, but Dirofilaria DNA was found only in abdomen pools for the study area. This study revealed new potential vector species for D. immitis. Mosquitoes with natural infections of D. repens were reported for the first time in Turkey.