Morphological and molecular description of Armillifer moniliformis larvae isolated from Sri Lankan brown palm civet (Paradoxurus montanus).

We report Armillifer moniliformis species infecting the endemic Sri Lankan brown palm civet (Paradoxurus montanus) from the Knuckles Range Forest Conservation Area, Sri Lanka. Larval stages of A. moniliformis were found during the postmortem of three civet cats found dead. Morphological studies were done by a light microscope and a scanning electron microscope (SEM). Histopathological examination was conducted using tissue samples obtained from the liver. For the molecular analysis, DNA was extracted from the isolated third-stage larvae. The NADH dehydrogenase subunit 5 (ND5) and the second internal transcribed spacer region (ITS-2), a portion of the large subunit nuclear ribosomal DNA (28S), a portion of 18S ribosomal rRNA gene (18S), and cytochrome c oxidase subunit 1 gene (COX1) were amplified using polymerase chain reaction (PCR). Excysted third-stage larvae were observed in the lungs, omentum, the pleural cavity, the abdominal cavity, and the surface of the spleen and the pericardium. Around 88 third-stage larvae were isolated from three civet cats. First-stage larvae in the liver were surrounded by outer fibrous layer over the inner germinal layer and filled with clear fluid. Slight hemorrhage, leukocyte infiltration, and mild hepatocellular degeneration in the liver were observed. The SEM examination indicated the unique oral apparatus comprises the oval-shaped mouth opening in between two pairs of curved, retractile hamuli. The sequences obtained for ND5, ITS-2, 28S, 18S, and COX1 were 301, 382, 325, 414, and 644 bp in length respectively. Morphology, sequence similarity search, sequence alignment, and phylogenetic analysis identified this parasite as A. moniliformis.

Visceral infection by Porocephalus spp. (Pentastomida) in Neotropical wild mammals.

Larval stages of pentastomids were collected from different organs of small mammals from the Peruvian Amazon. These parasitized mammals included: a western Amazonian oryzomys (Hylaeamys perenensis), an elegant oryzomys (Euryoryzomys nitidus), a lowland paca (Cuniculus paca), two kinkajous (Potos flavus), two silvery woolly monkeys (Lagothrix poeppigii) and a brown-mantled tamarin (Leontocebus fuscicollis). Pentastomids were found in the mesentery and parenchyma of the liver and lungs of these animals. All pentastomids were morphologically identified as nymphs of Porocephalus spp. Only the nymphs collected from select animals (the western Amazonian oryzomys, the elegant oryzomys and the brown-mantled tamarin) were analysed molecularly. Molecular analysis was performed amplifying the mitochondrial cytochrome c oxidase subunit I gene from select nymphs collected from the western Amazonian oryzomys, the elegant oryzomys and the brown-mantled tamarin. The nucleotide sequences exhibited 95.8-97.7% similarity between them. Also, these sequences showed an identity of 95.8-97.9% to Porocephalus crotali (GenBank accession numbers MG559647-MG559655). Molecular analysis indicated the presence of at least two Porocephalus species. These findings represent the first record of Porocephalus in these mammals, thus adding new intermediate hosts for this pentastomid genus. This work represents the first molecular data of Porocephalus in a Neotropical climate.

Using lizards to evaluate the influence of average abundance on the variance of endoparasites in semiarid areas: dispersion and assemblage structure.

The distribution of parasites within host populations and communities, and the mechanisms responsible for these patterns, are poorly understood aspects of wildlife parasitology. Here, we evaluate the influence of the average abundance of endoparasite variance, using endoparasites of lizards from the Caatinga domain (semiarid region), north-eastern Brazil. We hypothesized that, due to the high number of generalist endoparasite species, they may occur randomly throughout host populations in an aggregate pattern. In addition, we evaluated the degree to which sample variance is influenced by the average abundance of endoparasite species, patterns of co-occurrence and dominance among endoparasite species and similarities between abundance and the richness of endoparasite infracommunities in several host species. Between September 2015 and February 2016, 2141 lizards (1233 infected) from 16 species were collected from six Caatinga areas. In total, 25,687 endoparasites were collected, which belonged to 13 species including nematodes, pentastomids, cestodes, trematodes and acanthocephalans. Parasite-host associations documented here included 39 newly identified interactions. Endoparasites occurred in a typical aggregate pattern of distribution within their hosts; there was no measurable preference related to the acquisition of hosts by endoparasites. Despite the new records, endoparasites found were commonly associated with lizards in Caatinga environments, which may reflect fauna composed of generalist endoparasite species.

Co-infections in Visceral Pentastomiasis, Democratic Republic of the Congo.

Snakeborne Armillifer pentastomiasis is an emerging human parasitic infection in rural tropical areas where snake meat is eaten. After a series of severe ocular A. grandis larval infections and anecdotal abdominal infection in Sankuru District, Democratic Republic of the Congo, during 2014-2015, we systematically investigated possible pentastomid etiology in patients who underwent surgery in the region. Histologic and molecular analyses by established pentastomid 18S rDNA- and newly developed Armillifer-specific cytochrome oxidase PCRs revealed larval pentastomid lesions in 3.7% of patients. Some persons had A. armillatus and A. grandis co-infections. Another pentastomid larva, Raillietiella sp., was molecularly detected in 1 patient who had concomitant A. grandis and A. armillatus infection. The PCRs used were suitable for detecting pentastomid species even in highly necrotic tissues. Phylogenetic analyses of Armillifer cytochrome oxidase genes detected multiple local strains.

Pentastome assemblages of the Nile crocodile, Crocodylus niloticus Laurenti (Reptilia: Crocodylidae), in the Kruger National Park, South Africa.

Thirty-two specimens of the Nile crocodile, Crocodylus niloticus Laurenti (Reptilia: Crocodylidae), from the Kruger National Park, South Africa, and its vicinity were examined for pentastomid parasites during 1995 to 1999 and 2010 to 2011. Pentastomid parasites occurred throughout the year and were widespread in the study area with an overall prevalence of 97% and an overall mean abundance of 23.4 (0-81). Pentastome assemblages comprised six species in three sebekid genera: Alofia nilotici Riley et Huchzermeyer, 1995, A. simpsoni Riley, 1994, Leiperia cincinnalis Sambon, 1922, Sebekia cesarisi Giglioli in Sambon, 1922, S. minor (Wedl, 1861) and S. okavangoensis Riley et Huchzermeyer, 1995. The possible influence of host age, gender and geographic location (river system) on pentastome prevalence, abundance and species richness was investigated. Generally, neither host age, gender nor locality did affect infracommunities, likely because all hosts examined were adult or subadult and displayed comparable foraging behaviour, resulting in similar exposure pathways to fish intermediate hosts. Additionally, the longevity of pentastomids would contribute to accumulative infections as hosts mature. Structuring of pentastome assemblages was observed in as far as S. minor was the dominant species based on overall prevalence and abundance, followed by the equally common species S. cesarisi and L. cincinnalis. With an overall prevalence ranging from 34% to 41% and relatively low abundances, A. nilotici, A. simpsoni and S. okavangoensis form the rarer component of pentastome communities.

Molecular Diagnosis of Abdominal Armillifer grandis Pentastomiasis in the Democratic Republic of Congo.

Pentastomiasis is an emerging snake-borne parasitic zoonosis in the tropics. We describe a molecular and morphological study to diagnose a cluster of asymptomatic abdominal human infections caused by Armillifer grandis. The findings may indicate a silent epidemic in a rural area where severe symptomatic ocular cases with the same parasite species have recently surfaced. Molecular diagnostics are of increasing importance when patient material from remote areas cannot be thoroughly examined locally for logistic reasons.

An introduced pentastomid parasite (Raillietiella frenata) infects native cane toads (Rhinella marina) in Panama.

The pentastomid parasite, Raillietiella frenata, is native to Asia where it infects the Asian House gecko, Hemidactylus frenatus. This gecko has been widely introduced and recently R. frenata was found in introduced populations of cane toads (Rhinella marina) in Australia, indicating a host-switch from introduced geckos to toads. Here we report non-native adult R. frenata infecting the lungs of native cane toads in Panama. Eight of 64 toads were infected (median = 2.5, range = 1-80 pentastomids/toad) and pentastomid prevalence was positively associated with the number of buildings at a site, though further sampling is needed to confirm this pattern. We postulate that this pattern is likely due to a host shift of this parasite from an urban-associated introduced gecko. This is the first record of this parasite infecting cane toads in their native range, and the first instance of this parasite occurring in Central America.

Levisunguis subaequalis n. g., n. sp., a tongue worm (Pentastomida: Porocephalida: Sebekidae) infecting softshell turtles, Apalone spp. (Testudines: Trionychidae) in the southeastern United States.

A new tongue worm (Pentastomida) belonging to the Sebekidae Sambon, 1922 (Porocephaloidea Sambon, 1922) is described based on exemplars collected from softshell terrapins Apalone spinifera aspera (Agassiz) and Apalone ferox (Schneider) in the southeastern United States; a new genus is erected to accommodate the new species. The new species belongs in the Sebekidae because adults possess four simple hooks arranged in a trapezoid pattern on the ventral surface of the cephalothorax, a mouth opening between the anterior and posterior pairs of hooks, a terminal anus, an elongated uterus with preanal uterine pore, and a Y-shaped seminal vesicle. Nymphs possess geminate hooks, and the new species has an aquatic life-cycle in which nymphs become encapsulated in the body cavity of a freshwater fish and mature in the lungs of a terrapin. The new genus is distinct from other genera in the Sebekidae primarily by differences in hook morphology and the fact that representatives use a terrapin as a definitive host. Nymphs infecting fish and presumed to be the new species matured as postlarval juveniles conspecific with the new species when they were fed to the eastern mud turtle, Kinosternon subrubrum (Lacépède). Nymphs of the new species are anatomically similar to but larger than nymphs of Sebekia mississippiensis Overstreet, Self & Vliet, 1985 found in the mesentery of fishes captured in Florida, U.S.A. Adults of the new species differ from those of S. mississippiensis based on hook features, chloride cell pore pattern on annuli, body size, and use of a turtle rather than crocodilian definitive host. The new species is the third North American member of the Sebekidae.

Massive visceral pentastomiasis in a long-tailed macaque - an incidental finding.

BACKGROUND: An unusual case of visceral pentastomiasis in a male adult long-tailed macaque imported from China is reported. METHODS: The monkey was part of a toxicologic study. A massive accumulation of C-shaped parasites in various visceral organs was found post-mortem. RESULTS: Based on the morphology of the nymphs, pentastomiasis was diagnosed etiopathologically. The pentastome genus and species was identified as Armillifer agkistrodontis by PCR and respective sequencing. CONCLUSION: Molecular diagnostic methods are necessary tools to determine the exact species involved.

The nomenclature of the recent Pentastomida (Crustacea), with a list of species and available names.

The taxonomy of the Recent members of the crustacean subclass Pentastomida is based on nine accepted family names derived from 12 available names, 24 generic names derived from 37 available names (plus two incorrect subsequent spellings and one nomen nudum) and 124 accepted species names derived from 183 available names of which six remain incertae sedis as to their generic assignment. Compilation of this list has revealed that existing catalogues have included misspellings, wrong attributions and dates of the authors of taxa, and incorrectly nominated type-species. These are corrected here with reference to the original descriptions and diagnoses. Notably, all families except one were erected much earlier and by authors other than Fain (1961), who was credited by Martin & Davis (2001) and other authors before and afterwards with seven of the nine families they recognised. Other significant taxonomic anomalies are revealed. Raillietiellidae Sambon, 1922 is a senior synonym of Cephalobaenidae Heymons, 1922, the name in popular usage for the family including Cephalobaena Heymons, 1922 and Raillietiella Sambon, in Vaney & Sambon, 1910; here the two genera are placed in separate families following Almeida & Christoffersen (1999). Heymonsia Hett, 1934, considered a junior synonym of Raillietiella, is a nomen nudum. Raillietiella geckonis (Diesing, 1850) is a potential senior synonym of several SE Asian species of this genus. Raillietiella frenata Ali, Riley & Self, 1981 is a widely used species name but is a subjective junior synonym of R. hebitihamata Self & Kuntz, 1960 according to its own authors. Morphological and molecular evidence suggest that R. indica Gedoelst, 1921 is a subjective senior synonym of both species. The priority of Linguatulidae Haldeman, 1851 over Linguatulida Vogt, 1851, erected as a family in the same year, is established by applying the First Reviser rule. Linguatula serrata Frölich, 1789 is herein selected as the type-species of Prionoderma, making it an objective synonym of Linguatula Frölich, 1789. The priority of L. serrata over Taenia rhinaris Meyer, 1789 and T.capraea Abildgaard, 1789, all published in the same year, is established by applying the First Reviser rule. The purported synonymy of Netrorhynchus Zenker, 1827, also misspelled Nettorhynchus, with Armillifer Sambon, 1922 would seem to be ill-founded and without popular support. Armillifer australis Heymons, 1935, published as a subspecies of A. moniliformis (Diesing, 1836), is both a senior synonym and a homonym of A. australis Riley & Self, 1981. Humboldt (1812) is confirmed as the author of Porocephalus Humboldt, 1812 and P. crotali Humboldt, 1812. Pentastomidae Shipley, 1909 is an older family name than its subjective synonym Porocephalinae Sambon, 1922, but prevailing usage allows the latter to be retained as a family name. Cayerina mirabilis Kishida, 1927 is a genus and species from a Japanese frog that has not appeared in the more recent pentastome literature. Sebekia minor (Wedl, 1861) is an objective senior synonym of the more widely used S. wedli Gigioli, in Sambon, 1922. The importance of the many junior synonyms will become evident should refined morphological and molecular evidence reveal cryptic species or greater host-specificity than presently recognised.

Linguatula serrata in cattle in southeastern Iran: Epidemiological, histopathological and phylogenetic profile and its zoonotic importance.

This study aimed to determine the prevalence, histopathological observations, and phylogenetic analysis of L. serrata in cattle and its potential zoonotic and public health implications in southeastern Iran.The cross-sectional study was performed in cattle in southeastern Iran. Lymph nodes were collected from each cattle and examined by parasitological and histopathological techniques. A binary logistic regression and chi-square tests were implemented to analyze the data. Genomic DNA was randomly extracted from the nymphal stages of Linguatula isolates. Further characterization and phylogenetic relationships were done using two primers for amplification of partial DNA fragments of 18 s rRNA and cytochrome C oxidase subunit 1 (cox1), respectively.The results showed that 64 cattle of the total 404 were infected with L. serrata. There was no significant difference between linguatulosis infection and gender, while age was significantly different (P < 0.05). The cyst-like spaces containing the longitudinal and transverse sections of the L. serrata nymphs were surrounded by granulomatous reactions. The higher nucleotide variation in the cox1 region was supported by estimating the evolutionary divergence between L. serrata isolates and other Linguatula records of ruminants in Iran. The phylogenetic tree confirmed the close evolutionary relationships among all reported records of L. serrata in Iran.The high prevalence of linguatulosis caused by L. serrata declares the existence of a potential risk of FBPs for humans in southeastern Iran. This condition can advance more serious public health problems and requires a comprehensive control program and treatment strategies to prevent the disease.

Detection of a Larva of Armillifer armillatus in a Potto (Perodicticus potto) from the Republic of the Congo.

We determined the complete sequence of the mitochondrial DNA (mtDNA) of a parasite discovered between the subcutaneous tissue and the peritoneum of an African nocturnal non-human primate (NHP). The parasite and host sequences were obtained by a combination of Sanger sequencing and nanopore MinION techniques. Analyses of mtDNA gene arrangements and sequences unambiguously showed that the parasite investigated was the pentastomid Armillifer armillatus, also commonly named the tongue worm. The full-length mitochondrial genome of A. armillatus, measuring 16,706 bp in length, contains 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes, an arrangement identical to that of previously described pentastomid mitochondrial genomes. We describe here the second full mitochondrial genome of A. armillatus to date. To identify the NHP host, maximum likelihood phylogenetic analyses of a 441-bp fragment on the 12S rDNA gene and of a 1,140-bp fragment of the mitochondrial cytochrome b strongly support clustering with the African lorisid Perodicticus potto, a species that has rarely been reported as an intermediate host of this parasite.

A zoonotic parasite, Linguatula serrata, infection in a dog imported from Ethiopia to the United States.

A moderate number of oval-shaped, 114.7 × 61.3 µm in size, amber-colored, arthropod-like eggs that had chitinous, smooth, semi-thickened outer wall and 2-4 short appendages armed with 2 terminal hook-like structures were detected in multiple fecal samples from an approximately 9-month-old, intact female, collie-mixed dog that had been recently imported from Ethiopia to Oklahoma, United States. Initially the unusual arthropod-like eggs were considered to be a pseudoparasite, most likely mite eggs. However, based on the history of the dog, morphology of the eggs, and presence of the eggs in repetitive fecal flotations, a pentastomid, Linguatula serrata, was suspected. DNA extraction and PCR analysis of the partial 18S rRNA gene were performed on the eggs, and nucleic acid sequence showed 100% homology to L. serrata, a parasite of dogs, and L. arctica, a parasite of Norwegian reindeers. Rhinoscopy and head CT scan on the dog failed to demonstrate adult parasites or detect any pathologic changes. At this time, pentastomid eggs were no longer observed on fecal flotation. Due to the possibility of juvenile stages of the parasite still migrating in the dog, fluralaner (Bravecto®, Merck) was administered and continuing treatment recommended for at least 6 months. A follow-up fecal examination conducted a month after the treatment did not reveal any parasites or eggs. This is a case report of canine linguatuliasis diagnosed in Oklahoma, United States.

Morphological, Molecular, and Histopathological Data for Sebekia mississippiensis Overstreet, Self, and Vliet, 1985 (Pentastomida: Sebekidae) in the American Alligator, Alligator mississippiensis Daudin, and the Spotted Gar, Lepisosteus oculatus Winchell.

Novel molecular data from both mitochondrial ( cytochrome c oxidase subunit 1) and ribosomal regions ( 18S, ITS1-5.8S, ITS2, and 28S) are provided for Sebekia mississippiensis Overstreet, Self, & Vliet, 1985 , a pentastome infecting the American alligator, Alligator mississippiensis Daudin, 1801, and the spotted gar, Lepisosteus oculatus Winchell, 1864. Adult and nymphal pentastomes are described from the lungs and liver of the type host, A. mississippiensis, collected from Mississippi, while additional nymphs are described from the esophageal lining of L. oculatus specimens collected from Louisiana. This sequencing data will facilitate more accurate identification of various life cycle stages of S. mississippiensis, enabling future work to resolve many ambiguities in the literature regarding this species. Additionally, histopathological data are provided from both the definitive and intermediate hosts.

Molecular and Histopathological Data on Levisunguis subaequalis Curran, Overstreet, Collins & Benz, 2014 (Pentastomida: Eupentastomida: Porocephalida: Porocephaloidea: Sebekidae: Sebekinae) from Gambusia affinis in Alabama, USA.

Levisunguis subaequalis Curran, Overstreet, Collins & Benz, 2014 , was recently described from the lungs of the definitive hosts, softshell turtles, Apalone ferox (Schneider, 1783), and Apalone spinifera aspera (Agassiz, 1857) as well as the viscera of an intermediate host, the western mosquitofish, Gambusia affinis (Baird and Girard, 1853). However, the original account lacked molecular data. Furthermore, histological examination of infected host tissues in the original account of L. subaequalis did not reveal any pathological changes in the intermediate host. The present work provides a robust morphological description of the nymph and novel molecular data from the 18S and 28S ribosomal gene regions and the cytochrome c oxidase subunit 1 (COI) mitochondrial gene. Phylogenetic analyses using Bayesian inference and maximum likelihood analysis with concatenated sequence data from these 3 regions, as well as each region individually, placed the turtle pentastomid L. subaequalis as a sister clade to the crocodilian pentastomids of the genus Sebekia Sambon, 1922. While only concatenated phylogenetic analyses agreed with the currently accepted classification of the Eupentastomida and phylogenetic signal assessment indicated that the concatenated data set yielded the most phylogenetic signal, data from more taxa are still needed for robust phylogenetic inferences to be made. The intensity of infection ranged from 2 to 171 nymphs per fish, compared with the highest previously reported intensity of 6. These high-intensity infections with L. subaequalis were characterized by the nymphs occupying 5-50% of the coelomic cavity of G. affinis. However, despite this heavy parasite infection, fish exhibited minimal pathology. Observed pathology was characterized by compression or effacement of organs adjacent to the nymphs, particularly liver, swim bladder, and intestines, as well as the formation of granulomas around shed pentastomid cuticles. Nonetheless, the morphological and molecular data provided in the present work will bolster future efforts to identify this pentastomid in other hosts where pathology may be present in addition to aiding in the advancement of the field of molecular pentastomid systematics.

First report of molecular characterization and sequence phylogenetic analysis of Linguatula serrata from India.

Linguatula serrata is a pentastomid which is worldwide in distribution. However, a very few references are reported from India. In the present study, the cox I gene of L. serrata nymphs, originally isolated from mesenteric lymph nodes of buffaloes, was amplified and custom sequenced. Based on sequence analysis, two haplotypes were noticed and were subsequently submitted in NCBI database. The sequences were also compared with the other sequences available in the pubmed and phylogenetic analysis coupled with nucleotide homologies were commutated. The studied Indian isolates were found closer to Bangladesh and Iran isolates. This is the first report of molecular characterization of L. serrata from India.

Molecular characterization and phylogeny of Linguatula serrata (Pentastomida: Linguatulidae) based on the nuclear 18S rDNA and mitochondrial cytochrome c oxidase I gene.

Linguatula serrata, a cosmopolitan parasite, is commonly known as tongue worm belonging to the subclass Pentastomida.We collected the nymphal stage of the worm from mesenteric lymph nodes of cattle and identified these as L. serrata based on morphology and morphometry. The 18S rDNA sequences showed no intraspecific variation, although cox1 sequences showed 99.7-99.9% homology. In the phylogenies inferred from both gene loci, members of the genus Linguatula (order Porocephalida) were closer to those of the order Cephalobaenida than to those of Porocephalida, reflecting a mismatch with the corresponding morphology-based taxonomy. Accordingly, analyses of additional gene loci using a larger number of taxa across the Pentastomida should be undertaken to determine an accurate phylogenetic position within the Arthropoda.

First finding of nymphal stages of Linguatula serrata in a South American camelid, a vicuña from Peru.

Linguatula serrata, a pentastomid, was found parasitizing the lungs of a vicuña (Vicugna vicugna) from Cuzco, Peru. A total of 13 larvae were found encysted in the parenchymal tissue of the lungs. All larvae were identified as nymphal stages of L. serrata by morphological methods Diagnosis was confirmed by molecular analysis amplifying the cytochrome c oxidase 1 gene of three nymphs. Nucleotide sequences from the isolates were compared to previous sequences from GenBank, and it showed high similarity between them (>99%). This finding constitutes the first detection of L. serrata in a South American camelid.

Finding of pentastomes of genus Reighardia (Pentastomida) in the Belcher's gull (Larus belcheri).

This report describes the finding of Reighardia sp. (Pentastomida) infecting the air sac of two Belcher's gulls (Larus belcheri) found dead on the beaches of Pucusana, a district in southern Lima, Peru. Three pentastomes were collected from two Belcher's gulls. Then, they were morphologically and molecular analyzed. Molecular characterization of the parasite was achieved by amplifying a fragment of the small subunit ribosomal RNA gene (SSU rRNA). Based on both morphological and molecular data the pentastomes were identified as pentastomes of the genus Reighardia. This is the first report showing that the Belcher's gull is a new natural definitive host for this pentastome.