Natural history of the parasite Waddycephalus in the Townsville region of north-east Australia.

Waddycephalus is an understudied genus of pentastomids native to Australia and south-east Asia. The genus was recognized in 1922 but there has been little research on these pentastomid tongue worms over the last century. A few observations suggest a complex life cycle through 3 trophic levels. We aimed to add knowledge to the Waddycephalus life cycle in woodland habitats in the Townsville region of north-east Australia. We used camera trapping to identify the most likely first-intermediate hosts (coprophagous insects), we conducted gecko surveys to identify multiple new gecko intermediate host species and we dissected road-killed snakes to identify additional definitive hosts. Our study paves the way for further research into the intriguing life cycle of Waddycephalus, investigation of spatial variation in prevalence and impacts of the parasite on host species.

Systematic assessment of nymphs of Diesingiinae (Pentastomida: Sebekidae) parasitizing Palloceros harpagos (Cyprinodontiformes: Poeciliidae) from Parque Nacional Iguazú, Argentina: filling gaps in an incomplete phylogenetic framework.

During a search for parasites in fishes from Iguazu River, Argentina, a nymph of pentastomid was found encysted on mesenteries of Phalloceros harpagos. The 28S rDNA and COI mt-DNA were used to compare with the sequences deposited in the GenBank. Pentastomid nymphs belong to the subfamily Diesingiinae (Sebekidae) for having chloride cell pores distributed in a single row per annulus; also, the hooks and rows of spines of our material differ to other genera. Present specimens are most likely Diesingia sp., having 63-74 annuli, a U shaped oral cadre with fibers closing anteriorly and a peg like extension of the oral cadre. The 28S rDNA analysis places our samples into a sister group of Alofia sp., but the COI mt-DNA situate them on the base of the clade. In conclusion, our pentastomid positively belongs to Diesingia sp., however, indisputable assignation requires a reliable description of the nymph, or the availability of sequences linking nymphs and adults, which even could provide evidence of a new hitherto undescribed genus. Aditionally, the systematic position of Sebekia oxycephala previously described by P. harpagos is discussed on the basis of our results, allowing us to suggest a re-assignation of such specimens to the genus Diesingia.

Characterization of the Tongue Worm, Linguatula serrata (Pentastomida), Identified from Hares (Lepus europaeus) in Romania.

Linguatula serrata (Frölich, 1789) is a widespread parasite known as the tongue worm belonging to the family Linguatulidae. The adult form of the parasite is usually located in the upper respiratory tract of domestic and wild carnivores while the larval forms are located in the visceral organs of intermediate hosts (various herbivorous mammals). Twenty-four European brown hares (Lepus europaeus) were examined in this study, of which two were positive with L. serrata nymphs. The collected nymphs were examined morphologically using electron-microscopic analysis and molecularly by amplification of 18S rRNA and COX1 genes. Lung tissue samples were also collected and histopathological examination was performed. Histopathological examination revealed the following lesions: generalized inflammatory oedema, granulomas with necrosis, calcification and fibrosis in the bronchial tree. The results of molecular sequencing for L. serrata specimens collected from the European brown hares are deposited in GenBank. This study presents the first report on Linguatula serrata nymphs collected from L. europaeus in Romania, using molecular and morphological characterization simultaneously.

Characterization of tongue worms, Linguatula spp. (Pentastomida) in Romania, with the first record of an unknown adult Linguatula from roe deer (Capreolus capreolus Linnaeus).

Specimens of the pentastomid parasite, Linguatula serrata, have been reported from several animals in Romania, including some domestic dogs translocated to other parts of Europe. In this study, gray wolves (Canis lupus, n = 80), golden jackals (C. aureus, n = 115), red foxes (Vulpes vulpes, n = 236), and roe deer (Capreolus capreolus, n = 1) were examined for pentastomes. Overall, 17.5% of wolves were found to be infected with specimens of Linguatula, with a range of infections of one to five individuals per animal. Golden jackals and foxes had much lower infection levels, with 1.73% of golden jackals and 1.69% of foxes infected; both host species were found to be infected with one or two individual pentastomes per animal. The single deer specimen was infected with three individual pentastomes. The pentastomes collected from the wolves and golden jackals were determined to be immature and mature adult specimens of L. serrata based on morphological examination and molecular analysis using the 18S rRNA gene. No pentastomes collected from the red foxes were available for identification. The pentastomes collected from the roe deer were expected to be L. arctica but determined to be mature adult male specimens of an unknown Linguatula, herein, referred to as Linguatula sp. based on its morphology; the results of molecular sequencing for the Linguatula specimen collected from the deer were inconclusive, preventing a final species identification. This study presents the first report of L. serrata in any hosts from Romania through both morphological and molecular characterization, and also presents the first report of a Linguatula sp. in Ca. capreolus, utilizing morphological characterization. Issues of morphological variability are discussed, including the presence of spines in the hook pit of specimens of Linguatula. This study highlights the need to examine all specimens of Linguatula to confirm the stage of development. Despite the inconclusive molecular result for some specimens, the authors still urge future researchers to incorporate a combined molecular and morphological approach in identifying specimens of Linguatula.

28S rRNA sequences for Linguatula spp.

Identification of specimens belonging to the genus Linguatula (Pentastomida) is relatively easy due to their unique morphology. However, differentiation between species of Linguatula can be challenging for several reasons, including considerable differences between different developmental stages of the parasite within and between species. Currently, 18S rRNA and Cox1 sequences are the only available comparable sequences in GenBank, but recent research has discussed the utility of 28S rRNA for pentastomid phylogenetics. This study presents 28S rRNA gene sequences for two members of the genus Linguatula. Sequences of 28S rRNA were successfully obtained from well-identified samples of L. serrata (collected in Australia) and L. nuttalli (collected in South Africa), with voucher specimens. Phylogenetic analysis of the 28S rRNA region showed 6% difference between L. serrata and L. nuttalli, with low levels of intraspecific variation. In comparison, 18S rRNA and Cox1 sequences from the same specimens showed 0.23% and 13% interspecific differences, respectively. The results of this study show that 28S rRNA has greater genetic diversity to allow for improved differentiation between species of Linguatula than 18S rRNA but is on par with Cox1. Records that do not provide adequate morphological or molecular data to justify independent specific diagnoses must be regarded cautiously, and the need for continued research on species of Linguatula, using a combined morphological and molecular analysis, across a number of different hosts, development stages, geographical regions and molecular markers is highlighted.

Global status of neglected human Linguatula infection: a systematic review of published case reports.

Human linguatulosis is a rare but highly specialized pentastomid parasitic infection all around the world. The disease is caused by worm-like parasites which share some morphologic features of arthropods and annelids. The life cycle of Linguatula spp. is provided as an example of pentastomid biology. The adult forms of Linguatula spp. are found in the nasal passages and frontal sinuses of dogs and other canids, domestic animals, and humans. Infestation may occur in the viscera, where immature forms develop in the liver, spleen, lungs, eyes, and other organs, or in the nasopharyngeal area. This review includes previous studies on the genus Linguatula in Iran and other countries from 1940 to 2019. Considering a few reports of human infestation, a comprehensive search was performed on both English and Persian databases for publications reporting human cases all around the world. Finally, 30 papers of 62 human cases were included in this study. The mean age of the patients was 25.8 years (varied from 8 to 79 years). Altogether, 41 (66.1%) and 21 (33.9%) of the patients were female and male respectively. The maximum cases (n = 25) were from Sudan. Nasopharynx was the most frequently involved organ in human cases. Our findings offer that physicians should consider the Linguatula infection in patients with complaints like unexpected dysphagia, with coughing, sneezing, and vomiting, after consuming raw/undercooked contaminated ruminant's viscera.

Morphology and molecular taxonomy of the tongue worm, genus Raillietiella (Pentastomida) from the lungs of berber skinks Eumeces schneideri (Scincidae): First report.

In the present study, pentastomids belonging to the order Cephalobaenida were isolated from the lungs of Berber skinks Eumeces schneideri (Famiy: Scincidae), which were morphologically described by light and scanning electron microscopy and taxonomically justified by 18s rDNA molecular analyses of the parasites. Seventeen host specimens were collected from well-vegetated wadis at high altitudes, Jizan, Saudi Arabia as new type locality; twelve specimens (70.59%) were infected. All of the recovered parasites were adults, possessed small broadly triangular cephalothorax flattened on the ventral surface and merged smoothly with a uniformly thick and squat abdomen and terminated in a pair of divergent lobes. The results obtained indicated that the parasites belong to the sharp-tipped posterior-hook Raillietiella spp. distinguished from other raillietiedids of the same group some important characteristic features including annulus number, shape and dimensions of the buccal cadre, copulatory spicules, and anterior and posterior hooks. The anterior hook of the female specimens (n=5) had a blade length (AB) of 135±5 (110-146) µm and shank length (BC) 158±5 (150-169) µm while the posterior hook was much larger with AB measuring 221±5 (200-236) µm and BC 286±6 (280-289) µm. For the male specimens (n=5), the anterior hook had an AB of 73±3 (72-75) µm and a BC 102±5 (100-103) µm. The posterior hook was much larger with AB 190.6±5 (190-191) µm and BC 221±5 (280-289) µm. The morphological characterization of the recovered parasites was closely similar to R. aegypti previously isolated from the same host. Sequence alignment by the maximum likelihood analysis for the data obtained from the 18S rDNA analysis of the parasites exhibits identities ranging between 88-95% with pentastomid genera recovered from the GenBank. The phylogenetic tree supported the inclusion of the parasites within the monophyletic Pentastomida clade with maximum identity to the raillietiellid species. The recovered sequences from the present study were deposited in GenBank under Accession number MK970649.1. The present molecular analysis was the first to confirm the taxonomic position of R. aegypti isolated from the host examined.

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.

Verification of rabbits as intermediate hosts for Linguatula serrata (Pentastomida) in Australia.

We confirm the presence of nymphs of the introduced pentastomid, Linguatula serrata, in the introduced rabbit, Oryctolagus cuniculus, in Australia, based on morphological and molecular results. Two nymphs were collected from a single rabbit near the Kosciuszko National Park in New South Wales. Unlike reports of nymphs from domestic animals, these nymphs were not encapsulated, despite having the morphological features of infective nymphs. The possibility of different development pathways in lagomorphs is discussed. Examination of feral deer for L. serrata was unsuccessful and potential reasons for a lack of infection in these animals are postulated. Our results reiterate the need for a combined morphological and molecular approach to the identification of L. serrata. Further sampling of a range of feral and native animals is required to determine the true range of intermediate hosts and their relative importance in the transmission of L. serrata in Australia.

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.

Effect of age, sex, and season on the prevalence of Linguatula serrata infestation in mesenteric lymph nodes of goats slaughtered in Tabriz, Iran.

This research was conducted to determine the prevalence and intensity of infection with nymphs of Linguatula serrata in mesenteric lymph nodes of the goats from northwest of Iran. Moreover, the intensity of infection of mesenteric lymph nodes was compared based on gross appearance and the morphological changes in these nodes. In addition, the effects of age, sex, and season on the prevalence of Linguatula serrata were assessed. In this study, mesenteric lymph nodes (n = 16,284) were randomly collected from 2132 goats slaughtered in the abattoir of Tabriz, Iran, from September 2013 to September 2017. The samples were categorized based on their gross appearance (color and consistency) and then were cut into small pieces and immersed in normal saline (0.9% NaCl) solution and left for 5 to 6 h to allow nymphs to come out from the tissue. The results showed that 1241 out of total 2132 (58. 2%) goats and 9282 out of total 16,284 mesenteric lymph nodes (57%) were infested with the nymphal stage of Linguatula serrata. It was also revealed that the infestation rate was age-dependent: as the goats grow older, the infestation increases. Further, the prevalence of Linguatula serrata nymphs in the mesenteric lymph nodes in various seasons was not significant (P > 0.05). The prevalence of infestation rate in female goats was significantly higher than of male goats (P < 0.05). Besides, the infestation rate in the black-colored lymph nodes (75.88%) was significantly (P < 0.05) higher than those of hemorrhagic nodes (54.94%) and normal-colored nodes (22.65%). Moreover, the infestation rate of nymphs in the soft lymph nodes (83.91%) was significantly (P < 0.05) higher than those in normal (21.85%) and hard (32.43%) lymph nodes. Given the fact that the Linguatula serrata is a zoonotic parasite; thus, the inspection process should be meticulously done in an abattoir, especially in areas where residents consume raw or under-cooked liver and/or visceral organs of herbivores.

Unexpected Infection with Armillifer Parasites.

Visceral pentastomiasis is usually found incidentally during surgery. We describe a case of visceral pentastomiasis discovered during inguinoscrotal hernia surgery for a man from Benin, Africa. Because surgical removal of nymphs is needed for symptomatic patients only, this patient's asymptomatic pentastomiasis was not treated and he recovered from surgery uneventfully.

A Case of Pentastomiasis at the Left Maxilla Bone in a Patient with Thyroid Cancer.

Pentastomiasis, a zoonotic parasite infection, is typically found in the respiratory tract and viscera of the host, including humans. Here, we report for the first time an extremely rare case of intraosseous pentastomiasis in the human maxilla suffering from medication related osteonecrosis of the jaw (MRONJ). A 55-year-old male had continuously visited the hospital for MRONJ which had primarily developed after bisphosphonate and anti-neoplastic administration for previous bone metastasis of medullary thyroid cancer. Pain, bone exposure, and pus discharge in the right mandible and left maxilla were seen. Osteolysis with maxillary cortical bone perforation at the left buccal vestibule, palate, nasal cavity, and maxillary sinus was observed by radiologic images. A biopsy was done at the left maxilla and through pathological evaluation, a parasite with features of pentastome was revealed within the necrotic bone tissue. Further history taking and laboratory evaluation was done. The parasite was suspected to be infected through maxillary open wounds caused by MRONJ. Awareness of intraosseous pentastomiasis should be emphasized not to be missed behind the MRONJ. Proper evaluation and interpretation for past medical history may lead to correct differential diagnosis and therapeutic intervention for parasite infections.

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.

Prevalence and morphopathological characteristics of linguatulosis in one-humped camel (Camelus dromedarius) in Yazd, Iran.

Linguatula serrata is a cosmopolitan zoonotic parasite. Its adult form inhabit the nasal and respiratory passages of canids as the definitive hosts while its immature stages localize in the mesenteric lymph nodes or in other organs of herbivorous intermediate hosts. We examined the liver, mesenteric, and mediastinal lymph nodes of 272 camels slaughtered at the slaughterhouse of Yazd, Iran. Forty-one out of 272 camels (15.1 %) were infected with nymphs of L. serrata. Twenty-four out of 166 males (14.45 %) and 17 out of 106 females (16 %) were positive. The livers of five camels, which also had nymphs in their lymph nodes, were infected with the larval stage of this parasite. The infection rate increased with age and was highly significant, while sex did not play a significant role in the prevalence of this parasitic infection. The infected lymph nodes were grossly enlarged, edematous, and consisted of hemorrhagic and necrotic lesions. Histopathologically, the architecture of the infected lymph nodes was degraded, necrotic, and sectioned migrating stages of L. serrata were clearly visible. In some lymph nodes, parasitic granulomatous lymphadenitis with necrosis and in some cases, degenerated parasite in central area was observed. High prevalence of infection in camels suggests possibility of similar high rate of infection in other animals of this region. In view of the consumption of raw or undercooked visceral organs of camel by humans of this region, the importance of careful inspection at slaughterhouse needs to be emphasized.

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.

Complete Mitochondrial Genome of a Tongue Worm Armillifer agkistrodontis.

Armillifer agkistrodontis (Ichthyostraca: Pantastomida) is a parasitic pathogen, only reported in China, which can cause a zoonotic disease, pentastomiasis. A complete mitochondrial (mt) genome was 16,521 bp comprising 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, and 1 non-coding region (NCR). A phylogenetic tree drawn with the concatenated amino acid sequences of the 6 conserved PCGs (atp6, cox1-3, and nad2) showed that A. agkistrodontis and Armillifer armillatus constituted a clade Pentastomida which was a sister group of the Branchiura. The complete mt genome sequence of A. agkistrodontis provides important genetic markers for both phylogenetic and epidemiological studies of pentastomids.

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.