Description and molecular characterisation of Pelecitus copsychi Uni, Mat Udin & Martin n. sp. (Nematoda: Onchocercidae) from the white-rumped shama Copsychus malabaricus (Scopoli) (Passeriformes: Muscicapidae) of Pahang, Malaysia.

Species of the genus Pelecitus Railliet & Henry, 1910 the most widely distributed avian filariae in Africa and South America. Zoonotic cases in humans were reported in South America. While investigating the filarial fauna of wild animals in Malaysia, we discovered an undescribed filaria from the swollen footpad of the left leg of Copsychus malabaricus (Scopoli) in Pahang, Peninsular Malaysia. Adults of both sexes have a corkscrew-shaped body. Based on comparison of their morphological characteristics (i.e. pre-oesophageal cuticular ring distinct, oesophagus divided, vulva protuberant and situated at the level of anterior half of oesophagus, spicules strongly sclerotized and left spicule with broad blade) with other Pelecitus species, they are here described as Pelecitus copsychi Uni, Mat Udin & Martin n. sp. Multi-locus sequence analyses based on seven genes (12S rDNA, cox1, 18S rDNA, 28S rDNA, MyoHC, rbp1 and hsp70) were performed to determine the phylogenetic position of the new species. The calculated p-distance between the cox1 gene sequences for P. copsychi n. sp. and Pelecitus fulicaeatrae (Diesing, 1861) was 14.1%. Intraspecific genetic variation between two individuals of the new species was 0.4%. In both the Bayesian inference and maximum-likelihood trees, P. copsychi n. sp. was positioned in the second clade of ONC5, containing three genera of the subfamily Dirofilariinae (Foleyella Seurat, 1917, Pelecitus and Loa Stiles, 1905). Immunostaining and molecular analyses remained negative for the presence of Wolbachia endosymbionts. Our findings corroborate the division of the subfamily Dirofilariinae into ONC3 with Dirofilaria Railliet & Henry, 1911 and ONC5 with Pelecitus.

Prevalence of Onchocerca japonica and O. takaokai infections in the Japanese wild boar, Sus scrofa leucomystax, and the Ryukyu wild boar, S. s. riukiuanus, in Japan.

Reports of zoonotic infections with Onchocerca japonica (Nematoda: Filarioidea), which parasitizes the Japanese wild boar, Sus scrofa leucomystax, have recently increased in Japan. To predict the occurrence of infection in humans, it is necessary to determine the prevalence of O. japonica infection in the natural host animals. We investigated the presence of adult worms in the footpads, and of microfilariae in skin snips, taken from the host animals, between 2000 and 2018. Onchocerca japonica was found in 165 of 223 (74%) Japanese wild boars in Honshu and Kyushu. Among the nine regions studied, the highest prevalence of O. japonica infection was found in Oita, Kyushu, where 47 of 52 (90.4%) animals were infected. The ears were the predilection sites for O. japonica microfilariae. Adult worms of O. japonica were found more frequently in the hindlimbs than in the forelimbs of the host animals. Onchocerca takaokai was found in 14 of 52 (26.9%) Japanese wild boars in Oita. In Kakeroma Island among the Nansei Islands, both O. japonica and O. takaokai were isolated from the Ryukyu wild boar, S. s. riukiuanus. These observations could help predict future occurrences of human zoonotic onchocercosis in Japan.

Description and molecular characteristics of Morishitium polonicum malayense Urabe, Nor Hashim & Uni, n. subsp. (Trematoda: Cyclocoelidae) from the Asian glossy starling, Aplonis panayensis strigata (Passeriformes: Sturnidae) in Peninsular Malaysia.

We describe Morishitium polonicum malayense n. subsp. from Asian glossy starlings (Aplonis panayensis strigata) (Horsfield, 1821) (Passeriformis: Sturnidae) caught in Malaysia. The trematodes had parasitized the air sacs and the thoracic and body cavities of 40 out of 67 (59.7%) birds examined. The specimens each had an oral sucker, a postpharyngeal genital pore, and tandem testes, but lacked a ventral sucker. The morphological characteristics of our specimens were similar to those of M. polonicum polonicum (Machalska, 1980) from Poland. However, the anterior extremity of vitelline follicles of the present specimens sometimes extended to the level of pharynx. The oral sucker width, oral sucker width/pharynx width ratio, and intertesticular space metrics differed from those of M. p. polonicum. The maximum-likelihood trees based on the cytochrome c oxidase subunit I (COI) and the internal transcribed spacer 2 (ITS2) sequences indicated that the species from the present study formed a sister group with M. p. polonicum from the Czech Republic. The p-distances of COI and ITS2 sequences between the present specimens and M. p. polonicum from the Czech Republic were 6.9-7.5% and 0.6%, respectively. These genetic divergences indicate the border for intra- or interspecific variation of digeneans. The definitive host species and geographical distribution of the current specimens were distinct from those of M. p. polonicum from Europe. We thus concluded that the present specimens are ranked as a new subspecies of M. polonicum, namely M. polonicum malayense n. subsp.

Description, molecular characteristics and Wolbachia endosymbionts of Onchocerca borneensis Uni, Mat Udin & Takaoka n. sp. (Nematoda: Filarioidea) from the Bornean bearded pig Sus barbatus Müller (Cetartiodactyla: Suidae) of Sarawak, Malaysia.

BACKGROUND: The genus Onchocerca Diesing, 1841 includes species of medical importance, such as O. volvulus (Leuckart, 1893), which causes river blindness in the tropics. Recently, zoonotic onchocercosis has been reported in humans worldwide. In Japan, O. dewittei japonica Uni, Bain & Takaoka, 2001 from wild boars is a causative agent for this zoonosis. Many filarioid nematodes are infected with Wolbachia endosymbionts which exhibit various evolutionary relationships with their hosts. While investigating the filarial fauna of Borneo, we discovered an undescribed Onchocerca species in the bearded pig Sus barbatus Müller (Cetartiodactyla: Suidae). METHODS: We isolated Onchocerca specimens from bearded pigs and examined their morphology. For comparative material, we collected fresh specimens of O. d. dewittei Bain, Ramachandran, Petter & Mak, 1977 from banded pigs (S. scrofa vittatus Boie) in Peninsular Malaysia. Partial sequences of three different genes (two mitochondrial genes, cox1 and 12S rRNA, and one nuclear ITS region) of these filarioids were analysed. By multi-locus sequence analyses based on six genes (16S rDNA, ftsZ, dnaA, coxA, fbpA and gatB) of Wolbachia, we determined the supergroups in the specimens from bearded pigs and those of O. d. dewittei. RESULTS: Onchocerca borneensis Uni, Mat Udin & Takaoka n. sp. is described on the basis of morphological characteristics and its genetic divergence from congeners. Molecular characteristics of the new species revealed its close evolutionary relationship with O. d. dewittei. Calculated p-distance for the cox1 gene sequences between O. borneensis n. sp. and O. d. dewittei was 5.9%, while that between O. d. dewittei and O. d. japonica was 7.6%. No intraspecific genetic variation was found for the new species. Wolbachia strains identified in the new species and O. d. dewittei belonged to supergroup C and are closely related. CONCLUSIONS: Our molecular analyses of filarioids from Asian suids indicate that the new species is sister to O. d. dewittei. On the basis of its morphological and molecular characteristics, we propose to elevate O. d. japonica to species level as O. japonica Uni, Bain & Takaoka, 2001. Coevolutionary relationships exist between the Wolbachia strains and their filarial hosts in Borneo and Peninsular Malaysia.

A Philophthalmid Eyefluke from a Human in Japan.

Philophthalmid eyeflukes are cosmopolitan parasites of birds and occasionally of mammals, including humans. A gravid adult of Philophthalmus sp. was found from the bulbar conjunctiva of a 64-yr-old woman in Japan, who was diagnosed with acute conjunctivitis. The parasite was morphologically most similar to Philophthalmus hegeneri, but distinctive in lacking an esophagus and in having clearly lobed testes. The DNA sequence analysis of genes for nuclear 28S ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1 supported the identification at generic level. The morphological and molecular analyses strongly suggest that the eyefluke from a human in Japan should be treated as an undescribed species of Philophthalmus. The occurrence of human philophthalmosis is very rare. As far as we know, a total of 11 human cases have been reported worldwide to date.

Human case of Onchocerca dewittei japonica infection in Fukushima, Northeastern Honshu, Japan.

A 73-year-old man living in Kawamata-machi, Fukushima Prefecture, Northeastern Honshu, Japan, visited a hospital with complaints of a subcutaneous swelling that had developed on the back of his left hand. The nodule was surgically removed from the vagina fibrosa tendinis of his left forefinger. Based on the histopathological characteristics, the causative agent of this nodule was identified as a female Onchocerca dewittei japonica (Spirurida: Onchocercidae). The species identification was confirmed by cox1 gene sequencing of the worm tissues from paraffin-embedded sections of the nodule. Although 11 cases of zoonotic onchocercosis have previously been recorded in Kyushu and Western Honshu, Japan, the present findings represent the first human case of infection with O. dewittei japonica in Northeastern Honshu, Japan.

Epidemiological Investigation of Parasitic Infection of Schoolchildren from Six Elementary Schools in Sakon Nakhon Province, Thailand.

We conducted an epidemiological study of intestinal parasitic infection in 572 schoolchildren aged 4 to 12 years old from six elementary schools in Sakon Nakhon Province, Thailand from June 2013 to August 2014. We collected fecal, blood, and urine samples to investigate parasitic infection and conducted a questionnaire survey. Soil samples were examined for egg contamination. Fecal examination, using the formalin-ether sedimentation method, revealed that 39% of schoolchildren were infected with eight genera and eight species of parasites; three nematodes, two trematodes, one cestode, and two protozoa. Prevalence rates across the six schools (schools A through F) were: A (13%), B (15%), C (53%), D (11%), E (20%), and F (43%). Schools C and F showed significantly higher prevalence rates than the other schools (p<0.05). In school C, Necator americanus was detected in 49% of schoolchildren tested, while in school F a high prevalence of Opisthorchis viverrini and Heterophyes heterophyes, at a rate of 23% and 21%, respectively, was detected. The questionnaire survey revealed that health, hygiene practices and awareness were poor in school C. However, school F showed high levels of cognizance and practices relating to the prevention of infection. The schoolchildren ate a staple diet of undercooked river fish and the results revealed a high rate of fish-borne parasites. Soil samples showed Toxocara sp. contamination in and around the campus. Toxocara antibodies were detected in over 6% of schoolchildren. The use of urine samples, as opposed to serum samples, was found to be effective for antibody testing.

Modified method to enhanced recovery of Toxocara cati larvae for the purposes of diagnostic and therapeutic.

Human toxocariasis, extraintestinal-migration of Toxocara species, is a worldwide helminthic zoonosis in many places of the undeveloped countries. Toxocara cati is one of the common helminths in cats and it is a potentially preventable disease. Its diagnosis and treatment depend on the demonstration of specific excretory-secretory Toxocara antibodies from Toxocara larvae by immunological assays. This study provides a simple manual technique which can be performed in any laboratory for recovering a large number of Toxocara cati larvae from the thick-shelled eggs. The devices that are required contain a manual homogenizer and a filter membrane of 40 µm mesh; the rest of materials and solutions is standard laboratory ware. In the modified method the larval yields were 2.7 times higher (3000 larval/ml) and the time spent in performing the modified method was shorter (75 min). Further benefits over already techniques are the easy and repeatable, inexpensive and convenient materials, simplicity to perform and require less time for recovery of Toxocara cati larvae for subsequent cultivation and harvest of the larval excretory-secretory antigens for diagnostic or treatment purposes.

Modified flotation method, an effective technique for recovering helminth eggs in soil.

There is no established method for recovering helminth eggs in soil has yet to be established. Here, we introduce a novel method for their recovery based on a centrifugal flotation approach using a sucrose solution with a specific gravity of 1.20, with a coverslip being placed on the sucrose-filled centrifuge tube during centrifugation. The recovery of zoonotic Toxocara eggs from soil was more effective when this method was compared with the traditional flotation method. This method detects not only zoonotic Toxocara eggs, but Ascaris lumbricoides, Trichuris trichiura, and other human parasite eggs also, indicating that it can be used for epidemiological studies both in developed and developing countries.

Simple and effective method for observation of suspended parasite specimens using vaseline and paraffin mixture.

Parasitic specimens derived from protozoans and helminths have variable thickness. Therefore, microscopic observation of these specimens requires a preparation technique where the space between the coverslip and slide glass can be freely adjusted. However, standard suspension methods for parasites do not afford this flexibility due to the thickness of eggs and parasites. Mounting too large of a sample results in a floating coverslip, making observation difficult. In this article, we developed vaseline-paraffin solution (VPS) as a simple and effective mounting technique for observation of suspended parasite specimens. VPS placed between the coverslip and slide glass makes it possible to adjust the space between to accommodate specimens of variable thickness. For example, patterning of Toxocara egg surface protein layers can be observed using this improved method. Furthermore, VPS can be used as a sealing medium for long-term preservation. It is possible to keep suspended parasite specimens for more than two weeks.

Evaluation of Toxocara cati Excretory-Secretory Larval Antigens in Serodiagnosis of Human Toxocariasis.

BACKGROUND: Toxocariasis is the clinical term that is applied to infection in the human host with Toxocara species larvae. Serological tests are important tools for the diagnosis of toxocariasis. The aim of this study was to evaluate the excretory-secretory (ES) antigens of T. cati larvae using enzyme-linked immunosorbent assay (ELISA) and also Western blotting for serodiagnosis of human toxocariasis. METHOD: The ES antigens were prepared from T. cati third-stage larvae. Serum samples were obtained from 33 confirmed cases of toxocariasis, 35 patients infected with other parasitic diseases, and 30 from healthy individuals tested with ELISA and immunoblotting. RESULTS: The ELISA showed appropriate performance in term of specificity (96.7%) and sensitivity (97.0%). Electrophoretic analysis of T. cati ES antigens revealed a range of 20- to 150-kDa fractions. The highest sensitivity was achieved with 42- and 50-kDa fractions. CONCLUSION: The ELISA analyses using T. cati ES antigens demonstrated good sensitivity and specificity compared to T. canis ES as antigens for diagnosis of human toxocariasis. Accordingly, application of Western blotting, based on 42- and 50-kDa fractions of ES antigens, can be recommended for the accurate diagnosis of toxocariasis.

Differentiation between Viable and Dead Cryptosporidium Oocysts Using Fluorochrome Staining.

The use of nucleic acid staining with a fluorochrome dye to differentiate viable and dead (heat-killed) Cryptosporidium oocysts was assessed. The specificities (percentage of unstained viable oocysts) and sensitivities (percentage of stained dead oocysts) of the seven tested dyes (SYTO-17® and SYTO-59® to 64®) ranged from 65 to 76% (average 71%) and 83 to 95% (average 91%), respectively. SYTO-59 and SYTO-17 imparted greater color (4+) intensity than the other dyes (2+ or less). Of these two dyes, SYTO-17 exhibited more brightness and slower discoloration and was selected for use in further experiments. The optimum staining time for SYTO-17 at 37℃ was one hour or more (sensitivity of 96%). Dye concentrations of 20 and 30 µM resulted in maximal color intensity, and no further improvement was observed with further increases in dye concentration. Staining a mixture of viable and dead oocysts (1:1 ratio) with 20 µM dye at 37℃ for one hour yielded the expected results (approximately 50%), but no remarkable increase in the percent staining with time (up to 8 hours) was observed. In this study, no ghost oocysts were observed. The present study indicated that the fluorogenic nucleic acid dye SYTO-17 could be used to discriminate between live and dead Cryptosporidium oocysts.

Review of parasitic zoonoses in egypt.

This review presents a comprehensive picture of the zoonotic parasitic diseases in Egypt, with particular reference to their relative prevalence among humans, animal reservoirs of infection, and sources of human infection. A review of the available literature indicates that many parasitic zoonoses are endemic in Egypt. Intestinal infections of parasitic zoonoses are widespread and are the leading cause of diarrhea, particularly among children and residents of rural areas. Some parasitic zoonoses are confined to specific geographic areas in Egypt, such as cutaneous leishmaniasis and zoonotic babesiosis in the Sinai. Other areas have a past history of a certain parasitic zoonoses, such as visceral leishmaniasis in the El-Agamy area in Alexandria. As a result of the implementation of control programs, a marked decrease in the prevalence of other zoonoses, such as schistosomiasis and fascioliasis has been observed. Animal reservoirs of parasitic zoonoses have been identified in Egypt, especially in rodents, stray dogs and cats, as well as vectors, typically mosquitoes and ticks, which constitute potential risks for disease transmission. Prevention and control programs against sources and reservoirs of zoonoses should be planned by public health and veterinary officers based on reliable information from systematic surveillance.

Dynamics of Centrocestus armatus Transmission in Endemic River in Hyogo Prefecture, Japan.

Centrocestus armatus is an intestinal parasite belonging to the family Heterophyidae. We developed an apparatus for recovering cercariae and clarified the infection dynamics of this parasite. To clarify the circadian rhythm of cercarial shedding in the summer season, we filtrated 30 l of river water every 2 h for 24 h. Cercariae were first detected between 06:00 and 08:00 h, increased over time to reach peak at 16:00 h and decreased thereafter, thus showing a single-peak pattern. In a survey of seasonal change, approximately 200 cercariae were contained in 1 l of river water during the summer season, while none were found during the winter. This cercarial shedding pattern appeared to be related to sunrise/sunset and water/atmosphere temperature. Therefore, we examined whether cercarial shedding was affected by light or temperature changes under laboratory conditions, and confirmed that both light and temperature were important factors for cercarial shedding. Light was a stronger factor than water temperature. Cercarial shedding of C. armatus occurred in response to temperature and light. The change in the number of juvenile metacercariae detected in fish brain corresponded with monthly detection rates of cercariae; however, the incidence of new infections decreased in August. This suggests that Nipponocypris temminkii contains a defense mechanism against new infections that may have hindered the increase in parasite infectivity. These results clarified the smooth infection from the first to the second intermediate host of C. armatus in the endemic river. Throughout the study period, fecal samples were collected from 19 kites, 114 herons, and three unidentified species. However, our results using C. armatus showed a low value of 1% in herons and 5% in kites. The infection dynamics of final host to first intermediate host need to be further investigated.

Soil contamination by parasite eggs in rural village in the Philippines.

Infectious diseases caused by soil-transmitted helminths (STHs) are important diseases of humans, which affect about one third of the world's population. Examination of soil can be used to estimate the risk of STH infection in humans. We carried out this survey to clarify the current status of soil contamination by parasite eggs and to assess the risk of STH infection. During survey periods, we examined soil, faeces, and the lifestyle of residents. Six genera and eight species of parasite eggs including Ascaris lumbricoides, Toxocara cati, Toxocara canis, and Trichuris trichiura were recovered from 85 out of 120 soil samples (71%). Contamination of soil by parasite eggs had spread widely throughout the village, and 50% of eggs recovered had already developed into fertilized eggs. It is remarkable that Ascaris eggs were recovered from inside the houses. Prevalence of STH in school children was 63%. This may indicate that school or preschool children cause soil contamination. Some of the eggs recovered were not only from humans but also from dogs and cats. From the results obtained, the need for health education with regards to zoonoses was revealed because 77% of fertilized Toxocara spp. eggs were detected. We conclude that the risk of STH infection in residents was extremely high, because the soil in this village was highly contaminated by infective parasite eggs.

A comparative histopathology, serology and molecular study, on experimental ocular toxocariasis by Toxocara cati in Mongolian gerbils and Wistar rats.

The aim of this study was to compare the performance of three in-house diagnostic tests, that is, histopathology, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR), for the diagnosis after experimental infection with Toxocara cati. Twenty Mongolian gerbils and Wistar rats were divided into ten groups (n = 2/group). Toxocara cati infections were established in Mongolian gerbils and Wistar rats by administering doses of 240 and 2500 embryonated Toxocara cati eggs by gavage, respectively. Tissue sections were stained with Haematoxylin and Eosin and observed under the light microscope. Sera and vitreous fluid collected from separate infected groups were tested against Toxocara cati antigens, for 92 days postinfection. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) blocks, and aqueous fluids belong to the animals. The histopathology test gave negative results among the groups of animals examined between 5 and 92 days postinfection. The ELISA results showed that anti-Toxocara antibodies have risen between 7 and 61 days postinfection in sera and vitreous fluid in the animals infected, respectively. Analysis of PCR products revealed positive band (660 bp) in the orbital tissue infected Mongolian gerbils at 5 days postinfection. Of the three evaluated methods, the PCR could be recommended for scientific and laboratory diagnoses of toxocariasis in experimentally infected animals.

Development of free-living stages of Strongyloides ratti under different temperature conditions.

It is well known that the Strongyloides species have two different developmental courses-direct and indirect development-and selection of these courses is affected by various environmental factors. This study examined the effect of temperature on the development of first-stage larvae (L1s) of Strongyloides ratti, to clarify how larvae adapt and survive at unsuitable temperatures. It was revealed that L1s cultured at 4 or 10 °C for 120 h could not develop because of growth arrest or delay. However, L1s could develop after transfer to culture at 25 °C for 48 h. Although larvae cultured at 25 °C take indirect development, larvae subjected to low-temperature stimulation (at 4 or 10 °C) take direct development into infective third-stage larvae (L3s), and only 1 min of low-temperature stimulation was sufficient to induce direct development. Morphological study of low-temperature-stimulated L3s revealed that those stimulated at 4 °C (L3-4) showed less development, but those stimulated at 10 °C (L3-10) developed as well as the control (no low-temperature stimulation). Furthermore, we revealed that L3-10 showed similar infectivity to the control when they were injected subcutaneously into rats as the final host, which indicated that L3-10 grew normally. We conclude that S. ratti has a survival strategy of growth arrest or delay if excreted in cold conditions. Moreover, even if they start development after transfer to suitable conditions, they memorize low-temperature stimulation, which leads them to direct development thereafter so that they can immediately infect the final host.

Assessment of skin penetration of third-stage larvae of Strongyloides ratti.

Strongyloides stercoralis infection is caused by skin penetration of third-stage larvae (L3s). We studied skin penetration of L3s of Strongyloides ratti using an in vitro assay that has been used previously to study Angiostrongylus cantonensis, an agarose membrane with a temperature gradient, and scanning electron microscopy. Our results revealed that skin penetration of L3s depended on host skin temperature. When the target temperature of the outer liquid was 37°C, more than 80% of L3s penetrated the skin, but penetration was only 60% when the target temperature was 20°C. Thirdstage larvae moved rapidly on the agarose membrane toward optimum temperature area for this parasite, which indicates that L3 has a sensor that is sensitive to temperature changes. Penetration rate for hosts such as cat (36%), dog (32%), and bird (13%) were significantly lower than that for rat (82%). Although we could not establish the reason, L3s seemed to have an ability to differentiate these hosts at the time of penetration. By using scanning electron microscopy, penetration of L3s could be observed within 10 min. We demonstrated thermotaxis of L3 of S. ratti, and this peculiar characteristic seemed to have a close relationship with the process of searching for the host.

A human case of subcutaneous dirofilariasis caused by Dirofilaria repens in Vietnam: histologic and molecular confirmation.

Human dirofilariasis caused by infection with Dirofilaria worms has been frequently reported. The symptoms associated with infection by these filarial parasites, which are transmitted to humans by zooanthropophilic mosquitoes, are characterized by mainly pulmonary and subcutaneous nodules. Here, we report the first case in Vietnam of a subcutaneous dirofilariasis with a painful nodule in the right eyelid. An immature female worm was removed by excisional biopsy and identified as Dirofilaria repens by histology and DNA analysis.

Experimental Toxocara cati infection in gerbils and rats.

Mongolian gerbils and Wistar rats were inoculated orally with 240 and 2,500 Toxocara cati embryonated eggs, respectively, to evaluate the larval recovery in different tissues and organs, such as the liver, lungs, heart, kidney, and skeletal muscles after 5, 30, 49, 70, and 92 days post-infection (PI). Larval recovery rates were 1.7-30.0% in Mongolian gerbils on days 5-92 PI and 0.2-3.8% in rats on the same days. These results indicate that Mongolian gerbils and Wistar rats are suitable experimental paratenic hosts for the study of neurological toxocariasis as well as visceral toxocariasis.