Thelazia callipaeda in Slovakia - From sporadic cases to endemic areas.

Spirurid nematode Thelazia callipaeda, transmitted by the fruit fly Phortica variegata, is a causative agent of an ocular parasitic disease called also canine thelaziosis. Dogs, cats, and wild canids are considered the primary definitive hosts for the parasite, but humans may also serve as aberrant definitive hosts. For long decades the geographic range of T. callipaeda was strictly limited to the territory of Asia, but after the year 2000, the parasite began to spread rapidly through Europe. The first autochthonous infections of dogs and foxes in Slovakia were recorded in 2016. In the present study, the results of a whole-area surveillance for canine thelaziosis are reported. Altogether, 142 cases of infection caused by T. callipaeda were diagnosed by veterinarians in dogs between 2016 and the first quarter of 2021, and two cases of feline thelaziosis were recorded. The majority of the dogs showed mild ocular signs manifested by conjunctivitis; 8.5% of them suffered from more serious mucopurulent discharge, and in two dogs corneal ulceration was recorded. The screening revealed increasing trends in the occurrence of canine thelaziosis from both a temporal and spatial point of view and unambiguously confirms the endemic status of T. callipaeda in Slovakia with the prospect of its further expansion.

Echinocephalus inserratus sp. n. (Nematoda: Gnathostomatidae) from the stingray Pastinachus ater (Dasyatidae) and new records of congeneric and some other nematode larvae from teleost fishes off New Caledonia.

Based on light and electron microscopical studies, a new nematode parasite, Echinocephalus inserratus sp. n. (Spirurida: Gnathostomatidae), is described from the spiral valve of the broad cowtail stingray Pastinachus ater (Macleay) (Dasyatidae, Myliobatiformes) from off New Caledonia. The new species is morphologically and biometrically most similar to Echinocephalus overstreeti Deardorff et Ko, 1983, differing from it mainly in the absence of serrations on the posterior parts of pseudolabia and on interlabia, and in having a longer gubernaculum (150-299 µm long). Morphologically unidentifiable, mostly encapsulated larvae of Echinocephalus spp. were recorded from the following six species of teleost fishes collected in New Caledonian waters, serving as paratenic hosts: Perciformes: Acanthopagrus berda (Forsskål) (Sparidae) and Nemipterus furcosus (Valenciennes) (Nemipteridae); Tetraodontiformes: Abalistes stellatus (Anonymous), Pseudobalistes fuscus (Bloch et Schneider) (both Balistidae), Lagocephalus sceleratus (Gmelin) (Tetraodontidae) and Aluterus monoceros (Linnaeus) (Monacanthidae). Co-parasitising larvae of Ascarophis sp. and Hysterothylacium sp. were also collected from P. fuscus. All these findings represent new host and geographical records. A key to valid species of Echinocephalus Molin, 1858 is provided.

A Helminth Survey of Northern Bobwhite Quail (Colinus virginianus) and Passerines in the Rolling Plains Ecoregion of Texas.

The Northern bobwhite quail (Colinus virginianus) is a popular game bird that has been experiencing a well-documented decline throughout Texas since the 1960s. While much of this decline has been attributed to habitat loss and fragmentation, recent studies have identified other factors that may also contribute to decreasing quail populations. Parasites, in particular, have become increasingly recognized as possible stressors of quail, and some species, particularly the eyeworm (Oxyspirura petrowi) and cecal worm (Aulonocephalus pennula) are highly prevalent in Texas quails. Eyeworm infection has also been documented in some passerines, suggesting helminth infection may be shared between bird species. However, the lack of comprehensive helminth surveys has rendered the extent of shared infection between quail and passerines in the ecoregion unclear. Thus, helminth surveys were conducted on bobwhite, scaled quail (Callipepla squamata), Northern mockingbirds (Mimus polyglottos), curve-billed thrashers (Toxistoma curvirostre), and Northern cardinals (Cardinalis cardinalis) to contribute data to existing parasitological gaps for birds in the Rolling Plains ecoregion of Texas. Birds were trapped across 3 counties in the Texas Rolling Plains from March to October 2019. Necropsies were conducted on 54 individuals (36 quail and 18 passerines), and extracted helminths were microscopically identified. Nematode, cestode, and acanthocephalan helminths representing at least 10 helminth species were found. Specifically, A. pennula and O. petrowi had the highest prevalence, and O. petrowi was documented in all of the study species. This research adds to the body of knowledge regarding parasitic infections in quail and passerines of the Rolling Plains ecoregion and highlights the potential consequences of shared infection of eyeworms among these bird species.

[Ocular thelaziosis in a dog in Germany - an autochthonous case?] / Okuläre Thelaziose bei einem Hund in Deutschland ­ ein autochthoner Fall?

Thelazia callipaeda is a vector-borne nematode transmitted by Phortica spp. (fruit flies) and one of the causes of mild to severe conjunctivitis and keratitis in dogs, cats, rabbits and humans. It has been formerly known as the oriental eye worm based on its geographic occurrence. By now, it has been shown to be endemic in several southern and eastern European countries as well as extending its geographic distribution further throughout Europe. In the present case report, T. callipaeda infection was diagnosed in a female dog from Germany. The dog was referred by a local veterinarian due to a treatment-resistant conjunctivitis. A comprehensive ophthalmologic examination revealed 3 adult eye worms in the conjunctival sac as well as on the bulbar side of the nictitating membrane of the left eye. These were identified by morphological features and molecular techniques as T. callipaeda and represented the primary cause of the dog's unilateral blepharospasm, hyperemic conjunctiva and epiphora. Treatment consisted of manual collection of the adult worms and the administration of moxidectin/imidacloprid as spot on (Advocate®, Bayer). All clinical signs resolved within one week after treatment. In Germany, ocular thelaziosis still represents a rare disease. Usually, it is diagnosed in cats and dogs either imported from abroad or accompanying the owners on holiday travels to endemic countries. The dog presented in this case report was born in Germany. Except for a one-week stay in the Netherlands 11 months before the symptoms began, it had never traveled abroad. Considering the prepatency of T. callipaeda, an autochthonous transmission in this case is highly probable.

Molecular characterization of Oxyspirura mansoni and Philophthalmus gralli collected from the eyes of domestic chickens in Bangladesh.

A variety of helminths have been found in domestic chickens in Bangladesh, but little is known about their gene sequences. Here, parasitic nematodes and trematodes were collected from the eyes of domestic chickens and analyzed for their morphological and morphometric characteristics, and characterized molecularly. The helminths were identified as Oxyspirura mansoni and Philophthalmus gralli. The ITS1 and ITS2 sequences of O. mansoni were 532 bp and 306 bp in length, respectively, and showed low identity (50.7-62.7%) with those of O. petrowi and O. conjunctivalis. Furthermore, the O. mansoni CO1 sequences (393 bp) showed five haplotypes (97.5-99.5% similarity) that formed a monophyletic clade. With respect to P. gralli, the ITS1 (452 bp) and ITS2 (736 bp) sequences showed 100% similarity with the reference sequences in GenBank. Both the ND1 and CO1 phylograms showed that P. gralli from Bangladesh, Costa Rica and Peru form a monophyletic clade, distinct from the clades of P. lucipetus and P. lacrymosus. Our data show that, Philophthalmus gralli isolates from Bangladesh, Costa Rica and Peru are genetically close to each other.

Suspected autochthonous Thelazia callipaeda infection in a dog in northern Germany.

A 12-year old Elo dog was presented with recurring symptoms of conjunctivitis in November 2019. A single whitish nematode was found upon inspection of the eye and identified as a Thelazia callipaeda male. The morphological identification of the eye worm was supported by analysis of a partial cytochrome c oxidase I (cox1) gene sequence. The dog lived in Lower Saxony, northwestern Germany, and had not visited regions known to be endemic for T. callipaeda. This suggests that a local transmission cycle of this zoonotic nematode may exist in Germany.

First report of Spirocerca vulpis in red foxes (Vulpes vulpes) in Portugal.

Recent studies have described Spirocerca lupi-like nematodes in the stomach of red foxes (Vulpes vulpes) in Europe. A phylogenetic analysis of those specimens using mitochondrial DNA and their morphological reexamination allowed their characterization as a different species, Spirocerca vulpis. Between the years of 2010 and 2017, roundworms were collected from seven red foxes of northeastern Portugal found at necropsy with nodular lesions on their stomach wall. Histopathological analysis of four foxes revealed granulomatous lesions of the gastric nodules. On morphological assessment, by light microscopy, nematodes revealed the presence of six triangular teeth-like buccal capsule structures, which are absent in S. lupi. Polymerase chain reaction was run to amplify a 551 bp partial fragment of the cytochrome c oxidase subunit 1 gene. Sequences were 99% similar to S. vulpis (85% coverage) of red foxes from Spain and Bosnia and Herzegovina, 99% similar (99% coverage) to sequences of Spirocerca sp. of red foxes from Denmark and 93% similar (99% coverage) to S. lupi from South Africa. This is the first report of S. vulpis in foxes or any other host from Portugal.

Unravelling Spirocerca vulpis from red foxes from Switzerland: a 20-year-old record.

Spirocerca vulpis is a parasitic nematode of red foxes associated with gastric nodule formation. Since its description in 2018, this species has been reported in red foxes from Spain, Bosnia and Herzegovina, Italy, and Portugal. We present here the analysis of uncharacterized nematodes obtained from gastric nodules of a red fox from Switzerland in 1999. The specimens were identified as S. vulpis based on the observation of teeth-like structures in the buccal capsule and a 99.4% sequence identity to S. vulpis DNA from Spain. Phylogenetic analysis demonstrated the clustering of the Swiss sequences in a different group from specimens of other geographical locations. Altogether, this study constitutes the first report of S. vulpis in Switzerland and a report of the oldest specimen of this species in the world. Our findings highlight the widespread distribution of S. vulpis in Europe which may be facilitated by the free-roaming nature of red foxes.

A Case of Human Thelaziasis and Review of Chinese Cases.

PURPOSE: Human cases of thelaziasis caused by Thelazia callipaeda have increased in China in recent years. Although this species is of medical importance, our knowledge about the epidemiology of thelaziasis is still fragmentary. This study first reports a case of thelaziasis in central China. Then, the epidemiology of thelaziasis in China in the past 100 years (1917-2018) is reviewed. METHODS: A 5-year-old girl experienced discomfort in her left eye. Four thread-like worms were seen in the nasal upper eyelid of the left eye. The symptoms disappeared after these parasites were removed. In addition, we reviewed studies of Chinese human thelaziasis cited in articles or book chapters in all languages from inception to 31 Dec 2019. RESULTS: China is the nation with the most reports of thelaziasis (653 cases) in the world. More human cases were reported in central and eastern China than in other areas, and the majority of cases were from rural areas in poor socioeconomic settings. CONCLUSION: Special attention should be paid to this neglected disease in China. The use of a One Health approach is imperative for preventing eyeworm infections in humans.

[Screening and bioinformatic analysis of trehalase in Thelazia callipaeda].

OBJECTIVE: To characterize the trehalase gene in Thelazia callipaeda through screening the annotated data of the T. callipaeda genome, and to investigate the biological characteristics of the trehalase gene-coding protein. METHODS: The trehalase gene was screened from the T. callipaeda genome and subjected to validation by using a PCR assay. The structural features of the coding protein were analyzed with bioinformatics tools, including hydrophobicity, transmembrane region, signal peptides, conserved domains, as well as the secondary and tertiary structures and the antigen epitope. Homology analysis of the amino acid sequences was performed, and the phylogenetic tree was built by the MEGA X software. In addition, the protein-protein interaction network was deduced from the STRING database. RESULTS: The sequence of the trehalase gene with the complete CDS region was obtained from T. callipaeda genome, which had a length of 1 638 bp and encoded 545 amino acids. The encoded protein was predicted to have a molecular weight of 63 478.48 ku and be a secretory protein. The 5' domain of the encoded protein contained a signal peptide without transmembrane regions, and was predicted to contain 7 antigen epitopes. Based on the protein-protein interaction network of nematodes in the STRING database, the protein-protein interaction network of the trehalase gene of T. callipaeda was deduced, and 27 interactions covering 10 genes were identified. CONCLUSIONS: A trehalase gene is successfully identified in T. callipaeda genome and its coding protein receives a bioinformatics analysis, which provides insights into the research on the biological functions of the protein and the screening of vaccine candidates for thelaziasis callipaeda.

Molecular Identification of Oxyspirura Petrowi Intermediate Hosts by Nested PCR Using Internal Transcribed Spacer 1 (ITS1).

Recently, the heteroxenous eyeworm, Oxyspirura petrowi, has gained attention due to its prevalence in the declining game bird, Northern bobwhite (Colinus virginianus), but the intermediate hosts of many nematodes remain unknown. However, identifying the intermediate host of O. petrowi with traditional techniques would be difficult and time-consuming, especially considering there are more than 80 potential orthopteran hosts just in Texas. To screen a large number of samples quickly and effectively, primers for nested PCR (nPCR) were developed using the internal transcribed spacer 1 (ITS1) region. Then the nPCR was used to identify which of the 35 species collected from the Order Orthoptera were potential intermediate hosts of O. petrowi. With this technique, 18 potential intermediate hosts were identified. Later, we collected live specimens of species that tested positive to confirm the presence of larvae, but larvae were not found in the live specimens, nor in the extra tissue of the species that had tested positive for O. petrowi DNA. Despite this, this study demonstrated that nPCR is more sensitive than traditional techniques and can be a valuable tool in determining the intermediate hosts of parasites.

Insights on Spirocerca lupi, the Carcinogenic Dog Nematode.

Spirocerca lupi is a nematode transmitted by dung beetles that infects domestic and wild canids in tropical and subtropical regions and is associated with neoplasia. It produces a distinctive pathology with the formation of esophageal nodules classified as inflammatory, preneoplastic, or neoplastic with metastasis to distant organs. Aberrant central nervous system migration of this nematode is also responsible for severe neurological manifestations. Reports of spirocercosis have increased over the last two decades showing spread of this canine helminth in five continents. S. lupi from different geographical locations is genetically distinct with two genotypes, genotype I from Africa, Asia, and Australia, and genotype II from Europe, and recently separated from Spirocerca vulpis, a new species described in red foxes from Europe.

First report of canine ocular thelaziosis in the Republic of Moldova.

BACKGROUND: Countries of eastern Europe are considered, due to several risk factors, more vulnerable to infections with newly (re)emerging pathogens. During the last decade, in several European countries, reports of autochthonous cases of ocular thelaziosis due to Thelazia callipaeda have been published, posing a great concern from both veterinary and public health perspective. However, in the Republic of Moldova only limited epidemiological data are available regarding zoonotic vector-borne pathogens and, until now, no data exist on the zoonotic nematode T. callipaeda. METHODS: In September 2018, an 11-year-old dog, mixed-breed, intact male was referred to a private veterinary clinic from Chișinau, Republic of Moldova, with a history of 2 weeks of an ocular condition affecting the right eye. The ophthalmological exam revealed the presence of nematode parasites in the conjunctival sac and under the third eyelid. The collected parasites were identified by morphological techniques and molecular analysis. RESULTS: A total of 7 nematodes were collected, and 5 females and 2 males of T. callipaeda were identified morphologically. The BLAST analysis confirmed the low genetic variability of this parasite in Europe. The travel history of the patient allowed us to confirm the autochthonous character of the case. CONCLUSIONS: To the best of our knowledge, this is the first report of thelaziosis in dogs from the Republic of Moldova, which confirms the spreading trend of T. callipaeda and the existence of an autochthonous transmission cycle of this zoonotic parasite in the country.

First report of eyeworm infection by Thelazia callipaeda in gray wolf (Canis lupus) from Serbia.

Thelazia callipaeda, originally known as an "Oriental eyeworm," is a small nematode parasitizing the conjunctival sacs of domestic and wild animals and humans. Previous studies conducted in Serbia have reported the eyeworm infections in dogs, cats, and foxes, as well as in a human patient. As the data regarding thelaziosis from wildlife is still scarce, the aim of this study was to investigate the presence of T. callipaeda in gray wolf (Canis lupus) from Serbia. All collected nematodes were morphologically identified as T. callipaeda males (n = 64) or females (n = 225). Molecular characterization, conducted by PCR amplification followed by sequence analysis of partial cytochrome c oxidase subunit 1 gene (cox1), revealed only haplotype 1 of T. callipaeda. The overall prevalence of thelaziosis was 38.1% (8/21). In all positive animals, both eyes were affected, with a total parasitic load ranging from four to 132 worms per animal. Our results indicate the important epidemiological role of wolves as wildlife reservoirs of T. callipaeda, expanding geographic range of infection, as well as intra- and interspecies contact rates, although the role of other wild carnivore species (i.e., foxes and jackals) should be investigated in future studies.

[Advances in etiology, epidemiology and genetic diversity of Thelazia callipaeda].

Thelazia callipaeda, T. californiensis and T. gulosa are three causative agents of human thelaziasis. Most of the reported cases were caused by T. callipaeda, occurring in the old world, particularly in Asian and European countries. T. californiensis and T. gulosa have rarely been reported infecting humans in North America. T. callipaeda has long been called the oriental eye worm, referring to its traditional distribution across eastern and southeastern Asia (i.e., China, Korea, Japan, Indonesia, Thailand, and India) where infection is endemic in animals and humans, usually in poorer rural areas and mainly among children and the elderly. The identification of the parasite was mainly based on the characteristics of oral and genital organs. In Asia and Europe, vectors for this nematode are male Phortica okadai and P. variegata drosophilids respectively, which feed on ocular secretions of hosts and transmit infective stage larvae to domestic and wild carnivores, lagomorphs, and humans. China probably has the largest number of cases with thelaziasis in the world, and lots of cases have been existed in other Asian countries such as Japan and Korea. Although a few of human cases have been reported, there were high infection rates of wild animals and domesticated dogs and cats in most of European countries. Based on the cox1 gene, a total of 21 haplotypes were identified in the samples from worldwide, in which, one circulated only in European countries (h1), while the other 20 haplotypes were distributed in Korea, Japan and China. In general, the Chinese clinical isolates of T. callipaeda expressed high genetic diversity. The population differences between Europe and Asian countries were greater than those among China, Korea and Japan. The T. callipaeda populations from Europe and Asia should be divided into two separate sub-populations. These two groups started to diverge during the middle Pleistocene.

Phylogenetic analysis of Spirocerca lupi and Spirocerca vulpis reveal high genetic diversity and intra-individual variation.

BACKGROUND: Spirocerca lupi is a parasitic nematode of canids that can lead to a severe and potentially fatal disease. Recently, a new species, Spirocerca vulpis, was described from red foxes in Europe, suggesting a high genetic diversity of the Spirocerca spp. infecting canids. The genetic variation and phylogenetic relationships of S. lupi collected from naturally-infected domestic dogs from Australia, Hungary, Israel, Italy, India and South Africa, and S. vulpis from red foxes from Bosnia and Herzegovina, Italy and Spain, was studied using mitochondrial and rDNA markers. RESULTS: A high intra-individual variation was found in the first internal transcribed spacer (ITS1) locus in all Spirocerca spp., ranging between 0.37-2.84%, with up to six haplotypes per specimen. In addition, a combination of phylogenetic and haplotype analyses revealed a large variability between S. lupi specimens collected from different geographical locations using the ITS1 (0.37-9.33%) and the cytochrome c oxidase subunit 1 (cox1) gene (1.42-6.74%). This genetic diversity led to the identification of two S. lupi genotypes circulating among dogs (PTP support > 0.829), including genotype 1 found in S. lupi from Australia, India, Israel and South Africa, and genotype 2 represented by specimens from Hungary and Italy. These genotypes presented pairwise nucleotide distances of 0.14%, 8.06% and 6.48 ± 0.28% in the small rDNA subunit (18S), ITS1 and cox1 loci, respectively. Additionally, Nei's genetic distance in the ITS1 showed a further subdivision of genotype 1 worms into 1A (Israel and South Africa) and 1B (Australia and India). A morphological analysis of the anterior and posterior extremities of genotype 1 and genotype 2 worms using scanning electron microscopy did not show any differences between the specimens, contrary to the morphological differences between S. lupi and S. vulpis. CONCLUSIONS: These findings demonstrate the high genetic variability among Spirocerca spp. from different geographical locations, thereby expanding our understanding of the epidemiology, evolution and phylogenetic variability within the genus.

Phylogenetic Relationships of Spiruromorph Nematodes (Spirurina: Spiruromorpha) In North American Freshwater Fishes.

Nematodes are common in the parasite communities of North American freshwater fishes, and the majority of them belong to 1 conventional order, Spirurida Chitwood, 1933. Within the Spirurida, the superfamilies Habronematoidea Chitwood and Wehr, 1932 and Thelazioidea Sobolev, 1949 have undergone considerable diversification. The dominant families of these 2 superfamilies, Cystidicolidae Skrjabin 1946 and Rhabdochonidae Railliet, 1916, respectively, are particularly common, widely distributed, and diverse, especially in North America, yet their phylogenetic relationships remain largely unexplored. In this study, we use near complete sequences of the 18S rRNA genes ( SSU rDNA) from species in 6 genera ( Capillospirura Skrjabin, 1924, Cystidicola Fischer, 1798, Salmonema Moravec, Santos and Brasil-Sato, 2008, Rhabdochona Railliet, 1916, Spinitectus Fourment, 1883, and a putative new cystidicolid in mooneyes, Hiodontidae), along with a species of Hedruris Nitzsch, 1812 from newts as a surrogate for the fish parasite Hedruris tiara VanCleave and Mueller, 1932, to explore their phylogenetic relationships. These sequences, together with available sequences from a range of other nematodes, including fish nematodes in other groups (Camallanoidea and 'Seuratoidea'), were analyzed using Bayesian inference and maximum likelihood. The results from both analyses indicate, for the first time, support for the close relationships of the sturgeon parasite Capillospirura with Ascarophis van Beneden, 1871 and Cystidicola; the relationship of the cystidicolid from Hiodontidae with Salmonema of salmonid fishes; the monophyly of the 2 dominant spiruridan genera of fishes, Rhabdochona and Spinitectus; and for previous relationships among Nearctic Spinitectus spp. The results also indicate a closer relationship of Rhabdochona and Spinitectus than is suggested by their conventional classification and reject the monophyly of Habronematoidea, Thelazioidea, and Cystidicolidae. Hedruridae appears to be an early branching lineage of spirurins. Finally, the pattern of association between the fish parasites in this study and their hosts indicates, with few exceptions, ecologically driven diversification events involving host shifting not related to the phylogenetic relationships of their hosts.

Spirocerca vulpis sp. nov. (Spiruridae: Spirocercidae): description of a new nematode species of the red fox, Vulpes vulpes (Carnivora: Canidae).

Previous studies have reported nematodes of the Spirocercidae family in the stomach nodules of red foxes (Vulpes vulpes) described as Spirocerca sp. or Spirocerca lupi (Rudolphi, 1819). We characterized spirurid worms collected from red foxes and compared them to S. lupi from domestic dogs by morphometric and phylogenetic analyses. Nematodes from red foxes differed from S. lupi by the presence of six triangular teeth-like buccal capsule structures, which are absent in the latter. Additionally, in female worms from red foxes, the distance of the vulva opening to the anterior end and the ratio of the glandular-to-muscular oesophagus lengths were larger than those of S. lupi (P < 0.006). In males, the lengths of the whole oesophagus and glandular part, the ratio of the glandular-to-muscular oesophagus and the comparison of the oesophagus to the total body length were smaller in S. lupi (all P < 0.044). Phylogenetic analyses revealed that S. lupi and the red foxes spirurid represent monophyletic sister groups with pairwise nucleotide distances of 9.2 and 0.2% in the cytochrome oxidase 1 and 18S genes, respectively. Based on these comparisons, the nematodes from red foxes were considered to belong to a separate species, for which the name Spirocerca vulpis sp. nov. is proposed.

Population structure analysis of the neglected parasite Thelazia callipaeda revealed high genetic diversity in Eastern Asia isolates.

BACKGROUND: Thelazia callipaeda is the causative agent of thelaziasis in canids, felids and humans. However, the population genetic structure regarding this parasite remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we first explored the genetic variation of 32 T. callipaeda clinical isolates using the following multi-molecular markers: cox1, cytb, 12S rDNA, ITS1 and 18S rDNA. The isolates were collected from 13 patients from 11 geographical locations in China. Next, the population structure of T. callipaeda from Europe and other Asian countries was analyzed using the cox1 sequences collected during this study and from the GenBank database. In general, the Chinese clinical isolates of T. callipaeda expressed high genetic diversity. Based on the cox1 gene, a total of 21 haplotypes were identified. One only circulated in European countries (Hap1), while the other 20 haplotypes were dispersed in Korea, Japan and China. There were five nucleotide positions in the cox1 sequences that were confirmed as invariable among individuals from Europe and Asia, but the sequences were distinct between these two regions. Population differences between Europe and Asian countries were greater than those among China, Korea and Japan. The T. callipaeda populations from Europe and Asia should be divided into two separate sub-populations. These two groups started to diverge during the middle Pleistocene. Neutrality tests, mismatch distribution and Bayesian skyline plot (BSP) analysis all rejected possible population expansion of T. callipaeda. CONCLUSIONS: The Asian population of T. callipaeda has a high level of genetic diversity, but further studies should be performed to explore the biology, ecology and epidemiology of T. callipaeda.

Molecular detection of Oxyspirura larvae in arthropod intermediate hosts.

To determine potential intermediate hosts of Oxyspirura petrowi, a common nematode eyeworm of wild gallinaceous birds, various arthropod species including red harvester ants, beetles, wood cockroaches, crickets, grasshoppers, katydids, and desert termites were screened for the presence of O. petrowi using specific polymerase chain reaction (PCR) primers targeting the internal transcribed spacer 2 region (ITS2) of the eyeworm ribosomal deoxyribonucleic acid (rDNA). This is the first study to investigate the intermediate hosts of O. petrowi utilizing molecular techniques. We determined 38% (13/34) of the cockroaches, 27% (3/11) of the crickets, and 23% (68/289) of the grasshoppers which were positive for O. petrowi. Identifying potential intermediate hosts of O. petrowi is essential to better understanding the epizoology of the eyeworm's transmission mechanics and to controlling infections in wild gallinaceous birds.