Selection of suitable reference genes for normalization of RT-qPCR in three tissues of Northern bobwhite (Colinus virginianus) infected with eyeworm (Oxyspirura petrowi).

BACKGROUND: The Northern bobwhite (Colinus virginianus) is an economically important, and popular game bird in North America. Northern bobwhites have experiencing declines of > 3.5% annually in recent decades due to several factors. The eyeworm Oxyspirura petrowi is a nematode parasite frequently found in the eyes of bobwhites. Although reported frequently in wild bobwhites, there is no research to understand the host-parasite mechanism. Hence, it is important to investigate mechanisms of eyeworm invasion and immune modulation in bobwhite. Cytokine gene expression using RT-PCR is widely used to identify the innate immune response of a host to an infection. METHODOLOGY: In this study, we evaluated ten reference genes (HMBS, RPL19, RPL32, RPS7, RPS8, TATA, SDHA, YWHAZ, GAPDH, and ACTB) for their stability across three tissues (liver, spleen, and caecal tonsils) of control and O. petrowi infected Northern bobwhites. Primer efficiency and reference genes stability were assessed using GeNorm, NormFinder, and BestKeeper. RESULTS: Expression of these reference genes with respect to O. petrowi infection in bobwhites showed RPL32 and HMBS were the most stable genes in the liver, HMBS and SDHA were the most stable genes in the spleen, and HMBS and YWHAZ were equally stable reference genes in the caecal tonsils. CONCLUSION: Based on the geometric mean of all three analyses, our results indicate that the combination of RPL32 and HMBS for the liver, HMBS and SDHA for the spleen, and YWHAZ and HMBS for caecal tonsils might be used as reference genes for normalization in gene expression investigations on Northern bobwhites.

Emergence of Thelaziosis Caused by Thelazia callipaeda in Dogs and Cats, United States.

We report 2 autochthonous feline thelaziosis cases caused by the eyeworm Thelazia callipaeda and discuss the spread among dogs in the northeastern United States. Phylogenetic analysis suggests the parasite was introduced from Europe. Adopting a One Health approach is needed to limit further spread of T. callipaeda eyeworms in North America.

Occurrence of Thelazia callipaeda and its vector Phortica variegata in Austria and South Tyrol, Italy, and a global comparison by phylogenetic network analysis.

The zoonotic nematode Thelazia callipaeda infects the eyes of domestic and wild animals and uses canids as primary hosts. It was originally described in Asia, but in the last 20 years it has been reported in many European countries, where it is mainly transmitted by the drosophilid fruit fly Phortica variegata. We report the autochthonous occurrence of T. callipaeda and its vector P. variegata in Austria. Nematodes were collected from clinical cases and fruit flies were caught using traps, netting, and from the conjunctival sac of one dog. Fruit flies and nematodes were morphologically identified and a section of the mitochondrial cytochrome c oxidase subunit I gene (COI) was analysed. A DNA haplotype network was calculated to visualize the relation of the obtained COI sequences to published sequences. Additionally, Phortica spp. were screened for the presence of DNA of T. callipaeda by polymerase chain reaction. Thelazia callipaeda and P. variegata were identified in Burgenland, Lower Austria, and Styria. Thelazia callipaeda was also documented in Vienna and P. variegata in Upper Austria and South Tyrol, Italy. All T. callipaeda corresponded to haplotype 1. Twenty-two different haplotypes of P. variegata were identified in the fruit flies. One sequence was distinctly different from those of Phortica variegata and was more closely related to those of Phortica chi and Phortica okadai. Thelazia callipaeda could not be detected in any of the Phortica specimens.

Spirocerca lupi in the stomach of two Andean foxes (Lycalopex culpaeus) from Chile.

The genus Spirocerca includes nematodes that parasitize the stomach and the oesophagus of carnivores, chiefly canids. Herein, we provide new data about the morphological, histopathological, and molecular characterization of Spirocerca sp. in Andean foxes (Lycalopex culpaeus) in Chile. Intact immature worms, identified as Spirocerca sp., were recovered in the lumen of the stomach from two foxes. Histologically, worms morphologically consistent with spirurid nematodes were present within the wall of the stomach and surrounded by nodular areas of inflammation with central necrotic debris. Molecular analysis of the cox1 gene yielded 19 sequences and 5 nucleotide sequence types with 99.95 to 99.98% similarity, being shared between both foxes. Nucleotide similarity ranged from 93.1 (with genotype 2 of S. lupi and S. vulpis) to 95.8% (with genotype 1 of S. lupi), a higher similarity than noted from sequences of S. lupi from an Andean fox from Peru (91.0 to 93.3%). However, the Poisson Tree Processes for species delineation did not support the existence of a new species Spirocerca. Phylogenetic and nucleotide analyses suggest that these specimens belong to a new variant or genotype of S. lupi or to a cryptic species. Whether the presence of the worms in the stomach has to do with genotypic differences in parasites or host or some combination is uncertain. Spirocerca lupi has never been found in Chilean dogs and must be investigated.

Molecular characterization of oriental eyeworm (Thelazia callipaeda) detected from raccoon (Procyon lotor) and Japanese raccoon dog (Nyctereutes viverrinus) in Kanto region, Japan.

BACKGROUND: The oriental eyeworm Thelazia callipaeda (Spirurida: Thelaziidae) is an emerging parasitic ocular nematode of carnivores and humans. In domestic animals and humans, the infection causes varying degrees of inflammation and lacrimation, and wild carnivores represent an important reservoir. In this study we examined the infection status and molecular characterization of T. callipaeda in two urban carnivores, raccoons Procyon lotor and wild Japanese raccoon dogs Nyctereutes viverrinus, in the Kanto region of Japan. METHODS: From January 2020 to December 2021, 193 carcasses including 178 raccoons and 15 raccoon dogs were examined for the presence of worms in the eye. The worms from infected animals (one worm per host) were morphologically identified as T. callipaeda. Worms (1-5 worms per host) were subjected to genetic analysis using mitochondrial cytochrome c oxidase subunit I gene sequences. RESULTS: The prevalence of T. callipaeda in raccoons and Japanese raccoon dogs was 20.2% (36/178) and 13.3% (2/15), respectively. The cox1 sequences from 56 worms from 38 animals revealed three haplotypes (h9, h10, and h12). Analysis of multiple worms for five raccoons showed co-infection of two different haplotypes (h9 and h10) in a single host. Comparing our data with published sequences, three sequences obtained from raccoons and raccoon dogs shared the same haplotypes as those reported in humans, dogs, and cats in Japan. CONCLUSIONS: Our findings show a high prevalence of T. callipaeda in raccoons, suggesting that this invasive carnivore species serves as an important natural reservoir of T. callipaeda in the Kanto region of Japan, an area with the highest human population of the country.

The occurrence and molecular identification of Thelazia spp. in European bison (Bison bonasus) in the Bieszczady Mountains.

Infection with Thelazia nematodes results in eye disease in wild and domestic animals. The aim of the present study was to describe the occurrence of Thelazia nematodes in European bison, and to subject the isolated parasites to molecular identification and phylogenetical analysis. The eyeballs of 18 European bison from the Bieszczady Mountains, culled due to dysfunctional vision, were collected for study. The conjunctival sacs, tear ducts, corneal surface and nictitating membrane were rinsed with a saline solution. Any obtained nematodes were isolated under a stereoscopic microscope, and then identified as T. gulosa or T. skrjabini by molecular analysis of partial cox1 sequences. The prevalence of infection with Thelazia spp. was found to be 61%, with a 95% confidence interval (CI 95%) of 39-80%. Thelazia skrjabini was isolated from 56% (CI 95% 34-75%) of examined animals; T. gulosa was significantly less common (p = 0.038) with the prevalence of infection reaching 22% (CI 95% 9-45%). Three European bison were cross-infected with both T. gulosa and T. skrjabini. Phylogenetic analysis found the obtained sequences to be similar to those of Thelazia species from domestic ungulates in Europe. Infection intensity ranged from 1 to 16 nematodes per individual (median of three nematodes), and was significantly higher in females (6 nematodes) than in males (1 nematode; p = 0.019). A tendency for seasonal occurrence of nematodes in European bison was also observed. Our study provides further information regarding the patterns of Thelazia transmission in European bison in Poland.

Thelazia lacrymalis in horses from Romania: epidemiology, morphology and phylogenetic analysis.

BACKGROUND: Equine thelaziosis is a neglected vector-borne parasitic disease in modern veterinary medicine, lacking recent reports. It is transmitted by Musca autumnalis, and potentially other Muscidae species, by ingesting the lachrymal secretions of its equine host. The distribution of both Thelazia lacrymalis and its intermediate hosts remains largely unknown throughout Europe, with most studies dating back 20 years. The aim of this study was to assess the presence, prevalence and distribution of T. lacrymalis in horses from Romania. METHODS: The eyes of 273 horses, slaughtered at two abattoirs from the Northwestern and Western regions of Romania, were examined for the presence of T. lacrymalis between March and November 2021. Upon detection, the nematodes were collected and morphologically identified using the keys from literature. Following identification, one specimen from each animal was selected for molecular analysis while the rest underwent detailed morphometric measurements. Mapping and distribution, according to ecoregions, was done using the QGis 3.20 software, while sequences obtained were compared to those available in GenBank through BLAST analysis using the MEGA X software. RESULTS: Of the 273 animals sampled, 12 (4.39%) were positive for Thelazia spp. INFECTION: Eighty-seven nematodes were recovered, all morphologically identified as T. lacrymalis. The intensity of infestation varied between one and 33 nematodes/animal while five animals presented a bilateral infestation and seven a unilateral one. The highest prevalence was encountered in Pannonian ecoregion (12.12%) while the lowest was in the Alpine ecoregion (0%). Seventy-five intact specimens underwent detailed morphometric analysis, of the 18-20 parameters, resulting in notable differences in striation lengths compared to the data available in other reports. BLAST analysis identified a 96.46-98.60% similarity to the only other COI gene sequence available for T. lacrymalis. CONCLUSIONS: The current study represents the first report of T. lacrymalis in horses in Romania. The low prevalence rates are probably linked to the wide use of macrocyclic lactones.

Phortica oldenbergi (Diptera: Drosophilidae): A new potential vector of the zoonotic Thelazia callipaeda eyeworm.

Thelazia callipaeda is a zoonotic nematode parasitizing the eyes of many hosts species, primarily dogs. To date Phortica variegata and Phortica okadai are the only known vectors of this nematode in Europe and China, respectively. In this study we investigated the role played by a third species, Phortica oldenbergi, as vector of T. callipaeda in Europe. Drosophilid flies of this species were collected in central Italy and maintained in laboratory. One hundred forty P. oldenbergi were experimentally infected with T. callipaeda L1 recovered from field collected gravid females belonging to haplotype 1, which is that detected in several countries in Europe. Seventy-four (i.e., 60 females and 14 males) specimens died at 5 days post infection (d.p.i.) (±1) and scored negative for T. callipaeda larvae at the dissection. Sixty-six (i.e., 46 females and 20 males) P. oldenbergi survived and were dissected at 21 d.p.i. From those, T. callipaeda L3 were detected in the proboscis of two females (3.0%). Overall, at the molecular analysis, 11.4% (n = 16/140; 13 females and 3 males) scored positive for the presence of T. callipaeda DNA. Data herein reported brings further insights on the biology of T. callipaeda by adding P. oldenbergi as a new potential intermediate host under experimental conditions. The role of this drosophilid in the transmission cycle of T. callipaeda needs to be confirmed under natural conditions.

First report of human Thelazia callipaeda infection in Portugal.

The zoonotic parasitic nematode Thelazia callipaeda, also known as the oriental eye worm, is endemic in several European countries, including Portugal. Infections may result in ocular disease in domestic and wild animals as well as humans, with more or less severe manifestations. We report the first human case of ocular thelaziosis by T. callipaeda in Portugal, a country where the parasite had already been found to infect dogs, cats, red foxes, wild rabbits and a beech marten. An 80-year-old patient from east-central Portugal, who had been suffering from tearing for a few years, had whitish filiform fragments removed from the left eye. Polymerase chain reaction of partial cytochrome c oxidase subunit 1 and 18S small subunit rRNA genes followed by bidirectional sequencing and BLAST analysis confirmed T. callipaeda haplotype 1, the only haplotype previously reported in Europe. The endemicity of T. callipaeda in domestic and wild animals in east-central Portugal makes it very likely that infection of the human patient had occurred locally. In east-central and other geographical areas of Portugal, veterinarians and physicians, especially ophthalmologists, should regard T. callipaeda as a cause of ocular pathology in animals and humans.

Comparative mitogenomics of Spirocerca lupi from South Africa and China: Variation and possible heteroplasmy.

The complete mitochondrial genome of Spirocerca lupi isolated from a dog in South Africa was sequenced using next generation sequencing (NGS) technology and the 12 protein coding genes along with the two rRNA genes were compared to 18 other nematode species as well as S. lupi from China. The mitochondrial genome of S. lupi South Africa had a mean genetic diversity of 6.1 % compared to S. lupi China with some variation in nucleotide composition, gene positioning and size. Pairwise distance results indicated slightly higher variation when compared to the pairwise distances of other closely related species, however, this variation was not high enough for it to be considered a cryptic species. Phylogenetic analysis indicated that S. lupi from the two continents are very similar. In addition, single nucleotide polymorphisms were detected in the nad2 gene with ten sequence variants identified from 10 clones from a single nematode, suggesting possible heteroplasmy. The origin of the heteroplasmy is currently unknown but it is speculated to have arisen from accumulated mutations in the mitochondria during somatic replication.

Zoonotic Thelazia californiensis in dogs from New Mexico, USA, and a review of North American cases in animals and humans.

Thelazia californiensis is a spirurid nematode found in the conjunctival sac of domestic and wild animals, including humans, across the western United States of America. Herein, we report two cases of thelaziosis by T. californiensis in dogs from New Mexico, United States, based on integrated morphological and molecular approaches. Nematode specimens collected from the conjunctiva of both dogs were identified as T. californiensis based on morphology. Our study substantially expands the knowledge on morphometry of this nematode species. Therefore, these data will be useful for accurate diagnosis of thelaziosis in domestic animals, wildlife and humans in North America, using classical, microscopy-based methods. We characterized for the first time the mitochondrial cytochrome c-oxidase subunit 1 (COI) and 12S genes of T. californiensis. While these markers support the validity of T. californiensis, they were not very informative for elucidating the phylogenetic relationships among Thelazia species. Nevertheless, the characterization of these diagnostic markers for T. californiensis will be useful for studies on the epidemiology, molecular xenomonitoring of fanniid vectors, and population genetics of this multi-host, zoonotic parasite.

Morphological and molecular characterization of Spirocerca lupi (Nematoda: Spirocercidae) from domestic dogs in Vietnam with reference to Spirocerca vulpis.

Spirocerca lupi is a common parasitic nematode associated with esophageal cancer of canids. Recent surveys have revealed an increasing number of canids infected with Spirocerca spp. in Africa, the Americas, Europe and Western Asia, and described a new species, Spirocerca vulpis, from red foxes (Vulpes vulpes). However, in Southeast Asia, research on Spirocerca spp. is scarce. Therefore, the aim of this study is to explore Spirocerca infection in domestic dogs in Vietnam and to identify the Spirocerca species by analyzing their morphometric and molecular data. We found Spirocerca spp. specimens in 51 (17.7%) out of 287 dogs examined with the intensity of infection ranging from one to 29 nematodes per dog. These nematodes were morphologically and molecularly identified as S. lupi. For morphology, the presence/absence of teeth, the ratio of glandular to muscular esophagus lengths, and the position of the vulva opening of S. lupi and S. vulpis varied between reports, suggesting caution in identification of Spirocerca species based exclusively on morphological characteristics. The molecular analysis based on a partial cox1 sequence revealed that S. lupi from Vietnam is genetically close to those from India and China, but far different from those of Israel, South Africa, Peru and Hungary. Given high genetic and morphological variations, more extensive surveys on Spirocerca spp. from various mammalian hosts at a greater scale are necessary to elucidate the divergence of this nematode.

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.

A human corneal ulcer caused by Thelazia callipaeda in Southwest China: case report.

In this study, we describe a rare human case with corneal ulcer caused by thelaziosis in a 69-year-old man in Southwest China. A male nematode was discovered and removed from the patient's right eye with a long spicule and further identified by sequencing mitochondrial cox1 gene. The ophthalmologic and molecular biological evidence demonstrates the corneal ulcer caused by T. callipaeda infection, which is mainly distributed in Asian and European countries. Most T. callipaeda infections are emerged in the conjunctiva, leading to conjunctivitis. To the best knowledge of the authors, corneal ulcers caused by T. callipaeda have not been reported yet.

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.

Ophthalmic Thelazia callipaeda infections: first feline and new canine imported cases in Germany.

The first case of feline ocular Thelazia callipaeda infection and two new canine imported infections in West Germany are here described. The three animals had a history of recent travel to/from other countries. The young adult cat imported from Spain presented an intermittent unilateral ocular discharge. During in-depth ophthalmic examination, a single alive nematode was removed from the conjunctival compartment of the affected eye. Referring to the canine cases, an adult female dog originated from Kenya presented epiphora and mucous whitish-grey discharge of the right eye. During flushing of the nasolacrimal duct two small, thin and long nematodes were removed. Furthermore, a male Borzoi racing dog with regular visit to racing tracks in different countries presented ocular mucous discharge. At ophthalmologic examination, two transparent-whitish vital nematodes were removed. All nematode specimens of the three cases were morphologically identified as adult T. callipaeda parasites. The animals were treated orally with milbemycin oxime (2.0 mg/kg; cat) or milbemycin oxime/praziquantel (0.5 mg/kg and 5.0 mg/kg; dogs) twice with 1-week interval resulting in complete resolution of symptoms. The repeated introduction of patent T. callipaeda-infected animals, especially from southern and eastern endemic countries, will ease the establishment of ophthalmic thelaziosis in Northern Europe. The male fruit fly, Phortica variegata, an intermediate host of T. callipaeda, is endemic within European countries. Considering the clinical and zoonotic relevance of ophthalmic thelaziosis, enhanced disease awareness of European medical and veterinarian doctors and in-depth eye examination for proper detection of T. callipaeda are crucial for appropriate anthelmintic treatments and to limit spreading of the infection.

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.

First report of Thelazia callipaeda in a free-ranging Iberian wolf (Canis lupus signatus) from Spain.

Thelazia callipaeda (Spirurida, Thelaziidae) is a vector-borne zoonotic eyeworm able to infect a broad spectrum of carnivores. Here, we describe the first case of bilateral infection by T. callipaeda in the eyes of an adult female Iberian wolf (Canis lupus signatus) in central Spain. Nematodes collected were morphologically identified (n = 42), and two specimens were molecularly characterized. At the sequence analysis of the partial mitochondrial cytochrome c oxidase subunit 1 gene, T. callipaeda haplotype 1 (the only haplotype circulating in Europe) was detected. The role of the Iberian wolf as a natural reservoir for T. callipaeda in the life cycle of this emerging zoonosis and the implications in conservation are discussed.

[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.