Metabarcoding study to reveal the structural community of strongylid nematodes in domesticated horses in Thailand.

BACKGROUND: Mixed strongylid infections significantly impact equine health and performance. Traditional microscopy-based methods exhibit limitations in accurately identifying strongylid species. Nemabiome deep amplicon sequencing approach previously succeeded in describing the strongylid communities in livestock including equids. However, there are no available studies that describe the structural communities of strongylid parasites in horses in Thailand. Therefore, this study was undertaken encompassing the ITS-2 rDNA metabarcoding assay to characterize strongylid species within horse fecal samples collected from a cohort of yearlings at the largest domesticated stud farm in Thailand. In addition, to investigate the capability of ITS-2 rDNA in assessing the phylogenetic relationships among the identified strongylid species. RESULTS: The study identified 14 strongylid species in the examined equine populations, each with varying prevalence. Notably, Cylicocyclus nassatus and Cylicostephanus longibursatus were identified as the predominant species, with Strongylus spp. conspicuously absent. The phylogenetic analysis of 207 amplicon sequence variants (ASVs) displayed a complex relationship among the investigated cyathostomin species, with some species are positioned across multiple clades, demonstrating close associations with various species and genera. CONCLUSION: The ITS-2 nemabiome sequencing technique provided a detailed picture of horse strongylid parasite species in the studied population. This establishes a foundation for future investigations into the resistance status of these parasites and enables efforts to mitigate their impact.

Patterns of Equine Small Strongyle Species Infection after Ivermectin Intervention in Thailand: Egg Reappearance Period and Nemabiome Metabarcoding Approach.

The indiscriminate use of anthelmintics to control parasitic nematodes in horses has led to the emergence of anthelmintic resistance worldwide. However, there are no data available on using ivermectin for treating strongyle infections within domesticated horses in Thailand. Therefore, this study aimed to use the fecal egg count reduction (FECR) test to determine the strongylid egg reappearance period (ERP). Additionally, the nemabiome metabarcoding approach is incorporated to study patterns of strongyle species infection following ivermectin treatment. The study results indicate that, although ivermectin effectively eliminated adult strongyle parasites within two weeks post-treatment, the ERP was shortened to 6 weeks post-treatment with a mean FECR of 70.4% (95% CI 46.1-84.0). This potentially indicates a recent change in drug performance. In addition, nemabiome metabarcoding revealed that strongyle species have different levels of susceptibility in response to anthelmintic drugs. The reduction in ERP was associated with the early reappearance of specific species, dominated by Cylicostephanus longibursatus and Cylicocyclus nassatus, indicating the lower susceptibility of these species. In contrast, Poteriostomum imparidentatum, Triodontophorus nipponicus, and Triodontophorus serratus were not found post-treatment, indicating the high level of susceptibility of these species. This information is vital for comprehending the factors contributing to the emergence of resistance and for devising strategies to manage and control strongyle infections in horses.

New insight into genetic diversity of zoonotic-potential Ancylostoma ceylanicum in stray cats living in Bangkok, Thailand, based on deep amplicon sequencing.

AIMS: This study aimed to characterize feline hookworms from stray cats living in Bangkok. METHODS AND RESULTS: A total of 56 hookworm-positive faecal samples were identified for hookworm species by using PCR targeting the ITS1, 5.8S, and ITS2 fragment and qPCR targeting ITS2. Of 56 samples, 96.4% (54/56) were identified as Ancylostoma ceylanicum and 1.8% (1/56) as Ancylostoma caninum. With qPCR, 89.3% (50/56) were identified as single A. ceylanicum infection and 5.4% (3/56) as coinfection of A. ceylanicum and A. caninum. For genetic characterization of A. ceylanicum, 10 samples were pooled, and the partial COI gene was amplified, followed by deep amplicon sequencing. Five pooled samples were analysed, and 99.73% were identified with A. ceylanicum sequences, which were allocated into 19 haplotypes (AC01-AC19). Genetic diversity findings for A. ceylanicum in Asia revealed that three of eight haplotypes considered of zoonotic significance occurred in humans, dogs, and cats, including haplotypes H01, H20, and H21. The predominant haplotype in this study, AC01, was clustered with H01-a zoonotic haplotype. CONCLUSIONS: The diversity obtained by deep amplicon sequencing supported that the A. ceylanicum community had high genetic variation. Deep amplicon sequencing was a useful method to determine source, zoonotic potential, and host-parasite relationship.

Genetic diversity and characterization of Wolbachia endosymbiont in canine filariasis.

Canine filariasis is caused by nematodes from the family Onchocercidae, which is transmitted by arthropod vectors. The disease is commonly found in Southeast Asia and exists worldwide. Some filarial nematodes are associated with intracellular bacteria of the genus Wolbachia, which plays an important role in embryogenesis, molting, and the long-term survival of adult worms. This study aims to characterize Wolbachia sp. and determine the association between Wolbachia and canine filarial nematode species in Thailand. A total of 46 dog blood samples that were naturally infected with filarial nematodes were obtained to identify filarial nematode species by Giemsa stained under a light microscope and confirmed using the molecular technique. In order to characterize Wolbachia sp., the nested PCR assay targeting the 16S rRNA gene showed that all samples of Dirofilaria immitis and fifteen samples of Candidatus Dirofilaria hongkongensis were grouped into Wolbachia supergroup C. In addition, all samples of Brugia spp. and five samples of Candidatus Dirofilaria hongkongensis were classified into Wolbachia supergroup D. The genetic diversity analysis conducted using the 16S rRNA gene revealed a similar result when analyzed through phylogenetic tree analysis. This is the first genetic diversity study of Wolbachia of Candidatus Dirofilaria hongkongensis in infected dogs in Thailand.

Case report: Thromboembolic heartworm induced lower limb necrosis in a dog.

A 9-year-old spayed female boxer suffered from lameness in both hindlimbs with a perforated paw wound. Additionally, a linear, worm-like creature was penetrating out from the wound. On examination, the dog was emaciated and infected with heartworms, detected through a fresh blood smear, echocardiography, and transabdominal ultrasonography. Adult heartworms were detected at the right atrium (RA), right ventricle (RV), and pulmonary artery (PA), including the distal abdominal aorta, external iliac, and femoral arteries. During the surgery, adults heartworms were removed from both the heart (n = 8) and the femoral arteries (n = 5). Unfortunately, not all heartworms could be removed from these locations due to the extent of the heartworm infection. The opened, ischemic wounds in the distal limbs progressively expanded and the dog subsequently died, possibly due to caval syndrome complications and septicemia. The necropsy showed no evidence of an atrial septal defect, and a total of 25 adult heartworms were collected from the perforated paw, heart, pulmonary, and femoral arteries. All worms collected during the necropsy process were molecularly confirmed to be Dirofilaria immitis.

Application of a novel rectangular filtering microfluidic device for microfilarial detection.

The rectangular filtering microfluidic chip was invented using microfluidics device fabrication technology and can separate living microfilariae from blood samples without a syringe pump. The diagnostic results are highly effective. The device is based on the principle of separating millions of blood cells from microfilariae using a rectangular filter structure. It disperses fluid evenly into the flow-passage channel, and its rectangular filter structure is the key to success in reducing the pressure and separating blood cells from microfilariae effectively. The flow rate and blood cell concentration were optimized in our study. The chip is intended to be a point-of-care device that can reduce the use of superfluous instrumentation in the field. The technology is designed to be rapid, accurate, and easy-to-use for all users, especially those in remote areas.

Case Report: Molecular and Pathological Investigations of Zoonotic Anatrichosoma Spp.-Induced Ulcerative Pododermatitis in a Domestic Cat in Thailand.

Anatrichosoma spp. is a group of trichuroid nematodes that mainly infect non-human primates and domestic cats. The lifecycle of these nematodes remains unclear. In non-human primates, Anatrichosoma spp. were found in the nasal cavity. However, ulcerative dermatitis has been reported in infected cats. An adult, intact, female domestic short-haired cat was presented with ulcerative pododermatitis of all limbs. Punch biopsy was performed at the edge of the ulcerative wound for histopathological investigation and showed necrosis and infiltration of inflammatory cells around the nematode-like lesion. Eggs with Capillaria-like characteristics were present. Tissue sections were subjected to DNA extraction and PCR targeting 18S rRNA, using primers designed from Anatrichosoma 18S rRNA. The phylogenetic tree revealed that DNA obtained from the lesion of the domestic cat was grouped with Anatrichosoma spp. from the olive glass mouse (Abothirx olivacea), Capillaria plica and Eucoleus aerophilus, both from the red fox (Vulpes Vulpes). The study is the first report of feline anatrichosomiasis in Thailand, and we present both pathological findings and molecular evidence. The cat was successfully treated with emodepsine/praziquantel. The skin lesion recovered within 3 days after anthelmintic treatment. Because Anatrichosoma spp. have been reported in humans, the zoonotic potential of this parasite should be considered.

Prevalence of endoparasitic and viral infections in client-owned cats in metropolitan Bangkok, Thailand, and the risk factors associated with feline hookworm infections.

The prevalence of enteric parasites in cats in metropolitan Bangkok has not been updated in over 13 years. The main objectives of this study include updating the prevalence of endoparasitism in client-owned cats, status of retroviral infections and determining the association between feline hookworm infection and possible risk factors. A total of 509 fecal samples were collected from client-owned cats in 2014-2015 and examined by a wet fecal mount technique. If additional sample remained, a PBS-ethyl acetate sedimentation was done (n = 229), and ZnSO4 centrifugal flotation was also performed if there was sufficient remaining sample (n = 105). At least one parasite was observed in 32.0% (163/509) of cats, with Ancylostoma being the most common intestinal parasite detected in 21.6% (110/509) of cats. Other parasitic infections detected by fecal examinations included Toxocara (6.9%; 35/509), Platynosomum (3.7%; 19/509), Cystoisospora (3.5%; 18/509), Taenia (2.9%; 15/509), Spirometra (1.6%; 8/509), Dipylidium (0.4%; 2/509), and Opisthorchis-like trematode (0.2%; 1/509). Examination for Giardia infection was conducted with the SNAP® Giardia Test, a coproantigen test, on a subset of the fecal samples (233/509) and revealed a positive result on 3.9% (9/233) of samples. Plasma samples were analyzed using the SNAP® Triple Test detecting antigens of Feline Leukemia Virus (FeLV) and Dirofilaria immitis while also detecting antibodies to Feline Immunodeficiency Virus (FIV). Antigens of FeLV and antibodies to FIV were found in 7.1% (19/269) and 5.2% (14/269) of cats, respectively. None of the cats were found to have circulating antigen of Dirofilaria immitis using this test. No association between retroviral and endoparasitic infections was found. From multivariable logistic regression examining associated factors, the ability of cats to access the outdoors (adjusted OR = 3.22, 95% CI; 1.42-7.87) and having tapeworm segments or adult helminths in feces (adjusted OR = 3.31, 95% CI; 1.34-8.21) were significantly associated with the finding of hookworm eggs in feces. This work presents the most up-to-date data on enteric feline parasite prevalence in the metropolitan Bangkok area from which fecal samples were directly collected from cats. Consequently, this study emphasizes that diagnosis of parasitic infections and the routine use of antiparasitic medications should be encouraged by veterinarians and to owners in order to reduce the reservoir of potentially zoonotic parasites.

Seasonal distribution and environmental parameters associated with Brugia pahangi and Dirofilaria immitis in naturally infected dogs in Bangkok and vicinity, Thailand.

Dirofilaria immitis and Brugia pahangi are vector-borne parasites found in dogs and cats, including Thailand. In order to evaluate the effects of season and environmental parameters on the prevalence of these parasites, this retrospective study was conducted in 2019. A total of 79,506 canine blood samples were examined. B. pahangi was found in 0.55% of samples (438/79,506; 95% CI 0.50-0.61) while D. immitis was detected in 0.43% (345/79,506; 95% CI 0.39-0.48). One-way ANOVA found no effect of seasonal conditions on prevalence. For B. pahangi, the parameters rainfall, relative humidity and sunshine hours showed associations with p ≤ 0.20 and were included in multiple logistic regressions resulting in adjusted odds ratios of 0.53, 1.31 and 0.55, respectively. For D. immitis, only average temperature showed p ≤ 0.20, resulting in an odds ratio of 0.42. In conclusion, Thailand has environmental parameters that do not change very much during the year, so they might not affect the prevalence of two filarial nematodes. However, the threat of B. pahangi and D. immitis should not be ignored, especially in subtropical regions where their vectors are abundant. Both owners and veterinarians should be concerned about filarial prevention and control of D. immitis and B. pahangi.

Emergence of Dirofilaria repens (Spirurida: Onchocercidae) in dogs in Eastern Thailand.

BACKGROUND AND AIM: Dirofilaria repens is a zoonotic vector-borne parasite of dogs and cats. It is not commonly found in every part of Thailand, except the southern part. This study was conducted to investigate the prevalence of this parasite in Eastern Thailand in 2019. MATERIALS AND METHODS: A total of 8003 blood samples were collected from private veterinary clinics and animal hospitals in Eastern Thailand. Blood parasites were examined using buffy coat thin blood smears with Wright-Giemsa staining. D. repens was morphologically identified and confirmed using the acid phosphatase activity technique. RESULTS: The first case of D. repens was found in March 2019. The prevalence of D. repens from January to December 2019 was 0.44% (35/8003) (95% confidence interval 0.30-0.61). CONCLUSION: The prevalence data of D. repens in Eastern Thailand indicate that this parasitic infection should be considered as a zoonotic vector-borne disease. A strategic plan to control zoonotic transmission alongside a preventive program should be emphasized and encouraged among pet owners and veterinarians.