Unveiling zoonotic threats: molecular identification of Brugia sp. infection in a lion.

Brugia malayi and B. pahangi, potential zoonotic pathogens transmitted by mosquitoes, are believed to primarily infect dogs and cats as reservoir hosts. Although previous studies have indicated nematode infections in lions, particularly in zoo environments where human contact with these reservoirs is possible, limited documentation exists regarding Brugia sp. infections in lions in Thailand. This study aims to investigate a case of Brugia infection in a lion from a zoo in Thailand. The blood sample was collected and examined from a female lion, using staining methods to morphologically identify microfilaria at the genus level. Subsequently, the PCR was employed targeting specific genes, including mitochondrial 12S rDNA, 18S rDNA, cytochrome oxidase I (COI) and Wolbachia surface protein (wsp), to confirm the species of the filarial nematode parasite. The genetic sequencing results revealed a high similarity (99-100%) to B. malayi for the 12S rDNA, 18S rDNA, COI and wsp genes. Phylogenetic analysis based on nucleotide sequences from the 12S rDNA, 18S rDNA, COI and wsp genes showed that the sequences from this study belong to different clusters. This marks the inaugural documentation of molecular identification of Brugia infection in a lion, signifying that lions could function as reservoirs for this parasite and present a potential public health risk in the region. Our research underscores the effectiveness of molecular techniques and phylogenetic analysis in discerning and comprehending the evolution of filarial parasites. Additionally, it emphasizes the significance of these methods in enhancing the diagnosis, control, and prevention of zoonotic filarial nematode infections.

First molecular detection and genetic diversity of Hepatozoon sp. (Apicomplexa) and Brugia sp. (Nematoda) in a crocodile monitor in Nakhon Pathom, Thailand.

The crocodile monitor (Varanus salvator) is the most common monitor lizard in Thailand. Based on habitat and food, they have the potential to transmit zoonoses, with a high possibility of infecting ectoparasites and endoparasites. Diseases that could infect crocodile monitors and be transmitted to other animals, including humans. This research aims to identify and evaluate the phylogenetic relationships of Hepatozoon sp. and sheathed microfilaria in crocodile monitors. The phylogenetic analyses of Hepatozoon, based on 18S rRNA, and sheathed microfilaria, based on the COX1 gene, revealed that the Hepatozoon sp. were grouped with H. caimani, while sheathed microfilaria were grouped together with B. timori. This study provides insights into the genetic diversity and host-parasite interactions of hemoparasites in crocodile monitors in Thailand.

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.

Molecular identification and genetic diversity of equine ocular setariasis in Thailand based on the COI, 12S rDNA, and ITS1 regions.

Equine ocular setariasis is mainly caused by Setaria digitata, and the identification of this filarial nematode is based on morphology. However, morphological characterization alone is insufficient for the detection and differentiation of S. digitata from its congeners. In Thailand, the molecular detection of S. digitata is lacking and its genetic diversity is still unknown. This study aimed to phylogenetically characterize equine S. digitata from Thailand based on sequences derived from the mitochondrial cytochrome c oxidase subunit 1 (COI), the mitochondrial small subunit ribosomal DNA (12S rDNA), the nuclear internal transcribed spacer 1 (ITS1) and Wolbachia surface protein (wsp). Five samples of S. digitata were characterized, submitted to the NCBI database, and used for phylogenetic analysis as well as the assessment of similarity, entropy, and haplotype diversity. Phylogenetic analyses revealed that the S. digitata Thai strain was similar to S. digitata from China and Sri Lanka, with 99 to 100% similarity. The entropy and haplotype diversity indicated that the S. digitata Thai isolate was conserved and closely related to S. digitata worldwide. This is the first report on the molecular detection of equine ocular setariasis caused by S. digitata in Thailand.

Molecular detection of Mycoplasma wenyonii and its closely related hemotropic Mycoplasma sp. in blood-sucking flies from a buffalo farm in Chachoengsao province, Thailand.

Bovine hemoplasmosis is a disease in buffaloes and cattle caused by hemotropic mycoplasmas or hemoplasmas. Only two bovine hemoplasma species, Mycoplasma wenyonii and Candidatus Mycoplasma haemobos, have been described in several countries. Hemoplasmas induce acute hemolytic anemia or chronic infection, leading to production loss. Bovine hemoplasma DNA was also detected in blood-sucking arthropods, suggesting vector transmission in farms. To date, no studies of the molecular detection of bovine hemoplasmas in Thai buffaloes and arthropod vectors have been reported. This study aimed to study the 1-year diversity of hematophagous flies in a buffalo farm located in Chachoengsao province, Thailand, and to investigate the molecular occurrence of bovine hemoplasmas in those flies using a polymerase chain reaction (PCR) assay and sequence analyses. A total of 1,488 mosquitoes, 867 stable flies, and 312 tabanid flies were collected during this study. The most abundant mosquitoes, stable flies, and tabanid flies were Culex tritaeniorhynchus, Stomoxys calcitrans, and Tabanus megalops, respectively. A total of 249 genomic DNA samples of flies were tested using a PCR assay based on the 16S rRNA gene; 23.69% (59/249) of the insect samples were positive in this assay. Positive samples (n = 8) were subjected to bidirectional sequencing. The BLAST results showed that only three samples from Stomoxys calcitrans and two samples from Tabanus megalops showed 99.90% and 99.17% similarities to the M. wenyonii isolate B003 (MG948626/Water buffalo/Cuba) and the M. wenyonii isolate C124 (MG948625/Cattle/Cuba), respectively. This molecular occurrence of bovine hemoplasmas in blood-sucking flies suggested that those flies are the mechanical vectors for bovine hemoplasmas in Thailand. Based on the phylogenetic analysis of the 16S rRNA gene, the sequences of M. wenyonii were likely classified into two subgroups (A and B), suggesting closely related bovine hemoplasma species. Finally, the genetic analysis of the 23S rRNA gene from these two subgroups revealed that subgroup A could be M. wenynoii and subgroup B may be a subspecies of M. wenyonii or another putative novel species. However, further investigation should be conducted in buffaloes, cattle, and blood-sucking flies to gain more 16S rRNA and 23 rRNA gene sequences of bovine hemoplasmas.

Molecular detection and genetic analysis of porcine haemoplasmas in commercial pig farms from Thailand reveal a putative novel species.

Haemoplasma is a trivial name for haemotropic Mycoplasma spp., which can attach to the surface of red blood cells leading to deformity and anaemia in a wide range of mammalian animals, including pigs. In Thailand, there is only one study that reported the occurrence of Mycoplasma suis without other haemoplasma species. In this study, we examined the molecular occurrence and genetic diversity of porcine haemoplasmas in Thai domestic pigs (Sus scrofa domesticus) from commercial farms using a PCR assay targeting the 16S rRNA gene, DNA sequencing, nucleotide sequence type (ntST) analysis and phylogenetic analysis. A total of 665 blood samples were collected from pigs at thirteen farms located in eight provinces of Thailand during 2019-2020. Genomic DNA was extracted from blood samples and tested by PCR. The frequency of haemoplasma infection was 37.1% (247/665, 95% CI: 33.5%-40.9%) in all pigs. Among 247 PCR positive samples, 194 were sequenced and analysed by nucleotide BLAST, ntST diversity, phylogenetic trees and ntST networks. The results of this genetic analysis indicated that at least four species with 27 nucleotide sequence types (Mycoplasma suis, Mycoplasma parvum, Candidatus Mycoplasma haemosuis and a putative novel species) of porcine haemoplasmas were identified. Thus, it appears that haemoplasmas show a high genetic diversity in the Thai pig population. In addition, a putative novel species was genetically characterized by other markers, namely, the 23S rRNA and RNase P RNA (rnpB) genes. For phylogenetic analysis, Candidatus Mycoplasma haemosuis was placed into the Mycoplasma haemofelis group, and the three remaining species were placed into the Mycoplasma suis group in all trees containing the 16S rRNA, 23S rRNA and rnpB genes. Further studies, such as pathobiology and epidemiology, should be conducted to better characterize this putative novel species.

Serum protein profiles and C-reactive protein in natural canine filariasis.

BACKGROUND AND AIM: Canine filariasis is caused by several species of filarial worms. The pathophysiological response to infection is mainly due to the filaria lifecycle. Laboratory detection methods to assess the pathological alterations characteristic of filariasis are needed urgently. Serum protein profiles and C-reactive protein (CRP) levels are used widely to diagnose several animal diseases. This study aimed to determine the serum protein profiles and CRP levels in dogs infected with Dirofilaria immitis or Brugia pahangi or both parasites. MATERIALS AND METHODS: Blood samples were collected from 980 dogs presenting at animal hospitals and veterinary clinics in Bangkok and its vicinity. The presence of microfilaria in samples was determined using a buffy coat smear and staining with Wright-Giemsa. The sheathed and unsheathed microfilaria species were identified by acid phosphatase staining. Forty positive samples were tested. The serum protein profiles were identified by agarose gel electrophoresis. The CRP concentration was measured using a fluorescent immunoassay. RESULTS: Albumin levels and albumin-to-globulin ratios were significantly lower, and total protein, ß2 globulin, and γ globulin levels were significantly elevated in dogs infected with D. immitis and B. pahangi compared with reference values in normal dogs. The average CRP concentrations in dogs infected with D. immitis or B. pahangi were 69.9 and 12.9 mg/L, respectively. CONCLUSION: The total protein and γ globulin levels increased in canine filariasis compared with the normal reference range. The CRP concentration in dogs infected with D. immitis was extremely high, whereas that in dog infected with B. pahangi was normal.

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.