Histopathology and virulence of an in vitro-adapted Trypanosoma evansi TEDC 953 strain (Thailand isolate) in mice.

Background and Aim: Trypanosoma evansi is a blood and tissue protozoan parasite affecting domestic and wild animals. The T. evansi Thai strain, namely, T. evansi from dairy cattle number 953 (TEDC 953) strain, has been successfully isolated from dairy cattle and cultivated in vitro. The in vitro-cultivated parasite is useful for biological studies, evaluation of novel chemotherapeutic agents, and production of antigens for diagnostic tests. This study aimed to observe the histopathology and virulence of an in vitro-adapted T. evansi TEDC 953 strain in vivo. Materials and Methods: The histopathology and virulence of the TEDC 953 strain were clarified in mice. Six mice were infected with 1 × 105 trypomastigotes of TEDC 953 strain intraperitoneally, and four mice were in the negative control. Parasitemia was monitored daily, and the mice were euthanized on 30 days post-infection (DPI). Internal organs were collected for histopathological examination using hematoxylin and eosin staining. Results: Histopathological lesions were found in the liver, lung, heart, kidney, spleen, and brain of the inoculated mice. The main histopathological feature was lymphoplasmacytic inflammation in parenchyma and perivascular areas of multiple organs, and the severity of histopathological changes was related to the presence of trypomastigotes in the regional vessels. Granulomatous inflammation was seen in meninges, pleura, renal capsule, renal pelvis, and spleen of some infected mice. Four mice died at 17, 24, 26, and 27 DPI with an average parasitemia of 4.05 × 1011 trypomastigotes/mL. The average survival time was 23.5 DPI (mice = 4). Conclusion: This study confirmed that the TEDC 953 strain is infectious and pathogenic in mice after the continuously cultivated in vitro. To replace the use of experimental animals, the in vitro-cultivated parasite can be used instead in further studies.

Molecular Identification of Culicoides Species and Host Preference Blood Meal in the African Horse Sickness Outbreak-Affected Area in Hua Hin District, Prachuap Khiri Khan Province, Thailand.

African horse sickness (AHS) was reported as an outbreak in Thailand in 2020. Hematophagous insects from the genus Culicoides are the suspected vector responsible for AHS transmission. Horses in Hua Hin district, Prachuab Khiri Khan province, Thailand, were affected and died from AHS in 2020. However, the potential Culicoides species and its host preference blood meal in the affected areas are unknown. To investigate the potential vectors of AHS, Culicoides were collected using ultraviolet light traps placed near horse stables. Six horse farms, including five farms with AHS history and one farm without AHS history, were included in this study. Morphological and molecular identification of the Culicoides species was performed. Polymerase chain reaction (PCR) targeting the cytochrome b oxidase I (COXI) gene for confirmation of the Culicoides species, identification of the prepronociceptin (PNOC) gene for host preference blood meal, and bidirectional sequencing were conducted. Consequently, 1008 female Culicoides were collected, consisting of 708 and 300 samples captured at positions A and B at a distance of <2 and >5 m from the horse, respectively. Twelve Culicoides species identified by morphology were noted, including C. oxystoma (71.92%), C. imicola (20.44%), C. actoni (2.28%), C. flavipunctatus (1.98%), C. asiana (0.99%), C. peregrinus (0.60%), C. huffi (0.60%), C. brevitarsis (0.40%), C. innoxius (0.30%), C. histrio (0.30%), C. minimus (0.10%), and C. geminus (0.10%). The PCR detection of the Culicoides COXI gene confirmed Culicoides species in 23 DNA samples. PCR targeting the PNOC gene revealed that the Culicoides collected in this study fed on Equus caballus (86.25%), Canis lupus familiaris (6.25%), Sus scrofa (3.75%), and Homo sapiens (3.75%) for their blood meal. Human blood was identified from two samples of C. oxystoma and a sample of C. imicola. Three dominant species including C. oxystoma, C. imicola, and C. actoni that were reported in the Hua Hin area prefer to feed on horse blood. Moreover, C. oxystoma, C. imicola, and C. bravatarsis also feed on canine blood. This study revealed the species of Culicoides in Hua Hin district, Thailand, after the AHS outbreak.

Companion Vector-Borne Pathogens and Associated Risk Factors in Apparently Healthy Pet Animals (Dogs and Cats) in Khukhot City Municipality, Pathum Thani Province, Thailand.

Pet animals (dogs and cats) can be infected with several companion vector-borne pathogens (CVBPs). Morbidity and mortality have been reported in pet animals due to CVBP infections. Pet animals living in close proximity to humans are able to transmit zoonotic pathogens. This study used molecular techniques to investigate the prevalence of CVBPs in apparently healthy pet animals (dogs and cats) from Khukhot City Municipality, Pathum Thani province, Thailand. In total, 210 blood samples were randomly collected from 95 dogs and 115 cats for the detection of seven companion vector-borne pathogens (Anaplasma, Babesia, Bartonella, Ehrlichia, Hepatozoon, Mycoplasma, and Rickettsia) using polymerase chain reaction. The results showed that 10.5% (22/210) of apparently healthy pet animals were infected with at least one pathogen, comprising 6 dogs (6.3% of all dogs tested) and 16 cats (13.9% of all cats tested). Ehrlichia (6.3%) was present only in dogs; furthermore, 1.1% of the dogs were positive for Anaplasma. There was one dog case co-infected with two pathogens (1.1%). In cats, Mycoplasma (9.6%) was the predominant CVBP, followed by Rickettsia (4.4%). The DNA sequences of all positive animals were 97-99% homologous to those found in the GenBank™ database for all CVBPs identified, namely Ehrlichia canis, Anaplasma platys, Rickettsia felis, Mycoplasma haemofelis, and Candidatus Mycoplasma haemominutum. Additionally, the risk of infection with CVBPs in pets was significantly associated with age, with young dogs more likely to be infected with CVBPs than adult dogs (OR 8.5, 95% CI 1.4-50.1, p = 0.006), while adult cats were more likely to be infected with CVBPs than young cats (OR 3.8, 95% CI 1.0-14.0, p = 0.038). The detection of CVBPs demonstrated the potential risk of infection that may occur in apparently healthy pet animals in Pathum Thani province. These results confirmed that apparently healthy pet animals may still be at risk of vector-borne infections and could maintain the infection cycle in pet populations. Furthermore, sampling a greater number of apparently healthy pet animals may disclose predictors of CVBP positivity in domesticated animals in this area.

Molecular Detection and Genetic Characterization of Zoonotic Hookworm in Semi-Domesticated Cats Residing in Monasteries in Bangkok, Thailand.

Hookworms are the most common parasitic nematodes in the genus of Ancylostoma that infect both humans and animals in subtropical and tropical South East Asia. The common hookworm species in cats is Ancylostoma ceylanicum which is emerging in Thailand. However, the genetic characterization of hookworms in cats is outdated and insufficiently studied in Thailand. We aimed to investigate the prevalence, risk factors and genetic characterization of hookworm infection in semi-domesticated temple cats in Bangkok, Thailand. A total of 500 temple cat fecal samples were collected from 43 monasteries in 24 districts of Bangkok, Thailand. Polymerase Chain Reaction (PCR) was performed by amplifying the internal transcribed spacer (ITS) gene and mitochondrial cytochrome oxidase c subunit I (cox 1) gene. The infection prevalence of hookworm in temple cats was 13.2% (66/500). The highest prevalence was 34.6% in the Bang Khun Thian district, which is located in a suburban area. The risk factor analysis revealed that cats older than one year (OR 2.4, 95% CI 1.1-5.5, p < 0.05), lack of veterinary attention (OR 2.9, 95% CI 1.7-4.9, p < 0.001) and Bangkok zone (suburban vs. inner city; OR 2.9, 95% CI 1.6-5.4, p < 0.001) were significantly increasing hookworm infection risk. All hookworm positive samples were identified as A. ceylanicum by ITS gene. Moreover, genetic characterization of cox 1 gene in A. ceylanicum isolates indicated a mix of isolates from humans, cats and dogs. The findings show that temple cats can act as a potential source of zoonotic hookworm parasites for the human and animal population in Bangkok, Thailand. Therefore, appropriate control measures for hookworms in semi-domesticated temple cats as well as prevention measures for hookworms in pet cats and humans should be promoted.

Detection of Giardia duodenalis Zoonotic Assemblages AI and BIV in Pet Prairie Dogs (Cynomys ludovicanus) in Bangkok, Thailand.

Giardia is a flagellate protozoa that can be transmitted via direct contact and by consuming contaminated water. It is pathogenic in humans and various other animals, including exotic pets. Pet prairie dogs are popular in Thailand, but they have not been investigated regarding giardiasis. Giardia infection was measured, and genetic characterization was performed to investigate the zoonotic potential of Giardia carried by pet prairie dogs. In total, 79 fecal samples were examined from prairie dogs visiting the Kasetsart University Veterinary Teaching Hospital during 2017-2021. Simple floatation was conducted. Two Giardia-positive samples were submitted for DNA extraction, PCR targeting the Giardiassu rRNA, tpi and gdh genes was performed, and genetic characterization using sequencing analysis was conducted. Risk factors associated with Giardia infection were analyzed. Giardia infection was found in 11 out of the 79 pet prairie dogs (13.9%). Giardia infection was significantly higher in male prairie dogs (p = 0.0345). Coccidia cysts (12.7%), the eggs of nematodes (6.3%), and amoeba cysts (2.5%) were also detected. Genetic characterization of the two Giardia-positive samples revealed that they were G. duodenalis assemblage A, sub-genotypes AI and assemblage B, and sub-genotype BIV, the zoonotic assemblages. This was the first report of Giardia infection in pet prairie dogs in Bangkok, Thailand. The results revealed that these pet prairie dogs in Thailand were infected with zoonotic assemblages of G. duodenalis sub-genotype AI, which might have been derived from animal contaminants, whereas sub-genotype BIV might have been derived from human contaminants. Owners of prairie dogs might be at risk of giardiasis or be the source of infection to their exotic pets.

Preliminary Study on Comparative Efficacy of Four Light Sources for Trapping Culicoides spp. (Diptera: Ceratopogonidae) in Prachuap Khiri Khan Province, Thailand.

The light trap is an important tool to determine the presence and abundance of vectors in the field. However, no one has studied the efficiency of light traps for collecting Culicoides in Thailand. In the present study, the efficacy of four light sources was evaluated in Prachuap Khiri Khan province, Thailand. Incandescent (INCND) light, white fluorescent (WHT-FLR) light, ultraviolet fluorescent (UV-FLR) light, and UV light-emitting diode (UV-LED) light were tested using commercial traps. In total, 30,866 individuals of Culicoides species were collected from November 2020 to June 2021, of which 21,016 were trapped on site 1 and 6,731 were trapped on site 2. The two most abundant Culicoides species were C. imicola (54%) and C. oxystoma (31.2%). UV-FLR was highly effective, followed by UV-LED light, WHT-FLR light, and INCND light, respectively, for Culicoides collection. Significantly, more Culicoides species were collected in those traps baited with UV-FLR light, UV-LED light, or WHT-FLR light than for INCND light traps. Traps equipped with UV-FLR lights can be recommended to trap Culcoides biting midges for monitoring purposes.

Isolation and in vitro cultivation of Trypanosoma evansi Thai strains.

Trypanosoma evansi is a flagellate protozoan parasite responsible for "surra". To generate T. evansi antigens for serodiagnosis, parasites are generally propagated in laboratory animals before isolation. The alternation of animal models using axenic cultivation systems to produce trypomastigotes of various Trypanosoma species is currently available but has never been applied in Thailand. The isolation protocol for separation of live T. evansi trypomastigotes from animal blood components before in vitro cultivation has not been clearly documented. This study focused on validation of trypomastigote isolation method, in vitro cultivation of T. evansi Thai strains, and its virulence ability in vivo. In this study, two strains of T. evansi collected from Thailand were used. Trypanosoma evansi trypomastigotes were propagated in mice, and three different isolation methods, including: low-speed centrifugation, high-speed centrifugation, and ion exchange chromatography using diethylaminoethyl (DEAE) cellulose (or DE52), were compared. Four solutions of in vitro cultivation media, two different in vitro cultivation containers, and different trypomastigote densities for initiation of in vitro culture were compared. Virulence test using in vitro-adapted parasite for 100 days was conducted in vivo. The results showed that the DE52 isolation method was suitable for separation of live T. evansi trypomastigotes from animal blood components before conducting in vitro cultivation. Trypanosoma evansi Thai strains were successfully cultivated and multiplied in HMI-9 Solution I using 25 cm2 flasks and 12-well plates. The parasite was growing slowly at the initiation of in vitro culture for 15-16 days, and then rapidly increased to 10, 20, 50, 100, and 200 folds, approximately. The doubling times were varied from 11.95 ± 8 h to 41.18 ± 4.29 h in vitro. The maximum densities have reached from 0.14 × 106 to 4.63 × 106 trypomastigotes/ml. Virulence test showed that the in vitro-cultivated T. evansi was virulent in mice. In conclusion, T. evansi Thai strains were successfully isolated and cultivated in vitro for the first time. The isolation and in vitro cultivation protocols were clearly provided. The benefit of using the in vitro cultivation system helps in the production of T. evansi antigen, and replacing the use of experimental animals. It is also useful for the development of diagnostic tests in the future.

Detection of zoonotic Bartonella species in ticks and fleas parasitizing free-ranging cats and dogs residing in temples of Bangkok, Thailand.

Bartonellosis is a vector-borne disease caused by intraerythrocytic bacteria known as Bartonella spp. The potential vectors that transmit Bartonella spp. are fleas, ticks, sand flies, and lice. Several Bartonella spp. cause diseases in humans; however, there is few molecular evidence of Bartonella spp. in vectors in Thailand. The objectives of this study were to investigate Bartonella spp. and to evaluate the spatial distribution of Bartonella spp. prevalence in the ectoparasites parasitizing free-ranging cats and dogs in temple clusters of Bangkok, Thailand. In total, 343 ectoparasites were studied to extract their genomic DNA. Species of all specimens were identified using an identification key and conventional polymerase chain reaction (cPCR) was applied to confirm flea and tick species. Extracted DNA samples were processed using primers that targeted the gltA, rpoB, ftsZ, and ribC genes of Bartonella spp. Then, PCR-positive amplicons were sequenced and a phylogenetic tree was constructed. Recorded data were statistically analyzed using descriptive statistics, the chi-square test, Fisher's exact test, and the odds ratio. Area data were analyzed and a prevalence distribution map was plotted. The major parasitizing ticks and fleas in this study were Rhipicephalus sanguineus and Ctenocephalides felis, respectively. Overall, the prevalence of Bartonella spp. in ectoparasites was 7.00%. The gltA amplicons revealed the presence of B. henselae (4.78%) and B. clarridgeiae (4.78%) in C. felis, and B. koehlerae (1.25%) and B. phoceensis (1.25%) in R. sanguineus. Bartonella DNA was encountered in 16/39 (41.03%) districts and 28.57% of the temple clusters. Bang Khun Thian district had the highest positive proportion and Bang Bon district showed co-evidence of different Bartonella species. In addition, the intervening zones were a risk factor of Bartonella (p < 0.05), and the distribution map showed a scattered pattern of Bartonella-positive clusters. Finally, fleas showed to be important vector reservoirs for Bartonella spp., especially zoonotic species, however, experimental studies are needed to prove the Bartonella transmission in ticks.

Molecular Detection of Tick-Borne Pathogens in Stray Dogs and Rhipicephalussanguineus sensu lato Ticks from Bangkok, Thailand.

Canine tick-borne pathogens (CTBPs) such as Babesia vogeli, Ehrlichia canis, Anaplasma platys, Hepatozoon canis, and Mycoplasma haemocanis are important pathogens in dogs worldwide. Rhipicephalus sanguineus sensu lato, the main vector of several CTBPs, is the most common tick species found on dogs in Thailand. The present study identified CTBPs in dogs and ticks infested dogs. Samples (360 dog blood samples and 85 individual ticks) were collected from stray dogs residing in 37 temples from 24 districts in Bangkok and screened for CTBPs using molecular techniques. The most common CTBP found infecting dogs in this study was Ehrlichia canis (38.3%) followed by Mycoplasma haemocanis (34.2%), Hepatozoon canis (19.7%), Babesia vogeli (18.1%), and Anaplasma platys (13.9%), respectively. Furthermore, A. platys (22.4%) was the most common CTBP in ticks followed by M. haemocanis (18.8%), B. vogeli (9.4%), H. canis (5.9%), and E. canis (2.4%), respectively. The detection of CTBPs from the present study highlights the potential risk of infections that may occur in stray dogs and their ticks residing in Bangkok temples. These findings underline the importance of performing active surveys to understand the complexity of distributions of CTBPs in dogs and their ticks in Thailand.

Molecular detection of Giardia duodenalis and Cryptosporidium spp. from stray dogs residing in monasteries in Bangkok, Thailand.

Both Cryptosporidium spp. and Giardia duodenalis are enteric protozoan parasites that infect a wide variety of domestic animals as well as humans worldwide, causing diarrheal diseases. Giardia duodenalis assemblages C and D are specific to canine hosts and zoonotic assemblages A and B are also found in dogs as a reservoir host. In dogs, Cryptosporidium canis is the host-specific species while humans are infected by C. hominis and C. parvum and at least another 16 zoonotic Cryptosporidium species have been reported causing human infections, with C. meleagridis, C. viatorum, and C. ubiquitum being the most frequent. The objective of this study was to determine the prevalence of Cryptosporidium spp. and G. duodenalis from stray dogs in areas of Bangkok and to identify the species and assemblages. Fecal samples (540) were collected from dogs residing in 95 monasteries in 48 districts in the Bangkok metropolitan area. Nested Polymerase Chain Reaction (PCR) was performed using the ssu-rRNA gene for both parasites. In total, 3.0% (16/540) samples were positive for G. duodenalis, with most being G. duodenalis assemblage D (7/16) followed by assemblage C (7/16) and zoonotic assemblage A (2/16). The prevalence of Cryptosporidium spp. was 0.7% (4/540) based on the PCR results and all were the dog genotype C. canis. These results indicated that dogs residing in Bangkok monasteries poses a limited role as source of human giardiosis and cryptosporidiosis.

Evaluation of hematological alteration of vector-borne pathogens in cats from Bangkok, Thailand.

BACKGROUND: Cats can be carriers of infected arthropods and be infected with several vector-borne pathogens (VBPs) but there is limited knowledge about their pathogenic role in cats. This study aimed to assess the prevalence of some feline vector-borne agents by molecular technique and to characterize the hematological findings associated with these infections in a cat population from Bangkok Thailand. RESULTS: PCR was positive with at least one pathogen in 237 out of 372 subjects (63.7%), with prevalence of 39.5% (147/372) for Babesia spp., 36.9% (137/372) for hemoplasmas and 3.2% (12/372) for Hepatozoon spp. The cats older than 1 year were at significantly greater risk for VBPs infection (P = 0.001; OR = 1.43; 95% CI: 1.12 - 1.81) and hemoplasmas infection (χ2 = 10.8, df = 1; P < 0.0001; OR = 2.45; 95% CI: 1.49 - 4.01). A significant association between hematological findings and hemoplasma infection were identified in the present study. Besides, VBPs infection revealed more frequent in male cats (χ2= 6.38, df = 1, P = 0.01). Macrocytic hypochromic type of anemia was observed in cats infested with blood-sucking arthropods compared to the non-infested cats presented. CONCLUSIONS: The current study confirmed that Babesia, Hepatozoon and hemoplasmas had infected semi-domesticated cats in Bangkok, Thailand, with Babesia and hemoplasmas being dominant in prevalence. Some hematological findings were significantly associated with cats infected with vector-borne pathogens in this study including leukocyte count and platelets count that may help support veterinary technicians in diagnosis and appropriate treatment. Campaigns of VBPs monitoring in Bangkok emphasizing on the investigation of vectors and possible routes of the infection in animals should be conducted to prevent the transmission of the pathogens.

Seroprevalence of Toxoplasma gondii infection from water buffaloes (Bubalus bubalis) in northeastern and southern Thailand.

Toxoplasmosis is a parasitic disease resulting from infection with the apicomplexan Toxoplasma gondii (Nicolle et Manceaux, 1908), one of the world's most common parasites in warm-blooded animals, including humans. Sources of infection can be exposed to infected cat faeces, mother-to-child transmission during pregnancy, and notably, the consumption of undercooked contaminated meat of intermediate hosts. In Thailand, water buffaloes are highly valued for rice cultivation, traditional culture and meat production. Like several other mammalian species, these animals play a role as reservoirs of T. gondii, thus representing a threat to human health. The seroprevalence of T. gondii infection in swamp buffaloes was examined in southern and northeastern Thailand. In total, serum samples of 721 water buffaloes were collected from seven provinces (Ubon Ratchathani, Roi Et, Si Sa Ket, Surin, Buri Ram, Sakon Nakhon, and Songkhla) and examined for the presence of T. gondii infection using commercial latex agglutination test kits (TOXOCHECK-MT, Eiken Chemical Co., Tokyo, Japan). Of the 721 animals analysed, 49 (6.8%) were positive for T. gondii. Songkhla province had the highest seroprevalence (14.7%) among the seven provinces covered in this survey. There was a potential risk to local citizens of T. gondii infection identified by the present study, notably in northeastern Thailand, where despite lower seroprevalence consuming raw buffalo meat salad should be restricted to avoid the risk of zoonotic infections.

Prevalence of Tritrichomonas foetus infection in cats in Bangkok metropolitan area and in vitro drug sensitivity testing.

Tritrichomonas foetus is a causative agent of feline trichomonosis, resulting in large-bowel diarrhea in cats. Feline trichomonosis has been reported in the USA, Europe and some Asian countries but there is limited information for Thailand. This study investigated the prevalence of T. foetus infection in cats in the Bangkok metropolitan area and evaluated the in vitro efficacy of metronidazole (MDZ) and ronidazole (RDZ) against T. foetus Thai isolates. In total, 215 fecal samples were collected from 121 owned cats and 94 stray cats. All fecal samples were cultivated in InPouch™ TF-feline medium. Afterward, polymerase chain reaction (PCR) assays targeting the 5.8S rRNA gene, the ITS regions and DNA sequencing were used for the confirmation of T. foetus. The overall prevalence of T. foetus infection was 4.18% (9/215) based on cultivation and PCR. The sequencing results showed 100% homology to T. foetus sequences from GenBank. The average minimal lethal concentrations (MLCs) of MDZ were 333.33 and 66.67 µg/ml at 24 and 48 h, respectively. The average MLCs of RDZ were 29.16 and 12.5 µg/ml at 24 and 48 h, respectively. The MLC of the MDZ results revealed that T. foetus Thai isolates had a tendency to be MDZ-resistant. To the best of the authors' knowledge, this study was the first using in vitro cultivation and molecular techniques to report and confirm the presence of T. foetus in cats living in the Bangkok metropolitan area. Further studies are needed to determine the genuine infection rate of T. foetus in a greater population sample and the infection status in cats with signs of diarrhea in Thailand.

Cats as potential mammalian reservoirs for Rickettsia sp. genotype RF2125 in Bangkok, Thailand.

Rickettsia felis is an obligate intracellular alpha-proteobacteria and the cause of flea-borne spotted fever (FBSF), an emerging zoonosis of global public health importance, for which dogs and cats have been implicated as potential mammalian reservoirs hosts. The purpose of this study was to determine the prevalence and associated risk factors for R. felis-like species in semi-domesticated cats and their fleas in aim of understanding public health risks posed by cats and their fleas in Bangkok, Thailand. Single whole blood samples (n = 432) and where observed, fleas (n = 234), were collected from cats from 53 temple communities in Bangkok. Fleas were morphologically and genetically identified to a species level. Cat blood and fleas were subjected to a spotted fever group (SFG)-specific PCR targeting the partial outer membrane protein B (ompB). Those that were positive, were further characterised using an R. felis-specific nested PCR targeting the partial citrate synthase A (gltA) gene. All fleas were identified as Ctenocephalides felis felis. In total SFG Rickettsiae were detected in the blood of 82/482 (17.01%) cats and 3/234 fleas (1.28%). DNA sequencing of the partial ompB characterised all positive amplicons from cat blood and their fleas as 100% identical to Rickettsia endosymbiont of Ctenocephalides felis orientis isolate (Rickettsia sp. genotype RF2125) and Rickettsia asemboensis (GenBank accession no. KP256362 and KY650699, respectively). The gltA gene targeting R. felis was successfully amplified from 12/82 PCR-positive cat blood samples and these clustered with 99% bootstrap support with isolates within the Rickettsia sp. genotype RF2125 clade. Cats that were permitted to roam freely inside monasteries were more likely to be infected with R. felis compared with cats confined indoors. The results suggest that cats in Thailand are potential mammalian reservoir hosts for Rickettsia sp. genotype RF2125.

Molecular Identification of Cryptosporidium Species from Pet Snakes in Thailand.

Cryptosporidium is an important pathogen causing gastrointestinal disease in snakes and is distributed worldwide. The main objectives of this study were to detect and identify Cryptosporidium species in captive snakes from exotic pet shops and snake farms in Thailand. In total, 165 fecal samples were examined from 8 snake species, boa constrictor (Boa constrictor constrictor), corn snake (Elaphe guttata), ball python (Python regius), milk snake (Lampropeltis triangulum), king snake (Lampropeltis getula), rock python (Python sebae), rainbow boa (Epicrates cenchria), and carpet python (Morelia spilota). Cryptosporidium oocysts were examined using the dimethyl sulfoxide (DMSO)-modified acid-fast staining and a molecular method based on nested-PCR, PCR-RFLP analysis, and sequencing amplification of the SSU rRNA gene. DMSO-modified acid-fast staining revealed the presence of Cryptosporidium oocysts in 12 out of 165 (7.3%) samples, whereas PCR produced positive results in 40 (24.2%) samples. Molecular characterization indicated the presence of Cryptosporidium parvum (mouse genotype) as the most common species in 24 samples (60%) from 5 species of snake followed by Cryptosporidium serpentis in 9 samples (22.5%) from 2 species of snake and Cryptosporidium muris in 3 samples (7.5%) from P. regius.

Canine vector-borne pathogens in semi-domesticated dogs residing in northern Cambodia.

BACKGROUND: In Southeast Asia, the canine vector-borne pathogens Babesia spp., Ehrlichia canis, Anaplasma platys, Hepatozoon canis, haemotropic mycoplasmas and Dirofilaria immitis cause significant morbidity and mortality in dogs. Moreover, dogs have also been implicated as natural reservoirs for Rickettsia felis, the agent of flea-borne spotted fever, increasingly implicated as a cause of undifferentiated fever in humans in Southeast Asia. The objective of this study was to determine the prevalence and diversity of canine vector-borne pathogens in 101 semi-domesticated dogs from rural Cambodia using molecular diagnostic techniques. RESULTS: The most common canine vector-borne pathogens found infecting dogs in this study were Babesia vogeli (32.7 %) followed by Ehrlichia canis (21.8 %), Dirofilaria immitis (15.8 %), Hepatozoon canis (10.9 %), Mycoplasma haemocanis (9.9 %) and "Candidatus Mycoplasma haematoparvum" (2.9 %). A high level of co-infection with CVBD agents (23.8 %) was present, most commonly B. vogeli and E. canis. Naturally occurring R. felis infection was also detected in 10.9 % of dogs in support of their role as a natural mammalian reservoir for flea-borne spotted fever in humans. CONCLUSIONS: This study reports for the first time, the prevalence and diversity of CVBD pathogens in dogs in Cambodia. In total, five species of CVBD pathogens were found infecting semi-domesticated dogs and many were co-infected with two or more pathogens. This study supports the role of dogs as natural mammalian reservoirs for R. felis, the agent of flea-borne spotted fever in humans.

Prevalence and genotype of Giardia duodenalis in dairy cattle from Northern and Northeastern part of Thailand.

The aims of this study were to determine prevalence and genotype of Giardia duodenalis in feces of dairy cattle from the northern part and the northeastern part of Thailand. A total of 900 fecal samples were collected directly from rectum and examined by using zinc sulphate centrifugal flotation technique and polymerase chain reaction (PCR). The overall prevalence of G. duodenalis in dairy cows was 5.0 % (45/900) by zinc sulphate centrifugal flotation and 6.0 % (54/900) by PCR. Genotypes of G. duodenalis found in this study were Assemblage AI and E. The results indicated that dairy cattle may act as a potential risk of Giardia transmission among animals and humans (especially Assemblage AI).

Low risk for transmission of zoonotic Giardia duodenalis from dogs to humans in rural Cambodia.

BACKGROUND: A number of epidemiological studies have demonstrated Giardia as prevalent in both humans and dogs worldwide and have postulated the occurrence of anthroponotic, zoonotic and animal-specific cycles of transmission, which may be geographically and regionally unique in its epidemiology. The aim of this study was to utilise molecular tools to determine the prevalence and compare genotypes of Giardia duodenalis infecting humans and dogs living in a previously identified Giardia-endemic village in rural Cambodia in order to ascertain zoonotic transmission risk. FINDINGS: The prevalence of G. duodenalis in humans and dogs was 18.3% (40/218) and 10.6% (10/94) by PCR, respectively. Molecular characterisation of the small subunit of ribosomal RNA (SSU rRNA) gene, triose phosphate isomerase (TPI) gene and sub-assemblage characterisation of the glutamate dehydrogenase (gdh) gene placed 27.5% (11/40) of Giardia positive humans into assemblage AII and 72.5% (29/40) into assemblage BIII of G. duodenalis. In dogs, 20.0% (2/10) of Giardia-positive samples were characterised as G. duodenalis assemblage BIII, 40.0% (4/10) as assemblage C and 40.0% (4/10) as mix infection between assemblage C and D. CONCLUSION: Overall, just over 2% of dogs harboured potentially zoonotic assemblages of G. duodenalis in the studied communities and hence pose a minimal zoonotic risk for the transmission of Giardia to humans.

High prevalence of Ancylostoma ceylanicum hookworm infections in humans, Cambodia, 2012.

Ancylostoma ceylanicum, a hookworm of canids and felids in Asia, is becoming the second most common hookworm infecting humans. In 2012, we investigated the prevalence and infection dynamics of and risk factors for hookworm infections in humans and dogs in a rural Cambodian village. Over 57% of the population was infected with hookworms; of those, 52% harbored A. ceylanicum hookworms. The greatest intensities of A. ceylanicum eggs were in persons 21-30 years of age. Over 90% of dogs also harbored A. ceylanicum hookworms. Characterization of the cytochrome oxidase-1 gene divided isolates of A. ceylanicum hookworms into 2 groups, 1 containing isolates from humans only and the other a mix of isolates from humans and animals. We hypothesize that preventative chemotherapy in the absence of concurrent hygiene and animal health programs may be a factor leading to emergence of A. ceylanicum infections; thus, we advocate for a One Health approach to control this zoonosis.

The prevalence and diversity of intestinal parasitic infections in humans and domestic animals in a rural Cambodian village.

In Cambodia, intestinal parasitic infections are prevalent in humans and particularly in children. Yet, information on potentially zoonotic parasites in animal reservoir hosts is lacking. In May 2012, faecal samples from 218 humans, 94 dogs and 76 pigs were collected from 67 households in Dong village, Preah Vihear province, Cambodia. Faecal samples were examined microscopically using sodium nitrate and zinc sulphate flotation methods, the Baermann method, Koga Agar plate culture, formalin-ether concentration technique and Kato Katz technique. PCR was used to confirm hookworm, Ascaris spp., Giardia spp. and Blastocystis spp. Major gastrointestinal parasitic infections found in humans included hookworms (63.3%), Entamoeba spp. (27.1%) and Strongyloides stercoralis (24.3%). In dogs, hookworm (80.8%), Spirometra spp. (21.3%) and Strongyloides spp. (14.9%) were most commonly detected and in pigs Isospora suis (75.0%), Oesophagostomum spp. (73.7%) and Entamoeba spp. (31.6%) were found. Eleven parasite species were detected in dogs (eight helminths and three protozoa), seven of which have zoonotic potential, including hookworm, Strongyloides spp., Trichuris spp., Toxocara canis, Echinostoma spp., Giardia duodenalis and Entamoeba spp. Five of the parasite species detected in pigs also have zoonotic potential, including Ascaris spp., Trichuris spp., Capillaria spp., Balantidium coli and Entamoeba spp. Further molecular epidemiological studies will aid characterisation of parasite species and genotypes and allow further insight into the potential for zoonotic cross transmission of parasites in this community.