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Dog owners are greatly concerned about tick infestations in their pets. The prevalence and dispersion of ticks and their disease-causing microorganisms have been limited from the viewpoint of dog owners in Vietnam. This study investigated the presence of tick infestation and the pathogens associated with it in canines that were brought to veterinary hospitals in Vietnam. In the survey, 1,423 dogs participated from February to October 2022. Molecular and morphological methods were utilized to identify ticks and the associated pathogens. In addition,risk variables linked to tick infestation were documented and analyzed using statistical methods. The total exposure to the brown dog tick ( Rhipicephalus sanguineus sensu lato ) was 29.01%. Nam Dinh has the highest tick prevalence among the research areas. Tick infestation reached its highest point between June and September in the northern region of the country, with distinct seasons showing a strong correlation with tick infestation in dogs. Out of 177 tick pools examined, 146(82.49%) tested positive for at least one infection. Mycoplasma spp. (78.53%) was the most common, followed by Anaplasma spp. (37.29%), Rickettsia felis (5.08%), Babesia vogeli , and Hepatozoon canis (2.82%). In the current study, there was a statistically significant link between tick infestation and characteristics such as age, breed, body size, lifestyle, and bathing frequency. Understanding the seasonal behavior of vector ticks is crucial for identifying individuals or animals susceptible to tick-borne diseases. Studying the distribution of ticks and their ability to carry and disseminate zoonotic germs in specific places could assist veterinarians and policymakers in implementing effective strategies to manage zoonotic infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Do, Bui, Umemiya-Shirafuji, Inpankaew, Hasan, Zafar, Ma, Hang, Mohanta, Amer, El-Sayed, Xuan and Kamyingkird.)

Surra, a wasting disease caused by Trypanosoma evansi , is one of the major animal health burdens in camel-rearing countries, imposing significant economic losses due to reduced fertility and high mortality rates. The present study used inactivated T. evansi (from the Card Agglutination Test for Trypanosomes/ Trypanosoma evansi ; CATT/ T. evansi ) and flow cytometry to investigate their binding and activation potential toward camel leukocyte subsets. Labeling T. evansi with propidium iodide (PI) enabled their flow cytometric enumeration and identification with forward scatter (FSC; indicative for cell size) and side scatter (SSC; indicative for cell internal complexity) characteristics that are comparable with values reported for Trypanosoma cruzi . The incubation of PI-labeled non-opsonized T. evansi with camel leukocyte populations revealed that camel monocytes have the highest potential to bind T. evansi , followed by granulocytes and lymphocytes. The identification of pattern recognition receptors (PRRs) on camel immune cells and the pathogen-associated molecular patterns (PAMPs) in T. evansi that are responsible for this different binding capacity requires further studies. Stimulation of camel neutrophils with Trypanosoma evansi induced shape change, reactive oxygen species (ROS) production, and neutrophil extracellular traps (NET)-formation. To ensure that T. evansi , in the parasite concentration used in this study, is not apoptotic or necrotic to camel leukocytes, we evaluated cell apoptosis and necrosis after stimulation with T. evansi . The results revealed no impact of T. evansi stimulation for 2 h on the cell viability of camel leukocytes. Subsequent work may focus on the diagnostic employment of labeled T. evansi and flow cytometry for the detection of anti- Trypanosoma antibodies in camel serum. In addition, more efforts should be deployed to investigate the host-pathogen interaction mechanisms and the escape mechanisms of T. evansi in camels. To complete these data, further studies using the living or freshly killed parasites could also be implemented in camels and/or horses.

Tick-borne diseases (TBDs) massively impact bovine production. In endemic countries, animals are often subclinically infected, showing no signs of the illness. Anemia is a hallmark of TBDs, but there is inadequate information on its presence in infected Thai cattle. In the present study, 265 cattle from four provinces in Thailand were surveyed to identify tick-borne pathogens (TBPs) and to evaluate the changes in the packed cell volume (PCV) values associated with detection. Microscopy and polymerase chain reaction (PCR) were also compared for TBP detection. Babesia / Theileria / Hepatozoon was detected in 33.58% (89/265) of the cattle samples. Specifically, Babesia bovis (9/265), B. bigemina (12/265), Theileria orientalis (62/265), and Anaplasma marginale (50/265) were identified using species-specific assays. Significant decreases in the mean PCV levels were observed in cattle that were positive for at least one TBP ( p < 0.001), Babesia / Theileria / Hepatozoon ( p < 0.001), T. orientalis ( p < 0.001), and A. marginale ( p = 0.049). The results of PCR and microscopy for the detection of TBPs suggested slight and fair agreement between the two detection tools. The present findings contribute to a better understanding of TBDs in the field and shall facilitate the formulation of effective control for TBDs in Thailand.

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 × 10 5 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 × 10 11 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., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Phongphaew, et al.)

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.

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.

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.

Toxoplasmosis is one of the most common zoonotic parasitic diseases infecting nearly all warm-blooded animals, including poultry (geese, turkeys, chickens, and ducks). It is caused by Toxoplasma gondii ( T. gondii ), which is an obligate intracellular protozoan belonging to the Apicomplexa group. In Thailand, duck meat production for domestic consumption and international trade is mainly bred and produced in the central and western parts of the country. Free-grazing ducks in integrated duck-rice production have significant benefits in rice cultivation, accounting for the popularity of this farming system in Thailand. However, ducks are considered particularly susceptible to consuming T. gondii oocysts from water contaminated with cat feces due to the fact of their feeding habits of free-grazing and dabbling. Hence, the prevalence of this zoonotic parasite in a large-scale integrated farming context is particularly challenging with respect to the contamination of the food chain of humans and farm animals. In the present study, we examined the overall prevalence of T. gondii infection in slaughtered free-grazing ducks originating from Central and Western Thailand, setting the stage for an in-depth One Health approach to assess and manage the risks of integrated farming practices. A representative sample size of 161 ducks was calculated using a two-stage sampling method. Specifically, serum samples were collected from 217 slaughtered free-grazing ducks originating in six provinces in Central and Western Thailand. Serum antibodies against T. gondii were detected using an indirect fluorescent antibody test (IFAT). The positive control serum samples were prepared from ducks experimentally immunized with T. gondii . Sixty-eight (31.3%) of the two hundred and seventeen ducks were seropositive with T. gondii . Two groups of fattening ducks and spent layers showed similar seropositivity rates at 29% and 32.3%, with the majority of positive samples being found in the low titer. In addition, a wide distribution of positive serum samples was observed in all six provinces in the present study. To the best of our knowledge, this is the first report on a serological prevalence snapshot in commercially produced duck populations that have high interaction with farmed environments in Thailand, revealing a high infection pressure in areas of integrated duck-rice farming. Importantly, contaminated duck meat for commercial use, as well as offal and carcasses from slaughterhouses, completes the transmission of T. gondii from the environment into the food chain of humans and domestic animals. Hence, from a One Health perspective, it is important to clarify whether this transmission chain extends further to the wild, i.e., predator-prey cycles that are independent of duck farming or are self-contained.

Background and Aim: An apicomplexan protozoan parasite, namely, Theileria , primarily causes theileriosis in cattle worldwide. The virulence of the disease has been neglected because of it's low pathogenicity. However, the disease can have a substantial effect, depending on the virulence of the species, low host immunity, and coinfection. In Thailand, the molecular detection of Theileria infection in bullfighting cattle and its hematological alterations have not been reported. Thus, this study aimed to identify Theileria species in bullfighting cattle in Thailand., Materials and Methods: Blood samples were collected from bullfighting cattle presented at the Prince of Songkla University Animal Hospital and were determined on the basis of hematological evaluation and DNA extraction. Molecular detection using the 18s rRNA and merozoite surface antigen genes was conducted for Theileria spp. and Theileria orientalis , respectively. In addition, bidirectional sequencing of the positive samples was performed. Hematological alterations between Theileria infected and uninfected groups were statistically evaluated., Results: The levels of Theileria spp. and T. orientalis infection in bullfighting cattle were 44.62% (58/130) and 41.54% (54/130), respectively. Theileria orientalis , Theileria sinensis , and Theileria spp. infections were identified in bullfighting cattle samples. Hematological evaluation indicated that the red blood cell (RBC) level was significantly lower in Theileria- infected cattle., Conclusion: This study was the first to use molecular techniques in the identification of Theileria infection in bullfighting cattle in Thailand, with nearly one-half of the study population infected. Theileria infection in bullfighting cattle altered the RBC level, resulting in anemia. Therefore, tick control measures should be promoted., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Rakwong, et al.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 cm 2 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 × 10 6 to 4.63 × 10 6 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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 Giardia ssu 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.

Background and Aim: Toxoplasma gondii tachyzoite is the infective stage that causes acute infection, leading to severe toxoplasmosis. The tachyzoite stage has been extensively used for several inoculation purposes, including antigen production, immunological studies, nutrition mechanisms, and in vitro drug trials. The use of fresh tachyzoites is required for inoculation in either in vitro or in vivo studies. However, there is a lack of information on preserving live tachyzoites during transportation from laboratories to inoculation sites. Therefore, this study aimed to validate suitable preservative conditions for maintaining live parasites by determining the survival and viability of T. gondii tachyzoites on the basis of different media, temperatures, and incubation times., Materials and Methods: The free live T. gondii tachyzoites were evaluated on their viability when maintained in different media without 5% Carbon dioxide (CO 2 ). The purified tachyzoites of the RH and PLK strains were individually suspended in normal saline (NS), phosphate-buffered saline (PBS), minimum essential medium (MEM), and MEM with 10% fetal bovine serum (MEM-FBS) and incubated for 6 h at ice-cold (IC; 3-9°C) and room temperature (RT; 25°C). Parasite survival was measured at the 0, 1 st , 2 nd , 3 rd , 4 th , 5 th , and 6 th h post-incubation using the trypan blue exclusion test., Results: The viability was in the range of 85.0%-91.0% for IC using NS and 81.0%-85.1% (IC) and 75.3%-77.5% (RT) using PBS. The viability was approximately 75.0%-83.0% (IC) and 70.0%-79.0% (RT) using MEM and MEM-FBS. There was a significant difference in the viability between the seven periods on the basis of one-way repeated Analysis of variance and Friedman analyses. Parasite survival slightly reduced (20.0%-30.0%) in NS and MEM-FBS at both temperatures during incubation. Notably, PBS could not support tachyzoite viability after 3 h post-incubation., Conclusion: NS was a suitable preservative for maintaining purified T. gondii tachyzoites during transportation at IC and RT without 5% CO 2 supplementation. This could be a valuable medium for parasite transportation, especially when there is a large distance between the laboratory and inoculation site., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Nguyen, et al.)

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.

Background and Aim: Rabies is a highly infectious but neglected zoonotic disease. Almost 99% of rabies-related human deaths are caused by dog-mediated rabies. Although canine rabies vaccination is highly effective and provides protection, nationwide rabies vaccination campaigns have been insufficient in Cambodia, resulting in a limited number of rabies vaccinated dogs. This study aimed to explore the rabies knowledge, attitude, and practices (KAP) among participants from both dog rabies vaccinated (DRV) and dog rabies unvaccinated (DRUV) villages located in the Kandal and Prey Veng Provinces, Cambodia., Materials and Methods: A cross-sectional survey was conducted with dog owners in Kandal and Prey Veng Provinces, Cambodia, during August and September 2020. The structural questionnaire collected general sociodemographic information and the KAP associated with rabies transmission, clinical signs, management, and control. The data were then analyzed using Wilcoxon rank-sum test and Chi-square statistics., Results: In total, 312 participants were interviewed: 137 participants from DRV villages and 175 from DRUV villages. Among them, 99.4% (310/312) had previously heard about rabies. Out of these 310, 93.5% (290/310) were aware that rabies is a fatal disease, while 96.5% (299/310) were willing to vaccinate their dog against rabies if the vaccination was provided for free. However, 32.9% (102/310) indicated that they would be willing to sell their own dog if it bit someone or showed aggression. More than one-third (115/310) of all the respondents had poor overall KAP regarding rabies. The respondents from DRV villages had significantly higher overall scores with regard to rabies KAP than those from DRUV villages (p<0.0001). According to the factors related to overall KAP, village type and education level were significantly associated with overall KAP of the respondents (p<0.0001)., Conclusion: The rabies disease is recognized in Cambodia, and dog owners are willing to vaccinate their dogs if the vaccination is provided for free. The overall rabies-related KAP were poor among 30% of the respondents, and higher KAP scores were obtained for the DRV villages. The village type and education level were found to be associated with the different overall KAP of the participants., (Copyright: © Ung, et al.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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.

Background and Aim: Trypanosoma evansi infection has been reported in Thai livestock such as beef and dairy cattle. However, there is little information on T. evansi infection in bullfighting cattle in Southern Thailand. The aim of this study was to investigate the infection of T. evansi in bullfighting cattle presented for health checks at the Animal Hospital, Faculty of Veterinary Science, Prince of Songkla University, Thailand., Materials and Methods: Blood and serum samples were collected from 177 bullfighting cattle from April 2016 to February 2017 after bullfighting matches. Animal inspected showed signs of fever, weight loss, or exercise intolerance. Investigation of T. evansi infection was tested using polymerase chain reaction (PCR) with TBR primers and using indirect enzyme-linked immunosorbent assay with T. evansi crude antigen., Results: The seroprevalence of T. evansi in bullfighting cattle was 22.60% (40/177). The PCR results detected no parasite DNA in this study. However, bullfighting cattle may serve as T. evansi reservoirs., Conclusion: Health checking procedures for T. evansi should be promoted for bullfighting events so that infected animals can be quarantined in the preparatory stages of such events., (Copyright: © Kamyingkird, et al.)

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., Competing Interests: Declaration of Competing Interest The authors have no competing interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background and Aim: Stray cats are a reservoir for various zoonotic diseases relevant to public health in Thailand. The vector-borne infection, hemoplasmosis, also known as infectious anemia, is one such disease carried by domestic and wild felids. This study focuses on molecular characterization and phylogenetic analysis of hemoplasma in semi-domesticated cats in Bangkok, Thailand., Materials and Methods: In total, 473 blood samples were collected from 53 temple communities in 34 metropolitan districts and assayed using polymerase chain reaction (PCR) to amplify partial 16S rRNA genes for hemoplasma detection. Risk factors for hemoplasma infection (gender, age, free-roaming, and ectoparasite exposure) were analyzed using Chi-square tests, logistic regression, and odds ratios (OR) with 95% confidence intervals (95% CI) using R software (version 3.6.1). A phylogenetic tree was established from genetic characterization of hemoplasmas., Results: In total, 180 samples (38.05%) were positive for hemoplasma. Of half of the positive sequenced samples, 83.33% were infected with Candidatus Mycoplasma haemominutum ( CMhm ), 13.33% with Mycoplasma haemofelis ( Mhf ), and 3.33% with Candidatus Mycoplasma turicensis ( CMt ). Cats over 5 years old were more likely to be infected than younger cats (p<0.005, OR=3.8, 95% CI=1.64-4.78). Cats were diagnosed as positive based on PCR assays in 97% (33/34) of districts surveyed. The phylogenetic tree showed two majority clusters with three clades of feline hemotropic mycoplasma., Conclusions: Overall, the survey shows the prevalence (38.05%) and distribution of feline hemoplasma in semi-domesticated cats. This information will contribute to effective prevention and control strategies to minimize infections by feline vector-borne pathogens in Thailand., (Copyright: © Do, et al.)

Surra, caused by Trypanosoma evansi, is a widely distributed animal trypanosomosis; it affects both domestic and wild mammals with high economic impact. Clinical picture is moderate in bovines but severe in equids. Surra is also an important constraint for international animal trade and movements. Despite its impact, surra remains poorly diagnosed because of low sensitivity tests. To improve epidemiological knowledge of the disease and to secure international movement, efficient diagnosis tools are required. Here, we optimized and applied to equids the OIE-recommended indirect ELISA T. evansi that was validated in other species. Based on 96 positive and 1,382 negative horse reference samples from Thailand, a TG-ROC analysis was conducted to define the cutoff value. ELISA's sensitivity and specificity were estimated at 97.5% and 100%, respectively, qualifying the test to provide a reliable immune status of equids. The test was then applied on 1,961 horse samples from 18 Thai Provinces; the only scarce positives suggested that horses do not constitute a reservoir of T. evansi in Thailand. All samples from racing horses were negative. Conversely, two outbreaks of surra reported to our laboratory, originating from a bovine reservoir, exhibited high morbidity and lethality rates in horses. Finally, posttreatment follow-ups of infected animals allowed us to provide outbreak management guidelines., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Margot Camoin et al.)

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., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

[This corrects the article DOI: 10.1371/journal.pone.0185372.].

Human babesiosis is caused by the apicomplexan parasite Babesia microti, which is of major public health concern in the United States and elsewhere, resulting in malaise and fatigue, followed by a fever and hemolytic anemia. In this paper we focus on the characterization of a novel B. microti thrombospondin domain (TSP1)-containing protein (BmP53) from the new annotation of the B. microti genome (locus 'BmR1&#95;04g09041'). This novel protein (BmP53) had a single TSP1 and a transmembrane domain, with a short cytoplasmic tail containing a sub-terminal glutamine residue, but no signal peptide and Von Willebrand factor type A domains (VWA), which are found in classical thrombospondin-related adhesive proteins (TRAP). Co-localization assays of BmP53 and Babesia microti secreted antigen 1 (BmSA1) suggested that BmP53 might be a non-secretory membranous protein. Molecular mimicry between the TSP1 domain from BmP53 and host platelets molecules was indicated through different measures of sequence homology, phylogenetic analysis, 3D structure and shared epitopes. Indeed, hamster isolated platelets cross-reacted with mouse anti-BmP53-TSP1. Molecular mimicry are used to help parasites to escape immune defenses, resulting in immune evasion or autoimmunity. Furthermore, specific host reactivity was also detected against the TSP1-free part of BmP53 in infected hamster sera. In conclusion, the TSP1 domain mimicry might help in studying the mechanisms of parasite-induced thrombocytopenia, with the TSP1-free truncate of the protein representing a potential safe candidate for future vaccine studies.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), which affects equine production in various parts of the world. However, a safe and effective drug is not currently available for treatment of EP. Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine synthesis pathway and has been known as a novel drug target for several apicomplexan protozoan parasites. In this study, we evaluated four DHODH inhibitors; atovaquone (ATV), leflunomide (LFN), brequinar (Breq), and 7-hydroxy-5-[1,2,4] triazolo [1,5,a] pyrimidine (TAZ) on the growth of T. equi and B. caballi in vitro and compared them to diminacene aceturate (Di) as the control drug. The growth of T. equi and B. caballi was significantly hindered by all inhibitors except TAZ. The half maximal inhibitory concentration (IC 50 ) of ATV, LFN, Breq and Di against T. equi was approximately 0.028, 109, 11 and 40 μM, respectively, whereas the IC 50 of ATV, LFN, Breq and Di against B. caballi was approximately 0.128, 193, 5.2 and 16.2 μM, respectively. Using bioinformatics and Western blot analysis, we showed that TeDHODH was similar to other Babesia parasite DHODHs, and confirmed that targeting DHODHs could be useful for the development of novel chemotherapeutics for treatment of EP., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare that there are no conflicts of interest.

Background: Infections with Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale are endemic in Kenya yet there is a lack of adequate information on their genotypes. This study established the genetic diversities of the above tick-borne hemoparasites infecting cattle in Kenya., Methods: Nested PCR and sequencing were used to determine the prevalence and genetic diversity of the above parasites in 192 cattle blood samples collected from Ngong and Machakos farms. B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, A. marginale major surface protein 5, Theileria spp. 18S rRNA, T. parva p104 and T. orientalis major piroplasm surface protein were used as the marker genes., Results: B. bovis, B. bigemina, T. parva, T. velifera, T. taurotragi, T. mutans and A. marginale were prevalent in both farms, whereas T. ovis, Theileria sp. (buffalo) and T. orientalis were found only in Ngong farm. Co-infections were observed in more than 50 % of positive samples in both farms. Babesia parasites and A. marginale sequences were highly conserved while T. parva and T. orientalis were polymorphic. Cattle-derived T. parva was detected in Machakos farm. However, cattle and buffalo-derived Theileria were detected in Ngong farm suggesting interactions between cattle and wild buffaloes. Generally, the pathogens detected in Kenya were genetically related to the other African isolates but different from the isolates in other continents., Conclusions: The current findings reaffirm the endemicity and co-infection of cattle with tick-borne hemoparasites, and the role of wildlife in pathogens transmission and population genetics in Kenya.

Water buffalo are important draft animals for agriculture in resource-restricted areas worldwide. Water buffalo were shown to be experimentally susceptible to infection with Neospora caninum, potentially affected by neosporosis, and naturally exposed to the parasite in Asia. Although enzootic to Thailand, the distribution of N. caninum among Thai water buffalo is unclear. The objectives of this study were to determine the seroprevalence of N. caninum among water buffalo of northeast Thailand and to identify risk factors associated with their exposure to N. caninum. Sera from 628 water buffalo from 288 farms were tested with an indirect fluorescent antibody test (IFAT). A total of 57 samples from 48 herds contained antibodies to N. caninum, indicating overall seroprevalence of 9.1% and 16.7% among individual animals and herds, respectively. The overall seroprevalence was highest in provinces located in the Khorat Basin in the southern part of the region tested. Host age was also associated with seroprevalence, with the greatest seroprevalence (16.1%) among buffalo over 10 years of age, followed by 5-10 years of age (13.4%), 3-5 years (9.2%), and less than 3 years (1.2%). These results collectively suggested that horizontal transmission from canine definitive hosts was an important route of water buffalo exposure to N. caninum. These results also verified the importance of risk factor analysis for effective bovine neosporosis control strategies at the local level., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Surra, caused by Trypanosoma evansi, is a neglected disease due to frequent subclinical evolution, especially in bovines in Asia. However, acute and chronic signs are regularly observed, with significant sanitary and economic impacts. In this study, we evaluated and applied an indirect-ELISA test for the detection of anti-T. evansi immunoglobulin G in buffaloes using antibovine conjugate. Based on buffalo reference sera from the Philippines, a two-graph receiver operating characteristics analysis (TG-ROC) was conducted to define an optimal cut-off value; sensitivity and specificity were estimated at 92.5% and 94.2%, respectively. A cross-sectional serological survey was carried out in the major buffalo breeding areas of Thailand; 892 buffaloes from 8 provinces were sampled in North, Northeastern, and Southern Thailand. Seropositive buffaloes were found in all 8 provinces, on 20.3% of farms for an overall prevalence of 12.2% (95% CI 10.2-14.5%). Nearly one-third of the sampled population was exposed to infection. Broader sampling would be necessary but is not possible in the southern half-wild breeding systems. According to our results, buffaloes may constitute a large and robust reservoir for T. evansi, which is a permanent threat to other livestock such as cattle and horses as well as wild animals such as elephants in Southest Asia.

The resistance of Babesia parasites to current anti-babesiosis drugs is an issue of major concern. The inosine 5'-monophosphate dehydrogenase (IMPDH) of Babesia gibsoni has been identified and characterized as a molecular drug target in our previous studies. In the present study, inhibitory effects of IMPDH inhibitors (mycophenolate mofetil, mizoribine, ribavirin, 7-nitroindole, and mycophenolic acid) were evaluated in vitro or in vivo. In the inhibition assay of recombinant B. gibsoni IMPDH activity, mycophenolate mofetil was the most potent inhibitor (IC(50) = 2.58 ± 1.32 μM) while ribavirin was the least potent. The inhibitory effects of mycophenolate mofetil, mizoribine, ribavirin, and 7-nitroindole on the in vitro growths of B. gibsoni and Babesia bovis were also assessed. The results revealed that mycophenolate mofetil was the most potent inhibitor of the multiplications of both B. gibsoni (IC(50) = 0.13 ± 0.05 μM) and B. bovis (IC(50) = 0.97 ± 0.49 μM). Ribavirin was also the least potent for both B. gibsoni and B. bovis in vitro. Mycophenolic acid, a metabolite of mycophenolate mofetil, caused an inhibition of Babesia microti in mice with noticeable improvement in hematological parameters of the infected mice (ED(50) = 44.15 ± 12.53 mg/kg). Although the report provides a non-exhaustive view of potential treatment strategy without addressing the potential adverse effect of immune suppression on infections, these results indicated that the IMPDH might be a molecular target of MPA for B. microti . Altogether, we provide a basis for development of antibabesia prodrugs by targeting IMPDH of the parasites in the treatment of babesiosis.

Babesia microti is an emerging zoonotic protozoan organism that causes "malaria-like" symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD(+)) as a co-factor and lactate as a substrate. Inhibitory assay was also done using gossypol as BmLDH inhibitor to determine the inhibitory concentration 50 (IC50). The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite. In vitro enzyme kinetic studies further revealed that BmLDH is an active enzyme with a high catalytic efficiency at optimal pH of 10.2. The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively. The IC50 value for gossypol was 0.345 μM, while at 2.5 μM, gossypol caused 100% inhibition of BmLDH catalytic activity. These findings, therefore, provide initial evidence that BmLDH could be a potential drug target, although further in vivo studies are needed to validate the practical application of lactate dehydrogenase inhibitors against B. microti infection.

The emergence of drug resistance and adverse side effects of current bovine babesiosis treatment suggest that the search of new drug targets and development of safer and effective compounds are required. This study focuses on dihydroorotate dehydrogenase (DHODH), the fourth enzyme of pyrimidine biosynthesis pathway as a potential drug target for bovine babesiosis. Recombinant Babesia bovis DHODH protein (rBboDHODH) was produced in Escherichia coli and used for characterization and measurement of enzymatic activity. Furthermore, the effects of DHODH inhibitors were evaluated in vitro. The recombinant B. bovis DHODH histidine fusion protein (rBboDHODH) had 42.4-kDa molecular weight and exhibited a specific activity of 475.7 ± 245 Unit/mg, a Km = 276.2 µM for L-dihydroorotate and a Km= 94.41 µM for decylubiquinone. A 44-kDa band of native BboDHODH was detected by Western blot analysis and found in parasites mitochondria using a confocal microscope. Among DHODH inhibitors, atovaquone (ATV) and leflunomide (LFN) significantly inhibited the activity of rBboDHODH as well as the growth of B. bovis in vitro. The half maximal inhibitory concentration (IC50) of ATV and LFN was 2.38 ± 0.53 nM and 52.41 ± 11.47 µM, respectively. These results suggest that BboDHODH might be a novel target for development of new drug for treatment of B. bovis infection.

A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rvv) vectors expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. In this study, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a recombinant plasmid followed by vaccinia virus, both of which expressed the glutamic acid-rich protein (BgGARP) of Babesia gibsoni. The dogs immunized with the prime-boost regime developed a significantly high level of specific antibodies against BgGARP when compared with the control groups. The antibody level was strongly increased after a booster immunization with a recombinant vaccinia virus. Two weeks after the booster immunization with a recombinant vaccinia virus expressing BgGARP, the dogs were challenged with B. gibsoni parasite. The dogs immunized with the prime-boost regime showed partial protection, manifested as a significantly low level of parasitemia. These results indicated that this type of DNA/rvv prime-boost immunization approach may have use against B. gibsoni infection in dogs.

In order to determine the molecular and serological prevalence of Babesia bigemina and Babesia bovis, a total of 247 blood samples were collected from cattle and water buffalos in Beheira and Faiyum Provinces in Egypt and examined by nested polymerase chain reaction (nPCR) and enzyme-linked immunosorbent assay (ELISA). In cattle, the prevalence of B. bigemina and B. bovis was 5.30% and 3.97% by nPCR and 10.60% and 9.27% by ELISA, respectively, whereas those of water buffalos were 10.42% and 4.17% by nPCR and 15.63% and 11.46% by ELISA, respectively. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and health status. Sequencing analysis revealed two genotypes for B. bovis spherical body protein-4. In conclusion, the current data provide valuable information regarding the epidemiology of B. bigemina and B. bovis infections in cattle and water buffalos from Egypt, which can be employed in developing future strategies for disease management and control., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The inosine monophosphate dehydrogenase (IMPDH) enzyme has been characterized and validated as a molecular drug target in other apicomplexans but not in the genus Babesia. Subsequently, we cloned and expressed a Babesia gibsoni IMPDH (BgIMPDH) cDNA in Escherichia coli. We also determined the inhibitory effect of mycophenolic acid (MPA) on recombinant BgIMPDH (rBgIMPDH) activity and the Babesia-growths in vitro. The translated BgIMPDH peptide contained thirteen amino acid residues responsible for substrate and cofactor binding in its catalytic domain with Gly374 in BgIMPDH being replaced by Ser388 in mammalian IMPDH. The native BgIMPDH enzyme in the parasite was approximately 54-kDa a mass similar to His-tag rBgIMPDH protein. The Km values of the rBgIMPDH were 8.18±0.878 (mean±standard error of the mean) μM and 360.80±43.41μM for IMP and NAD(+), respectively. MPA inhibited the rBgIMPDH activity yielding a Ki value of 20.93±1.83μM with respect to NAD(+). For Babesia growths, the IC50s were 0.95±0.21 and 2.88±0.49μM for B. gibsoni and B. bovis, respectively. Therefore, our results suggest that MPA may inhibit the replication of Babesia parasites by targeting IMPDH enzyme of the purine pathway., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Trypanosoma evansi is responsible for the most largely distributed animal trypanosomosis, affecting a wide range of wild and domestic animals. Its surveillance requires the implementation of standardized and reliable diagnostic tools. Although the development of polymerase chain reaction (PCR) tools has greatly improved our diagnostic capacity, factors affecting their sensitivity need to be acknowledged and accounted for in the interpretation of results. The targeted gene and the primer design have already been shown to greatly affect the sensitivity of a PCR, and the best-performing sets of primers have been previously identified. However, the sensitivity of the PCR is also largely influenced by the DNA extraction or sample preparation method. In this paper, we selected 6 DNA extraction or blood sample preparation methods representative of what would be used in a budget-constrained setting: phenol-chloroform, Chelex(®), Flexigen (Qiagen(®)) kit, Genekam(®) kit and two original protocols using sodium hydroxide. We studied the effects of the preparation method on the detection limit of the subsequent PCR. Our results show that the extraction method strongly affects the PCR sensitivity. The classical phenol-chloroform extraction method allowed for the PCR with the lowest detection limit. Some combinations of extraction method and primer set had detection limits that were not compatible with their use as a reliable diagnostic test, and would severely reduce the performance of a surveillance program. Therefore, we encourage laboratories to carefully select their sample preparation and PCR protocols, depending on the aimed sensitivity, cost, safety, time requirement and objectives., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Ovine theileriosis is a tick-borne disease that restricts the development of small ruminant husbandry in northern China. In this study, we report on a molecular epidemiological survey of ovine Theileria spp. in 198 blood samples taken from sheep in northern China. The DNA samples were screened by a nested polymerase chain reaction (PCR) targeting the 18S rRNA gene of ovine Theileria spp. The prevalence of ovine Theileria spp. in Yanji, Nongan, Longjing, Toudao and Jinchang was 80%, 40%, 37%, 24% and 32%, respectively. The sequencing analyses approved the present of the T. orientalis and/or T. luwenshuni in these regions. Taken together, we have demonstrated a high incidence of Theileria spp. in northern China that calls for the need to design effective control programs for ovine theileriosis.

A recombinant C-terminal antigen derived from Babesia caballi 48-kDa rhoptry protein (rBc48/CT) was made for the development of a serologically diagnostic test. Antiserum raised against the rBc48/CT reacted specifically with the corresponding native protein by Western blotting and the indirect fluorescent antibody test (IFAT). Next, an indirect enzyme-linked immunosorbent assay (Bc48/CT-ELISA) and an immunochromatographic test based on the Bc48/CT (Bc48/CT-ICT) were constructed and employed for the detection of an antibody to B. caballi in a variety of equine sera. The results of Bc48/CT-ELISA and Bc48/CT-ICT were highly concordant with those of IFAT and ELISA, with full-length protein of Bc48 used as the reference tests. Our results demonstrate the success of Bc48/CT as antigen for the serological diagnosis of B. caballi infection in horses., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Although Babesia bovis and Babesia bigemina infections cause economic losses in the cattle industry in northern Thailand, there is inadequate information on Babesia isolates present in the area. Therefore, to determine the prevalence and genetic relationship between Babesia isolates, we screened 200 blood samples of cattle from Chiang Rai, Chiang Mai, and Lumpang provinces of northern Thailand. A nested polymerase chain reaction using primers targeting B. bovis spherical body protein 2 (BboSBP2) and B. bigemina rhoptry-associated protein 1a (BbiRAP-1a) genes revealed a prevalence of 12 and 21 % for B. bovis and B. bigemina, respectively, while that of mixed infections was 6.5 % samples. The prevalences of B. bovis in Chiang Rai, Chiang Mai, and Lumpang were 9.5, 3.7, and 25.5 %, respectively. For B. bigemina, the prevalences were 15.8, 12.9, and 39.2 % in Chiang Rai, Chiang Mai, and Lumpang, respectively. Mixed infections with B. bovis and B. bigemina were 6.3 % in Chiang Rai, 1.9 % in Chiang Mai, and 13.7 % in Lumpang. The identical sequences of either BboSBP2 gene or BbiRAP-1a gene were shared among the Babesia isolates in the three provinces of northern Thailand. Further analysis using the internal transcribed spacer gene revealed at least four genotypes for B. bovis and five genotypes for B. bigemina in northern Thailand, while the sequences present great genetic diversities in the different isolates. Overall, we have demonstrated a high prevalence and polymorphism of Babesia parasites in northern Thailand calling for the need to design effective control programs for bovine babesiosis.

A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) targeting secreted antigen 1 of Babesia microti (BmSA1) was developed for detection of B. microti infection. The optimized DAS-ELISA was sensitive enough to detect circulating BmSA1 by day 2 post-infection, in sequential sera of a hamster infected with B. microti. This detection was 4 days earlier than antibody detection by indirect ELISA. The kinetics of circulating BmSA1 coincided with the profile of parasitemia. The specificity of this assay was evaluated using sera from animals experimentally infected with different species of Babesia. The DAS-ELISA had a higher sensitivity than the microscopic examination of Giemsa-stained blood smears for detection of the infection in hamsters. Taken together, these results indicated that BmSA1 could be a potential marker for surveillance of human babesiosis., (Copyright © 2011 Elsevier Inc. All rights reserved.)