Satellite telemetry tracks flyways of Asian Openbill storks in relation to H5N1 avian influenza spread and ecological change.

BACKGROUND: Asian Openbills, Anastomus oscitans, have long been known to migrate from South to Southeast Asia for breeding and nesting. In Thailand, the first outbreak of H5N1 highly pathogenic avian influenza (HPAI) infection in the Openbills coincided with the outbreak in the poultry. Therefore, the flyways of Asian Openbills was determined to study their role in the spread of H5N1 HPAI virus to poultry and wild birds, and also within their flocks. RESULTS: Flyways of 5 Openbills from 3 colonies were monitored using Argos satellite transmitters with positioning by Google Earth Programme between 2007 and 2013. None of the Openbills tagged with satellite telemeters moved outside of Thailand. Their home ranges or movement areas varied from 1.6 to 23,608 km2 per month (95% utility distribution). There was no positive result of the viral infection from oral and cloacal swabs of the Openbills and wild birds living in the vicinity by viral isolation and genome detection during 2007 to 2010 whereas the specific antibody was not detected on both Openbills and wild birds by using microneutralization assay after 2008. The movement of these Openbills did not correlate with H5N1 HPAI outbreaks in domestic poultry but correlated with rice crop rotation and populations of the apple snails which are their preferred food. Viral spread within the flocks of Openbills was not detected. CONCLUSIONS: This study showed that Openbills played no role in the spread of H5N1 HPAI virus, which was probably due to the very low prevalence of the virus during the monitoring period. This study revealed the ecological factors that control the life cycle of Asian Openbills.

A LONG-TERM SEROSURVEY OF AVIAN INFLUENZA H5 AMONG WILD BIRDS IN NAKHON SAWAN PROVINCE, THAILAND.

An outbreak of HPAIV H5N1 in Nakhon Sawan province, Thailand, in 2004 caused sporadic deaths of Asian openbill storks ( Anastomus oscitans). An investigation was undertaken to determine if this virus occurs and circulates in wild birds in Nakhon Sawan province. Following the outbreak, a widespread serosurvey was conducted using the hemagglutination inhibition assay and microneutralization assay to detect antibodies against AIV H5. From 2007 to 2014, blood was collected from a total of 753 wild birds, representing 10 orders and 44 species. The results reveal that 10 serum samples were positive for AIV H5 antibodies. These seropositive results, found in the orders Ciconiiformes and Anseriformes, demonstrate that waterfowl serve as a reservoir host of AIV. Moreover, the seroprevalences in streak-eared bulbul showed habitat sharing with waterfowl or duck.

Molecular detection and genetic diversity of bovine Babesia spp., Theileria orientalis, and Anaplasma marginale in beef cattle in Thailand.

Babesia spp., Theileria orientalis, and Anaplasma marginale are significant tick-borne pathogens that affect the health and productivity of cattle in tropical and subtropical areas. In this study, we used PCR to detect the presence of Babesia bovis, Babesia bigemina, and T. orientalis in 279 beef cattle from Western Thailand and A. marginale in 608 beef cattle from the north, northeastern, and western regions. The PCRs were performed using species-specific primers based on the B. bovis spherical body protein 2 (BboSBP2), B. bigemina rhoptry-associated protein 1a (BbiRAP-1a), T. orientalis major piroplasm surface protein (ToMPSP), and A. marginale major surface protein 4 (AmMSP4) genes. To determine the genetic diversity of the above parasites, amplicons of B. bovis and B. bigemina ITS1-5.8s rRNA gene-ITS2 regions (B. bovis ITS, B. bigemina ITS), ToMPSP, and AmMSP4 genes were sequenced for phylogenetic analysis. PCR results revealed that the prevalence of B. bovis, B. bigemina, T. orientalis, and A. marginale in the Western region was 11.1, 12.5, 7.8, and 39.1 %, respectively. Coinfections of two or three parasites were observed in 17.9 % of the animals sampled. The study revealed that the prevalence of A. marginale in the western region was higher than in the north and northeastern regions (7 %). Sequence analysis showed the BboSBP2 gene to be more conserved than B. bovis ITS in the different isolates and, similarly, the BbiRAP-1a was more conserved than B. bigemina ITS. In the phylogenetic analysis, T. orientalis MPSP sequences were classified into types 3, 5, and 7 as previously reported. A. marginale MSP4 gene sequences shared high identity and similarity with each other and clustered with isolates from other countries. This study provides information on the prevalence and genetic diversity of tick-borne pathogens in beef cattle and highlights the need for effective strategies to control these pathogens in Thailand.

Molecular study of feline hemoplasmas in free- ranging fishing cats (Prionailurus viverrinus) in Thailand.

Feline hemoplasmas, consisting of Mycoplasma haemofelis (M. haemofelis), Candidatus Mycoplasma haemominutum (Ca. M. haemominutum), and Candidatus Mycoplasma turicensis (Ca. M. turicensis), cause feline infectious anemia and zoonoses. Using multiplex PCR and phylogenetic analysis based on 16S rRNA, 22 blood samples from fishing cats (Prionailurus viverrinus) living in Khao Sam Roi Yot National Park, Thailand were determined positive for M. haemofelis (13.6%)and Ca. M. haemominutum (22.7%). M. haemofelis and Ca. M. haemominutum infection can result severe anemia and asymptomatic, respectively. However, not all positive cases exhibit anemia symptoms. Future study of hemoplasma infection in wild felids is necessary for conservation and the preservation of public health in Thailand.

Molecular evidence for genetic distinctions between Chlamydiaceae detected in Siamese crocodiles (Crocodylus siamensis) and known Chlamydiaceae species.

Chlamydiosis, caused by Chlamydiaceae, is a zoonotic disease found in humans and several species of animals, including reptiles and amphibians. Although chlamydiosis in saltwater crocodiles has been previously reported in South Africa and Papua New Guinea, the reported strains have not been identified or confirmed. Therefore, the main aim of this study was to sequence and characterize Chamydiaceae isolated from Siamese crocodiles. Results showed the 16S ribosomal (r) RNA and the 16S/23S rRNA gene of the crocodile isolates were closely related to the genus Chlamydophila with matched identity greater than 98%. The phylogenetic tree constructed from the 16S/23S rRNA gene showed the crocodile cluster diverges far from Cp. caviae with a 100% bootstrap value. The tree based on the ompA gene loci distinguished the crocodile strains into genotypes I, II, and III. The present study is the first report on Chlamydophila detected in Siamese crocodiles that is genetically distinct from the known species of Chlamydiaceae.

Susceptibility of openbill storks (Anastomius oscitans) to highly pathogenic avian influenza virus subtype H5N1.

This investigation detailed the clinical disease, gross and histologic lesions in juvenile openbill storks (Anastomus oscitans) intranasally inoculated with an avian influenza virus, A/chicken/Thailand/vsmu-3 (H5N1), which is highly pathogenic for chickens. High morbidity and mortality were observed in openbill storks inoculated with HPAI H5N1 virus. Gross lesions from infected birds were congestion and brain hemorrhage (10/20), pericardial effusions, pericarditis and focal necrosis of the cardiac muscle (2/20), pulmonary edema and pulmonary necrosis, serosanguineous fluid in the bronchis (16/20), liver congestion (6/20), bursitis (5/20), subcutaneous hemorrhages (2/20) and pinpoint proventiculus hemorrhage (2/20). Real time RT-PCR demonstrated the presence of viral RNA in organs associated with the lesions: brain, trachea, lungs, liver, spleen and intestines. Similar to viral genome detection, virus was also isolated from these vital organs. Antibodies to influenza virus detected with a hemagglutination inhibition test, were found only in the openbill storks who died 8 days post-inoculation.

Distribution and abundance of Stomoxyini flies (Diptera: Muscidae) in Thailand.

Stomoxyini flies (Diptera: Muscidae) include species of parasitic flies of medical and veterinary importance. The adult flies feed on the blood of mammals and may transmit several parasites and pathogens. We conducted an entomological survey of Stomoxyini flies from different sites in Thailand. Stomoxyini flies were collected at four major types of sites: zoos, livestock farms, wildlife conservation areas and a national park using vavoua traps between November 2010 and April 2011. A total of 3,314 Stomoxyini flies belonging to the genera Stomoxys, Haematobosca, Haematostoma and Haematobia were collected. Eight species were identified: S. calcitrans (46.6%), S. uruma (26.8%), S. pulla (4.3%), S. indicus (0.7%), S. sitiens (0.1%), H. sanguinolenta (11.2 %), H. austeni (0.5%) and H. irritans exigua (9.8%). The diversity of Stomoxyini flies in the livestock farms was higher than the other sites. Altitude correlated with the number of flies. This study provides information that may be useful for Stomoxyini flies control.

Detection and phylogenetic characterization of hepatitis E virus genotype 3 in a captive wild boar in Thailand.

Hepatitis E virus (HEV) was studied in different types of wild boar captive settings in Thailand, including a wildlife breeding research station, zoo, and commercial wild boar farm, which were located in different locations of Thailand. Fifty-one fecal samples were collected and screened for HEV RNA and then analyzed. One sample obtained from a wildlife breeding research station in Ratchaburi province was HEV positive. Phylogenetic characterization revealed that the virus was HEV genotype 3 and belongs to subgroup 3e, which is closely related to HEV recently isolated from domestic pigs and humans in the country. It was hypothesized that HEV is shared among wild boars, domestic pigs, and humans in Thailand.

Immobilization of captive plains zebras (Equus quagga) with a combination of etorphine hydrochloride, acepromazine, and xylazine hydrochloride.

The plains zebra (Equus quagga) is a zebra species commonly kept in zoos around the world. However, they are not tame like their domestic relatives and are difficult to immobilize. We immobilized 30 captive plains zebra with a combination of etorphine hydrochloride (2-4 mg), acepromazine (8 mg), and xylazine hydrochloride (30 or 50 mg) to perform physical examination and blood sample collection for disease diagnostics. Physiological parameters including heart rate, respiratory rate, body temperature, and hemoglobin oxygen saturation were recorded. All zebras exhibited satisfactory anesthesia and fully recovered without re-narcotization. The results suggest that etorphine hydrochloride-acepromazine-xylazine hydrochloride combination for plains zebra immobilization is a safe and sufficient regimen for short procedures such as wellness examinations and sample collection.

Occurrence of ectoparasites on rodents in Sukhothai Province, northern Thailand.

A survey of ectoparasites on rodents was carried out bimonthly from April 2008 to March 2009 in 3 districts of Sukhothai Province, northern Thailand. A total of 130 rodents comprising 8 species of hosts were captured and examined for ectoparasites. The hosts examined were Bandicota indica, Bandicota savilei, Rattus losea, Rattus rattus, Rattus exulans, Rattus norvegicus, Menetes berdmorei and Tamiops mcclellandii. Ninety-seven ectoparasites were collected: 1 species of tick (Hemaphysalis bandicota), 2 species of mites (Laelaps nuttali and Laelaps echidninus), and 1 species of flea (Xenopsylla cheopis) were identified. The infestation rates by ticks, mites and fleas on the rodents were 0.77, 5.38 and 6.15%, respectively. Monitoring the rodent population and their ectoparasites is important for future planning of prevention and control of zoonotic diseases in the area.

Ectoparasitic fauna of birds, and volant and non-volant small mammals captured at Srinakarin Dam, Kanchanaburi, Thailand.

The investigation of ectoparasitic fauna on birds, and volant and nonvolant small mammals at Srinakarin Dam, Kanchanaburi Province, Thailand was carried out under a national biodiversity and disease surveillance program for four consecutive months: January, February, May and June 2009. A total of 122 animals, comprised of 15 species of birds, 9 species of volant small mammals and 8 species of non-volant small mammals were examined for ectoparasite infestation. Of these animals, 1 genus of hard ticks (Ixodidae), 2 species of mesostigmatid mites (Laelapidae), 4 genera in three families of astigmatid mites (Proctophyllodidae, Pteronyssidae and Trouessartiidae), 4 species in three families of lice (Philopteridae, Polyplacidae and Trichodectidae) and 2 families of batflies (Nycteribiidae and Streblidae) were collected. This is the first survey conducted to determine ectoparasites infesting birds and small mammals living in the reserved forest of Srinakarin Dam, Thailand. A lower infestation rate of ectoparasites was observed in mammals, ranging from 3.5% to 10.3% than birds, with infestation rates between 7.3% and 34.2%. No major potential health risks to people who lived in this area were found.

Antimicrobial Resistance in Swine Fecal Specimens Across Different Farm Management Systems.

Antimicrobial use in agricultural animals is known to be associated with increases in antimicrobial resistance. Most prior studies have utilized culture and susceptibility testing of select organisms to document these phenomena. In this study we aimed to detect 66 antimicrobial resistance (AMR) genes for 10 antimicrobial agent classes directly in swine fecal samples using our previously developed antimicrobial resistance TaqMan array card (AMR-TAC) across three different swine farm management systems. This included 38 extensive antimicrobial use (both in treatment and feed), 30 limited antimicrobial use (treatment only), and 30 no antimicrobial use farms. The number of resistance genes detected in extensive antimicrobial use farms was higher than in limited and no antimicrobial use farms (28.2 genes ± 4.2 vs. 24.0 genes ± 4.1 and 22.8 genes ± 3.6, respectively, p < 0.05). A principal component analysis and hierarchical clustering of the AMR gene data showed the extensive use farm samples were disparate from the limited and no antimicrobial use farms. The prevalence of resistance genes in extensive use farms was significantly higher than the other farm categories for 18 resistance genes including bla SHV, bla CTX-M1 group, bla CTX-M9 group, bla VEB, bla CMY2-LAT, aac(6')-lb-cr, qnrB1, gyrA83L-E. coli, armA, rmtB, aac(3)-IIa, mphA, 23S rRNA 2075G-Campylobacter spp., mcr-1, catA1, floR, dfrA5-14, and dfrA17. These genotypic findings were supported by phenotypic susceptibility results on fecal E. coli isolates. To examine the timing of AMR gene abundance in swine farms, we also performed a longitudinal study in pigs. The results showed that AMR prevalence occurred both early, presumably from mothers, as well as after weaning, presumably from the environment. In summary, detection of AMR genes directly in fecal samples can be used to qualitatively and quantitatively monitor AMR in swine farms.

Comparison of outbreaks of H5N1 highly pathogenic avian influenza in wild birds and poultry in Thailand.

Wild bird surveillance for highly pathogenic avian influenza (HPAI) H5N1 virus from 2004 to 2007 in Thailand indicated that the prevalence of infection with avian influenza H5N1 virus in wild birds was low (1.0%, 95% confidence interval [CI]: 0.7-1.2, 60/6,263 pooled samples). However, the annual prevalence varied considerably over this period, with a peak of 2.7% (95% CI: 1.4, 4.1) in 2004. Prevalence dropped to 0.5% (95% CI: 0.3, 0.8]) and 0.6% (95% CI: 0.3, 1.0) in 2005 and 2006, respectively, and then increased to 1.8% (95% CI: 1.0, 2.6) in 2007. During this period, 16 species from 12 families of wild birds tested positive for H5N1 virus infection. All samples from juvenile birds were negative for H5N1 virus, whereas 0.6% (95% CI: 0.4, 0.9) of pooled samples from adult birds were positive. Most positive samples originated from peridomestic resident species. Infected wild bird samples were only found in provinces where poultry outbreaks had occurred. Detection of H5N1 virus infection in wild birds was reported up to 3 yr after eradication of the poultry outbreaks in those provinces. As observed with outbreaks in poultry, the frequencies of H5N1 outbreaks in wild birds were significantly higher in winter. Further understanding of the mechanisms of persistence and ongoing HPAI H5N1 transmission between wild birds and domestic poultry is needed.

A survey of ectoparasitic arthropods on domestic animals in Tak Province, Thailand.

In July 2008 a survey of ectoparasites on domestic animals was conducted in the Royal Thai Army areas of operation along the Thai-Myanmar Border, Tak Province, Thailand. Eleven different ectoparasites were collected: two species of hard ticks (Ixodidae), three species of fleas (Siphonaptera) and 6 species of sucking or chewing lice (2 species each in the suborders Anoplura, Ischnocera and Amblycera) were collected. Domestic dogs (Canis lupusfamiliaris) (n = 94) were found infested with 2 species of flea Ctenocephalides felis orientis (86.2%) and Echidnophaga gallinacea (1.1%), one species of tick, Rhipicephalus sanguineus (21.3%), and one louse species, Heterodoxus spiniger (7.4%). Domestic cats (Felis catus) (n = 6) were found infested with only flea species, Ctenocephalides felis felis (100%) and E. gallinacea (33.3%). Cattle (Bos indicus) (n = 11) had Rhipicephalus (Boophilus) microplus (72.7%), Linognathus vituli (27.3%), and Solenopotes capillatus (9.1%) present, while chickens (Gallus domesticus) (n = 10) had infestations with E. gallinacea (20%), and 3 lice species, Lipeurus caponis (10%), Goniodes dissimilis (10%) and Menopon gallinae (60%). This is believed to be the first report of S. capillatus collected in Thailand.

Genotypic antimicrobial resistance assays for use on E. coli isolates and stool specimens.

Antimicrobial resistance (AMR) is an emerging public health problem and methods for surveillance are needed. We designed 85 sequence-specific PCR reactions to detect 79 genes or mutations associated with resistance across 10 major antimicrobial classes, with a focus on E. coli. The 85 qPCR assays demonstrated >99.9% concordance with sequencing. We evaluated the correlation between genotypic resistance markers and phenotypic susceptibility results on 239 E. coli isolates. Both sensitivity and specificity exceeded 90% for ampicillin, ceftriaxone, cefepime, imipenem, ciprofloxacin, azithromycin, gentamicin, amikacin, trimethoprim/sulfamethoxazole, tetracycline, and chloramphenicol phenotypic susceptibility results. We then evaluated the assays on direct stool specimens and observed a sensitivity of 97% ± 5 but, as expected, a lower specificity of 75% ± 31 versus the genotype of the E. coli cultured from stool. Finally, the assays were incorporated into a convenient TaqMan Array Card (TAC) format. These assays may be useful for tracking AMR in E. coli isolates or directly in stool for targeted testing of the fecal antibiotic resistome.

Molecular epidemiological analysis of highly pathogenic avian influenza H5N1 subtype isolated from poultry and wild bird in Thailand.

A comprehensive molecular epidemiological analysis was performed on highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype derived from poultry and wild bird during 2004-2007 in Thailand. Sequence analysis followed by phylogenetic analysis was applied to all eight segments of the viruses. Viruses belonging to clades 1 and 2.3.4 in the HA phylogenetic tree have been shown to circulate in Thailand. Our analysis revealed differential evolution of the HPAI viruses among clade 1 strains. Isolates from Phichit province in 2006 resided in two distinct branches, designated 1.p1 and 1.p2. A hemagglutination inhibition test with a panel of monoclonal antibodies demonstrated a possible antigenic drift between the Phichit isolates. Involvement of free-grazing duck practice in the area was discussed as a cause of the differential evolution among the Phichit isolates. A branch, designated 1-TGWB and consisting exclusively of isolates from zoological tigers and wild birds, was evident in all phylogenetic trees constructed in the study. The branch's existence indicated that the HPAI viruses could have been maintained in the wild bird population for a certain period, although no involvement of wild birds in HPAI transmission to poultry was evident in this study.

Serosurveillance for pandemic influenza A (H1N1) 2009 virus infection in domestic elephants, Thailand.

The present study conducted serosurveillance for the presence of antibody to pandemic influenza A (H1N1) 2009 virus (H1N1pdm virus) in archival serum samples collected between 2009 and 2013 from 317 domestic elephants living in 19 provinces situated in various parts of Thailand. To obtain the most accurate data, hemagglutination-inhibition (HI) assay was employed as the screening test; and sera with HI antibody titers ≥20 were further confirmed by other methods, including cytopathic effect/hemagglutination based-microneutralization (microNT) and Western blot (WB) assays using H1N1pdm matrix 1 (M1) or hemagglutinin (HA) recombinant protein as the test antigen. Conclusively, the appropriate assays using HI in conjunction with WB assays for HA antibody revealed an overall seropositive rate of 8.5% (27 of 317). The prevalence of antibody to H1N1pdm virus was 2% (4/172) in 2009, 32% (17/53) in 2010, 9% (2/22) in 2011, 12% (1/8) in 2012, and 5% (3/62) in 2013. Notably, these positive serum samples were collected from elephants living in 7 tourist provinces of Thailand. The highest seropositive rate was obtained from elephants in Phuket, a popular tourist beach city. Young elephants had higher seropositive rate than older elephants. The source of H1N1pdm viral infection in these elephants was not explored, but most likely came from close contact with the infected mahouts or from the infected tourists who engaged in activities such as elephant riding and feeding. Nevertheless, it could not be excluded that elephant-to-elephant transmission did occur.

Investigation on predominant Leptospira serovars and its distribution in humans and livestock in Thailand, 2010-2015.

BACKGROUND: Leptospirosis is a worldwide zoonotic bacterial disease caused by infection with leptospires. Leptospirosis in humans and livestock is an endemic and epidemic disease in Thailand. Livestock may act as reservoirs for leptospires and source for human infection. METHODOLOGY/PRINCIPAL FINDINGS: Data on leptospirosis infection in humans and livestock (Buffaloes, Cattle, and Pigs) species during 2010 to 2015 were analyzed. Serum samples were examined using Microscopic Agglutination Test (MAT) to identify antibodies against Leptospira serovars using a cut-off titer ≥ 1:100. The seroprevalence was 23.7% in humans, 24.8% in buffaloes, 28.1% in cattle, and 11.3% in pigs. Region specific prevalence among humans and livestock was found in a wide range. The most predominant serovars were Shermani, followed by Bratislava, Panama, and Sejroe in human, Shermani, Ranarum, and Tarassovi in buffaloes, and Shermani and Ranarum in cattle and pigs. Equally highest MAT titers against multiple serovars per one sample were found mainly in buffaloes and cattle showing equally titers against Ranarum and Shermani. The correlations of distribution of serovars across Thailand's regions were found to be similar in pattern for cattle but not for buffaloes. In humans, the serovar distribution in the south differed from other regions. By logistic regression, the results indicated that livestock is more susceptible to infection by serovar Shermani when compared to humans. CONCLUSIONS/SIGNIFICANCE: This study gives a detailed picture of the predominance of Leptospira serovars in relation to region, humans and typical livestock. The broad spatial distribution of seroprevalence was analyzed across and within species as well as regions in Thailand. Our finding may guide public health policy makers to implement appropriate control measures and help to reduce the impact of leptospirosis in Thailand.

Pathogenicity of Genetically Similar, H5N1 Highly Pathogenic Avian Influenza Virus Strains in Chicken and the Differences in Sensitivity among Different Chicken Breeds.

Differences in the pathogenicity of genetically closely related H5N1 highly pathogenic avian influenza viruses (HPAIVs) were evaluated in White Leghorn chickens. These viruses varied in the clinical symptoms they induced, including lethality, virus shedding, and replication in host tissues. A comparison of the host responses in the lung, brain, and spleen suggested that the differences in viral replication efficiency were related to the host cytokine response at the early phase of infection, especially variations in the proinflammatory cytokine IL-6. Based on these findings, we inoculated the virus that showed the mildest pathogenicity among the five tested, A/pigeon/Thailand/VSMU-7-NPT/2004, into four breeds of Thai indigenous chicken, Phadu-Hung-Dang (PHD), Chee, Dang, and Luang-Hung-Khao (LHK), to explore effects of genetic background on host response. Among these breeds, Chee, Dang, and LHK showed significantly longer survival times than White Leghorns. Virus shedding from dead Thai indigenous chickens was significantly lower than that from White Leghorns. Although polymorphisms were observed in the Mx and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV.

Molecular epidemiology of bovine Babesia spp. and Theileria orientalis parasites in beef cattle from northern and northeastern Thailand.

Beef cattle production represents the largest cattle population in Thailand. Their productivity is constrained by tick-borne diseases such as babesiosis and theileriosis. In this study, we determined the prevalence of Babesia bigemina, Babesia bovis and Theileria orientalis using polymerase chain reaction (PCR). The genetic markers that were used for detection of the above parasites were sequenced to determine identities and similarity for Babesia spp. and genetic diversity of T. orientalis. Furthermore the risk factors for the occurrence of the above protozoan parasites in beef cattle from northern and northeastern parts of Thailand were assessed. A total of 329 blood samples were collected from beef cattle in 6 provinces. The study revealed that T. orientalis was the most prevalent (30.1%) parasite in beef cattle followed by B. bigemina (13.1%) and B. bovis (5.5%). Overall, 78.7% of the cattle screened were infected with at least one of the above parasites. Co-infection with Babesia spp. and T. orientalis was 30.1%. B. bigemina and T. orientalis were the most prevalent (15.1%) co-infection although triple infection with the three parasites was observed in 3.0% of the samples. Sequencing analysis revealed that B. bigemina RAP1 gene and B. bovis SBP2 gene were conserved among the parasites from different cattle samples. Phylogenetic analysis showed that the T. orientalis MPSP gene from parasites isolated from cattle in north and northeast Thailand was classified into types 5 and 7 as reported previously. Lack of tick control program was the universal risk factor of the occurrence of Babesia spp. and T. orientalis infection in beef cattle in northern and northeastern Thailand. We therefore recommend training of farmers on appropriate tick control strategies and further research on potential vectors for T. orientalis and elucidate the effect of co-infection with Babesia spp. on the pathogenicity of T. orientalis infection on beef in northern and northeastern Thailand.