Background and Aim: Dog blood parasites are important tick-borne diseases causing morbidity and mortality in dogs worldwide. Four dog blood parasites species are commonly found in Thailand: Babesia canis, Hepatozoon canis, Ehrlichia canis, and Anaplasma platys. They are transmitted easily by tick species. However, there is little prevalence data available in Thailand. Diseases presentation of blood parasites infection is similar, but the treatment of each species is different. Current diagnosis mainly relies on microscopic examination of a stained blood smear, which has low sensitivity. Therefore, accurate diagnosis is important. This study aims to evaluate the efficacy of the conventional polymerase chain reaction (PCR) method and routine blood smears in the detection of four blood parasites species in dogs from Buriram Province, Thailand. Materials and Methods: In total, 49 EDTA-blood samples were collected from dogs in Buriram Province, Thailand. Blood parasite infection was compared using the Giemsa-stained blood smear technique to identify the parasite under a 100× oil immersion with PCR amplification of the 18S rDNA gene of B. canis and H. canis and the 16S rDNA gene of E. canis and A. platys. Results: Only one dog out of 49 was positive for H. canis based on microscopic examination whereas the PCR results showed that 2.04% (1/49), 4.08% (2/49), 36.73% (18/49), and 30.61% (15/49) of dogs were positive for B. canis, H. canis, E. canis, and A. platys, respectively. Moreover, coinfection was found in 16.33% (8/49) of dogs. Conclusion: This study is the first report to demonstrate the molecular prevalence of blood parasites in domestic dogs in Buriram Province. The results indicated that the PCR method exhibited much higher sensitivity and reliability for blood parasites diagnosis in dogs. Therefore, our data support serious concern regarding the diagnostic technique used in routine blood testing and also provide prevalence data for the management and control of blood parasites in this area.

Canine monocytic ehrlichiosis caused by Ehrlichia canis infection is a life-threatening vector-borne disease in dogs worldwide. Routine blood smear has very low sensitivity and cannot accurately provide a quantitative result. Conventional PCR (cPCR) and real-time PCR (qPCR) are widely used as molecular methods for E. canis detection. qPCR is quantitative but relies on standard curves of known samples. To overcome this difficulty, this study developed a new E. canis quantitative detection method, using droplet digital polymerase chain reaction (ddPCR). ddPCR was evaluated against cPCR and blood smears. PCR amplicons and genomic DNA (gDNA) from 12 microscopic positive samples were used to identify the limits of detection (LODs) in ddPCR and cPCR. Our ddPCR was assessed in 92 field samples, it was compared with cPCR and blood smears. ddPCR showed LOD = 1.6 copies/reaction, or 78 times more sensitive than cPCR (LOD = 126 copies/reaction), using PCR amplicons as a template, whereas both ddPCR and cPCR had equal LODs at 0.02 ng gDNA/reaction. In addition, ddPCR had 100% sensitivity and 75% specificity for E. canis detection compared to cPCR and no cross-reaction with other blood pathogens was observed. ddPCR identified more positive samples than cPCR and blood smear. ddPCR improved the overall performance of E. canis detection, with a better LOD and comparable sensitivity and specificity to cPCR. The technique might be helpful for diagnosis of E. canis in light infection, evaluating the number of E. canis and follow-up after treatment.

Background and Aim: Toxoplasma gondii is recognized as a zoonosis causing toxoplasmosis in animals globally. Cat is a definitive host of T. gondii and sheds oocyst through feces, which can infect human beings and animals through contaminated food ingestion. A precise diagnostic test is essential to prevent T. gondii infection in both humans and animals. This study aimed to develop and evaluate the pETite- dense granule antigen 7(GRA7)-based indirect enzyme-linked immunosorbent assay (ELISA) to detect T. gondii infection in cats. Materials and Methods: T. gondii-GRA7 was cloned and expressed in the Expresso® small ubiquitin-related modifier (SUMO) T7 Cloning and Expression System. The recombinant pETite-GRA7 was purified using HisTrap affinity chromatography and confirmed using Western blot analysis. The recombinant protein was used to develop and evaluate the indirect ELISA for T. gondii infection detection. In total, 200 cat sera were tested using pETite-GRA7-based indirect ELISA and indirect fluorescent antibody test (IFAT). The statistical analysis based on Kappa value, sensitivity, specificity, positive predictive value, negative predictive value, χ2 test, and receiver operating characteristic (ROC) curve was used to evaluate the performance of the test. Results: A 606 bp GRA7 polymerase chain reaction (PCR) product was obtained from T. gondii RH strain genomic DNA. The gene was cloned into the pETite™ vector and transformed to HI-Control Escherichia coli BL21 (DE3) for protein expression. Approximately 35 kDa of recombinant pETite-GRA7 was observed and Western blot analysis showed positive bands against anti-6-His antibody and positive-T. gondii cat serum. A sample of 0.5 μg/mL of pETite-GRA7 was subjected to indirect ELISA to detect T. gondii infection in the cat sera. The results showed sensitivity and specificity of pETite- GRA7-based indirect ELISA at 72% and 96%, respectively. An acceptable diagnostic performance was characterized by high concordant results (94%) and substantial agreement (Kappa value=0.65) with IFAT. The seroprevalence levels of ELISA and IFAT were 10% and 9%, respectively, and were not significantly (p>0.05) different. The expected performance of ELISA at different cutoff points using the ROC curve analysis revealed 89% sensitivity and 92% specificity at the cutoff value of 0.146, with a high overall assay accuracy (area under the curve=0.94). Conclusion: In this study, the pETite™ vector, N-terminal 6xHis SUMO fusion tag, was used to improve the solubility and expression level of GRA7. The recombinant pETite-GRA7 showed enhanced protein solubility and purification without special condition requirements. This pETite-GRA7-based indirect ELISA showed high concordant results and substantial agreement with IFAT. ELISA revealed an acceptable sensitivity and specificity. These initial data obtained from cats' sera demonstrated that pETite-GRA7-based indirect ELISA could be a useful method for local serological diagnosis of T. gondii infection in cats in Thailand.

Background and Aim: Ehrlichia canis and Anaplasma platys are tick-borne, Gram-negative bacteria that cause canine monocytic ehrlichiosis and canine cyclic thrombocytopenia, respectively. These diseases are of great importance and are distributed globally. This study aimed to create new primers for the identification of E. canis and A. platys in naturally infected dogs using polymerase chain reaction (PCR), DNA sequencing, and phylogenetic analysis using the 16S rDNA and gltA genes. Materials and Methods: In total, 120 blood samples were collected from dogs in three different locations (Saraburi, Buriram, and Nakhon Ratchasima provinces) in Central and Northeast Thailand. The molecular prevalence of E. canis and A. platys was assessed using PCR targeting the 16S rDNA and gltA genes. All positive PCR amplicons were sequenced, and phylogenetic trees were constructed based on the maximum likelihood method. Results: Ehrlichia canis had an overall molecular prevalence of 15.8% based on the 16S rDNA gene, compared to 8.3% based on the gltA gene. In addition, the overall molecular prevalence of A. platys using the 16S rDNA gene was 10.8%, while the prevalence rate was 5.8% using the gltA gene. Coinfection was 0.8% in Saraburi province. The partial sequences of the 16S rDNA and gltA genes of E. canis and A. platys in dogs in Central and Northeast Thailand showed 96.75%–100% identity to reference sequences in GenBank. Phylogenetic analysis of the 16S rDNA and gltA genes revealed that E. canis and A. platys sequences were clearly grouped into their own clades. Conclusion: This study demonstrated the molecular prevalence of E. canis and A. platys in Central and Northeast Thailand. The 16S rDNA and gltA genes were useful for the diagnosis of E. canis and A. platys. Based on the phylogenetic analysis, the partial sequences of the 16S rDNA and gltA genes in E. canis and A. platys were related to prior Thai strains and those from other countries.

Background and Aim: Hemoplasmas are defined as small, epicellular parasitic bacteria that can infect the red blood cells of several mammalian species. Diseases caused by these bacteria range from asymptomatic infections to acute hemolytic anemia. However, data on hemoplasmas in non-human primates in Thailand remain to be limited. Therefore, this study aims to determine the occurrence and genetic diversity of hemoplasmas among long-tailed macaques in Thailand. Materials and Methods: Blood samples were collected from 339 long-tailed macaques in three provinces of Thailand. DNA was then extracted from the blood samples and tested for hemoplasma using broad-range nested polymerase chain reaction (PCR) based on the 16S rRNA gene. PCR-positive samples were sequenced, and phylogenetic analysis for species identification was conducted. Results: In total, 38 (11.2%) out of the 339 samples were found to be positive for hemoplasmas, based on the broad-range nested PCR assay of the 16S rRNA gene. The 16S rRNA sequences of Mycoplasma spp. were highly similar (98-99% identity) to "Candidatus Mycoplasma haemomacaque." Furthermore, phylogenetic analysis using maximum likelihood demonstrated that the sequences were located in the same cluster of "Ca. M. haemomacaque." Conclusion: The detection of hemoplasmas among long-tailed macaques in Thailand is reported. Genetic characterization confirmed that these hemoplasmas are closely related to "Ca. M. haemomacaque." These results indicate that long-tailed macaques in several locations in Thailand may be infected and serve as reservoirs for this parasite.

Background and Aim: Keratoconjunctivitis sicca (KCS) is a chronic inflammatory ocular disease that occurs in many dog breeds worldwide. This study aimed to investigate the tear protein pattern of healthy dogs, KCS dogs, and KCS dogs after treatment with cyclosporine A (CsA). Materials and Methods: Twenty-eight dogs of any breed were enrolled in the study. The subjects were divided into three groups: Healthy, KCS, and CsA-treated dogs. Tear samples were collected using Schirmer strips. Tear proteins extracted from the strips were analyzed using two-dimensional electrophoresis. For the first dimension, total protein from tears was separated by isoelectric focusing. The second dimension was performed using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel images were analyzed and the protein spots of differential expression were manually cut for protein annotation using mass spectrometry. Results: In total, 12 protein spots were excised and subjected to protein identification. Associated with KCS, six protein spots were a downregulated protein, namely, lysozyme. The other six protein spots were upregulated in KCS dogs, consisting of heat shock protein beta-1, protein S100-A12, and keratin type II cytoskeletal 1 and 5. After treatment with CsA for 45 days, the lysozyme protein was still decreasing and the inflammation protein (S100-A12) was not identified. Conclusion: Inflammatory tear proteins and proteins involved in cellular stress were present in KCS dogs and appeared to be reduced in medicated eyes. Treatment with topical CsA in the short term may not improve the activity of antibacterial proteins. Changes in the expression patterns of these four proteins might be useful for disease severity and progression assessment, as well as for exploring a novel method for dry eye management in dogs.