Molecular Detection and Characterization of Hemotropic Mycoplasma in Assamese Macaques (Macaca assamensis) of Northern Thailand.

Hemotropic mycoplasmas, also known as hemoplasmas, are parasitic bacteria that infect red blood cells, potentially leading to varying degrees of anemia across numerous mammalian species, including nonhuman primates. The present study aims to investigate the prevalence of hemoplasma infection and identify the species involved among free-ranging Assamese macaques (Macaca assamensis) inhabiting northern Thailand. A total of 133 blood samples were collected from Assamese macaques in Chiang Rai province, Thailand, and subjected to screening for hemoplasma infection utilizing nested PCR amplification targeting the 16S rRNA gene. Positive samples were subsequently analyzed through nucleotide sequencing and phylogenetic analysis for putative species identification. Current study results revealed that 17.3% (23/133; 95% CI 11.29-24.81) of Assamese macaques tested positive for hemoplasma infection using the nested PCR assay. Partial 16S rRNA sequences derived from hemoplasma isolates in Assamese macaques exhibited 99% homology, forming a cluster within the same phylogenetic clade as "Candidatus Mycoplasma haematomacacae," previously identified in long-tailed macaques, rhesus macaques, and Japanese macaques. These findings suggest the presence of "Ca. M. haematomacacae" not only in long-tailed macaques and rhesus macaques but also in Assamese macaques in Thailand. To our knowledge, this marks the first molecular detection of "Ca. M. haematomacacae" in Assamese macaques in Thailand. These results hold significance as they enhance our understanding of hemoplasma infection distribution among macaque populations in Thailand.

Systematic evaluation of TaqMan real-time polymerase chain reaction assays targeting the dsb and gltA loci of Ehrlichia canis in recombinant plasmids and naturally infected dogs.

Background and Aim: Because of the diversity of local genotypes of Ehrlichia canis, genes targeted by TaqMan real-time polymerase chain reaction (RT-PCR) assays should be systematically evaluated. This study evaluated the amplification efficiency, linearity, precision, and sensitivity of two TaqMan RT-PCR assays targeting the dsb and gltA loci of E. canis in recombinant plasmids and naturally infected dogs. Materials and Methods: Thirty blood samples were collected from dogs showing clinical signs of canine monocytic ehrlichiosis at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand. The dsb and gltA genes were amplified by conventional PCRs (cPCRs) on the blood samples and were then sequenced. Meanwhile, RT-PCR was used to detect dsb and gltA genes in 10-fold dilutions of the recombinant plasmids. Results: Both dsb and gltA were amplified with a high degree of linearity (R2 ≥0.975 and 0.993, respectively) in all dilutions, although the mean percentage of relative standard deviation of gltA was lower, but the difference was non-significant. The detection limits of RT-PCR and cPCR were 10-7 and 10-6, respectively, for both loci. RT-PCR targeting dsb (22/30; 73.3%) and gltA (15/30; 50%) yielded a number of positive results that did not differ significantly (p=0.06). The RT-PCR positive results of the dsb gene (22/30) differed significantly from that of cPCR (11/30) (p=0.004). In contrast, the RT-PCR positive results of the gltA gene (15/30) did not differ significantly from that of cPCR (12/30) (p=0.43). The mean Ct value (30.2) based on dsb RT-PCR of 22 positive cases was higher than that of gltA RT-PCR (Ct=27.4) on 15 positive cases. The Ct values from dsb RT-PCR were >30 in all seven discordant samples that were not detected by the gltA RT-PCR. Conclusion: RT-PCR targeting the dsb gene was more sensitive for detecting E. canis in naturally infected dogs. This study suggested that TaqMan RT-PCR of the dsb gene should be selected for E. canis research in this region.

Molecular detection and phylogeny of Ehrlichia canis and Anaplasma platys in naturally infected dogs in Central and Northeast 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.

Expression of recombinant 35 kDa fragment of VP2 protein of canine parvovirus using Escherichia coli expression system.

BACKGROUND AND AIM: Canine parvovirus (CPV) is one of the most common viral infections in dogs, causing acute hemorrhagic gastroenteritis and high mortality. Vaccination effectively prevents CPV infection. However, the currently available CPV vaccines have concerns such as maternal immunity interference, shedding of virus vaccine, and false-positive result based on polymerase chain reaction after vaccination. A subunit vaccine can overcome these problems. This study aimed to express the recombinant 35 kDa fragment of the VP2 protein (consisting of epitopes 1-7) and the recombinant full-length VP2 protein (consisting of epitopes 1-10) and to study the ability of these two recombinant proteins to react with rabbit anti-CPV polyclonal antibodies. MATERIALS AND METHODS: The full length and 35 kDa fragment of VP2 gene of CPV were cloned into the pBAD202 Directional TOPO™ expression vector and expressed in E. coli. The recombinant full-length and the recombinant 35 kDa fragment proteins of VP2 were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. RESULTS: The recombinant full-length and the recombinant 35 kDa fragment VP2 genes were successfully cloned and expressed. The optimum concentrations of arabinose and induction time for the recombinant full-length and the recombinant 35 kDa fragment VP2 proteins were 0.2% for 6 h and 0.02% for 6 h, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 protein molecular weights were approximately 81 and 51 kDa, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 proteins specifically interacted with rabbit anti-CPV polyclonal antibodies. CONCLUSION: These results suggest that the recombinant 35 kDa fragment and the recombinant full-length VP2 proteins may be useful in developing a CPV diagnostic test or vaccine.

Molecular identification of Trichuris trichiura and Hymenolepis diminuta in long-tailed macaques (Macaca fascicularis) in Lopburi, Thailand.

BACKGROUND AND AIM: Trichuris trichiura and Hymenolepis diminuta are helminthic intestinal parasites that infect humans and other animals, including non-human primates. However, molecular detection of these parasites remains scarce in long-tailed macaques (Macaca fascicularis), which coexist with human communities in Thailand. Thus, this study aimed to molecularly confirm the occurrence of Trichuris spp. and Hymenolepis spp. infection and determine the species of both parasites that were found in long-tailed macaques. MATERIALS AND METHODS: A total of 200 fecal samples were randomly collected from long-tailed macaques living in Lopburi, Thailand, and tested based on polymerase chain reaction (PCR) assays for Trichuris spp. and Hymenolepis spp. infections. The PCR products were submitted for DNA purification and sequencing. Phylogenetic analysis was performed using the maximum likelihood method. RESULTS: Of 200 tested samples, three (1.5%) were positive for Trichuris spp. Sequence analysis of all positive samples revealed the presence of T. trichiura, while eight samples (8/200, 4%) positive for Hymenolepis spp. were classified as H. diminuta. No significant associations were found between parasite infection and sex of macaques. CONCLUSION: This study revealed that long-tailed macaques harbor T. trichiura and H. diminuta. These results suggested that local residents and tourists must pay attention to limiting contact with long-tailed macaques and take hygienic precautions to reduce the risk of zoonotic and anthroponotic transmission of these parasites between humans and long-tailed macaques.

Detection and genetic characterization of "Candidatus Mycoplasma haemomacaque" infection among long-tailed macaques (Macaca fascicularis) in Thailand using broad-range nested polymerase chain reaction assay.

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.

Molecular Detection of Bartonella quintana among Long-Tailed Macaques (Macaca fascicularis) in Thailand.

Bartonella quintana is a zoonotic pathogen with a worldwide distribution. Humans and non-human primates are considered to be natural reservoir hosts for B. quintana. However, information on the molecular epidemiology of this organism is very limited in regard to long-tailed macaques (Macaca fascicularis) in Thailand. Therefore, this study aimed to investigate the occurrence and genetic diversity of Bartonella spp. among long-tailed macaques in Thailand. In total, 856 blood samples were collected from long-tailed macaques in Thailand. All specimens were screened for Bartonella spp. using a polymerase chain reaction (PCR) assay targeting the 16S rRNA, gltA and ftsZ genes. All positive samples were further analyzed based on nucleotide sequencing, phylogenetic analysis and multiple sequence alignment analysis. Only one macaque showed a positive result in the PCR assays based on the 16S rRNA, gltA and ftsZ genes. Nucleotide sequencing and phylogenetic analysis revealed that the obtained sequences were closely related to B. quintana previously detected in non-human primates. Single-nucleotide polymorphisms (SNPs) were detected in the gltA and ftsZ gene sequences. This study revealed that long-tailed macaques in Thailand carried B. quintana. Despite the low infection rate detected, long-tailed macaques may be a reservoir of B. quintana.

Molecular detection and prevalence of Giardia duodenalis and Cryptosporidium spp. among long-tailed macaques (Macaca fascicularis) in Thailand.

Giardia duodenalis and Cryptosporidium spp. are divergent protozoal intestinal parasites that infect human beings and other animals, including non-human primates. Although long-tailed macaques (Macaca fascicularis) reside in human communities in Thailand, the prevalence of Giardia spp. and Cryptosporidium spp. in these primates has not been previously investigated. The objective of this study was to evaluate long-tailed macaques living near human communities as possible hosts of these intestinal parasites. In 2014, 200 fecal samples were randomly collected from long-tailed macaques living in different areas of Lopburi province, Thailand, and tested with a panel of PCR assays for Giardia spp. and Cryptosporidium spp. G. duodenalis assemblage B was most frequently detected (6%), while assemblage A and an inconclusive assemblage were detected in single samples, for a total G. duodenalis infection rate of 7%. Two samples (1%) tested positive for Cryptosporidium spp., which were both classified as monkey genotypes. No significant associations were found between G. duodenalis infection and sex or location of macaques. This study indicates that long-tailed macaques can carry G. duodenalis and, to a lesser extent, Cryptosporidium spp. monkey genotype. These results warrant education of residents and tourists to limit contact with long-tailed macaques and to take hygienic precautions to mitigate risk of zoonotic and anthroponotic transmission of these parasites between people and macaques.