Serological detection of Rickettsia spp. and evaluation of blood parameters in pet dogs and cats from Bangkok and neighboring provinces.

Rickettsiosis is caused by Orientia spp. and Rickettsia spp., arthropod-borne zoonotic intracellular bacteria. The close relationships between pet dogs, cats and owners increase the risk of rickettsial transmission, with limited studies on the seroprevalence in pets. This study investigated the prevalence of rickettsia exposure among dogs and cats in Bangkok and neighboring provinces. The samples from 367 dogs and 187 cats used in this study were leftover serum samples from routine laboratory testing stored at the Veterinary Teaching Hospital. In-house Enzyme-linked immunosorbent assay (ELISA) tests included IgG against the scrub typhus group (STG), typhus group (TG), and spotted fever group (SFG). The seroprevalence in pet dogs was 30.25% (111/367), including 21.53% for STG, 4.36% for TG, and 1.09% for SFG. Co-seroprevalence consisted of 2.72% for STG and TG, 0.27% for STG and SFG, and 0.27% for pangroup infection. The prevalence in cats was 62.56% (117/187), including 28.34% for STG, 4.28% for TG, and 6.42% for STG. Co-seroprevalence in cats consisted of STG and TG (4.28%), STG and SFG (5.35%), TG and SFG (3.21%), and three-group infection (10.69%). No significant difference in seroprevalence for the three serogroups was observed in any of the 64 districts sampled. The mean hematocrit level significantly decreased in seropositive dogs (P<0.05). Seropositive dogs and cats were detected in significantly greater numbers of anemia cases than nonanemia cases (P<0.05) (odds ratio: 7.93, 0.44, p = 0.00, p = 0.01). A significantly higher number of seropositive cats had decreased hemoglobin levels (P<0.05) (odds ratio: 3.63, p = 0.00). The seropositive samples significantly differed among older cats (P<0.05). These high exposures in pet dogs and cats could constitute important relationship dynamics between companion animals and rickettsial vectors. Significantly decreased hematocrit and hemoglobin levels indicated anemia in the exposed dogs and cats. The study findings will raise awareness of this neglected disease among pet owners and veterinary hospital personnel and aid in future public health preventative planning.

Bacterial DNA and osteoarthritis in dogs with patellar luxation and cranial cruciate ligament rupture.

Background and Aim: The association between bacterial DNA in stifle joints, including those with cranial cruciate ligament rupture (CCLR) and medial patellar luxation (MPL), and osteoarthritis in dogs remains elusive. This study investigated the potential association between the detection of bacterial DNA and osteoarthritis in dogs using a broad-range polymerase chain reaction technique targeting the 16S ribosomal RNA gene. Materials and Methods: Synovial fluid (35 samples) and knee tissue samples (32 samples) were obtained from 35 dogs diagnosed with CCLR (n = 20; 11 males and nine females) or MPL (n = 15; five males and 10 females) who underwent a surgical operation between October 2014 and April 2015. Results: Dogs with CCLR had a higher average osteoarthritis score than those with MPL (2.0 ± 0.9 vs. 0.5 ± 0.9; p = 0.005). Bacterial DNA was detected in the stifle joints of 60.71% of dogs with MPL. Pelomonas spp. (25.00%), Halomonas spp. (17.86%), and 5 other species (17.86%) were the most frequently identified bacteria. Bacterial DNA was detected in 41.03% of dogs with CCLR. Pelomonas spp. (15.38%), Sphingomonas spp. (10.26%), Halomonas spp. (5.13%), and 4 other species (10.26%) were the most frequently identified bacteria. No significant difference was observed in the prevalence of bacterial DNA obtained from tissue samples (46.88%) or joint fluid samples (51.43%). The presence of bacterial DNA was not associated with the type of knee injury (MPL or CCLR; p = 1.000). There was a higher prevalence of bacterial DNA in samples from dogs with moderate-to-severe osteoarthritis (94.44%) than in those with minimal osteoarthritis (41.18%), and a significant association between the presence of bacterial DNA and moderate-to-severe osteoarthritis was identified (p < 0.01). Conclusion: Dogs with moderate-to-severe osteoarthritis were more likely to have bacterial DNA in their stifle joints than those with no or minimal osteoarthritis. These findings provide valuable insight into the potential role of bacterial DNA in joint tissue or joint fluid and the development of osteoarthritis in dogs.

Characterization and Homology Modeling of Catalytically Active Recombinant PhaCAp Protein from Arthrospira platensis.

Polyhydroxybutyrate (PHB) is a biocompatible and biodegradable polymer that has the potential to replace fossil-derived polymers. The enzymes involved in the biosynthesis of PHB are ß-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaC in Arthrospira platensis is the key enzyme for PHB production. In this study, the recombinant E. cloni®10G cells harboring A. platensis phaC (rPhaCAp) was constructed. The overexpressed and purified rPhaCAp with a predicted molecular mass of 69 kDa exhibited Vmax, Km, and kcat values of 24.5 ± 2 µmol/min/mg, 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. The catalytically active rPhaCAp was a homodimer. The three-dimensional structural model for the asymmetric PhaCAp homodimer was constructed based on Chromobacterium sp. USM2 PhaC (PhaCCs). The obtained model of PhaCAp revealed that the overall fold of one monomer was in the closed, catalytically inactive conformation whereas the other monomer was in the catalytically active, open conformation. In the active conformation, the catalytic triad residues (Cys151-Asp310-His339) were involved in the binding of substrate 3HB-CoA and the CAP domain of PhaCAp involved in the dimerization.

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.

Development and evaluation of indirect enzyme-linked immunosorbent assay using recombinant dense granule antigen 7 protein for the detection of Toxoplasma gondii infection in cats in Thailand.

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, χ 2 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.

Antibacterial Activity of Solanum torvum Leaf Extract and Its Synergistic Effect with Oxacillin against Methicillin-Resistant Staphyloccoci Isolated from Dogs.

Methicillin-resistant staphylococci (MRS) have been considered a veterinary and public health threat that needs to be addressed, as they are known to cause serious infections, with limited therapeutic options. Thus, in this study, we aimed to examine the potential antibacterial activity of the leaf extract of Solanum torvum against MRS isolated from clinically healthy dogs. In total, seven mecA-positive Staphylococcus isolates tested in this study were identified using 16S rRNA gene sequencing, and all of them were classified as multidrug-resistant using disk diffusion tests. According to gas chromatography-mass spectrometry analysis, the main phytochemical components found in the leaf extract were hexadecanoic acid and its ethyl ester and 9,12,15-octadecatrienoic acid, ethyl ester, (Z,Z,Z). The minimum inhibitory concentration (MIC) breakpoints for the leaf extract against all tested isolates ranged from 2 to 16 mg/mL, while the MIC breakpoints for oxacillin were from 2 to 512 mg/L. Although varying effects were found, the positive effects of the leaf extract were most evident in combination with oxacillin. These results suggested that S. torvum leaf extract may complement classical antibiotics and may potentially drive the development of an effective therapeutic option for MRS.

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.

Investigation of hemotropic Mycoplasma spp. genotypes in client-owned cats in Thailand.

The genetic information for three feline hemoplasmas is limited in Southeast Asia. According to the limited genetic data, this study modified a nested-PCR method targeting the 16S rRNA gene by designing a novel primary forward degenerate primer. Two hundred and thirty-one archived DNA extracts from the blood of client-owned cats with a variety of diseases were used. The modified nested PCR detected feline hemoplasma DNA in 64 of 231 (27.7 %) samples. Sanger DNA sequencing, BLAST, and phylogenetic analyses revealed nine nucleotide sequences of Mycoplasma haemofelis (Mhf) (3.9 %, 9/231), fifty-three nucleotide sequences of Candidatus Mycoplasma haemominutum (CMhm) (22.94 %, 53/231) and two nucleotide sequences of Candidatus Mycoplasma turicensis (CMtc) (0.86 %, 2/231). The phylogenetic analysis demonstrated separate genotypes of 30 DNA sequences of Thai CMhm. In addition, this analysis elucidated distinct genotypes of CMhm in Thai fishing cats (Prionailurus viverrinus). The domestic cat and Thai fishing cat groups were the two major groups separating Thai CMhm genotypes based on the 16S rRNA. One CMhm sequence in Thai fishing cats was also present in domestic cat CMhm genotypes. This result suggests that transmission of CMhm between domestic cats and Thai fishing cats has likely occurred. One Mhf sequence had low genetic identity (82 % similarity). The phylogenetic analysis confirmed that this sequence was still very closely related to Mhf reference sequences. This Mhf-like genotype could be a candidate novel Mhf genotype. This new genetic information for feline hemotropic Mycoplasma provides valuable information for future feline-related clinical studies.

Detection of specific IgM and IgG antibodies in acute canine monocytic ehrlichiosis that recognize recombinant gp36 antigens.

The efficacy of antibody detection tools for all stages of Ehrlichia canis infections and for various genotypes remains unclear. We produced recombinant gp36 (rgp36) antigens from different isolates of Thai E. canis to confirm the immunoreactivities to these recombinant proteins from naturally infected dogs. Sera and blood samples were taken from 21 dogs naturally infected with E. canis and in the clinical stages of acute phase ehrlichiosis. The expression vectors and competent E. coli produced two isolates of rgp36. These two major rgp36s were recognized by the dogs' sera in Western blotting, with both anti-dog IgM and IgG used as secondary antibodies. The two different genotypes of these local recombinant immunoreactive proteins were gp36 subgroup A (isolate 1055) and subgroup B (isolate 533). The Western blot analyses successfully identified both specific IgM and IgG from the dogs' sera. Of all 21 cases, five dogs presented specific IgM, twenty dogs presented specific IgG, and the commercial test used found fifteen seropositive dogs. There were four dogs that presented both specific IgM and IgG. Only one dog presented specific IgM only. This report is the first identification of a specific IgM in dogs in response to acute infections with E. canis. The recombinant gp36 isolates may be useful as potential antigenic material for subsequent serological tests that have a high possibility for differentiating between acute, chronic, primary, and nonprimary infections with E. canis.

Molecular detection and genetic diversity of Anaplasma marginale based on the major surface protein genes in Thailand.

Anaplasma marginale is the rickettsial agent of anaplasmosis, a tick-borne disease, which affects cattle and other ruminants in tropical and subtropical areas of the world, and causing huge economic losses because of decreasing meat and milk production. In the present study, molecular methods have been used to determine the occurrence and genetic diversity of A. marginale, based on the genes encoding the major surface proteins (msps) genes, in blood samples from 520 cattle and 121 buffaloes in the north and northeastern regions of Thailand. The polymerase chain reaction (PCR) results based on the msp4 gene indicated that 66 (10.30%) cattle were positive for A. marginale, whereas no positive result was obtained from buffaloes. The phylogenetic analysis based on the maximum likelihood method using 13, 29 and 27 nucleotide sequences from msp2, msp4, msp5 clones, respectively, revealed that the sequences detected in this study are obviously distributed in different clusters. The sequence analysis demonstrated that msp2 gene is genetically diverse, while msp4 and msp5 genes are conserved in Thailand. These findings corroborated the diversity analysis of the same sequences, which showed 13, 27 and 27 haplotypes of the msp2, msp4 and msp5 genes, respectively. In addition, the entropy analyses of amino acid sequences exhibited 127, 75 and 51 high entropy peaks with values ranging from 0.27119 to 2.45831, from 0.14999 to 2.17552 and from 0.15841 to 1.05453 for MSP2, MSP4 and MSP5, respectively. Therefore, the results indicate a low molecular occurrence of A. marginale in cattle blood samples in Thailand. From these results; however, a high degree of genetic diversity was observed in the analyzed A. marginale population. Hence, our finding could be used to improve the immunodiagnostics and vaccination programs for anaplasmosis.

Dynamic and structural insights into tick serpin from Ixodes ricinus.

Ixodid ticks have a crucial impact on people and domestic animals worldwide. These parasites also pose a serious threat to livestock. To date, vaccination of hosts against ticks is a safer, more sustainable alternative to chemical control of ticks and the disease agents they transmit. Because of their roles in tick physiology, serpins (serine protease inhibitors) from tick saliva are among the candidates for anti-tick vaccines. Inhibitory serpins employ a suicide inhibition mechanism to inhibit proteases, where the serpin reactive centre loop (RCL) is cleaved, by the targeted protease, and then inserted into the main ß-sheet of the serpin. This causes a massive conformational change called the 'stressed to relaxed' (S→R) transition, leading to the breakdown of serpin into two regions (core domain and cleaved polypeptide). Recently, the first tick serpin crystal structure from Ixodes ricinus in R-state was reported. We thus employed molecular dynamics simulations to better understand serpin structure and dynamics in atomic detail. Overall, R-state serpin showed high rigidity, especially the core domain. The most flexible region is the terminal of the cleaved polypeptide, due to its high-water exposure, while the rest of the cleaved polypeptide is stably trapped behind the core domain. T363, D367 and N375 are found to play a vital role in protein-protein attachment. This finding can be used to explain the high stability of the R-state serpin at the atomic level and provides insight into this tick serpin which will be useful for rational anti-tick vaccine development. AbbreviationsMDMolecular DynamicsRCLReactive centre loopCommunicated by Ramaswamy H. Sarma.

Feline panleukopenia virus as the cause of diarrhea in a banded linsang (Prionodon linsang) in Thailand.

A banded linsang (Prionodon linsang) presented at our hospital with clinical signs of acute diarrhea. Fecal samples were positive for canine parvovirus (CPV) as determined by polymerase chain reaction with primers specific for both CPV and feline panleukopenia virus (FPV). The full-length VP2 was cloned, sequenced, and compared with sequences of FPV and CPV strains reported in GenBank. The amino acids that determined the host range were similar to those of FPV. Moreover, amino acid analysis of VP2 revealed over 98% homology to FPV. The FPV isolate was closely related with FPV isolates from Japan, South Korea, and China. To the best of our knowledge, this is the first study to report that banded linsang can be infected with FPV.

Contamination of Streptococcus suis in pork and edible pig organs in central Thailand.

BACKGROUND AND AIM: Streptococcus suis is an important zoonotic pathogen that can cause serious diseases in both swine and humans worldwide, especially in Asian countries. Since the majority of human cases reported in Thailand were infected by the consumption of a raw pork dish, the microbial food safety hazard associated with raw meat has been a matter of concern. Therefore, this study aimed to investigate the contamination by S. suis in pork and edible pig organs sold in central Thailand. MATERIALS AND METHODS: In total, 88 raw pork and pig organ samples were purchased from markets, butcher shops, and supermarkets in central Thailand. The samples were examined using the loop-mediated isothermal amplification (LAMP) technique. LAMP reactions used for the detection of the DNA of S. suis (LAMPSS) and S. suis serotype 2 or 1/2 (LAMPSS2) were carried out according to previous studies. RESULTS: The percentage of LAMPSS-positive samples was as high as 85.23% (75/88) while the percentage of LAMPSS2-positive samples was 17.05% (15/88). The percentages of LAMPSS- and LAMPSS2-positive samples were relatively high in both pig organs (lung and heart) and meat (sliced pork and minced pork) compared with the previous report. Except one supermarket, LAMPSS-positive samples were found in all sources investigated in this study. The pork and pig organs obtained from the markets and the butcher shops additionally gave positive results for LAMPSS2. CONCLUSION: Using LAMP techniques, high rate contamination of S. suis was found in raw pork and edible pig organs sold at different sources in central Thailand. The cross-contamination could have occurred through slaughtering, meat cutting, and meat handling processes. Therefore, consumers and people involved in the pig production industry should be aware of the potential hazards of S. suis infection; food safety education is crucial to prevent further infection.

High rate misidentification of biochemically determined Streptococcus isolates from swine clinical specimens.

In this study, 22 bacterial isolates from swine necropsy specimens, which were biochemically identified as Streptococcus suis and other Streptococcus species, were re-examined using species-specific PCR for authentic S. suis and 16S rRNA gene sequencing for the verification of the former judge. Identification of S. suis on the basis of biochemical characteristics showed high false-positive (70.6%) and false-negative (60%) rates. The authentic S. suis showed various capsular polysaccharide synthesis gene types, including type 2 that often isolated from human cases. Five of 22 isolates did not even belong to the genus Streptococcus. These results suggested that the misidentification of the causative pathogen in routine veterinary diagnosis could be a substantial obstacle for the control of emerging infectious diseases.

Potentially hazardous Streptococcus suis strains latent in asymptomatic pigs in a major swine production area of Thailand.

PURPOSE: Carrier pigs have been considered as the major reservoir of Streptococcus suis and couldbe a significant source of human infection. Therefore, we investigated the prevalence and characteristics of latent S. suis in asymptomatic pigs in the pig-farming area of central Thailand, and compared the data to those previously reported in other regions. METHODOLOGY: We collected samples from 340 asymptomatic pigs. S. suis isolates from the samples were confirmed by species-specific PCR (recN PCR). The capsular polysaccharide synthesis gene (cps) types, virulence-associated gene profiles and sequence types (STs) of the isolates were investigated.Results/Key findings. The prevalence of S. suis found in this study was 37 % (125/340 pigs). The most prevalent genotype was mrp-/epf-/sly-. Among the 16 cps-types identified in 135 isolates, cps-type 16 was the most frequent (11 %), whereas 44 % of the isolates were non-typable. In common with the strains causing human sepsis in Thailand, two cps-type 9 isolates and a cps-type 24 isolate from slaughtered pigs belonged to ST16 and ST221, respectively. All the isolated cps-type 2 strains were confirmed as serotype 2 by co-agglutination tests, and these belonged to ST104, the unique ST commonly found in Thai patients; however, in contrast to the endemic areas, the prevalence of serotype 2 strains was relatively low (2 %) and no ST1 isolate was found. CONCLUSION: Our results showed the population structure differences between S. suis in central Thailand and other regions; however, zoonotic S. suis is certainly latent in asymptomatic pigs in this intensive swine production area.

Association of Ehrlichia canis, Hemotropic Mycoplasma spp. and Anaplasma platys and severe anemia in dogs in Thailand.

Canine tick-borne bacteria; Ehrlichia canis, hemotropic Mycoplasma spp. and Anaplasma spp., are organisms transmitted by Rhipicephalus sanguineus ticks. However, only a few clinical studies evaluating dogs infected with these organisms and anemia condition have been published. In this study, the potential tick-borne bacteria linked to anemia were investigated in eighty-one blood samples selected from anemic dogs using a broad range nested-PCR of the 16S rRNA gene. Positive results were shown in 12/81 blood specimens (14.81%). Nucleotide sequences from the PCR products were analyzed using BLAST and resulted in identification of Ehrlichia canis (8), Candidatus Mycoplasma haematoparvum (1) and Anaplasma platys (3). Two other PCR assays were used to detect and identify the positive results of these pathogens including a specific PCR for Ehrlichia canis (gp36) and a specific nested-PCR for hemoplasma species (16S rRNA) and the phylogenetic analyses of E. canis and canine hemoplasmas were performed using these two loci. These specific PCRs revealed co-infection of E. canis and Mycoplasma haemocanis in two cases. These two male dogs had presented with jaundice, severe hemolytic anemia, severe thrombocytopenia, leukocytosis, mild azotemia and hepatitis. Ehrlichia canis was detected in a significantly greater number of severe anemia cases (PCV<15%) than moderate or mild anemia cases (PCV 16-29%) (P<0.05) and these severe anemia cases were 7-fold more at risk of having E. canis infections (odds ratio: 7.11, p=0.020). However, no statistical differences were detected between E. canis detection and degrees of thrombocytopenia or leukopenia. From the results of this study, we conclude that the severity of anemia is associated with E. canis infections rather than the severity of thrombocytopenia.