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African Swine Fever (ASF) control is severely limited by a diagnostic gap, as laboratory-based quantitative polymerase chain reaction (qPCR) is highly sensitive but slow, whereas field-deployable immunochromatographic assays (ICAs) are rapid but unreliable. To address this limitation, this study evaluated a novel, rapid isothermal assay, RNase hybridization-assisted amplification (RHAM), as a high-sensitivity, point-of-need diagnostic solution. This study compared the performance of RHAM and a conventional p72-based ICA against the qPCR reference standard using 106 diverse clinical field samples, including oral swabs, blood, serum, and organs, collected from suspected ASF cases in Thailand. The ICA exhibited markedly low diagnostic performance, achieving only 56.76% sensitivity and showing moderate agreement (κ = 0.421) with qPCR, highlighting the need for a more reliable alternative. In contrast, the RHAM assay achieved 94.59% sensitivity and 96.88% specificity, providing results rapidly within 35 min. This statistically superior performance (McNemar’s test, p < 0.0001) demonstrated almost perfect agreement (κ = 0.891) with the qPCR reference standard, missing only four samples with very high Ct values (>30). In conclusion, RHAM is a powerful, accurate, and field-deployable diagnostic tool that effectively bridges the diagnostic gap, offering qPCR-like sensitivity for the rapid containment of ASF outbreaks.

African Swine Fever (ASF) control is severely hampered by the reliance on slow, laboratory-bound diagnostics. While rapid, field-deployable lateral flow assays (LFAs) are urgently needed, the comparative performance of key single-antigen targets remains poorly characterized. This study aimed to develop and systematically evaluate the diagnostic performance of three in-house single-antigen LFAs targeting ASF virus P30, P54, and P72, using swine field samples from Thailand, including a panel of 143 quantitative polymerase chain reaction-negative swine serum samples. The performance of each LFA was compared against a commercial multi-antigen (P32/P62/P72) indirect ELISA, which served as the reference standard, classifying 64 samples as positive and 79 as negative. The P72-based LFA demonstrated perfect diagnostic performance (100% sensitivity, 100% specificity) and perfect agreement (κ = 1.0) with the enzyme-linked immunosorbent assay (ELISA). Similarly, the P30 LFA demonstrated high performance (100% sensitivity, 98.7% specificity) with ‘Almost Perfect’ agreement (κ = 0.9859). In contrast, the P54 LFA was unsuitable, achieving 100% sensitivity but unacceptably low specificity (88.6%) due to a high rate of false positives. Overall, the single-antigen P72 and P30 LFAs demonstrated excellent concordance with the multi-antigen ELISA, supporting their reliable for detecting antibodies against ASFV. Although these assays do not replace molecular methods for acute infection detection, they represent valuable complementary tools for serosurveillance.

Canine monocytic ehrlichiosis is cause by Ehrlichia canis resulting in hematologic disorders and severe clinical signs. The aim of this study was to scrutinize the molecular detection and genetic diversity of E. canis based on the trp36 gene in dogs from Thailand’s northern and central regions. A total of 120 dogs blood samples were amplified for trp36 gene of E. canis using the polymerase chain reaction (PCR). Forty-seven out of 120 dog blood samples (39.16%, 47/120) were positive for E. canis the trp36 DNA with 790 bp of PCR amplicon size. The factor significantly associated with E. canis infection is animal housing status ( p  < 0.05). Sequence and phylogenetic analysis showed that E. canis trp36 gene of Thailand isolates was clustered into 1st clade with similarity ranging from 95.65 to 100% together with the US genogroup. The 14 haplotypes of the trp36 gene shown in TCS network exhibited that haplotype #1–4 was found in Thailand. The entropy analysis of the trp36 gene illustrated 751 polymorphic sites and 271 entropy peaks of nucleic and amino acid sequences, respectively. Hence, these findings are crucial for better understanding the epidemiology of Ehrlichia infection and could be helpful for implementing control measures in Thailand.

Background Canine circovirus (CanineCV) is a single-stranded circular DNA virus that infects domestic and wild canids in many countries. CanineCV is associated with gastroenteritis and diarrhea, respiratory disease, and generalized vasculitis leading to a fatal event. The Capsid protein (Cap) is a structural protein of the virus which has high genetic variability and plays a role in the canine immune response. In this study, we cloned the full-length CanineCV Capsid gene ( Cap ). In-silico analyses were used to explore the genomic and amino acid variability and natural selection acting on the Cap gene. The immune relevance for T-cell and B-cell epitopes was predicted by the immunoinformatic approach. Results According to the Cap gene, our results showed that CanineCV was separated into five phylogenetic groups. The obtained CanineCV strain from this study was grouped with the previously discovered Thai strain (MG737385), as supported by a haplotype network. Entropy analyses revealed high nucleotide and amino acid variability of the Capsid region. Selection pressure analysis revealed four codons at positions 24, 50, 103, and 111 in the Cap protein evolved under diversifying selection. Prediction of B-cell epitopes exhibited four consensus sequences based on physiochemical properties, and eleven peptide sequences were predicted as T-cell epitopes. In addition, the positive selection sites were located within T-cell and B-cell epitopes, suggesting the role of the host immune system as a driving force in virus evolution. Conclusions Our study provides knowledge of CanineCV genetic diversity, virus evolution, and potential epitopes for host cell immune response.

Leucocytozoon sabrazesi is the intracellular protozoa of leucocytozoonosis, which is transmitted by the insect vectors and affects chickens in most subtropical and tropical regions of the globe, except South America, and causing enormous economic losses due to decreasing meat yield and egg production. In this study, L. sabrazesi gametocytes have been observed in the blood smears, and molecular methods have been used to analyse the occurrence and genetic diversity of L. sabrazesi in blood samples from 313 chickens raised in northern, western and southern parts of Thailand. The nested polymerase chain reaction (nested PCR) assay based on the cytb gene revealed that 80.51% (252/313) chickens were positive of L. sabrazesi . The phylogenetic analysis indicated that L. sabrazesi cytb gene is conserved in Thailand, showed 2 clades and 2 subclades with similarity ranged from 89.5 to 100%. The diversity analysis showed 13 and 18 haplotypes of the sequences from Thailand and from other countries, respectively. The entropy analyses of nucleic acid sequences showed 26 high entropy peaks with values ranging from 0.24493 to 1.21056, while those of amino acid sequences exhibited 5 high entropy peaks with values ranging from 0.39267 to 0.97012. The results; therefore, indicate a high molecular occurrence of L. sabrazesi in chicken blood samples with the associated factors that is statistically significant ( p  < 0.05). Hence, our results could be used to improve the immunodiagnostic methods and to find appropriate preventive control strategies or vaccination programs against leucocytozoonosis in order to mitigate or eliminate the harmful impact of this infection on chicken industry.

Babesia bovis and B. bigemina are the most common tick-borne parasites that cause bovine babesiosis which effects livestock production, leading to economic losses in tropical and subtropical areas of the world. The aims of this study were to determine the molecular detection, genetic diversity and antigenicity prediction of B. bovis based on spherical body protein 2 ( sbp-2 ) gene and B. bigemina based on rhoptry-associated protein 1a ( rap-1a ) gene in cattle in Thailand. By PCR assay, the molecular detection of B. bovis and B. bigemina infection revealed levels of 2.58% (4/155) and 5.80% (9/155), respectively. The phylograms showed that B. bovis sbp-2 and B. bigemina rap-1a sequences displayed 5 and 3 clades with similarity ranging between 85.53 to 100% and 98.28 to 100%, respectively, when compared within Thailand strain. Diversity analysis of sbp-2 and rap-1a sequences showed 18 and 4 haplotypes, respectively. The entropy analysis illustrated 104 and 7 polymorphic sites of sbp-2 and rap-1a nucleic acid sequences, respectively, while those of sbp-2 and rap-1a amino acid sequences showed 46 and 4 high entropy peaks, respectively. Motifs analysis exhibited the distribution and conservation among sbp-2 and rap-1a sequences. The continuous and discontinuous B-cell epitopes have also been evaluated in this work. Therefore, our findings may be used to ameliorate the understanding inputs of molecular phylogeny, genetic diversity and antigenicity of B. bovis and B. bigemina Thailand stains.

Leucocytozoon sabrazesi is an intracellular haemoprotozoan parasite responsible for leucocytozoonosis, which is transmitted by insect vectors and affects chickens in tropical and subtropical areas in many countries. It causes huge economic losses due to decreased meat and egg production. In the present study, we used nested PCR to determine the genetic diversity of L. sabrazesi based on the cytb, coxI, coxIII and concatenated genes in chickens in Thailand. In addition, we found co-infections between L. sabrazesi and Plasmodium spp. ( P. gallinaceum or P. juxtanucleare ) in chickens that were not identified by microscopic examination of blood smears. The phylogenetic analysis indicated that L. sabrazesi cytb and coxIII genes were conserved with similarity ranging from 99.9 to 100% and 98 to 100%, respectively whereas the coxI gene was diverse, with similarities ranging from 97 to 100%. These findings ascertained the nucleotide analysis of the cytb, coxI, coxIII and concatenated sequences in which 4, 8, 10 and 9 haplotypes were found, respectively. In addition, it was found that the large number of synonymous substitutions and conservative amino acid replacements in these mitochondrial genes occurred by non-synonymous substitution. The evolutionary analysis of the K a / K s ratio supported purifying selection and the negative values of both Fu’s F s and Tajima’s D indicate selective sweep especially for the coxI gene. The entropy and Simplot analysis showed that the genetic variation in populations of Plasmodium spp. was higher than in Leucocytozoon. Hence, the nucleotide sequences of three mitochondrial genes could reflect the evolutionary analysis and geographic distribution of this protozoan population that switches hosts during its life cycle. Leucocytozoon sabrazesi est le parasite hémoprotozoaire intracellulaire responsable de la leucocytozoonose, qui est transmise par des insectes vecteurs et affecte les poulets dans les zones tropicales et subtropicales de nombreux pays. Il provoque d’énormes pertes économiques en raison de la diminution de la production de viande et d’œufs. Dans la présente étude, nous avons utilisé la PCR nichée pour déterminer la diversité génétique de L. sabrazesi sur la base des gènes cytb, coxI, coxIII et concaténés chez des poulets en Thaïlande. De plus, nous avons trouvé des co-infections entre L. sabrazesi et Plasmodium spp. ( P. gallinaceum ou P. juxtanucleare ) chez des poulets, qui n’ont pas été identifiées par l’examen microscopique de frottis sanguins. L’analyse phylogénétique a indiqué que les gènes cytb et coxIII de L. sabrazesi étaient conservés avec une similarité allant respectivement de 99,9 à 100 % et de 98 à 100 %, alors que le gène coxI était diversifié, avec des similarités allant de 97 à 100 %. Ces découvertes ont confirmé l’analyse des nucléotides des séquences cytb, coxI, coxIII et concaténées dans lesquelles 4, 8, 10 et 9 haplotypes ont été trouvés, respectivement. De plus, il a été constaté que le grand nombre de substitutions synonymes et de remplacements conservateurs d’acides aminés dans ces gènes mitochondriaux se produisaient par substitution non synonyme. L’analyse évolutive du rapport K a / K s a soutenu la sélection purificatrice et les valeurs négatives des Fs de Fu et D de Tajima indiquent un balayage sélectif, en particulier pour le gène coxI. L’entropie et l’analyse Simplot ont montré que la variation génétique de la population de Plasmodium spp. était plus élevée que pour Leucocytozoon . Par conséquent, les séquences nucléotidiques de trois gènes mitochondriaux pourraient refléter l’analyse évolutive et la répartition géographique de cette population de protozoaires qui changent d’hôte au cours de leur cycle de vie.

There was little information regarding the occurrence of canine vector-borne disease (CVBDs) in shelter dogs in Thailand. This work is the first report regarding a molecular method used to determine the occurrence and genetic diversity of three canine tick-borne pathogens (TBPs) (Hepatozoon canis, Anaplasma platys and Ehrlichia canis) in blood samples from 275 shelter dogs in the north and central areas of Thailand. The PCR results based on the 18S rRNA and 16S rRNA genes showed that 71 (25.82%) dogs were positive for at least a TBP. The overall occurrence rates of H. canis, A. platys and E. canis infections were 1.81, 16.36 and 7.64%, respectively. For the phylogenetic analysis, A. platys 16S rRNA gene was genetically diverse, while H. canis 18S rRNA and E. canis 16S rRNA genes were conserved. The haplotype diversity exhibited 12 and 2 haplotypes as well as 78 and 178 polymorphic sites of A. platys and E. canis 16S rRNA genes, respectively. Our findings could be used to improve the understanding of phylogeny and genetic diversity of TBP rRNA genes and used to ameliorate the diagnosis and control programmes for the diseases in Thailand.

The purpose of this study was to evaluate the potential use of deciduous teeth stem cells from horses for wound healing in animal wound model. This study assessed the efficacy and safety of foal deciduous teeth stem cells (fDSCs) administration in rabbit excisional wound model. Deciduous teeth stem cells were harvested from horses and full thickness excisional wound was made on rabbits by surgical removal of epidermis and dermis to the depth of subcutaneous fat. New Zealand White rabbits (n=8) were divided into a control group (n=4) and a treatment group (n=4). Multiple injections of fDSCs suspension were intravenously administered into the rabbits with excisional wounds in the treatment group. Wound sizes were recorded on days 3, 5, 7, 10, 14, 21 and 28. The administration of fDSCs enhanced wound healing in the rabbit wound model. The scar sizes were significantly smaller compared with the control group (p<0.05). The rabbits exhibited immediate recovery with no complications. The transplantation of fDSCs provided good choice in terms of wound regeneration and healing. Therefore, our results suggested that fDSCs might provide a new approach to clinical treatment of wounds in veterinary patients, especially in horses in the future.