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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.

Canine circovirus (CanineCV) is a contagious virus that causes severe gastroenteritis, diarrhea, respiratory disease, and vasculitis, often resulting in fatality among infected dogs. In this study, a recombinant Capsid protein (rCap) of CanineCV was expressed in the Escherichia coli ( E. coli ) Rosetta (DE3) pLysS host cell, followed by affinity purification, and then analyzed by SDS-PAGE, revealing a molecular weight of approximately 31 kDa. The antigenicity of the CanineCV rCap protein was confirmed through recognition by a rabbit anti-CanineCV rCap protein polyclonal antibody (PoAb). Additionally, the reactivity and specificity of this PoAb were assessed using indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis before applying in an immunohistochemistry (IHC), namely, immunoperoxidase detection. The immunoperoxidase assay using rabbit anti-CanineCV rCap protein PoAb demonstrated that the CanineCV Cap protein was predominantly located in immune cells, especially lymphocytes and macrophages, within the spleen, lung, tracheobronchial lymph nodes, small intestine, and kidney. Similarly, the Cap protein was also found in pneumocytes in the lung and renal tubular epithelial cells in the kidney. These findings reflected the biological activity and cell tropism of the virus. Therefore, the recombinant Cap protein and its PoAb could be used for the development of a valuable diagnostic tool for CanineCV detection.

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.

Gastrointestinal (GI) parasitic diseases play an important role in the major constraints on goat production in Thailand and worldwide. The aim of this study was to assess the occurrence of GI parasitic infections among goats in Ratchaburi province of Thailand. In this study, fecal samples (394) were collected and tested by means of the floatation, sedimentation and direct smear methods to discriminate eggs or oocyst of the parasites. The overall occurrence of GI parasitic infections was 86.54%. The respective occurrences for the raising distance, farm system, water source nearby, type of feed and aquatic plants were found to vary significantly (p < 0.01), while a significant difference was not observed counting upon the goats' gender and age. The result disclosed that the highest occurrence of nematode, trematode, cestode and protozoa were Strongyloides sp. (57.77%), Paramphistomum cervi (33.43%), Moniezia sp. (29.91%) and Eimeria sp. (1.75%), respectively. Both single infection (18.78%) and mixed infection (67.67%) were evaluated in the examined goats. And so, on top of this, GIS results exhibited the movements or raising distance of goats indicating parasitic disease distribution. We intensively noted the need to find appropriate preventive control strategies against parasitic diseases in order to reduce or eliminate the harmful impact of parasitic infections on the goat industry.