Background and Aim: Staphylococcus pseudintermedius is a zoonotic bacterium commonly found in animals, especially dogs. These bacteria can survive on environmental surfaces for several months. The infection of S. pseudintermedius from the environment is possible, but properly cleaning surface objects can prevent it. This study aimed to investigate the prevalence of methicillin-resistant S. pseudintermedius (MRSP) in the environment of a recently constructed veterinary hospital in Southern Thailand, where we hypothesized that the prevalence of MRSP might be very low. Materials and Methods: At three different time points, 150 samples were collected from different environmental surfaces and wastewater across the veterinary hospital. The collection was done after the hospital's cleaning. Bacteria were purified in the culture before being identified as species by biochemical tests and polymerase chain reaction (PCR). Next, the antimicrobial-resistant profile was performed using an automated system (Vitek 2). Finally, the antimicrobial resistance genes were identified using PCR. Results: Fifteen colonies of S. pseudintermedius were isolated from the surfaces of eight floors, four tables, two chairs, and one rebreathing tube. Fourteen colonies (93.3%) were multidrug-resistant (MDR) and carried the blaZ gene (93.3%). The majority of colonies were resistant to benzylpenicillin (93.3%), cefovecin (93.3%), ceftiofur (93.3%), kanamycin (93.3%), and neomycin (93.3%). Notably, only four colonies (26.7%) were methicillin-susceptible S. pseudintermedius, whereas 11 colonies (73.3%) were MRSP and carried both the mecA and blaZ genes. Five MRSP (45.5%) were resistant to at least 14 antimicrobial drugs, represented as extensively drug-resistant (XDR) bacteria. Ten of eleven MRSP (90.9%) were Staphylococcal chromosomal mec type V, while another displayed untypeable. Despite the routine and extensive cleaning with detergent and disinfectant, MRSP isolates were still detectable. Conclusion: Many isolates of MRSP were found in this veterinary hospital. Almost all of them were MDR, and nearly half were XDR, posing a threat to animals and humans. In addition, the current hospital cleaning procedure proved ineffective. Future research should be conducted to determine the bacterial biofilm properties and bacterial sensitivity to certain detergents and disinfectants.

Avian malaria and leucocytozoonosis can cause fatal diseases, whereas avian trypanosomiasis is reported to be harmless in chickens. Backyard chickens can be infected by several pathogens, including blood parasites, that may shed to industrial poultry production, with a consequently higher economic impact. This study aimed to investigate the presence of several blood parasites (Plasmodium, Leucocytozoon and Trypanosoma) in backyard chickens raised in Southern Thailand, using PCR-based detection and microscopic methods. From June 2021 to June 2022, 57 backyard chickens were sampled. Fresh thin blood smears were prepared from 11 individuals, and buffy coat smears were prepared from 55 of them. Both thin blood smears and buffy coat smears were used for microscopic analysis. Two nested PCR protocols that amplify a fragment of cytochrome b (cytb) and small subunit rRNA (SSU rRNA) genes were used to identify Haemosporida and Trypanosoma parasites, respectively. The number of positive samples was higher with the application of nested PCR than when buffy coat smears were used. Three new Plasmodium lineages (GALLUS47-49) and thirteen Leucocytozoon lineages (GALLUS50-62) were found. Trophozoites, meronts and gametocytes of Plasmodium gallinaceum (GALLUS01) were present in one thin blood smear. All thin blood smears revealed Leucocytozoon infections, but only three samples were a single infection. These three samples revealed the presence of fusiform host cell–parasite complexes, of which the morphological features resembled those of Leucocytozoon macleani (possible synonym is Leucocytozoon sabrazesi), while the cytb showed that this parasite is closely related to the lineage GALLUS06-07, described as Leucocytozoon schouteni. The Trypanosoma prevalence was 33.33%; it was present in only one of the thin blood smears, and it resembles Trypanosoma calmettei. This study showed the prevalence of a high diversity of Plasmodium (64.91%) and Leucocytozoon (89.47%) in Thai chickens. Both nested-PCR and buffy coat smear can be used as the diagnostic tool for the testing of Plasmodium, Leucocytozoon and Trypanosoma for parasitic control in backyard chickens and poultry farms. The information on the parasite species that can be found in chickens raised in Southern Thailand was also considered as the baseline information for further study.

Background and Aim: Staphylococci are commensal bacteria and opportunistic pathogens found on the skin and mucosa. Sports animals are more prone to injury and illness, and we believe that antimicrobial agents might be extensively used for the treatment and cause the existence of antimicrobial-resistant (AMR) bacteria. This study aimed to investigate the diversity and AMR profile of staphylococci in sports animals (riding horses, fighting bulls, and fighting cocks) in South Thailand. Materials and Methods: Nasal (57 fighting bulls and 33 riding horses) and skin swabs (32 fighting cocks) were taken from 122 animals. Staphylococci were cultured in Mannitol Salt Agar and then identified species by biochemical tests using the VITEK® 2 card for Gram-positive organisms in conjunction with the VITEK® 2 COMPACT machine and genotypic identification by polymerase chain reaction (PCR). Antimicrobial susceptibility tests were performed with VITEK® 2 AST-GN80 test kit cards and VITEK® 2 COMPACT machine. Detection of AMR genes (mecA, mecC, and blaZ) and staphylococcal chromosomal mec (SCCmec) type was evaluated by PCR. Results: Forty-one colonies of staphylococci were isolated, and six species were identified, including Staphylococcus sciuri (61%), Staphylococcus pasteuri (15%), Staphylococcus cohnii (10%), Staphylococcus aureus (7%), Staphylococcus warneri (5%), and Staphylococcus haemolyticus (2%). Staphylococci were highly resistant to two drug classes, penicillin (93%) and cephalosporin (51%). About 56% of the isolates were methicillin-resistant staphylococci (MRS), and the majority was S. sciuri (82%), which is primarily found in horses. Most MRS (82%) were multidrug-resistant. Almost all (96%) of the mecA-positive MRS harbored the blaZ gene. Almost all MRS isolates possessed an unknown type of SCCmec. Interestingly, the AMR rate was notably lower in fighting bulls and cocks than in riding horses, which may be related to the owner's preference for herbal therapy over antimicrobial drugs. Conclusion: This study presented many types of staphylococci displayed on bulls, cocks, and horses. However, we found a high prevalence of MRS in horses that could be transmitted to owners through close contact activities and might be a source of AMR genotype transmission to other staphylococci.

Multiple blood pathogen infections are increasingly found in many areas, particularly in tropical regions. This study aimed to investigate the effectiveness and safety of using doxycycline monotherapy to treat triple blood pathogen infection in sheltered dogs. A total of 375 sheltered dogs were screened for blood pathogen using polymerase chain reaction technique (PCR). There were 34 dogs with triple infection (Babesia vogeli, Ehrlichia canis, and Anaplasma platys), and most dogs displayed anemia and thrombocytopenia. These dogs were treated with doxycycline (10 mg/kg/day) orally for four weeks, and their blood profiles were monitored. Almost all pathogens were undetectable by PCR by day 14 and pathogens were all cleared by day 28. Most blood profiles significantly improved after 14 days. The improvement continued after 28 days. The red blood cell and platelet count (PLT) were increased in similar trends between mild-to-moderate and severe thrombocytopenia groups. The average PLT in the mild-to-moderate thrombocytopenia group raised to normal level by day 14, whereas in the severe group, it was gradually increased to normal level by day 70. We propose using doxycycline monotherapy for 28 days to eliminate all pathogens, and facilitate recovery and welfare in dogs suffering with triple blood pathogen infections.

Background and Aim: Mammaliicoccus sciuri, formerly known as Staphylococcus sciuri, is an opportunistic pathogen in the environment, human and animal mucosa, and skin. Although this pathogen is becoming more resistant to drugs and harmful to animals and humans, basic knowledge of this pathogen remains limited. This study aimed to investigate a new multilocus sequencing type (MLST) related to the antibiotic resistance pattern of M. sciuri from animals in southern Thailand. Materials and Methods: We used 11 methicillin-resistant M. sciuri (MRMS) isolates in this study which were obtained from six horses, four cows, and one chicken of the previous study. Antimicrobial resistance (AMR) was re-evaluated based on the minimum inhibitory concentration using the VITEK® 2 automated system. Three AMR genes were examined, namely mecA, mecC, and blaZ. Staphylococcal chromosomal cassette mec (SCCmec) gene detection was performed through the multiplex polymerase chain reaction (PCR). Internal segments of the seven housekeeping genes, ack, aroE, ftsZ, glpK, gmk, pta1, and tpiA, were used for multilocus sequence typing. The population of resistant bacteria and the types of multidrug-resistant, extensively drug-resistant, and pandemic drug-resistant bacteria were classified through descriptive analysis. Results: mecA and blaZ genes were detected in all isolates; however, the mecC gene was not observed in any isolate based on the PCR results. All MRMS isolates revealed a non-typable SCCmec. Seven MLSTs (71, 81, 120, 121, 122, 199, and 200) were identified in this study. Conclusion: The characteristics of MRMS in Southern Thailand were variable, particularly in cattle and horses. The antibiogram and SCCmec types of this pathogen remain concerns with regard to antibiotic-resistant gene transmission among Staphylococcus and Mammaliicoccus species. All MLSTs in Thailand revealed the distribution among clones in Asia, including the virulence of a zoonotic clone in Southern Thailand.

Background and Aim: Filarial nematode typically produces a larval stage (microfilariae) in the bloodstream of vertebrate hosts, where microfilariae reside in the blood or subcutaneous tissues. Filarial nematodes cause human diseases, such as river blindness and elephantiasis, which are widely studied. However, in avian species, they are overlooked because they are nonpathogenic. In Thailand, microfilaria can be found in wild birds and domestic chickens. Recently, an increase in the number of blood samples submitted to veterinary diagnostic laboratories may have increased the number of microfilariae. Therefore, knowledge about filarial species and reliable detection methods are important. Therefore, this study aimed to investigate the efficacy of buffy coat smear and polymerase chain reaction (PCR)-based methods for the detection of microfilaria in domestic chickens. In addition, parasites were identified using the sequence of the cytochrome c oxidase subunit 1 (COX1) gene. Materials and Methods: Giemsa-stained buffy coat smears from a previous study were reanalyzed. These available buffy coat smears were prepared from 55 domestic chickens raised as backyard free-ranging in Southern Thailand. Fifty-seven frozen genomic DNA extracted from chicken blood were used to detect the presence of the COX1 gene in Onchocercidae nematodes. The nested PCR protocol for amplification of the OnchoCOI_ R2-OnchoCOI_ R2 fragment of the COX1 gene was applied from a previous report. Sequences of COX1 were analyzed to identify Onchocercidae nematodes and if they were single or mixed infections. We constructed Bayesian phylogenetics to identify parasites and assessment of the relationship between filarial nematodes in avian species and other vertebrate hosts. Results: Buffy coat smears from 15 samples revealed microfilaria. Of these 15 samples, only eight were positive for COX1 nested-PCR amplification. The other two buffy coat-negative samples were also positive for nested-PCR. Sequencing of these 11 nested PCR-positive samples revealed that almost all of them were Onchocercidae nematodes. Bayesian phylogenetic analysis showed that chicken Onchocercidae spp. were grouped with other avian filarial nematodes. However, all chickens Onchocercidae spp. showed a double peak in the sequencing chromatogram, indicating mixed filarial infection (species or haplotypes). Therefore, no chicken Onchocercidae sequence was deposited on National Center for Biotechnology Information, GenBank. Conclusion: Giemsa-stained buffy coat smear was a reliable method for the detection of chicken microfilaria in routine veterinary diagnostic laboratories. Development of a new PCR-based method is necessary. This method may provide greater sensitivity and specificity of detection. In addition, the PCR method allowed us to access the genetic characteristics of nematodes, which helped us maximize our knowledge of nematodes. Further investigations, such as the pathogenicity of filarial nematodes in chickens and their potential vectors, are required. Keywords: Buffy coat smear, Chickens, Cytochrome c oxidase I, Microfilaria, Nested-polymerase chain reaction.