Methicillin-resistant Staphylococci in canine pyoderma in Thailand.

Background and Aims: Methicillin-resistant Staphylococci (MRS) seriously threatens animal and human health. Repeated antibiotic use allows the bacteria to develop resistance to several antibiotic classes and become multidrug-resistant (MDR). Canine pyoderma, a common skin condition in dogs, is mainly caused by Staphylococci, including MRS. Detecting this infection in all canine populations is crucial to develop a proper preventive plan. This study estimated the prevalence, antibiogram, and risk factors of MRS in canine patients at a referral animal hospital in Khon Kaen, Thailand. Materials and Methods: Skin swabs and relevant information were collected from 56 client-owned dogs that visited the hospital from September 2019 to September 2020. Staphylococci colonies were subjected to molecular identification and antibiotic susceptibility tests using an automated system (VITEK® 2). These colonies were also genetically identified using multiplex-polymerase chain reaction (PCR) and sequencing. The mecA gene, encoding methicillin resistance, was detected using simplex-PCR. The risk factors of MRS infection and their association with MRS infection were analyzed using logistic regression and the Chi-square test, respectively. Results: The prevalence of MRS was found to be 35.7% (20/56 dogs). By species, methicillin-resistant Staphylococcus pseudintermedius was found in 24 of 104 isolates (23.1%), and all samples were MDR. Receiving systemic antibiotics in the past 6 months was a major risk factor associated with MRS infection (p < 0.05; odds ratio (OR) > 1). In addition to the MRS isolates, the mecA gene was also detected in methicillin-susceptible Staphylococci isolates. This might be because of the high expression of blaI, and mutations in c-di-AMP cyclase DacA, RelA, and Fem proteins. Conclusion: A high prevalence of MRS and MDR was observed in the studied population, which might be potentially due to improper antibiotic use by the owners and horizontal transfer of drug-resistance genes.

Molecular detection and genetic characterization of porcine circovirus 4 (PCV4) in Thailand during 2019-2020.

Porcine circovirus 4 (PCV4) is considered a novel PCV, firstly found in China in 2019 and later discovered in Korea. This present study investigated the prevalence and genetic characteristics of PCV4 from high pig-density areas in Thailand during 2019-2020. From 734 samples, three samples (0.4%) from aborted fetuses and porcine respiratory disease complex (PRDC) cases were found positive for PCV4, two of the PCV4-positive samples were coinfected with both PCV2 and PRRSV, and the other PCV4-positive sample was found coinfected with PCV2. In situ hybridization (ISH) revealed the presence of PCV4 in the bronchial epithelial cells and in lymphocytes and histiocyte-like cells in the lymphoid follicles of the PRDC-affected pig. The complete Thai PCV4 genome had over 98% nucleotide identity with other PCV4 strains and was closely related to the Korean and Chinese PCV4b strains. Importantly, the amino acid residue at position 212 of the Cap gene is recommended for differentiating PCV4a (212L) from PCV4b (212M) based on currently available PCV4 genome sequences. These findings provide important clues for the pathogenesis, epidemiology, and genetic characteristics of PCV4 in Thailand.

Pathology and molecular characterization of Leucocytozoon caulleryi from backyard chickens in Khon Kaen Province, Thailand.

AIM: The aim of this study was to characterize Leucocytozoon caulleryi from backyard chickens in Khon Kaen Province, Thailand. MATERIALS AND METHODS: Tissue samples were collected from backyard chickens suspected to have leucocytozoonosis and subjected to histopathology examination. The BLAST Tool at NCBI GenBank (Basic Local Alignment Research Tools) (http://www.ncbi.nlm.nih.gov/BLAST) was used to identify the nucleotide sequence of the partial cytochrome c oxidase subunit I (cox I) gene. A Phylogenetic tree for analysis of L. caulleryi was constructed by using MEGA 7.0 software (https://www.megasoftware.net/). RESULTS: The necropsy results revealed the subcutaneous hemorrhages of pectoral muscles, multifocal hemorrhages of the thymus and pectoral muscles, hemorrhage of the proventriculus and peritoneal cavity, and megaloschizonts of the pancreas and intestine, including subcapsular hemorrhages of the liver. Microscopic examination revealed numerous megaloschizonts of Leucocytozoon spp. in the pectoral muscles, intestine, pancreas, and thymus. Molecular analysis of the partial cox I gene showed that the causal agent was closely related to L. caulleryi reported in Japan. CONCLUSION: From these results, L. caulleryi was determined to be the causal agent of leucocytozoonosis and was closely associated with L. caulleryi reported in Japan.

Opisthorchis viverrini antigens up-regulates the expression of CD80 and MHC class II in JAWSII mouse dendritic cells and promotes IL-10 and TGF-ß secretions.

Dendritic cells (DCs) are antigen-presenting cells (APC) involved in the initiation of immune responses. Maturation of DCs is characterized by the high expression of major histocompatibility complex (MHC) class II and co-stimulatory clusters of differentiation (CD) 40, CD80, and CD86 molecules. Matured DCs are required for T cell differentiation and proliferation. However, the response of DCs to Opisthorchis viverrini antigens has not yet been understood. Therefore, this study sought to determine the expression of surface molecules of JAWSII mouse DCs stimulated by crude somatic (CS) and excretory-secretory (ES) antigens of O. viverrini. ES antigen significantly induced only mRNA expression of CD80 and MHC class II in JAWSII mouse DCs, while CS antigen promoted up-regulation of both mRNA and protein levels of CD80 and MHC class II, indicating relative maturation of JAWII mouse DCs. Moreover, the secreted cytokines from the co-cultures of O. viverrini antigens stimulated JAWSII DC with naïve CD4+ T cells was determined. Significantly increased levels of immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-ß) were found. The up-regulation of these cytokines may indicate the response of regulatory T cells (Treg) to CS antigen-stimulated JAWSII DC. These findings may lead to a better understanding of the role that DCs play in O. viverrini infection.

Development of injectable chitosan/biphasic calcium phosphate bone cement and in vitro and in vivo evaluation.

Injectable biphasic calcium phosphate bone cements (BCPCs) composed of ß-tricalcium phosphate (ß-TCP) and hydroxyapatite (HA) have been intensively investigated because of their high rate of biodegradation, bioactivity and osteoconductivity, which can be adjusted by changing the ratio between ß-TCP and HA phases after setting. The aim of this study was to evaluate the performance of 1 wt% chitosan fiber additive with biphasic calcium phosphate as an injectable bone cement both in vitro and in vivo. In vitro evaluation of compressive strength, degradation rate, morphology, and cell and alkaline phosphatase activities was done by comparison with bone cement without ß-TCP. The in vivo results for micro-CT scanning and histological examinations for three groups (control, BCPC and commercial biphasic calcium phosphate granules) were characterized and compared. After the addition of 20 wt% ß-TCP to calcium phosphate cement, the initial and final setting times of the sample were 3.92 min and 11.46 min, respectively, which were not significantly different from cement without ß-TCP. The degradation time of the BCPC material was longer than that of calcium phosphate cement alone. The healing process was significantly faster for BCPC than for the control and commercial product groups. Therefore, this is the first evidence that BCPC is an attractive option for bone surgery due to its faster stimulation of healing and faster degradation rate.

Porcine circovirus type 3 (PCV3) shedding in sow colostrum.

The major objective of this work was to investigate the shedding of porcine circovirus type 3 (PCV3) in sow colostrum. PCV3 titers in the serum and colostrum samples of 38 sows were determined using qPCR. Interestingly, this is the first report regarding the identification of PCV3 from the colostrum samples. In the studied farm, the prevalence of PCV3 in the colostrum samples was 44.74% (17/38). When sows were grouped based on the PCV3 titers in the serum into the "High-viremic", "Low-viremic" and "Non-viremic" sows, it was shown that the High-viremic sows showed significantly higher PCV3 colostrum prevalence (100%; 9/9) with the PCV3 titers ranging from 4.01 to 7.33 genomic copies/mL. The results indicated that PCV3 in the colostrum might be partly influenced by the viremic stage of the infection. However, the results also showed that approximately 41% of sows shedding PCV3 with low titers in the colostrum (7/17) were non-viremic sows. In conclusion, this study identified the presence of PCV3 in sow colostrum. Clinical impacts and mechanisms of colostrum shedding of PCV3 should be further investigated.

Porcine circovirus type 3 (PCV3) infection in grower pigs from a Thai farm suffering from porcine respiratory disease complex (PRDC).

Porcine circovirus type 3 (PCV3) is a newly emerging virus with unknown pathogenesis. The major objective of this study was to investigate the presence of PCV3 in pigs from a farm in Thailand suffering from porcine respiratory disease complex (PRDC). Initially, a Thai PCV3 strain (PCV3/Thailand/PB01/17) was identified from a pig originated from a farm with PRDC problem during grower period and whole genome analysis showed that the Thai PCV3 shared highest nucleotide identity of 99.60% with the South Korean strain PCV3/KU-1602. The presence of PCV3 infection in PRDC-affected pigs was then investigated in this farm. Serum samples from clinically healthy pigs and pigs showing PRDC-related clinical signs during 5-18 weeks were used in PCV3 detection by PCR. The results showed that the PRDC-affected pigs exhibited higher prevalence of PCV3 infection and higher PCV3 titers comparing with the clinically healthy pigs. These results confirmed the presence of PCV3 in a Thai farm with PRDC problem. The pathogenesis of PCV3 on PRDC should be clarified in further studies.

Efficacy of Fostera® PRRS modified live virus (MLV) vaccination strategy against a Thai highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection.

Recently, the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) (HP-PRRSV) belonging to lineage 8 causes severe symptom with high morbidity and high mortality rates to the Asian pig industry. A recent study showed that pigs immunized with Fostera® PRRS modified live virus (MLV) of lineage 8 could provide a degree of protection against a Vietnamese HP-PRRSV infection. It should be noted that PRRSV commonly found after weaning causes porcine respiratory disease complex (PRDC). Vaccination strategy should be evaluated in each farm scenario. Eighty-one PRRSV-free piglets obtained from a PRRS-free herd were divided into two experiments with the major difference of infection timing after vaccination, 42 days in experiment 1 (n = 42) and 28 days in experiment 2 (n = 39). Each experiment had similar protocol containing three groups including a negative control, unvaccinated challenged, and vaccinated challenged groups. Pigs in vaccination groups were immunized with Fostera® PRRS MLV vaccine at 3 weeks of age. Then, unvaccinated challenged and vaccinated challenged groups were intranasally inoculated with a Thai HP-PRRSV (10PL01). Vaccinated challenged pigs showed significantly lower levels of mean rectal temperatures, clinical severity, lung lesion scores, and viral titers in serum and lung tissue compared to the unvaccinated challenged pigs (p < 0.05). Vaccinated challenged pigs had higher survival rate than those of unvaccinated challenged pigs in both experiments. It should be noted that pigs challenged 42 days after vaccination showed a better performance than pigs challenged 28 days after vaccination. In conclusion, Fostera® PRRS MLV vaccine was able to improve the survival rate against the Thai HP-PRRSV infection in both 42- and 28-day vaccination-to-infection protocols.

Genetic characterization of Thai swine influenza viruses after the introduction of pandemic H1N1 2009.

Pandemic H1N1 2009 (pH1N1), influenza virus containing triple reassortant internal genes (TRIG) from avian, human, and swine influenza viruses emerged in 2009 as a highly infectious virus that was able to be transmitted from humans to pigs. During June 2010-May 2012, influenza virus surveillance was conducted in Thai pig population. Twenty-three samples (1.75%) were successfully isolated from total of 1,335 samples. Interestingly, pH1N1 (7 isolates, 30.34%), reassortant pH1N1 (rH1N1) (1 isolate, 4.35%), Thai endemic H1N1 (enH1N1) (3 isolates, 13.04%), reassortant H3N2 with pH1N1 internal genes (rH3N2) (9 isolates, 39.13%), and reassortant H1N2 with pH1N1 internal genes (rH1N2) (3 isolates, 13.04%) were found. It should be noted that rH1N1, rH1N2, and rH3N2 viruses contained the internal genes of pH1N1 virus having a TRIG cassette descendant from the North American swine lineage. Although all isolates in this study were obtained from mild clinically sick pigs, the viruses were still highly infective and possibly may play an important role in human-animal interfacing transmission. In addition, the TRIG cassette may have an influence on antigenic shift resulting in emergence of novel viruses, as seen in this study. Continuing surveillance of influenza A natural hosts, particularly in pigs is necessary.

Experimental infection with a Thai reassortant swine influenza virus of pandemic H1N1 origin induced disease.

BACKGROUND: Following the emergence of the pandemic H1N1 influenza A virus in 2009 in humans, this novel virus spread into the swine population. Pigs represent a potential host for this virus and can serve as a mixing vessel for genetic mutations of the influenza virus. Reassortant viruses eventually emerged from the 2009 pandemic and were reported in swine populations worldwide including Thailand. As a result of the discovery of this emergent disease, pathogenesis studies of this novel virus were conducted in order that future disease protection and control measures in swine and human populations could be enacted. METHODS: The pandemic H1N1 2009 virus (pH1N1) and its reassortant virus (rH1N1) isolated from pigs in Thailand were inoculated into 2 separate cohorts of 9, 3-week-old pigs. Cohorts were consisted of one group experimentally infected with pH1N1 and one group with rH1N1. A negative control group consisting of 3 pigs was also included. Clinical signs, viral shedding and pathological lesions were investigated and compared. Later, 3 pigs from viral inoculated groups and 1 pig from the control group were necropsied at 2, 4, and 12 days post inoculation (DPI). RESULTS: The results indicated that pigs infected with both viruses demonstrated typical flu-like clinical signs and histopathological lesions of varying severity. Influenza infected-pigs of both groups had mild to moderate pulmonary signs on 1-4 DPI. Interestingly, pigs in both groups demonstrated viral RNA detection in the nasal swabs until the end of the experiment (12 DPI). CONCLUSION: The present study demonstrated that both the pH1N1 and rH1N1 influenza viruses, isolated from naturally infected pigs, induced acute respiratory disease in experimentally inoculated nursery pigs. Although animals in the rH1N1-infected cohort demonstrated more severe clinical signs, had higher numbers of pigs shedding the virus, were noted to have increased histopathological severity of lung lesions and increased viral antigen in lung tissue, the findings were not statistically significant in comparison with the pH1N1-infected group. Interestingly, viral genetic material of both viruses could be detected from the nasal swabs until the end of the experiment. Similar to other swine influenza viruses, the clinical signs and pathological lesions in both rH1N1 and pH1N1 were limited to the respiratory tract.

Retrospective swine influenza serological surveillance in the four highest pig density provinces of Thailand before the introduction of the 2009 pandemic Influenza A virus subtype H1N1 using various antibody detection assays.

Genetic characterization of the hemagglutinin gene of the 6 selected Thai Swine influenza virus (SIV) isolates (4 H1 and 2 H3 isolates) used in the establishment of a hemagglutination inhibition (HI) assay was analyzed. Based on the phylogenetic analysis, Thai SIVs could be divided into 3 clusters of the H1 viruses (clusters I and II belonging to classical swine H1α, and cluster III belonging to classical swine H1γ), and 2 clusters of the H3 viruses both belonging to human-like 1970s. The serological results indicated that swH1N1-06 (H1 cluster I) is a suitable representative SIV for the HI test antigen to detect H1 SIV-specific antibodies in the Thai swine population, while both swH3N2-05 and swH3N2-07 should be used for Thai H3 SIV-specific antibody detection. The HI test results of swine sera collected from pigs in the 4 highest pig population provinces of Thailand indicated that the percentage of pigs seropositive to swH3N2-07 was highest compared to swH1N1-06, swH1N1-09, and swH3N2-05 (85.4%, 50.1%, 18.6%, and 15.8%, respectively). It should be noted that countries lacking SIV genetic information should be concerned with determining the most suitable HI test antigens to use when performing the tests due to the genetic variation and limited cross-reaction of SIVs. The results of the current study demonstrated that HI tests should be implemented with the suitable field strains as the representative test antigen to ascertain accurate SIV serostatus in Thailand and that test antigens should be genetically analyzed and compared with circulating strains regularly.

An indirect enzyme-linked immunosorbent assay using a recombinant truncated capsid protein of Porcine circovirus-2.

Porcine circovirus-2 (PCV-2) serology is commonly used for PCV-2 herd status determination and optimal timing of PCV-2 vaccination programs. The objectives of the current study were to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) using a recombinant nuclear localization signal truncated capsid (rntCap) protein expressed in an Escherichia coli system and to determine the diagnostic performance of the developed rntCap indirect ELISA in comparison with immunoperoxidase monolayer assays (IPMAs). Based on a receiver operating characteristic (ROC) curve analysis of the rntCap indirect ELISA (n = 90), an optimum cutoff optical density (OD) of 0.330 was determined, which resulted in diagnostic sensitivity, diagnostic specificity, and accuracy of 98.33%, 93.33%, and 96.67%, respectively. Average OD values of the positive (n = 8) and negative sera (n = 8) tested by either purified glutathione-S-transferase (GST) protein or the rntCap protein as the coating antigen revealed that the mean OD values tested by the rntCap indirect ELISA were significantly different from using GST alone (P < 0.005). The correlation between the established rntCap indirect ELISA and the IPMA results demonstrated as the linear regression (Spearman correlation coefficient = 0.772, P < 0.005) indicated that the OD ratio obtained from the rntCap indirect ELISA could be used to predict the levels of the IPMA titers. More samples are needed for enhancing the diagnostic sensitivity, specificity and accuracy. In conclusion, the establishment of the rntCap indirect ELISA could be used as a serodiagnostic assay for large-scale detection of PCV-2 antibodies in swine and has the capability to be produced commercially for routine use in diagnostic laboratories.