Development of a latex agglutination test based on VH antibody fragment for detection of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is an important porcine pathogen that causes diseases in both swine and human. For rapid SS2 identification, a novel latex agglutination test (LAT) based on heavy-chain variable domain antibody (VH) was developed. Firstly, the soluble 47B3 VH antibody fragment from a phage display library, in which cysteine residues were engineered at the C-terminus, was expressed in Escherichia coli. The purified protein was then gently reduced to form monomeric soluble 47B3 VH subsequently used to coat with latex beads by means of site-specific conjugation. The resulting VH-coated beads gave a good agglutination reaction with SS2. The LAT was able to distinguish S. suis serotype 2 from serotype 1/2, which shares some common sugar residues, and showed no cross-reaction with other serotypes of S. suis or other related bacteria. The detection sensitivity was found to be as high as 1.85x106 cells. The LAT was stable at 4°C for at least six months without loss of activity. To the best of our knowledge, this is the first LAT based on a VH antibody fragment that can be considered as an alternative for conventional antibody-based LAT where VHs are the most favored recombinant antibody.

Ratiometric electrochemical lateral flow immunoassay for the detection of Streptococcus suis serotype 2.

An electrochemical lateral flow immunoassay (eLFIA) strip with high reproducibility was developed to rapidly and accurately detect Streptococcus suis serotype 2. This proposed strip was fabricated by integrating ratiometric electrochemical detection and LFIA (R-eLFIA). The R-eLFIA exhibited excellent reproducibility, which was improved by 3.8 times compared to a single electrode. A dual-working screen-printed graphene electrode (SPGE) was designed by tuning the working electrode with electroactive species in the biosensing system. Ferrocene carboxylic acid (Fc) was used as a signal probe, and sunset yellow (SY) at one working electrode was used as an internal reference signal to provide a built-in correction for reducing the effects of inherent background current. S. suis serotype 2-specific antibodies were immobilized on a nitrocellulose membrane of LFIA, which is located on the position of Fc-SPGE. In the presence of the analyte, an immunocomplex formed on the region of Fc-SPGE, causing a decrease in Fc current while SY current remained constant. The current ratio's decrease was proportional to S. suis serotype 2's concentration. Under optimization, this biosensor showed good linearity in the range of 102-1010 CFU/mL with a limit of detection of 10 CFU/mL and achieved a rapid detection time (15 min). Moreover, the R-eLFIA biosensor exhibited excellent reproducibility and high selectivity and was applied in human serum samples. Thus, this study successfully matched the advantages of the ratiometric strategy and LFIA and has great potential to be used as an effective tool for rapidly detecting S. suis serotype 2 in clinical samples.

Unlocking the Secrets of Streptococcus suis: A peptidomics comparison of virulent and non-virulent serotypes 2, 14, 18, and 19.

Streptococcus suis (S. suis) is an important bacterial pathogen, that causes serious infections in humans and pigs. Although numerous virulence factors have been proposed, their particular role in pathogenesis is still inconclusive. The current study explored putative peptides responsible for the virulence of S. suis serotype 2 (SS2). Thus, the peptidome of highly virulent SS2, less prevalent SS14, and rarely reported serotypes SS18 and SS19 were comparatively analyzed using a high-performance liquid chromatography-mass spectrometry method (LC-MS/MS). Six serotype-specific peptides, 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-acetyltransferase (DapH), alanine racemase (Alr), CCA-adding enzyme (CCA), peptide chain release factor 3 (RF3), ATP synthase subunit delta (F0F1-ATPases) and aspartate carbamoyltransferase (ATCase), were expressed moderately to highly only in the SS2 peptidome with p-values of less than 0.05. Some of these proteins are responsible for bacterial cellular stability; especially, Alr was highly expressed in the SS2 peptidome and is associated with peptidoglycan biosynthesis and bacterial cell wall formation. This study indicated that these serotype-specific peptides, which were significantly expressed by virulent SS2, could serve as putative virulence factors to promote its competitiveness with other coexistences in a particular condition. Further in vivo studies of these peptides should be performed to confirm the virulence roles of these identified peptides.

Prediction of the spread of African swine fever through pig and carcass movements in Thailand using a network analysis and diffusion model.

Background: African swine fever (ASF) is a serious contagious viral disease of pigs that affects the pig industry. This study aimed to evaluate the possible African swine fever (ASF) distribution using network analysis and a diffusion model through live pig, carcass, and pig product movement data. Material and Methods: Empirical movement data from Thailand for the year 2019 were used, and expert opinions were sought to evaluate network properties and the diffusion model. The networks were presented as live pig movement and carcass movement data at the provincial and district levels. For network analysis, a descriptive network analysis was performed using outdegree, indegree, betweenness, fragmentation, and power law distribution, and cutpoints were used to describe movement patterns. For the diffusion model, we simulated each network using spatially different infected locations, patterns, and initial infection sites. Based on expert opinions, the initial infection site, the probability of ASF occurrence, and the probability of the initial infected adopter were selected for the appropriated network. In this study, we also simulated networks under varying network parameters to predict the infection speed. Results and Conclusions: The total number of movements recorded was 2,594,364. These were divided into 403,408 (403,408/2,594,364; 15.55%) for live pigs and 2,190,956 (2,190,956/2,594,364; 84.45%) for carcasses. We found that carcass movement at the provincial level showed the highest outdegree (mean = 342.554, standard deviation (SD) = 900.528) and indegree values (mean = 342.554, SD = 665.509). In addition, the outdegree and indegree presented similar mean values and the degree distributions of both district networks followed a power-law function. The network of live pigs at provincial level showed the highest value for betweenness (mean = 0.011, SD = 0.017), and the network of live pigs at provincial level showed the highest value for fragmentation (mean = 0.027, SD = 0.005). Our simulation data indicated that the disease occurred randomly due to live pig and carcass movements along the central and western regions of Thailand, causing the rapid spread of ASF. Without control measures, it could spread to all provinces within 5- and 3-time units and in all districts within 21- and 30-time units for the network of live pigs and carcasses, respectively. This study assists the authorities to plan control and preventive measures and limit economic losses caused by ASF.

Rapid visual Candidatus Liberibacter asiaticus detection (citrus greening disease) using simple alkaline heat DNA lysis followed by loop-mediated isothermal amplification coupled hydroxynaphthol blue (AL-LAMP-HNB) for potential local use.

An outbreak of citrus greening or Huanglongbing disease bacteria occurs in many areas. We sampled and identified an ongoing ~year 2020 orange tree endemic in northern Thailand as Candidatus Liberibacter asiaticus. We thereby developed a plant greening disease (C. Liberibacter asiaticus) detection assay using simple alkaline heat DNA lysis and loop-mediated isothermal amplification coupled hydroxynaphthol blue (AL-LAMP-HNB), and evaluated the developed assay for its feasibility as point-of-care detection on 65 plant leaf samples with 100-1×104 copies of C. Liberibacter asiaticus or mocked injection compared with commercial DNA lysis kit and PCR-GE. Our assay is sensitive to 5-8.9 copies of omp (equaling 0.0056-0.01 fg) compatible with PCR-GE limit of detection. This ultra sensitive limit of detection could allow the disease detection before clinical apparent state of disease when C. Liberibacter asiaticus infection number is few, i.e. fewer than 100 copies of C. Liberibacter asiaticus. The assay is also specific with 6 degenerate primers targeting every strain of C. Liberibacter asiaticus omp from GenBank database, rapid (40 min total assay time), inexpensive (~2-3 USD/reaction), does not require sophisticated instrumentation, and has comparable assay accuracy (93.85-100% accuracy, 100% specificity, and 89.74-100% sensitivity) to bacterial DNA extraction by a commercial kit followed by PCR and gel electrophoresis (92.31% accuracy, 100% specificity, and 87.18% sensitivity) based on the real sample tests. Hence, the technique could be used in local or laboratory resource-restricted settings. The test result could be read by naked eyes through the color change from violet (negative) to sky blue (positive) for a C. Liberibacter asiaticus-infected specimen. Furthermore, this assay uses safe chemical reagents and, thus, is safe for the users.

Quantitative Risk Assessment of Susceptible and Ciprofloxacin-Resistant Salmonella from Retail Pork in Chiang Mai Province in Northern Thailand.

The adverse human health effects as a result of antimicrobial resistance have been recognized worldwide. Salmonella is a leading cause of foodborne illnesses while antimicrobial resistant (AMR) Salmonella has been isolated from foods of animal origin. The quantitative risk assessment (RA) as part of the guidelines for the risk analysis of foodborne antimicrobial resistance was issued by the Codex Alimentarius Commission more than a decade ago. However, only two risk assessments reported the human health effects of AMR Salmonella in dry-cured pork sausage and pork mince. Therefore, the objective of this study was to quantitatively evaluate the adverse health effects attributable to consuming retail pork contaminated with Salmonella using risk assessment models. The sampling frame covered pork at the fresh market (n = 100) and modern trade where pork is refrigerated (n = 50) in Chiang Mai province in northern Thailand. The predictive microbiology models were used in the steps where data were lacking. Susceptible and quinolone-resistant (QR) Salmonella were determined by antimicrobial susceptibility testing and the presence of AMR genes. The probability of mortality conditional to foodborne illness by susceptible Salmonella was modeled as the hazard characterization of susceptible and QR Salmonella. For QR Salmonella, the probabilistic prevalences from the fresh market and modern trade were 28.4 and 1.9%, respectively; the mean concentrations from the fresh market and modern trade were 346 and 0.02 colony forming units/g, respectively. The probability of illness (PI) and probability of mortality given illness (PMI) from QR Salmonella-contaminated pork at retails in Chiang Mai province were in the range of 2.2 × 10-8-3.1 × 10-4 and 3.9 × 10-10-5.4 × 10-6, respectively, while those from susceptible Salmonella contaminated-pork at retails were in the range 1.8 × 10-4-3.2 × 10-4 and 2.3 × 10-7-4.2 × 10-7, respectively. After 1000 iterations of Monte Carlo simulations of the risk assessment models, the annual mortality rates for QR salmonellosis simulated by the risk assessment models were in the range of 0-32, which is in line with the AMR adverse health effects previously reported. Therefore, the risk assessment models used in both exposure assessment and hazard characterization were applicable to evaluate the adverse health effects of AMR Salmonella spp. in Thailand.

Thermal inactivation of African swine fever virus in feed ingredients.

African swine fever virus (ASFV) causes a fatal infectious disease affecting domestic pigs and wild boars. ASFV is highly stable and easily transmitted by consumption of contaminated swine feed and pork products. Heat treatment of feed ingredients is a means to minimize the risk of contamination through swine feed consumption. The objectives of this study were to determine the thermal inactivation of ASFV in non-animal and animal origin feed ingredients. The rate of thermal inactivation is represented by decimal reduction time (DT) or time required to reduce ASFV per 1 log at temperature T. The mean D60, D70, D80 and D85 of meat and bone meal (MBM), soybean meal (SBM), and maize grain (MZ) are in the ranges 5.11-6.78, 2.19-3.01, 0.99-2.02, and 0.16-0.99 min, respectively. DT is used to compare the heat resistance of ASFV in the feed ingredient matrices. The mean DT of ASFV in MBM, SBM and MZ was not statistically significant, and the heat resistance of ASFV in MBM, SBM, and MZ was not different at 60, 70, 80, or 85 °C. The multiple DT was used to develop a DT model to predict DT at various inactivation temperatures. The DT models for MBM, SBM, and MZ are log DT = - [Formula: see text] + 2.69, log DT = - [Formula: see text] + 2.55, and log DT = - [Formula: see text] + 4.01. To expand and ease the field applications, a spreadsheet predicting the DT and the inactivation time (with 95% confidence interval) from these DT models is available to download.

Inactivation rate of African swine fever virus by a formaldehyde-based product.

African swine fever (ASF) is an important transboundary animal disease with a high mortality rate. The high African swine fever virus (ASFV) titers in the excretion of infected wild boar possibly contaminate the feed ingredient. Once contaminated, it could support persistent residual titer of the ASFV. The chemical inactivation of imported feed ingredients is a precautionary risk management measure to restrict the import risk of ASFV through international trade. The log ASFV titers were linearly reduced as a function of the inactivation time after exposure to 0.03%, 0.05%, 0.1%, and 0.2% formaldehyde-based product (FBP). A four-log reduction of ASFV titer was achieved after exposure to 0.2% FBP and 0.03%-0.1% FBP for 30-min and 60-min inactivation times, respectively. The decimal reduction time or D value is defined as the time required to inactivate the virus titer by 1 log. The ASFV inactivation rate from the independent experiment of FBP concentration was converted to a D value. The observed mean D0.2%, D0.1%, D0.05%, and D0.03% of FBP were 13.4, 44.9, 45.0, and 45.3 min per log reduction of ASFV, respectively. The interpretation of D0.2% of FBP is that the ASFV titer is inactivated by 1 log after being exposed to 0.2% FBP for every 13.4 min. A more effective chemical has a lower D value because of a shorter inactivation time required to achieve the same 1-log reduction. In addition, the hypothetical inactivation time by any chemical additive is scenario-specific and is calculated by the product of D value (at a certain concentration) and log titers of residual ASFV. This study introduces the concept and application of the D value to compare the virucidal activity of chemicals and to determine the hypothetical inactivation time of chemicals depending on the chemical concentration including the virus titer in the feed.

This study demonstrated the virucidal activity of 0.03%­0.2% formaldehyde-based product (FBP) against the African swine fever virus with cell culture. This product may have the potential to inactivate African swine fever (ASF) in feed or feed ingredients. The D value concept was lately introduced to compare the virucidal activity across various types of chemicals and different concentrations of FBP. Even though the concentrations of FBP tested were in a limited range, the decimal reduction time curve (equation) allows users to determine the exposure time of the desired virucidal activity of some other concentrations of FBP. Future studies are needed to verify viricidal activity in feed or feed ingredients along with pig bioassay to show it may or may not prevent ASF infection by feed.

Persistence of African swine fever virus on porous and non-porous fomites at environmental temperatures.

BACKGROUND: African swine fever (ASF) is a lethal contagious disease affecting both domestic pigs and wild boars. Even though it is a non-zoonotic disease, ASF causes economic loss in swine industries across continents. ASF control and eradication are almost impossible since effective vaccines and direct antiviral treatment are not available. The persistence of ASFV on fomites plays an important role in the indirect transmission of ASFV to pigs encountering ASFV-contaminated fomites. ASFV persistence on porous and non-porous fomites (glass, metal, rubber, and cellulose paper) at different environmental temperatures was determined. The persistence of ASFV of fomites was determined by the rate of ASFV inactivation in terms of DT, or the time required to reduce ASFV per 1 log at each selected environmental temperature (T). DT is used to compare the persistence of ASFV on the fomites. RESULTS: The mean D25, D33, and D42, of dried infectious ASFV on glass, metal, rubber, and paper were in the ranges 1.42-2.42, 0.72-1.94, and 0.07-0.23 days, respectively. The multiple DT were used to develop a DT model to predict the DT for some other environmental temperatures. The DT models to predict the persistence of dried infectious ASFV on glass, metal, rubber, and paper are log DT = (- T/21.51) + 1.34, log DT = (- T/20.42) + 1.47, log DT = (- T/14.91) + 2.03, and log DT = (- T/10.91) + 2.84, respectively. A spreadsheet as a quick and handy tool predicting the persistence time of dried infectious ASFV on fomites at various environmental temperatures based on these DT models is available for public to download. CONCLUSION: Persistence of dried infectious ASFV on paper are significantly the longest at lower environmental temperatures whereas that of dried infectious ASFV on paper is significantly the shortest at higher environmental temperature.

Thermal Inactivation of African Swine Fever Virus in Swill.

The indirect transmission of the African swine fever virus (ASFV) is through contaminated fomite, feed ingredients, pork- and pig-derived products, including swill, as ASFV is highly stable within suitable organic material. Some previous studies have indicated that ASFV outbreaks were associated with swill feeding, particularly in smallholder pig farms. These outbreaks emphasize the significance of the appropriate heat treatment of swill to eliminate ASFV residual titer. The World Organization for Animal Health (OIE) recommended the heat treatment of swill at a temperature of at least 90°C for at least 60 min, with continuous stirring, while the Food and Agriculture Organization (FAO) recommended heat treatment at 70°C for 30 min. The lack of scientific evidence regarding ASFV inactivation by heat treatment of swill leads to such inconsistent recommendations. Therefore, the objectives of this study were to assess the thermal inactivation of ASFV in three swill formulae and to develop a D T model to predict D T at some other inactivation temperatures. The significant reduction of ASFV in swill occurred at temperatures as low as 60°C. D T or decimal reduction time (DRT) is defined as the time required to reduce the virus titer by 1 log, and this was also used as a comparative index of heat resistance. The mean D 60, D 70, D 75, and D 80 of ASFV in three swill formulae were in the ranges 23.21-33.47, 5.83-10.91, 2.15-2.22, and 1.36-1.47 min, respectively. These D T could be widely used for any nutritive composition of swill other than the three swill formulae in this study since there was no statistical difference of all D T of ASFV across three swill formulae. Based on D 70 and the predicted D 90 from the D T model in this study, including the highest ASFV titer in pork products, the calculated inactivation times at 70 and 90°C were 119 and 4 min, respectively.

Cross-sectional risk assessment of zoonotic Streptococcus suis in pork and swine blood in Nakhon Sawan Province in northern Thailand.

A cross-sectional study evaluated the risk of zoonotic Streptococcus suis (S. suis) illness from consuming raw pork and swine blood in Nakhon Sawan Province. A four-step risk assessment recommended by the Codex Alimentarius Commission was used to evaluate the risk along the pork supply chain. A total of 480 pork and swine blood samples were collected from the abattoir (n = 120) and retail (n = 360) during December 2020 and January 2021. Streptococcus suis in samples was enumerated using a culture-based technique and then confirmed by the biochemical and molecular technique. Streptococcus suis was serotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Two positive swine blood samples were contaminated with non-zoonotic S. suis serotype 23 at retail. In the case of all negative samples, the deterministic prevalence becomes zero and then the risk could not be estimated. Otherwise, the beta probability distribution was used to describe the probabilistic prevalence, while the maximum likelihood estimator was applied to estimate the upper limit of a probability distribution of concentration. The district averages of probabilistic prevalences of zoonotic S. suis in pork products at abattoir and retail were 9.9% and 4.1%, respectively. The district averages of concentrations of zoonotic S. suis in pork and blood samples from abattoir were 6.8 × 10-3  cfu/g and 6.83 cfu/ml and in pork and blood samples from retail were 2.3 × 10-3  cfu/g and 2.30 cfu/ml, respectively. The overall annual risk estimate per 100,000 population in pork and swine blood from abattoir and retail were 9.8 × 10-11 , 2.2 × 10-6 , 5.4 × 10-13 , and 8.3 × 10-8 . These risk estimates were negligible (<10-6 ) except for the annual risk estimate in swine blood from the abattoir. The results from this cross-sectional risk assessment should prompt the food safety regulator to cautiously sample by taking into account the duration of sampling and sample size.

Phenotypic and molecular characterization of ß-lactamase and plasmid-mediated quinolone resistance genes in Klebsiella oxytoca isolated from slaughtered pigs in Thailand.

Background and Aim: Over recent years, antimicrobial-resistant Klebsiella species in humans, animals, food animals, food products, and agricultural environments have been the center of attention due to its role in the evolution of antimicrobial resistance. The emergence of resistance to fluoroquinolones and cephalosporins of third and higher generations in Klebsiella oxytoca has not received much attention in animal husbandry compared to that in Klebsiella pneumoniae. Reports on K. oxytoca are limited in the study area. Therefore, we investigated the antimicrobial susceptibility and resistance genes in K. oxytoca isolated from slaughtered pigs in Thailand. Materials and Methods: Microbiological examination was conducted on 384 Klebsiella spp. isolates recovered from slaughtered pigs in ten provinces of Thailand. Seventy-two K. oxytoca isolates (18.75%) were examined for antimicrobial-resistant genes (ß-lactamase [bla TEM, bla CTX-M, and bla SHV]) and fluoroquinolone-resistant genes (qnrA, qnrB, qnrC, qnrD, qnrS, oqxAB, aac(6')-Ib-cr, and qepA). Results: The most common genotype was bla CTX-M (58/72, 80.55%), followed by bla TEM with bla CTX-M (7/72, 9.72%) and bla TEM (6/72, 8.33%). The most common bla CTX-M group was bla CTX-M-1 (19/58, 32.76%), followed by bla CTX-M-9 (1/58, 1.72%). Plasmid-mediated quinolone resistance genes were identified in 13 (18.05%) isolates: qnrS (16.70%) and qnrB (1.4%). All 13 isolates had qnrS transferable to an Escherichia coli recipient, whereas qnrB was not detected in any transconjugants. Either bla CTX-M or bla TEM harbored by one K. oxytoca strain was transferable to an E. coli recipient. Analysis of antimicrobial susceptibility revealed that more than 90% of the bla CTX-M-carrying K. oxytoca isolates were susceptible to chloramphenicol, trimethoprim, ceftazidime, cefepime, cefotaxime, amoxicillin-clavulanic acid, piperacillin-tazobactam, and fosfomycin. All K. oxytoca isolates (13) harboring qnr were susceptible to carbapenem and ceftriaxone; however, 43 (74.13%) of the K. oxytoca isolates harboring bla CTX-M exhibited extended-spectrum ß-lactamase activity. Most of the K. oxytoca isolates from pigs were highly resistant to ampicillin, azithromycin, and gentamicin. Conclusion: To prevent further transmission of Klebsiella spp. Between food animals and humans, strict control of antibiotic use in clinical and livestock settings is necessary along with routine disinfection of the livestock environment and efforts to increase awareness of antimicrobial resistance transmission.

Quantitative Risk Assessments of Hepatitis A Virus and Hepatitis E Virus from Raw Oyster Consumption.

A quantitative risk assessment of hepatitis A virus (HAV) and hepatitis E virus (HEV) from raw oyster consumption from farm and retail was evaluated over three seasons. This risk assessment comprises four steps: hazard identification, dose-response assessment, exposure assessment, and risk characterization. We used probabilistic models for prevalence, concentration, and oyster consumption. HEV dose-response (DR) model based on HEV dosing in chimpanzees and used to perform a dose-response assessment of HEV was proposed. Both HAV and HEV were simultaneously enumerated by real-time PCR to determine viral doses. The probabilistic prevalences of HAV and HEV were in the ranges of 8-20% and 8-40%, respectively. The best-fit DR model was the beta-Poisson with alpha and N50 equal to 216.9 and 3.03 × 107 , respectively. After running the Monte Carlo simulation, the annual cases of foodborne hepatitis A and hepatitis E from raw oyster consumption from farms were 9,264-17,526 and 1-604, respectively, while those at retail were 7,694-14,591 and 1-204, respectively. This study suggested that consuming farm oysters poses a significantly higher risk of hepatitis A than hepatitis E. The best-fit DR model for HEV developed in this study could determine risks of hepatitis E from raw oyster consumption in Thailand.

Klebsiella pneumoniae Complex Harboring mcr-1, mcr-7, and mcr-8 Isolates from Slaughtered Pigs in Thailand.

Dissemination of the mobile colistin resistance gene mcr in Enterobacterales among humans, animals, and the environment is a public health issue. We characterized mcr genes in the Klebsiella pneumoniae complex (KpnC) isolated from slaughtered pigs in Thailand. The 280 KpnCs consisted of K. pneumoniae (85%), Klebsiella quasipneumoniae (8.21%), and Klebsiella variicola (6.79%). mcr genes were detected in 6.79% (19/280) of KpnC isolates, consisting of mcr-8 (n = 9; 3.21%), mcr-7 (n = 7; 2.50%), mcr-7 + mcr-8 (n = 2; 0.71%), and mcr-1 + mcr-7 (n = 1; 0.36%). K. pneumoniae predominantly carried the mcr-7 and mcr-8 genes, while K. variicola and K. quasipneumoniae harbored mcr-7 and mcr-8, respectively. Six of the nineteen mcr-harboring KpnC isolates exhibited colistin resistance, and five had mcr-1 or mcr-8 transferable to an Escherichia coli recipient. Antimicrobial susceptibility analysis revealed that all mcr-carrying KpnC isolates were susceptible to carbapenems, cefotaxime, cefepime, amoxicillin/clavulanic acid, piperacillin/tazobactam, amikacin, and fosfomycin, and had high resistance to azithromycin. Multilocus sequence analysis demonstrated that the mcr-harboring KpnC isolates were genetically diverse. A 'One-Health' approach is useful to combat antimicrobial-resistant bacteria through coordinating the human, animal, and environmental sectors. Hence, continuous monitoring and surveillance of mcr-carrying KpnCs throughout the pork supply chain is crucial for ensuring public health.

Application of phage display technology for the production of antibodies against Streptococcus suis serotype 2.

Streptococcus suis (S. suis) serotype 2 infection is a problem in the swine industry and responsible for most cases of human infection worldwide. Since current multiplex PCR cannot differentiate between serotypes 2 and 1/2, then serotype-specific antibodies (Abs) are required for serotype identification to confirm infection by serotype 2. This study aimed to generate Abs specific to S. suis serotype 2 by phage display from a human heavy chain variable domain (VH) antibody library. For biopanning, whole cells of S. suis serotype 2 were used as the target antigen. With increasing selection stringency, we could select the VH Abs that specifically bound to a S. suis serotype 2 surface antigen, which was identified as the capsular polysaccharide (CPS). From ELISA analysis, the specific phage clone 47B3 VH with the highest binding activity to S. suis serotype 2 was selected and shown to have no cross-reactivity with S. suis serotypes 1/2, 1, and 14 that shared a common epitope with serotype 2 and occasionally cause infections in human. Moreover, no cross-reactivity with other bacteria that can be found in septic blood specimens was also observed. Then, 47B3 VH was successfully expressed as soluble 47B3 VH in E. coli TG1. The soluble 47B3 VH crude extract was further tested for its binding ability in a dose-dependent ELISA assay. The results indicated that the activity of phage clone 47B3 was still retained even when the Ab occurred in the soluble form. A quellung reaction demonstrated that the soluble 47B3 VH Ab could show bioactivity by differentiation between S. suis serotypes 2 and 1/2. Thus, it will be beneficial to use this VH Ab in the diagnosis of disease or discrimination of S. suis serotypes Furthermore, the results described here could motivate the use of phage display VH platform to produce serotyping antibodies.

Peptidomics Analysis of Virulent Peptides Involved in Streptococcus suis Pathogenesis.

Streptococcus suis (S. suis) is a zoonotic pathogen causing severe streptococcal disease worldwide. S. suis infections in pigs and humans are frequently associated with the virulent S. suis serotype 2 (SS2). Though various virulence factors of S. suis have been proposed, most of them were not essentially accounted for in the experimental infections. In the present study, we compared the peptidomes of highly virulent SS2 and SS14 in humans, the swine causative serotypes SS7 and SS9, and the rarely reported serotypes SS25 and SS27, and they were cultured in a specified culture medium containing whole blood to simulate their natural host environment. LC-MS/MS could identify 22 unique peptides expressed in the six S. suis serotypes. Under the host-simulated environment, peptides from the ABC-type phosphate transport system (SSU05_1106) and 30S ribosomal protein S2 (rpsB) were detected in the peptidome of virulent SS2 and SS14. Therefore, we suggest that these two proteins or their derived peptides might be involved in the survival of S. suis when simulated with a blood environment.

Fluoroquinolone resistance in non-typhoidal Salmonella enterica isolated from slaughtered pigs in Thailand.

Introduction. The emergence and spread of non-typhoidal Salmonella enterica (NTS) serovars resistant to fluoroquinolones and third- and higher-generation cephalosporins is a matter of great concern. Antimicrobial-resistant NTS is increasingly being discovered in humans, animals, food animals, food products, and agricultural environments. Pigs are considered a major reservoir of antimicrobial-resistant Salmonella spp.Hypothesis/Gap Statement. Fluoroquinolone-resistant Salmonella spp. warrant further surveillance and characterization for a better understanding of the bacteria isolated from animals.Aim. NTS isolated from pork from slaughterhouses across Thailand were characterized in terms of their serovars; resistance to fluoroquinolones, third-generation cephalosporins, and carbapenems; and antimicrobial resistance genes.Methodology. A total of 387 NTS isolates, collected from slaughtered pigs in ten provinces across Thailand between 2014 and 2015, were characterized based on their serovars, antimicrobial resistance genes, and susceptibility to fluoroquinolones, third-generation cephalosporins, and carbapenems.Results. Among all NTS isolates, S. enterica serovar Rissen was predominant. Antimicrobial resistance was exhibited in 93/387 isolates (24 %). Although 24 (6.2 %) isolates were susceptible to all the tested antimicrobials, they were found to possess ß-lactamase genes, such as bla TEM, bla SHV, or bla CTX-M. Mobilized colistin-resistant genes (mcr) and resistance to colistin were not observed in any tested isolate. Carbapenem resistance was detected in ten isolates (10.7 %); however, bla KPC, bla NDM, bla OXA-48-like, and bla IMP were not present. Among the 93 antimicrobial-resistant isolates, 87.1 % showed fluoroquinolone resistance with the quinolone resistance gene (qnrS) combined with topoisomerase genes parC (T57S) or gyrA (S83E/Y and D124E/G) substitutions, or topoisomerase gene substitutions alone.Conclusion. We found high fluoroquinolone resistance rates among the NTS isolates from pigs from slaughterhouses. The fluoroquinolone resistance mechanism in NTS was associated with the combination of qnrS and substitutions in gyrA, parC, or both. To prevent the transmission of antimicrobial-resistant NTS between animals and humans, continuous monitoring, surveillance, and regulation of Salmonella in the pork supply chain are pivotal.

Streptococcus suis serotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

Distribution and Molecular Characterization of Escherichia coli Harboring mcr Genes Isolated from Slaughtered Pigs in Thailand.

The resistance of Enterobacteriaceae to colistin mediated by plasmid-borne mobile mcr genes is an emerging public health concern. This study aimed to explore the distribution and characteristics of Escherichia coli isolates harboring mcr genes from slaughtered pigs in Thailand from 2014 to 2015. A total of 779 E. coli isolates were assessed, of which 61 (7.8%) were found to carry mcr genes, including mcr-1, mcr-3, mcr-6, mcr-7, mcr-8, and mcr-9, together with co-occurrences of mcr-1+mcr-3, mcr-1+mcr-9, and mcr-3+mcr-6+mcr-7. In these mcr-harboring E. coli isolates, mcr-1 (40.9%) and mcr-9 (32.8%) were predominant. Colistin resistance was mainly mediated by the mcr-1 gene, whereas intermediate resistance was noted in isolates that harbored mcr-9, mcr-6, mcr-7, and mcr-8 genes. Most E. coli isolates harboring mcr genes were susceptible to third-generation cephalosporins and all of these isolates were susceptible to carbapenems. Clermont phylotyping demonstrated that mcr-harboring isolates mainly belonged to phylogroup A (44.3%), followed by phylogroups B1 (34.4%), D (14.8%), and B2 (6.6%). Multilocus sequence typing revealed that 25 sequence types (STs) were assigned to 45 mcr-harboring E. coli isolates, whereas the remaining 16 isolates were novel STs. The mcr-1 and mcr-9 genes were mostly predominant in ST101 and ST8900, respectively. This study provides a comprehensive insight into the prevalence and diversity of mcr-harboring E. coli isolates obtained from slaughtered pigs across Thailand. Strengthening of surveillance systems by the government for controlling and preventing mcr dissemination from animals to humans or vice versa is urgently needed. No clinical trial registration number.

Prevalence and quantification of pathogenic Vibrio parahaemolyticus during shrimp culture in Thailand.

Vibrio parahaemolyticus is a major cause of seafood-borne gastroenteritis. The human pathogenic strains possess tdh or trh or both genes. In Thai shrimp farming, the level of pathogenic V. parahaemolyticus contamination has not been completely characterized, although it has been identified as a risk for people who consume undercooked shrimp. In this study, the prevalence and concentration of V. parahaemolyticus (total Vp) and pathogenic V. parahaemolyticus (tdh+ Vp and trh+ Vp) were investigated during shrimp culture cycles using the most probable number (MPN) method and were confirmed by PCR and the loop-mediated isothermal amplification (LAMP) techniques. The prevalence and concentration of total Vp were high in broodstock and egg samples at the start of the hatchery cycle, but the organism decreased in the subsequent larval and postlarval stages. In contrast, total Vp was low at the beginning of the pond cycle and dramatically increased during the later stages of culture. Broodstock and fresh feed were important sources of V. parahaemolyticus. Numbers of tdh+ Vp and trh+ Vp detected by the LAMP technique were much greater than those detected by the PCR technique, especially in the late stages of the pond cycle. A direct correlation between total Vp and pathogenic Vp was demonstrated only during the harvest stage. This study will be useful as a guideline to establish levels of V. parahaemolyticus presence which can be considered as safe during shrimp culture. In addition, it could be used to identify the source of V. parahaemolyticus, which has recently been reported to be one of the etiologic agents of acute hepatopancreatic necrosis disease.