Ter Hsin Chen

Mutations in the Salmonella enterica serovar Choleraesuis cAMP-receptor protein gene lead to functional defects in the SPI-1 Type III secretion system.

Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis) causes a lethal systemic infection (salmonellosis) in swine. Live attenuated Salmonella Choleraesuis vaccines are effective in preventing the disease, and isolates of Salmonella Choleraesuis with mutations in the cAMP-receptor protein (CRP) gene (Salmonella Choleraesuis ∆crp) are the most widely used, although the basis of the attenuation remains unclear. The objective of this study was to determine if the attenuated phenotype of Salmonella Choleraesuis ∆crp was due to alterations in susceptibility to gastrointestinal factors such as pH and bile salts, ability to colonize or invade the intestine, or cytotoxicity for macrophages. Compared with the parental strain, the survival rate of Salmonella Choleraesuis ∆crp at low pH or in the presence of bile salts was higher, while the ability of the mutant to invade intestinal epithelia was significantly decreased. In examining the role of CRP on the secretory function of the Salmonella pathogenicity island 1 (SPI-1) encoded type III secretion system (T3SS), it was shown that Salmonella Choleraesuis ∆crp was unable to secrete the SPI-1 T3SS effector proteins, SopB and SipB, which play a role in Salmonella intestinal invasiveness and macrophage cytotoxicity, respectively. In addition, caspase-1 dependent cytotoxicity for macrophages was significantly reduced in Salmonella Choleraesuis ∆crp. Collectively, this study demonstrates that the CRP affects the secretory function of SPI-1 T3SS and the resulting ability to invade the host intestinal epithelium, which is a critical element in the pathogenesis of Salmonella Choleraesuis.

Transmission of Salmonella between swine farms by the housefly (Musca domestica).

The domestic pig is an important source of human salmonellosis, and houseflies are potential mechanical vectors of foodborne Salmonella pathogens. In 2005, we recovered 144 Salmonella isolates from flies and swine stool samples from 11 farms in Taoyuan County and Hsin Chu County (northwestern Taiwan). A total of 71.5% of the isolates were resistant to at least three antibiotics. There were a total of 14 serotypes, and 8 of these serotypes were present in both flies and swine stool samples. Some multidrug-resistant Salmonella strains coming from different swine farms were found to have identical pulsed-field gel electrophoresis (PFGE). Among four common serotypes, we identified 18 PFGE patterns, 8 of which were present in flies and swine stools. The similarity in PFGE profiles between isolates from swine and flies in different farms indicate the potential of flies to serve as a vector for transmission.

Molecular characterization of enrofloxacin resistant Actinobacillus pleuropneumoniae isolates.

Enrofloxacin (ER) resistant Actinobacillus pleuropneumoniae strains emerged in Taiwan in 2002. The mechanism of ER resistance in A. pleuropneumoniae has not yet been reported. A total of 48 A. pleuropneumoniae isolates were obtained from the lungs of pigs with pleuropneumonia in Taiwan between September 2007 and April 2008. Twenty-nine isolates were found to be resistant to enrofloxacin. To understand the mechanisms of A. pleuropneumoniaes resistance to ER, enrofloxacin susceptibility of the isolates along with the mutations of the quinolone resistance-determining region (QRDR) of DNA gyrase and topoisomerase IV, qnr genes were analyzed. Enrofloxacin resistant isolates were found to carry at least one mutation in the QRDR of gyrA, leading to amino acid changes at codon 83 or 87. Efflux pump inhibitor (Phe-Arg-beta-naphthylamide) decreased enrofloxacin minimum inhibitory concentration 2-16-fold, suggesting participation of efflux in ER resistance. Plasmid mediated quinolone resistance genes qnr were not detected in these isolates. In conclusion, enrofloxacin resistance of A. pleuropneumoniae may be linked to multiple target gene mutations and active effluxs.

Serotype occurrence and antimicrobial susceptibility of Salmonella isolates recovered from pork carcasses in Taiwan (2000 through 2003)

One hundred fifty-eight Salmonella strains isolated from pork carcasses in a nationwide screening program in Taiwan from 2000 through 2003 were analyzed for serotype distribution and antimicrobial susceptibility. Twenty Salmonella serotypes were obtained, among which Derby, Anatum, Typhimurium, and Schwarzengrund were the most frequently isolated, accounting for 76% of the strains. Antimicrobial susceptibility tests with the microdilution method were performed on these serotypes to determine the MIC. All strains tested were sensitive to ceftriaxone, with an MIC90 (minimum concentration inhibiting 90% of isolates tested) of 0.25 to 8 microg/ml. More than 60% of the strains were resistant to ampicillin, chloramphenicol, tetracycline, nalidixic acid, and sulfamethoxazole, with MIC90 values of 128 to >512 microg/ml. More than 80% of the Salmonella Schwarzengrund strains were resistant to ciprofloxacin (MIC90 = 8 microg/ml) and enrofloxacin (MIC90 = 16 microg/ml). The Salmonella Typhimurium strains exhibited 17 and 23% resistance to ciprofloxacin and enrofloxacin, respectively, with an MIC90 of 8 microg/ ml, and these two antibiotics also were active against Salmonella Derby and Salmonella Anatum. Cephalothin, gentamicin, and trimethoprim had limited activity against Salmonella Anatum and Salmonella Schwarzengrund, with MIC90 values of 256 to >512 microg/ml. Cephalothin and gentamicin were moderately active against Salmonella Derby and Salmonella Typhimurium, but 30 to 40% of these strains were resistant to trimethoprim. The Salmonella strains isolated from pork carcasses in Taiwan were relatively resistant to the antimicrobial agents tested, with the exception of ceftriaxone. Although a variety of MIC values were obtained, generally these values were high.

Actinobacillus pleuropneumoniae serotype 10 derived ApxI induces apoptosis in porcine alveolar macrophages

Actinobacillus pleuropneumoniae (AP) is the causative agent of swine pleuropneumonia, a fibrinous, exudative, hemorrhagic, necrotizing pleuropneumonia affecting all ages of pigs. Actinobacillus pleuropneumoniae exotoxins (Apx) are one of the major virulence factors of AP. Due to the complex nature of Apx toxins produced by AP, little is known regarding the interactions of individual species of Apx toxin with target cells. The objective of this study was to examine whether AP serotype 10-derived exotoxin, ApxI, caused apoptosis in porcine alveolar macrophages (PAMs) and to delineate the underlying signaling pathways. Isolated PAMs were stimulated with different concentrations of native ApxI and monitored for apoptosis using Hoechst staining, TUNEL, and DNA laddering assays. The ApxI-stimulated PAMs exhibited typical morphological features of apoptosis, including condensation of chromatin, formation of apoptotic bodies and DNA laddering. ApxI-induced apoptosis in a concentration- and time-dependent manner. Furthermore, to delineate the signaling events involved in ApxI-induced apoptosis, it was observed that caspase 3 was activated in ApxI-stimulated PAMs. Ablation of caspase 3 activity via specific inhibitors protected PAMs from apoptosis by ApxI. This study is the first to demonstrate that native ApxI causes apoptosis in PAMs at low concentrations and that these apoptotic events are mediated via a caspase 3-dependent pathway. These findings suggest a role of ApxI in AP infection as it might impair the host defense system through the induction of apoptosis in PAMs.

Efficacy of a novel Pasteurella multocida vaccine against progressive atrophic rhinitis of swine

The efficacy of a novel vaccine composed of three short recombinant subunit Pasteurella multocida toxin (PMT) proteins in combination with a bi-valent P. multocida whole-cell bacterin (rsPMT-PM) was evaluated in field studies for prevention and control of progressive atrophic rhinitis (PAR) of swine at 15 conventional farrow-to-finish farms. Experimental piglets that were immunized twice with the rsPMT-PM vaccine developed detectable titers of neutralizing antibodies (greater than 1:8) that prevented the growth retardation and pathological lesions typically observed following challenge with authentic PMT. A total of 542 sows were vaccinated once or twice prior to parturition and serum neutralizing antibody titers were evaluated. Both single and double vaccination protocols induced neutralizing antibody titers of 1:16 or higher in 62% and 74% of sows, respectively. Notably, neither sows nor piglets at a farm experiencing a severe outbreak of PAR at the time of the vaccination trial had detectable antibody titers, but antibody titers increased significantly to 1:16 or higher in 40% of sows following double vaccination. During the year after vaccination, clinical signs of PAR decreased in fattening pigs and growth performance improved sufficiently to reduce the rearing period until marketing by 2 weeks. Collectively, these results indicate that the rsPMT-PM vaccine could be used to provide protective immunity for controlling the prevalence and severity of PAR among farm-raised swine.

Kidney stone distribution caused by melamine and cyanuric acid in rats

BACKGROUND Melamine (M), which is composed of multi-amine, has been used as a food additive to falsely increase protein contents. Furthermore, cyanuric acid (CA) is a derivative of melamine. It is known that these mixtures can cause renal toxicity. METHODS The objective of this study was to investigate the possible target cells during acute renal toxicity of melamine and cyanuric acid (MCA) mixture crystals in vivo. Rats were provided with a lethal dose of MCA (1:1; 400mg/kg) and observed after 0.5, 1, 3, 12, 24, and 48-h intervals. RESULTS MCA caused degeneration/necrosis in the proximal tubules starting at 12h and increased at 24 and 48 h. A small number of yellow-green crystals were observed in the dilated distal renal tubules at 48 h post-treatment. Ultrastructurally, pyknosis, mitochondrial vesicles, and cellular swelling were found in the proximal tubular cells at 0.5h. Small needle-like crystals in the cytoplasm and large crystals in the lumen of tubules indicated physical damage to the renal cells. CONCLUSION These results clearly reveal that in the MCA-induced renal toxicity model, crystals are distributed to both the proximal and distal tubules in rats. The proximal tubular cells may be initially injured and subsequently block the distal tubules with MCA crystals during early acute intoxication.

Analysis of Ciprofloxacin-Resistant Salmonella Strains from Swine, Chicken, and Their Carcasses in Taiwan and Detection of parC Resistance Mutations by a Mismatch Amplification Mutation Assay PCR

One hundred thirty-three Salmonella strains isolated from the viscera of swine, chicken, and carcasses of swine and chicken in Taiwan from 2004 to 2006 were identified to serotype level and analyzed for their susceptibility to ciprofloxacin. In total, 76 (57%) strains of the Salmonella isolates exhibited high-level resistance to ciprofloxacin, having MICs ranging from 16 to 64 microg/ml. DNA sequence analysis revealed that mutations in the quinolone resistance-determining regions of gyrA (Ser83Phe, Asp87Gly or Asp87Asn), parC (Ser80Arg, or Ser80Ile or Glu84Lys), and parE (Ser458Pro) genes were associated with the Salmonella strains that demonstrated resistance to ciprofloxacin. A mismatch amplification mutation assay (MAMA)-PCR was developed to identify mutations in parC at codons 80 and 84. Specific PCR products were only recovered from ciprofloxacin-resistant Salmonella strains but not from the susceptible strains. MAMA-PCR targeting the mutations in parC correlated with what DNA sequencing revealed. In conclusion, monitoring ciprofloxacin-resistant Salmonella from animal sources should be performed on a regular basis. MAMA-PCR targeting parC could provide a fast method for those laboratories interested in quickly characterizing the resistance profile and with little access to DNA sequencing facilities.

A low-pH medium in vitro or the environment within a macrophage decreases the transcriptional levels of fimA, fimZ and lrp in Salmonella enterica serovar Typhimurium.

Many Salmonella Typhimurium isolates produce type 1 fimbriae and exhibit fimbrial phase variation in vitro. Static broth culture favours the production of fimbriae, while solid agar medium inhibits the generation of these appendages. Little information is available regarding whether S. Typhimurium continues to produce type 1 fimbriae during in vivo growth. We used a type 1 fimbrial phase-variable strain S. Typhimurium LB5010 and its derivatives to infect RAW 264.7 macrophages. Following entry into macrophages, S. Typhimurium LB5010 gradually decreased the transcript levels of fimbrial subunit gene fimA, positive regulatory gene fimZ, and global regulatory gene lrp. A similar decrease in transcript levels was detected by RT-PCR when the pH of static broth medium was shifted from pH 7 to a more acidic pH 4. A fimA-deleted strain continued to multiply within macrophages as did the parental strain. An lrp deletion strain was unimpaired for in vitro growth at pH 7 or pH 4, while a strain harboring an lrp-containing plasmid exhibited impaired in vitro growth at pH 4. We propose that acidic medium, which resembles one aspect of the intracellular environment in a macrophage, inhibits type 1 fimbrial production by down-regulation of the expression of lrp, fimZ and fimA.

Association of MRC-1 and IL-28B with the treatment outcome of hepatitis C: a case control study

BackgroundThe aim of this study was to evaluate whether polymorphisms of the mannose receptor C type 1 (MRC-1) and interleukin 28B (IL-28B) genes are associated with the treatment outcome of patients infected with hepatitis C virus genotypes 1 and 2 (HCV-1 and HCV-2, respectively) who are treated with peginterferon plus ribavirin (PEG-IFNα-RBV).MethodsWe analyzed the association of the patients’ sustained viral responses (SVRs) to PEG-IFNα-RBV therapy with 2 single nucleotide polymorphisms (SNPs) in MRC-1 and 3 SNPs in IL-28B. We selected patients infected with either HCV-1 (n = 265) or HCV-2 (n = 195) with or without SVR.ResultsAmong the MRC-1 SNPs, rs691005 was found to be associated with SVR in HCV-1-infected patients (P < 0.0001). The IL-28B rs8099917 SNP was found to be associated with SVR in HCV-1- and HCV-2-infected patients (HCV-1, P < 0.0001; HCV-2, P = 0.002), while IL-28B rs955155 and rs10853728 SNPs were found to be associated with SVR in HCV-1-infected patients (P = 0.003) and HCV-2-infected patients (P = 0.02), respectively. We also identified an interaction between MRC-1 rs691005 and IL-28B rs8099917 (P = 0.001). The C-T haplotype was shown to have a positive effect on SVR in HCV-1-infected patients (OR = 1.77, 95% CI = 1.2, 2.62), whereas the T-G haplotype was shown to have a negative effect on SVR in HCV-1-infected patients (OR = 0.28, 95% CI = 0.14, 0.58).ConclusionsThese results suggest that SNPs of IL-28B and MRC-1 can be used as genetic markers for predicting the outcome of PEG-IFNα-RBV treatment of HCV infections.