Feline mammary carcinoma-derived extracellular vesicle promotes liver metastasis via sphingosine kinase-1-mediated premetastatic niche formation.

BACKGROUND: Feline mammary carcinoma (FMC) is one of the most prevalent malignancies of female cats. FMC is highly metastatic and thus leads to poor disease outcomes. Among all metastases, liver metastasis occurs in about 25% of FMC patients. However, the mechanism underlying hepatic metastasis of FMC remains largely uncharacterized. RESULTS: Herein, we demonstrate that FMC-derived extracellular vesicles (FMC-EVs) promotes the liver metastasis of FMC by activating hepatic stellate cells (HSCs) to prime a hepatic premetastatic niche (PMN). Moreover, we provide evidence that sphingosine kinase 1 (SK1) delivered by FMC-EV was pivotal for the activation of HSC and the formation of hepatic PMN. Depletion of SK1 impaired cargo sorting in FMC-EV and the EV-potentiated HSC activation, and abolished hepatic colonization of FMC cells. CONCLUSIONS: Taken together, our findings uncover a previously uncharacterized mechanism underlying liver-metastasis of FMC and provide new insights into prognosis and treatment of this feline malignancy.

Reevaluation of Hemoparasites in the Black Spiny-Tailed Iguana (Ctenosaura similis) with the First Pathological and Molecular Characterizations of Lankesterella desseri n. sp. and Redescription of Hepatozoon gamezi.

Hemoprotozoa are microorganisms that parasitize the blood and possess intricate life cycles. Despite the complexity of their nature, little is known about the biology of hemoprotozoa in reptilian hosts. In this study, we conducted disease surveillance on blood samples collected from six black spiny-tailed iguanas (Ctenosaura similis) exhibiting clinical signs. We found two different types of hemoparasites in the blood films and further confirmed they belong to the genera Lakesterella and Hepatozoon through molecular methods. In the tissue section from a dead iguana infected only with Lakesterella sp., parasites were also found in melanomacrophages of the liver and kidney. Since Lakesterella sp. infection has not been reported in C. similis, we propose this hemococcidian as a new species, Lankesterella desseri n. sp. The Hepatozoon parasites discovered in this study were classified as Hepatozoon gamezi based on their morphological characteristics, particularly the notable deformation of all infected erythrocytes, and this classification was further corroborated through molecular biological and phylogenetic analyses. This is the first hemoprotozoa investigation in C. similis with pathological and molecular characterization of these pathogens. We suggest that more studies are needed to understand the epidemiology, transmission, and impact of these parasites on their hosts and ecosystems.

Gastrointestinal Mast Cell Tumor in an African Dormouse (Graphiurus sp.).

Mast cell tumors (MCTs) are well-known neoplasms derived from either mucosal or connective tissue mast cells. While well studied in several domestic species, MCTs are rarely documented in rodents. A three-year-old, male African dormouse (Graphiurus sp.) presented with a history of vomiting and anorexia for 3 months. Sonography revealed thickened gastric mucosa and hyperperistalsis. The patient died after receiving symptomatic treatment for 2 months. At necropsy, locally extensive, pale, thickened mucosal foci obscuring the first half of the stomach lumen was noted. Histological examination revealed moderately polymorphic, round, oval to spindle cells with amphophilic cytoplasmic granules infiltrating the mucosa to tunica muscularis, with moderate numbers of eosinophils. The mucosa was severely ulcerated with the proliferation of granulation tissue. The granules in most tumor cells exhibited metachromasia with the toluidine blue stain. Neoplastic cells revealed positive membranous immunoreactivity to KIT. Herein, we report the first case report of MCT in dormouse but also the first gastrointestinal MCT in a rodent species.

Antimicrobial resistance genes and genetic characteristics of multidrug-resistant Escherichia coli in a veterinary hospital in Taiwan.

Introduction. Antimicrobial resistance associated with animal hosts is easily transmitted to humans either by direct contact with resistant organisms or by transferring resistance genes into human pathogens.Gap statement. There are limited studies on antimicrobial resistance genes and genetic elements of multidrug-resistant (MDR) Escherichia coli in veterinary hospitals in Taiwan.Aim. The aim of this study was to investigate antimicrobial resistance genes in multidrug-resistant Escherichia coli from animals.Methodology. Between January 2014 and August 2015, 95 multidrug-resistant Escherichia coli isolates were obtained from pigs (n=66), avians (n=18), and other animals (n=11) in a veterinary hospital in Taiwan. Susceptibility testing to 24 antimicrobial agents of 14 antimicrobial classes was performed. Antimicrobial resistance genes, integrons, and insertion sequences were analysed by polymerase chain reaction and nucleotide sequencing. Pulsed-field gel electrophoresis (PFGE), and multi-locus sequence typing were used to explore the clonal relatedness of the study isolates.Results. Different antimicrobial resistance genes found in these isolates were associated with resistance to ß-lactams, tetracycline, phenicols, sulfonamides, and aminoglycosides. Fifty-five of 95 E. coli isolates (55/95, 57.9 %) were not susceptible to extended-spectrum cephalosporins, and bla CTX-M-55 (11/55, 20.0 %) and bla CMY-2 (40/55, 72.7 %) were the most common extended-spectrum ß-lactamase (ESBL) and AmpC genes, respectively. Both bla CTX-M and bla CMY-2 were present on conjugative plasmids that contained the insertion sequence ISEcp1 upstream of the bla genes. Plasmid-mediated FOX-3 ß-lactamase-producing E. coli was first identified in Taiwan. Forty isolates (40/95, 42 %) with class 1 integrons showed seven resistance phenotypes. Genotyping of 95 E. coli isolates revealed 91 different XbaI pulsotypes and 52 different sequence types. PFGE analysis revealed no clonal outbreaks in our study isolates.Conclusion. This study showed a high diversity of antimicrobial resistance genes and genotypes among MDR E. coli isolated from diseased livestock in Taiwan. To our knowledge, this is the first report of plasmid-mediated ESBL in FOX-3 ß-lactamase-producing E. coli isolates in Taiwan. MDR E. coli isolates from animal origins may contaminate the environment, resulting in public health concerns, indicating that MDR isolates from animals need to be continuously investigated.

Establishment and Characterization of Feline Mammary Tumor Patient-Derived Xenograft Model.

Feline mammary tumor is a relatively commonly diagnosed neoplasm in the cat. Development of new veterinary cancer therapies is limited by the shortage of in vivo models that reproduce tumor microenvironment and metastatic progression. Four feline mammary tumor orthotopic patient-derived xenograft model (PDX) successfully established in NOD-SCID gamma (NSG) mice. The overall success rate of PDX establishment was 36% (4/11). Histological, immunohistochemical, and short tandem repeat analysis showed a remarkable similarity between patient's tumor and xenograft. The tumor grafts conserve original tumor essential features, including distant metastasis. Primary FMT-1807 cell line isolated from FMT-1807PDX tumor tissue. Tumorigenicity of FMT-1807 cells expanded from PDX was assessed by orthotopic injection into NSG mice. Mice yielded tumors which preserve the lung and liver metastasis ability. This work provides a platform for FMT translational investigation.

Toll-like receptor 4 prevents AOM/DSS-induced colitis-associated colorectal cancer in Bacteroides fragilis gnotobiotic mice.

Bacteroides fragilis (BF) plays a critical role in developing and maintaining the mammalian immune system. We previously found that BF colonization could prevent inflammation and tumor formation in a germ-free (GF) colitis-associated colorectal cancer (CAC) mouse model. The role of Toll-like receptor 4 (TLR4) in CAC development has not been clearly elucidated in BF mono-colonized gnotobiotic mice. The wild-type (WT) and TLR4 knockout (T4K) germ-free mice were raised with or without BF colonization for 28 days (GF/WT, GF/T4K, BF/WT, and BF/T4K) and then CAC was induced under azoxymethane (AOM)/dextran sulfate sodium (DSS) administration. The results showed that tumor formation and tumor incidence were significantly inhibited in the BF/WT group compared to those observed in the GF/WT group. However, the tumor prevention effect was not observed in the BF/T4K group unlike in the BF/WT group. Moreover, the CAC histological severity of the BF/WT group was ameliorated, but more severe lesions were found in the GF/WT, GF/T4K, and BF/T4K groups. Immunohistochemistry showed decreased cell proliferation (PCNA, ß-catenin) and inflammatory markers (iNOS) in the BF/WT group compared to those in the BF/T4K group. Taken together, BF mono-colonization of GF mice might prevent CAC via the TLR4 signal pathway.

Autochthonous Lactic Acid Bacteria Isolated From Dairy Cow Feces Exhibiting Promising Probiotic Properties and in vitro Antibacterial Activity Against Foodborne Pathogens in Cattle.

Bovine enteric bacterial pathogens are a major cause of health decline in agricultural cattle populations. The identification of host-derived microbiota with probiotic characteristics is key for the development of treatments utilizing pathogen displacement and recolonization by commensal flora. In this study, intestinal microbiota in fecal samples from four Holstein dairy cows were analyzed using 16S ribosomal RNA gene next-generation sequencing, leading to the identification of three Lactobacillus isolates (Lactobacillus gasseri, Lactobacillus reuteri, and Lactobacillus salivarius). By taking advantage of the preferential growth in acidified culture media, bacterial characteristics examination, and restriction fragment length polymorphism analysis of 16S rRNA genes, the three lactic acid bacteria (LAB) strains were successfully isolated. The three LAB isolates possess the prerequisite growth tolerances for probiotic functionality, as well as exhibit effective antimicrobial potency against enteric bacterial pathogens of cattle, including Escherichia coli O157:H7, Mycobacterium avium subspecies paratuberculosis, and Salmonella species (Salmonella enteritidis, Salmonella typhimurium, and Salmonella Dublin). Moreover, the LAB isolates showed significant adhesion to cattle intestine, implying greater survivability potential due to their species specificity when administered in the same host species. The LAB isolates were sensitive to most antibiotics with notable resistances of L. gasseri to streptomycin and L. salivarius to kanamycin. Genes attributed to specific antibiotic resistances demonstrated a low risk of lateral transfer in a conjugation study. Our in vitro results demonstrate the promising probiotic characteristics of these newly identified Lactobacillus strains and their considerable potential to serve as probiotics feed supplements for cows.

Housing condition-associated changes in gut microbiota further affect the host response to diet-induced nonalcoholic fatty liver.

Diet-induced obesity is the most widely used animal model for studying nonalcoholic fatty liver disease (NAFLD). However, the physiological effects of a high-fat diet (HFD) are inconsistent between different studies. To elucidate this mystery, mice raised with conventional (CONV), specific pathogen-free (SPF) and gentamicin (G) treatments and fed with standard diet (STD) or HFD were analyzed in terms of their physiology, gut microbiota composition, hepatic steatosis and inflammation. Serum biochemistry showed increased levels of cholesterol and aspartate aminotransferase in the G-STD and CONV-HFD groups, respectively. The CONV-HFD group exhibited more inflammatory foci compared to the SPF-HFD and G-HFD groups. Furthermore, immunohistochemistry staining revealed the infiltration of Kupffer cells in the liver, consistent with increased mRNA levels of MCP-1, CD36 and TLR4. Principal coordinate analysis and the cladogram of LEfSe showed that the distinguished clusters of gut microbiota were dependent on housing conditions. The Rikenellaceae, F16 and Desulfovibrionaceae were strongly correlated with hepatic inflammation. Otherwise, higher NAFLD activity score correlated with altered relative abundances of Bacteroidetes and Firmicutes. In conclusion, gut microbiota varying with housing condition may be pivotal for the host response to HFD.

Gnotobiotic mice inoculated with Firmicutes, but not Bacteroidetes, deteriorate nonalcoholic fatty liver disease severity by modulating hepatic lipid metabolism.

Nonalcoholic fatty liver disease (NAFLD) is a serious liver disorder and characterized by the hepatic accumulation of excess fatty acids. Clinical studies and animal models have shown a shift of gut microbiota from bacteroidetes to firmicutes in NAFLD patients and a diet-induced NAFLD mouse model. Therefore, we hypothesized that these 2 groups of bacteria may have differential effects on lipid metabolism in the liver, which further contributed to pathogenesis of NAFLD. To elucidate these effects, we inoculated two species of Bacteroidetes (B-group) or five species of Firmicutes (F-group) which were isolated from healthy individuals into germ-free mice. We found that the F-group induced elevated body weight, liver weight, and hepatic steatosis compared to the B-group under high-fat diet (HFD) conditions. The mRNA expression level of cluster of differentiation 36 (CD36) was elevated in the F-group compared to that in the B-group. Increased mRNA expression levels of fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD1), and diacylglycerol O-acyltransferase 2 (DGAT2) were also seen under HFD conditions in the F-group compared to that in the B-group. In addition, the expression level of miR802-5p was only elevated in the F-group under HFD conditions. Taken together, our results suggested that these specific species of Firmicutes may induce more hepatic steatosis by modulating fatty acid influx and lipogenesis compared to those of Bacteroidetes. These results may provide more understanding of the effects of gut microbiota on NAFLD.

Functional and structural investigations of fibronectin-binding protein Apa from Mycobacterium tuberculosis.

BACKGROUND: Alanine and proline-rich protein (Apa) is a secreted antigen of Mycobacterium spp. which involves in stimulating immune responses and adhering to host cells by binding to fibronectin (Fn). Here, we report the crystal structure of Apa from Mycobacterium tuberculosis (Mtb) and its Fn-binding characteristics. METHODS: The crystal structure of Mtb Apa was determined at resolutions of 1.54 Å. The dissociation constants (KD) of Apa and individual modules of Fn were determined by surface plasmon resonance and enzyme-linked immunosorbent assay. Site-directed mutagenesis was performed to investigate the putative Fn-binding motif of Apa. RESULTS: Mtb Apa folds into a large seven-stranded anti-parallel ß-sheet which is flanked by three α-helices. The binding affinity of Mtb Apa to individual Fn modules was assessed and the results indicated that the Mtb Apa binds to FnIII-4 and FnIII-5 of Fn CBD segment. Notably, structure analysis suggested that the previously proposed Fn-binding motif 258RWFV261 is buried within the protein and may not be accessible to the binding counterpart. CONCLUSIONS: The structural and Fn-binding characteristics we reported here provide molecular insights into the multifunctional protein Mtb Apa. FnIII-4 and FnIII-5 of CBD are the only two modules contributing to Apa-Fn interaction. GENERAL SIGNIFICANCE: This is the first study to report the structure and Fn-binding characteristics of mycobacterial Apa. Since Apa plays a central role in stimulating immune responses and host cells adhesion, these results are of great importance in understanding the pathogenesis of mycobacterium. This information shall provide a guidance for the development of anti-mycobacteria regimen.

Significance of sphingosine kinase 1 expression in feline mammary tumors.

BACKGROUND: Sphingosine kinase 1 (SPHK1) is an enzyme that converts pro-apoptotic ceramide and sphingosine into anti-apoptotic sphingosine-1-phosphate. There is growing evidence that SPHK1 activation promotes oncogenic transformation, tumor growth, chemotherapy resistance, and metastatic spread. High SPHK1 expression has been associated with a poor prognosis in several human cancers. RESULTS: In the present study, the expression level of SPHK1 was examined in feline mammary tumor (FMT) specimens, and the IHC expression level of SPHK1 was associated with the histological grade of FMTs. IHC analysis of 88 FMT cases revealed that the expression level of SPHK1 was upregulated in 53 tumor tissues (60.2%) compared to adjacent mammary tissues. SPHK1 expression in FMTs was significantly associated with histological grade, presence of lymphovascular invasion, and estrogen receptor negativity. Treatment of primary FMT cells with SPHK1 inhibitors reduced cell viability, indicating that SPHK1 acts to promote FMT cell survival. These results indicate that SPHK1 may play an important role in FMTs and may be a therapeutic target in cats with FMT. CONCLUSIONS: SPHK1 over-expression in breast cancer tissues is associated with a poor prognosis in humans. SPHK1 over-expression in more aggressive FMTs provides support for a potential role of SPHK1 inhibitors for the treatment of FMTs. Targeting SPHK1 has potent cytotoxic effects in primary FMT cells. These findings suggest that further examination of the role SPHK1 plays in FMTs will pave the way for the investigation of SPHK1 inhibitors in future clinical applications.

The germ-free mice monocolonization with Bacteroides fragilis improves azoxymethane/dextran sulfate sodium induced colitis-associated colorectal cancer.

Objective: Inflammatory bowel disease (IBD) is generally considered as a major risk factor in the progression of colitis-associated colorectal cancer (CAC). Previous studies have indicated that the composition of gut microflora may be involved in CAC induction and progress. Bacteroides fragilis (BF) is a Gram-negative anaerobe belonging to colonic symbiotic bacteria of the host. This study was aimed to investigate the protective role of BF in a colorectal cancer (CRC) model induced by azoxymethane (AOM) and dextran sulfate sodium (DSS) in germ-free (GF) mice. Materials and methods: Total 22 GF mice were divided into two groups: GF and BF group. Half of the GF mice were colonized with BF for 28 days before CRC induction by AOM/DSS. Results: BF colonization increased animal survival (100%). Cecum weight and cecum/body weight ratio significantly decreased in BF/AOM/DSS group. Interestingly, there was a significant decrease in tumor number and tumor incidence in the BF/AOM/DSS group as compared to the GF/AOM/DSS group. The adenocarcinoma/adenoma incidence and histologic score were also decreased in the BF/AOM/DSS group. In addition, immunohistochemistry staining found decreased numbers of cell proliferation (PCNA) and inflammatory cell (granulocytes) infiltration in the colon mucosa of the BF group. The ß-catenin staining in the BF/AOM/DSS group had fewer and weaker positive signal expressions. Taking together, the BF colonization significantly ameliorated AOM/DSS-induced CRC by suppressing the activity of cell proliferation-related molecules and reducing the number of inflammatory cells. Conclusions: Symbiotic BF may play a pivotal role in maintaining the gastrointestinal immunophysiologic balance and regulating anti-tumorigenesis responses.

Supplementation with Beef Extract Improves Exercise Performance and Reduces Post-Exercise Fatigue Independent of Gut Microbiota.

Beef extract (BE) is a nutritional supplement obtained by cooking beef meat. Compared with traditional chicken essence or clam extract, BE is cheaper to produce and may be used for wound healing, as a chemotherapy supplement, or to prevent fatigue. In this study, we evaluated the potential beneficial effects of BE on exercise performance and the related role of the gut microbiota. Pathogen-free male BALB/c mice were divided into three groups to receive vehicle or BE (0, 12.3, or 24.6 mL/kg) by oral gavage for 28 days. Exercise performance was evaluated using forelimb grip strength, swimming time to exhaustion, and physiological levels of fatigue-related biomarkers (serum lactate, blood urea nitrogen, and glucose levels) after physical challenges. BE supplementation elevated endurance and grip strength in a dose-dependent manner; significantly decreased lactate and blood urea nitrogen levels after physical challenge; and significantly increased muscle glycogen content. The germ-free mice supplemented with BE or an equal-calorie portion of albumin did not show significant differences from the other groups in exercise performance and levels of related biomarkers. Therefore, BE supplementation improved endurance and reduced fatigue, which might be related to BE composition, but had no correlation with the gut microbiota.

Effects of L-arabinose efflux on λ Red recombination-mediated gene knockout in multiple-antimicrobial-resistant Salmonella enterica serovar Choleraesuis.

In this study, six swine-derived multiple-antimicrobial-resistant (MAR) strains of Salmonella Choleraesuis (S. Choleraesuis) were demonstrated to possess higher efflux pump activity than the wild-type (WT). L-Arabinose, a common inducer for gene expression, modulated S. Choleraesuis efflux pump activity in a dose-dependent manner. At low L-arabinose concentrations, increasing L-arabinose led to a corresponding increase in fluorophore efflux, while at higher L-arabinose concentrations, increasing L-arabinose decreased fluorophore efflux activity. The WT S. Choleraesuis that lacks TolC (ΔtolC), an efflux protein associated with bacterial antibiotic resistance and virulence, was demonstrated to possess a significantly reduced ability to extrude L-arabinose. Further, due to the rapid export of L-arabinose, an efficient method for recombination-mediated gene knockout, the L-arabinose-inducible bacteriophage λ Red recombinase system, has a reduced recombination frequency (~ 12.5%) in clinically isolated MAR Salmonella strains. An increased recombination frequency (up to 60%) can be achieved using a higher concentration of L-arabinose (fivefold) for genetic manipulation and functional analysis for MAR Salmonella using the λ Red system. The study suggests that L-arabinose serves not only as an inducer of the TolC-dependent efflux system but also acts as a competitive substrate of the efflux system. In addition, understanding the TolC-dependent efflux of L-arabinose should facilitate the optimization of L-arabinose induction in strains with high efflux activity.

Effects of plant- and animal-based high-fat diets on lipid storage and distribution in environmental bacteria-colonized gnotobiotic mice.

Different edible oils such as lard and soybean oil have been reported to interact with the gut microbiota, affecting host lipid metabolism. However, whether bacteria derived from the environment influence host lipid metabolism remains unclear. This study aimed to clarify the roles of environmental bacteria in host lipid storage and distribution with various edible oils. Gnotobiotic C57BL/6JNarl mice were inoculated with Lysinibacillus xylanilyticus and Paenibacillus azoreducens and then fed either a normal diet (LabDiet 5010, control group) or a diet containing 60% lard (L-group) or soybean oil (S-group) for 18 months. Interestingly, the S-group accumulated massive amounts of white adipose tissue compared to the L- and control groups, while the L-group displayed more hepatic steatosis and fatty droplets than the other groups. The expression of fatty acid synthase (FAS), hydroxymethylglutaryl-coenzyme A reductase (HMGCR), sterol regulatory element-binding protein 2 (SREBP2), and peroxisome proliferator-activated receptor gamma (PPARγ) in the livers of the L-group were markedly elevated compared to the S-group. FAS and PPARγ protein levels were also markedly elevated. However, there were no differences in the expression of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-α between the groups. Our results suggest that environmental bacteria may affect host hepatic inflammation and lipid distribution in the presence of high-fat diets, with different effects depending on the fat type consumed.

High Diversity of Antimicrobial Resistance Genes, Class 1 Integrons, and Genotypes of Multidrug-Resistant Escherichia coli in Beef Carcasses.

Multidrug-resistant Escherichia coli can contaminate food meat during processing and cause human infection. Phenotypic and genotypic characterization of the antimicrobial resistance were conducted for 45 multidrug-resistant E. coli isolates from 208 samples of beef carcasses. The mechanisms of resistance were evaluated using polymerase chain reaction and sequencing methods, and the clonal relationship among isolates was evaluated using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Different variants of bla, tet, flo, dfrA, and aadA genes were detected in most of the strains resistant to ß-lactam, tetracycline, chloramphenicol, sulfonamides, and aminoglycosides, respectively. Extended-spectrum ß-lactamase (ESBL)-producing E. coli was found in 42.2% of the 45 E. coli isolates and the most commonly detected ESBL genotypes were CTX-M group 1 and 9. Class 1 integrons with nine different arrangements of gene cassettes were present in 28 of 45 E. coli isolates. Twenty-nine PFGE groups and 24 MLST types were identified in their clonal structure. This study revealed that E. coli isolates from beef contained high diversity of antimicrobial resistance genes, integrons, and genotypes. These results highlighted the role of beef meat as a potential source for multidrug-resistant E. coli strains and the need for controlling beef safety.

Determination of the quality of stripe-marked and cracked eggs during storage.

OBJECTIVE: Stripe marks, which occasionally occur on the shell, do not cause breakage to the shell and shell membranes of eggs. This study investigated the quality of intact eggs (IEs), minor stripe-marked eggs (MEs), severe stripe-marked eggs (SEs), and cracked eggs (CEs) during 3-week storage at 25°C. METHODS: Shell eggs were collected the day after being laid and were washed. Among them, eggs without any visual cracks or stripe marks on the shells were evaluated as IEs by the plant employees using candling in a darkened egg storage room; the remaining eggs exhibited some eggshell defects. At day 3, the eggs were further categorized into IEs, MEs, SEs, CEs, and broken eggs (BEs) on the basis of the description given. Except BEs, which were discarded, the remaining eggs were stored at 25°C (approximate relative humidity 50%) and then analyzed. RESULTS: Stripe marks were observed primarily within the first 3 days after washing. At day 3, CEs had significantly (p<0.05) lower Haugh unit values, but all eggs had grades AA or A, according to the United States Department of Agriculture standard. As storage time increased, differences in egg quality between groups were more obvious. IEs had the highest eggshell breaking strength. During storage, the total plate counts and pathogens, namely Escherichia coli, Campylobacter spp., Staphylococcus aureus, and Salmonella spp., were not detectable in the internal content of IEs and SEs. CONCLUSION: In conclusion, cracks degraded egg quality severely and minor stripe marks only slightly influenced the egg quality.

Prevalence and molecular characterization of Clostridium difficile isolates from a pig slaughterhouse, pork, and humans in Taiwan.

Clostridium difficile causes antibiotic-associated diarrhea in both humans and animals. The ribotype 078, predominant in food animals, is associated with community-acquired C. difficile infection, and C. difficile is suggested to be a foodborne pathogen. Recently, the C. difficile ribotype 078 lineage emerged in patients and pigs in Taiwan. This study aimed to investigate the prevalence and molecular characterization of C. difficile isolated from a pig slaughterhouse, retail meat, ready-to-eat meals, and humans in Taiwan. We collected samples from one slaughterhouse (n=422), 29 retail markets (raw pork, n=62; ready-to-eat pork, n=65), and one hospital (non-diarrheal humans, stool, n=317) in 2015. The isolated C. difficile were subjected to ribotyping and multilocus variable-number tandem-repeat analysis (MLVA). In the slaughterhouse, the isolation rate from carcasses was high (23%, 21/92) and ribotype 126 dominated. Scalding water was found to have C. difficile contamination (44%, 4/9), and two of the seven isolates were ribotype 126. The isolation rates from raw pork and ready-to-eat pork were between 20% and 29%. Ribotypes 126, 127, and 014 were found in raw pork, whereas ribotype 078 was not identified in this study. Eight isolates-seven non-toxigenic isolates and one ribotype 017-were found in non-diarrheal human samples. Notably, MLVA showed that ribotype 126 isolates from the slaughterhouse, pig stool, colons, carcasses, and scalding water were closely genetically related, indicating serious risk for cross-contamination. However, the genetic evidence of foodborne transmission from carcasses to food and humans is still lacking.

Involvement of NF-κB in regulation of Actinobacillus pleuropneumoniae exotoxin ApxI-induced proinflammatory cytokine production in porcine alveolar macrophages.

Actinobacillus pleuropneumoniae is a crucial respiratory pathogen that causes fibrinous, hemorrhagic, necrotizing pleuropneumonia in pigs. A. pleuropneumoniae exotoxins (ApxI to IV) are the major virulence factors contributing to A. pleuropneumoniae pathogenesis. Previously, we demonstrated that ApxI induces the expression of proinflammatory cytokines in porcine alveolar macrophages (PAMs) via the mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK). Nonetheless, the role of nuclear factor (NF)-κB-a transcription factor widely implicated in immune and inflammatory responses-in ApxI-elicited cytokine production has yet to be defined. In the present study, we examined the involvement of NF-κB in ApxI-elicited production of interleukin (IL)-1ß, IL-8, and tumor necrosis factor (TNF)-α in PAMs and investigated the correlation between NF-κB and MAPK (p38 and JNK) pathways in this event. The results of Western blot analysis, confocal microscopy, and a DNA binding activity assay revealed that the classical NF-κB pathway was activated by ApxI, as evidenced by the decreased levels of IκB and subsequent NF-κB translocation and activation in ApxI-stimulated PAMs. Moreover, the blocking of ApxI-induced NF-κB activation significantly attenuated the levels of mRNA and protein secretion of IL-1ß, IL-8, and TNF-α in PAMs. Notably, the attenuation of JNK activation by a specific inhibitor (SP600125) reduced ApxI-induced NF-κB activation, whereas a p38 blocker (SB203580) had no effect on the NF-κB pathway. Further examination revealed that the level of phosphorylation at serine 536 on the NF-κB p65 subunit was dependent on JNK activity. Collectively, this study, for the first time, demonstrates a pivotal role of NF-κB in ApxI-induced IL-1ß, IL-8, and TNF-α production; JNK, but not p38, may positively affect the activation of the classical NF-κB pathway.

TolC is important for bacterial survival and oxidative stress response in Salmonella enterica serovar Choleraesuis in an acidic environment.

The outer membrane protein TolC, which is one of the key components of several multidrug efflux pumps, is thought to be involved in various independent systems in Enterobacteriaceae. Since the acidic environment of the stomach is an important protection barrier against foodborne pathogen infections in hosts, we evaluated whether TolC played a role in the acid tolerance of Salmonella enterica serovar Choleraesuis. Comparison of the acid tolerance of the tolC mutant and the parental wild-type strain showed that the absence of TolC limits the ability of Salmonella to sustain life under extreme acidic conditions. Additionally, the mutant exhibited morphological changes during growth in an acidic medium, leading to the conflicting results of cell viability measured by spectrophotometry and colony-forming unit counting. Reverse-transcriptional-PCR analysis indicated that acid-related molecules, apparatus, or enzymes and oxidation-induced factors were significantly affected by the acidic environment in the null-tolC mutant. The elongated cellular morphology was restored by adding antioxidants to the culture medium. Furthermore, we found that increased cellular antioxidative activity provides an overlapping protection against acid killing, demonstrating the complexity of the bacterial acid stress response. Our findings reinforce the multifunctional characteristics of TolC in acid tolerance or oxidative stress resistance and support the correlative protection mechanism between oxygen- and acid-mediated stress responses in Salmonella enterica serovar Choleraesuis.