Investigation of biofilm formation and the associated genes in multidrug-resistant Salmonella pullorum in China (2018-2022).

The study explored the biofilm (BF) formation capacity, BF-related gene profiles, and the trends in antimicrobial resistance (AMR) of Salmonella pullorum (SP) strains over several years. A total of 627 SP strains were isolated from 4,540 samples collected from chicken farms in Guangxi, China during 2018-2022. The BF-forming capacity of these isolates was assessed using crystal violet staining, and the presence of eight BF-related genes (csgA, csgB, csgD, ompR, bapA, pfs, luxS, and rpoS) in BF formation-positive strains was determined through Polymerase Chain Reaction (PCR) analysis. Antimicrobial susceptibility test was conducted to investigate the AMR of the isolates. Minimum Inhibitory Concentration (MIC) and Minimal Biofilm Eradication Concentration (MBEC) of nine SP-BF strains were determined using the broth microdilution method to assess the impact of BF formation on AMR. Additionally, the Optimal Biofilm Formation Conditions (OBFC) were investigated. The results indicated that 36.8% (231/627) of the strains exhibited a positive BF-formation capacity. Among these, 24.7% (57/231) were strong BF producers, 23.4% (54/231) were moderate BF producers, and 51.9% (120/231) were weak BF producers. Analysis of the eight BF-related genes in SP-BF strains revealed that over 90% of them were positive for all the genes. Antimicrobial susceptibility test conducted on the isolates showed that 100% (231/231) of them exhibited resistance to at least one antibiotic, with 98.3% (227/231) demonstrating multidrug resistance (MDR). Both MIC and MBEC measurements indicated varying degrees of increased AMR after BF formation of the bacteria. The optimal conditions for BF formation were observed at 37°C after 48 h of incubation, with an initial bacterial concentration of 1.2 × 106 CFU/mL. Notably, NaCl had a significant inhibitory effect on BF formation, while glucose and Trypticase Soy Broth (TSB) positively influenced BF formation. The results of the study emphasized the need for effective preventive and control strategies to address the challenges posed by the BF formation and MDR of SP in the field.

The Use of Star Anise-Cinnamon Essential Oil as an Alternative Antibiotic in Prevention of Salmonella Infections in Yellow Chickens.

Salmonella is capable of harming human and animal health, and its multidrug resistance (MDR) has always been a public health problem. In addition, antibiotic-free or antibiotic-reduced policies have been implemented in poultry production. Therefore, the search for antibiotic alternatives is more urgent than ever before. The aim of this study was to assess the antibacterial activity of star anise-cinnamon essential oil (SCEO) in vitro and its prophylactic effect against the infections of Salmonella pullorum, Salmonella give, and Salmonella kentucky in vivo. The results demonstrated that SCEO is effective against Salmonella pullorum, Salmonella give, and Salmonella kentucky in vitro. Supplementation with SCEO could significantly decrease the infections of Salmonella pullorum and Salmonella give, whereas it could slightly but not significantly decrease the infection of Salmonella kentucky, while also significantly alleviating the body weight (BW) loss caused by the infections of Salmonella pullorum, Salmonella give, and Salmonella kentucky in Yellow chickens. The SCEO had the best prophylactic effect against the infection of Salmonella give in Yellow chickens, followed by the infection of Salmonella pullorum and the infection of Salmonella kentucky. The SCEO, used as an antibiotic alternative, could be an effective prevention strategy against the infections of Salmonella pullorum, Salmonella give, and Salmonella kentucky in Yellow chickens.

Rapid, Sensitive, Specific, and Visual Detection of Salmonella in Retail Meat with Loop-Mediated Isothermal Amplification, Targeting the invA Gene.

ABSTRACT: Salmonella is one of the major pathogenic bacteria causing foodborne diseases. The rapid detection of Salmonella in food is of great significance to food safety. In this study, the loop-mediated isothermal amplification (LAMP) method was developed, and primers were designed targeting the invA gene of Salmonella. Standard samples of recombinant invA-plasmid and 100 retail meat samples were tested by LAMP and compared with the results tested by conventional PCR and the routine Chinese National Food Safety Standard-Microbiological Examination of Food-Examination of Salmonella, respectively. The results showed that Salmonella strains of eight different serotypes were amplified successfully by the developed LAMP assay, and it was 1,000-fold more sensitive than conventional PCR, with the analytical sensitivity of 8 × 102 copies per µL of the standard sample of invA-plasmid. The results were visualized directly by adding calcein and MnCl2 in the LAMP reaction tube, and the positively amplified products turned green after an incubation of 2 min. In parallel detection, the positive rate of Salmonella by the LAMP assay was highly correlated with the routine Chinese national standard method. The results of the study demonstrated that the developed LAMP assay is a simple, rapid, strongly specific, highly sensitive, and visual detection method for Salmonella.

Identification of a quantitative trait loci (QTL) associated with ammonia tolerance in the Pacific white shrimp (Litopenaeus vannamei).

BACKGROUND: Ammonia is one of the most common toxicological environment factors affecting shrimp health. Although ammonia tolerance in shrimp is closely related to successful industrial production, few genetic studies of this trait are available. RESULTS: In this study, we constructed a high-density genetic map of the Pacific white shrimp (Litopenaeus vannamei) using specific length amplified fragment sequencing (SLAF-seq). The constructed genetic map contained 17,338 polymorphic markers spanning 44 linkage groups, with a total distance of 6360.12 centimorgans (cM) and an average distance of 0.37 cM. Using this genetic map, we identified a quantitative trait locus (QTL) that explained 7.41-8.46% of the phenotypic variance in L. vannamei survival time under acute ammonia stress. We then sequenced the transcriptomes of the most ammonia-tolerant and the most ammonia-sensitive individuals from each of four genetically distinct L. vannamei families. We found that 7546 genes were differentially expressed between the ammonia-tolerant and ammonia-sensitive individuals. Using QTL analysis and the transcriptomes, we identified one candidate gene (annotated as an ATP synthase g subunit) associated with ammonia tolerance. CONCLUSIONS: In this study, we constructed a high-density genetic map of L. vannamei and identified a QTL for ammonia tolerance. By combining QTL and transcriptome analyses, we identified a candidate gene associated with ammonia tolerance. Our work provides the basis for future genetic studies focused on molecular marker-assisted selective breeding.

Construction of a High-Density Genetic Map and Identification of Quantitative Trait Loci for Nitrite Tolerance in the Pacific White Shrimp (Litopenaeus vannamei).

Nitrite is a major environmental toxin in aquaculture systems that disrupts multiple physiological functions in aquatic animals. Although nitrite tolerance in shrimp is closely related to successful industrial production, few genetic studies of this trait are available. In this study, we constructed a high-density genetic map of Litopenaeus vannamei with 17,242 single nucleotide polymorphism markers spanning 6,828.06 centimorgans (cM), with an average distance of 0.4 cM between adjacent markers on 44 linkage groups (LGs). Using this genetic map, we identified two markers associated with nitrite tolerance. We then sequenced the transcriptomes of the most nitrite-tolerant and nitrite-sensitive individuals from each of four genetically distinct L. vannamei families (LV-I-4). We found 2,002, 1,983, 1,954, and 1,867 differentially expressed genes in families LV-1, LV-2, LV-3, and LV-4, respectively. By integrating QTL and transcriptomics analyses, we identified a candidate gene associated with nitrite tolerance. This gene was annotated as solute carrier family 26 member 6 (SLC26A6). RNA interference (RNAi) analysis demonstrated that SLC26A6 was critical for nitrite tolerance in L. vannamei. The present study increases our understanding of the molecular mechanisms underlying nitrite tolerance in shrimp and provides a basis for molecular-marker-assisted shrimp breeding.

Prevalence and antimicrobial resistance of retail-meat-borne Salmonella in southern China during the years 2009-2016: The diversity of contamination and the resistance evolution of multidrug-resistant isolates.

Salmonella, one of the most important foodborne pathogens, can be the cause of bacterial food-borne illness and is commonly associated with the consumption of retail meat. Multidrug-resistant Salmonella isolates with high adaptability, have been responsible for many foodborne disease outbreaks. Here we present an investigation on the contamination and the antimicrobial resistance of Salmonella in retail meat obtained from supermarkets and from open markets in Guangxi, China. From the years 2009 to 2016, a total of 604 Salmonella isolates were recovered from a total of 3340 meat samples including 797 beef, 911 pork, 942 chicken and 690 duck, representing 18.08% of the samples tested. Pork was the most contaminated meat. Salmonella was detected in 322 samples from supermarkets and the positive rate of 21.03% was higher than that of 15.70% in 284 samples from open markets (P<0.05). The prevalence of Salmonella in retail meat in the summer and fall months: June (2015, 40.63%), October (2012, 34.6%; 2016, 43.75%) was higher than in other seasons of the year. One hundred and twenty-seven serotypes were identified among the 604 Salmonella enterica isolates, and S. Derby (28.48%), S. Agona (9.77%), S. London (4.97%) and S. Enteritidis (4.47%) were the most common serotypes. Tests of susceptibility to 21 antimicrobial agents showed that 87.58% of the isolates were resistant to at least one antimicrobial, and 57.79% exhibited multidrug resistance (MDR), as they were resistant to at least three antimicrobials. The presence of most of the antimicrobial-resistant genes tested was consistent with the resistant phenotypes found. Among all the antimicrobial resistant genes (ARGs) examined in this study, blaTEM-1, aadA1, cmlA, tetA, sul1 and sul2 were the most prevalent resistant genes in the multidrug resistant isolates. Our findings show that there was a trend that the Salmonella contamination in retail meat had increased and isolates showed an MDR phenotype and that the MDR had become more and more serious. Twenty-one isolates of S. Agona were randomly analyzed by using the enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and six different types were found, indicating the existence of cross-contamination in the food market. The results indicate that the hazard analysis of the critical control points (HACCP) system for the whole food chain of retail meat should be further analyzed and improved.

The diagnosis and successful replication of a clinical case of Duck Spleen Necrosis Disease: An experimental co-infection of an emerging unique reovirus and Salmonella indiana reveals the roles of each of the pathogens.

Duck spleen necrosis disease (DSND) is an emerging infectious disease that causes significant economic loss in the duck industry. In 2018, a duck reovirus (named DRV/GX-Y7) and Salmonella indiana were both isolated from the spleens and livers of diseased ducks with DSND in China. The DRV/GX-Y7 strain could propagate in the Vero, LMH, DF-1 and DEF cells with obvious cytopathic effects. The genome of DRV/GX-Y7 was 23,418 bp in length, contained 10 dsRNA segments, ranging from 3959 nt (L1) to 1191 nt (S4). The phylogenetic analysis showed that the DRV/GX-Y7 strain was in the same branch with the new waterfowl-origin reovirus cluster, but was obviously far distant from the clusters of other previous waterfowl-origin reoviruses Muscovy duck reovirus (MDRV) and goose-origin reovirus (GRV), broiler/layer-origin reovirus (ARV) and turkey-origin reovirus (TRV). The RDP and SimPlot program analysis revealed that there were two potential genetic reassortment events in the M2 and S1 segments of the genome. In order to have a clear insight into the pathogenic mechanism of DRV/GX-Y7 and S. Indiana in clinical DSND, an infection experiment was further conducted by challenging commercial ducklings with the two isolates individually and with both. The results showed that DRV/GX-Y7 produced severe hemorrhagic and/or necrotic lesions in the immune organs (thymus, spleen, and bursae) of experimentally infected ducklings. And, that the co-infection of DRV/GX-Y7 and S. Indiana could greatly enhance the pathogenesis by increasing the morbidity and mortality in ducklings whose clinical symptoms and lesions were similar to the natural clinical DSND cases. In summary, the results suggested that the pathogen causing duck spleen necrosis was an emerging unique genetic reassortment strain of duck Orthoreovirus that was significantly different from any previously reported waterfowl-derived Orthoreovirus and the co-infection with the Salmonella isolate could increase the severity of the disease.

The effect of a selected yeast fraction on the prevention of pullorum disease and fowl typhoid in commercial breeder chickens.

A selected yeast fraction (SYF) was tested for the purpose of preventing pullorum disease and fowl typhoid in breeder chickens. In a challenge-protection experiment, commercial Three-Yellow breeder chicks were initially divided into groups A, B (challenged, treated), C (challenged, untreated), and D (unchallenged, untreated). The group A diet was supplemented with SYF and group B was supplemented with Acidipure via drinking water. At 7 D, birds of groups A, B, and C were divided into 2 equal subgroups (A1-A2, B1-B2, and C1-C2). Subgroups A1, B1, and C1 were challenged with Salmonella pullorum (SP), while subgroups A2, B2, and C2 were challenged with Salmonella gallinarum (SG). Clinical signs and mortality were recorded daily. At intervals, antibodies against SP and SG were detected by a plate agglutinate test (PAT). At 42 D, all birds were weighed and necropsied, lesions were recorded and challenge pathogens were isolated. Results showed that SP and SG isolation positive rates of groups A1-A2 were significantly lower (P < 0.05) than those of B1-B2 and C1-C2, respectively. The average body weight (BW) of groups A1-A2 was significantly higher (P < 0.05) than that of B1-B2 and C1-C2, respectively. In the field trial, chicks were randomly divided into 3 groups. Group 1 birds were fed a diet supplemented with SYF, group 2 diet was supplemented with Acidipure via drinking water, and group 3 was fed the same but un-supplemented diet as the control group. Antibodies against SP and SG were detected by PAT at 120 D. The antibodies positive rate of group 1 was significantly lower (P < 0.05) than those of groups 2 and 3, while no significant difference (P > 0.05) was found between groups 2 and 3. The results demonstrated that SYF supplementation could significantly decrease SP and SG infection rates, improve the BW of birds challenged with SP and SG, and was more effective than Acidipure via drinking water.

Cathepsin L in the orange-spotted grouper, Epinephelus coioides: molecular cloning and gene expression after a Vibrio anguillarum challenge.

The orange-spotted grouper, Epinephelus coioides, is an important fish maricultured in many Asian countries. In the present study, the full-length cDNA of cathepsin L, an immunity related gene of fishes, was isolated from E. coioides using rapid amplification of cDNA ends (RACE). It is 1,443 bp in length, including an open reading frame (ORF) of 1,011 bp. The open reading frame encoded a preproprotein of 336 amino acids (aa), which consisted of a signal peptide of 16 aa, a proregion peptide of 98 aa and a mature peptide of 222 aa. The preproprotein contained an oxyanion hole (Gln), a catalytic triad formed by Cys, His and Asn, and the conserved ERWNIN, GNFD and GCNGG motifs, all characteristic of cathepsin L. Homology analysis revealed that the deduced amino acid sequence of E. coioides cathepsin L shared 80.1-94.8 % identity with those of reported fishes. Tissue-dependent mRNA expression analysis showed that the cathepsin L transcript was expressed in all the examined tissues of the healthy E. coioides, being highest in the liver and moderate in the heart, gonad and intestine. After Vibrio anguillarum stimulation, the mRNA expression of cathepsin L in E. coioides was significantly increased in the skin, fin, gills, liver, blood, spleen, head kidney and intestine, with the highest observed in the spleen (10.6-fold) at 12 h post-injection and the next in blood (7.5-fold) at 8 h post-injection. These results provided initial information for further studies on the physiological and immunological roles of the cathepsin L gene in the orange-spotted grouper.