A biphasic accelerated strand exchange amplification strategy for culture-independent and rapid detection of Salmonella enterica in food samples.

Salmonella enterica is a common foodborne pathogen that can cause food poisoning in humans. The organism also infects and causes disease in animals. Rapid and sensitive detection of S. enterica is essential to prevent the spread of this pathogen. Traditional technologies for the extraction and detection of this pathogen from complex food matrices are cumbersome and time-consuming. In this study, we introduced a novel strategy of biphasic assay integrated with an accelerated strand exchange amplification (ASEA) method for efficient detection of S. enterica without culture or other extraction procedures. Food samples are rapidly dried, resulting in a physical fluidic network inside the dried food matrix, which allows polymerases and primers to access the target DNA and initiate ASEA. The dried food matrix is defined as the solid phase, while amplification products are enriched in the supernatant (liquid phase) and generate fluorescence signals. The analytical performances demonstrated that this strategy was able to specifically identify S. enterica and did not show any cross-reaction with other common foodborne pathogens. For artificially spiked food samples, the strategy can detect 5.0 × 101 CFU mL-1S. enterica in milk, 1.0 × 102 CFU g-1 in duck, scallop or lettuce, and 1.0 × 103 CFU g-1 in either oyster or cucumber samples without pre-enrichment of the target pathogen. We further validated the strategy using 82 real food samples, and this strategy showed 92% sensitivity. The entire detection process can be finished, sample-to-answer, within 50 min, dramatically decreasing the detection time. Therefore, we believe that the proposed method enables rapid and sensitive detection of S. enterica and holds great promise for the food safety industry.

Chlamydia psittaci detected at a live poultry wholesale market in central China.

BACKGROUND: We investigated the presence of Chlamydia psittaci in poultry and the environment in live poultry wholesale markets in Changsha during 2021-2022 and conducted a phylogenetic analysis to understand its distribution in this market. METHODS: In total, 483 samples were analyzed using real-time polymerase chain reaction and 17 C. psittaci-positive samples using high-throughput sequencing, BLAST similarity, and phylogenetic analysis. RESULTS: Twenty-two out of 483 poultry and environmental samples were positive for C. psittaci (overall positivity rate: 4.55%) with no difference in positivity rates over 12 months. Chlamydia psittaci was detected at 11 sampling points (overall positivity rate: 27.5%), including chicken, duck, and pigeon/chicken/duck/goose shops, with pigeon shops having the highest positivity rate (46.67%). The highest positivity rates were found in sewage (12.5%), poultry fecal (7.43%), cage swab (6.59%), avian pharyngeal/cloacal swab (3.33%), and air (2.29%) samples. The ompA sequences were identified in two strains of C. psittaci, which were determined to bear genotype B using phylogenetic analysis. Thus, during monitoring, C. psittaci genotype B was detected in the poultry and environmental samples from the poultry wholesale market in Changsha. CONCLUSIONS: To address the potential zoonotic threat, C. psittaci monitoring programs in live poultry markets should be enhanced.

Does Salmonella diarizonae 58:r:z53 Isolated from a Mallard Duck Pose a Threat to Human Health?

Salmonella diarizonae (IIIb) is frequently isolated from reptiles and less frequently from birds and mammals. However, its isolation from invasive human infections has not been widely reported. Migratory mallard ducks are excellent bioindicators of pathogen presence and pathogen antibiotic resistance (AMR). We present the first isolation from a mallard duck in central Europe of the antibiotic-resistant Salmonella enterica subsp. diarizonae with the unique antigenic pattern 58:r:z53 and report its whole-genome sequencing, serosequencing, and genotyping, which enabled the prediction of its pathogenicity and comparison with phenotypic AMR. The isolated strain was highly similar to S. diarizonae isolated from humans and food. Twenty-four AMR genes were detected, including those encoding aminoglycoside, fluoroquinolone, macrolide, carbapenem, tetracycline, cephalosporin, nitroimidazole, peptide antibiotic, and disinfecting agent/antiseptic resistance. Six Salmonella pathogenicity islands were found (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, and SPI-13). An iron transport system was detected in SPI-1 centisome C63PI. Plasmid profile analyses showed three to be present. Sequence mutations in the invA and invF genes were noted, which truncated and elongated the proteins, respectively. The strain also harbored genes encoding type-III secretion-system effector proteins and many virulence factors found in S. diarizonae associated with human infections. This study aims to elucidate the AMR and virulence genes in S. enterica subsp. diarizonae that may most seriously threaten human health.

Metagenomic insights into the relationship between intestinal flora and residual feed intake of meat ducks.

In this study, we sought to determine the effects of intestinal flora on the feed efficiency of meat ducks by evaluating the correlation between intestinal flora and residual feed intake. The F2 generation of Cherry Valley ducks × Runzhou Crested White ducks was used as the study subjects, and feed consumption being recorded from d 21 to 42. RFI was calculated based on growth performance, and 20 low RFI and 20 high RFI ducks were randomly selected to characterize the effect of RFI on growth performance. To analyze the intestinal flora affecting RFI, 16s rDNA sequencing was performed on the contents of 5 intestinal segments from the HR and LR groups, and macrogenomic sequencing was performed on the cecal contents. Feed intake, average daily feed intake, feed conversion ratio, and residual feed intake were lower in low RFI. Analysis of the intestinal flora revealed the cecum to be more highly enriched in the carbohydrate metabolism pathway and less enriched with potentially pathogenic taxa than the other assessed intestinal regions. Further analysis of the cecal microbiota identified nine significantly differentially enriched intestinal flora. In this study, we accordingly identified a basis for the mechanisms underlying the effects of the intestinal flora on meat duck feed efficiency.

The novel probiotic preparation based on Lactobacillus spp. mixture on the intestinal bacterial community structure of Cherry Valley duck.

The development and utilization of probiotics have many environmental benefits when they are used to replace antibiotics in animal production. In this study, intestinal lactic acid bacteria were isolated from the intestines of Cherry Valley ducks. Probiotic lactic acid bacterial strains were screened for antibacterial activity and tolerance to produce a Lactobacillus spp. mixture. The effects of the compound on the growth performance and intestinal flora of Cherry Valley ducks were studied. Based on the results of the antibacterial activity and tolerance tests, the highly active strains Lactobacillus casei 1.2435, L. salivarius L621, and L. salivarius L4 from the intestines of Cherry Valley ducks were selected. The optimum ratio of L. casei 1.2435, L. salivarius L621, and L. salivarius L4 was 1:1:2, the amount of inoculum used was 1%, and the fermentation time was 14 h. In vivo experiments showed that compared with the control group, the relative abundances of intestinal Lactobacillus and Blautia were significantly increased in the experimental group fed the lactobacilli compound (P < 0.05); the relative abundances of Parabacteroides, [Ruminococcus]_torques_group, and Enterococcus were significantly reduced (P < 0.05), and the growth and development of the dominant intestinal flora were promoted in the Cherry Valley ducks. This study will provide more opportunities for Cherry Valley ducks to choose microecological agents for green and healthy breeding.

Integrated transcriptome and microbiome analyses of residual feed intake in ducks during high production period.

Residual feed intake (RFI) is a crucial parameter for assessing the feeding efficiency of poultry. Minimizing RFI can enhance feed utilization and reduce costs. In this study, 315 healthy female ducks were individually housed in cages. Growth performance was monitored during the high laying period, from 290 to 325 d of age. The cecal transcriptome and microbiome of 12 ducks with high RFI and 12 with low residual feed intake (LRFI) were analyzed. Regarding growth performance, the LRFI group exhibited significantly lower RFI, feed conversion ratio (FCR), and feed intake (Fi) compared to the HRFI group (p < 0.01). However, there were no significant differences observed in body weight (BW), body weight gain (BWG), and egg mass (EML) between the groups (p > 0.05). Microbiome analysis demonstrated that RFI impacted gut microbial abundance, particularly affecting metabolism and disease-related microorganisms such as Romboutsia, Enterococcus, and Megamonas funiformis. Transcriptome analysis revealed that varying RFI changed the expression of genes related to glucose metabolism and lipid metabolism, including APOA1, G6PC1, PCK1, and PLIN1. The integrated analysis indicated that host genes were closely linked to the microbiota and primarily function in lipid metabolism, which may enhance feeding efficiency by influencing metabolism and maintaining gut homeostasis.

Lactobacillus acidophilus and Bacillus subtilis significantly change the growth performance, serum immunity and cecal microbiota of Cherry Valley ducks during the fattening period.

This study explored the effects of a Bacillus subtilis and Lactobacillus acidophilus mixture containing the co-fermented products of the two probiotics on growth performance, serum immunity and cecal microbiota of Cherry Valley ducks. This study included 480 one-day-old Cherry Valley ducks divided into four feeding groups: basal diet (control group) and basal diet supplemented with 300, 500, or 700 mg/kg of the probiotic powder; the ducks were raised for 42 days. Compared with the control group, body weight on day 42 and the average daily gain on days 15-42 significantly increased (p < 0.05), and the feed conversion rate significantly decreased (p < 0.05) in the experimental groups. Furthermore, the serum immunoglobulin (Ig) A, IgG, IgM, and interleukin (IL)-4 levels increased significantly (p < 0.05), and IL-1ß, IL-2, and tumor necrosis factor-α decreased significantly (p < 0.05) in the experimental groups. Finally, Sellimonas, Prevotellaceae NK3B31 group, Lachnospiraceae NK4A136 group and Butyricoccus played an important role in the cecal microbiota of the experimental group. Thus, the probiotic powder has impacts on the growth performance, serum immunity and cecal microbiota of Cherry Valley Ducks.

New evidence for gut-muscle axis: Lactic acid bacteria-induced gut microbiota regulates duck meat flavor.

The interaction between gut microbiota and muscles through the gut-muscle axis has received increasing attention. This study attempted to address existing research gaps by investigating the effects of gut microbiota on meat flavor. Specifically, lactic acid bacteria were administered to ducks, and the results of e-nose and e-tongue showed significantly enhanced meat flavor in the treatment group. Further analyses using GC-MS revealed an increase in 6 characteristic volatile flavor compounds, including pentanal, hexanal, heptanal, 1-octen-3-ol, 2,3-octanedione, and 2-pentylfuran. Linoleic acid was identified as the key fatty acid that influences meat flavor. Metagenomic and transcriptomic results further confirmed that cecal microbiota affects the duck meat flavor by regulating the metabolic pathways of fatty acids and amino acids, especially ACACB was related to fatty acid biosynthesis and ACAT2, ALDH1A1 with fatty acid degradation. This study sheds light on a novel approach to improving the flavor of animal-derived food.

Genetic Characteristics of Extended-Spectrum Beta-Lactamase-Producing Salmonella Isolated from Retail Meats in South Korea.

Earlier studies have validated the isolation of extended-spectrum beta-lactamase-producing Salmonella (ESBL-Sal) strains from food. While poultry is recognized as a reservoir for Salmonella contamination, pertinent data regarding ESBL-Sal remains limited. Consequently, the Ministry of Food and Drug Safety has isolated Salmonella spp. from retail meat and evaluated their antibiotic susceptibility and genetic characteristics via whole-genome sequencing. To further elucidate these aspects, this study investigates the prevalence, antibiotic resistance profiles, genomic characteristics, and homology of ESBL-Sal spp. obtained from livestock-derived products in South Korean retail outlets. A total of 653 Salmonella spp. were isolated from 1,876 meat samples, including 509 beef, 503 pork, 555 chicken, and 309 duck samples. The prevalence rates of Salmonella were 0.0%, 1.4%, 17.5%, and 28.2% in the beef, pork, chicken, and duck samples, respectively. ESBL-Sal was exclusively identified in poultry meat, with a prevalence of 1.4% in the chicken samples (8/555) and 0.3% in the duck samples (1/309). All ESBL-Sal strains carried the blaCTX-M-1 gene and exhibited resistance to ampicillin, ceftiofur, ceftazidime, nalidixic acid, and tetracycline. Eight ESBL-Sal isolates were identified as S. Enteritidis with sequence type (ST) 11. The major plasmid replicons of the Enteritidis-ST11 strains were IncFIB(S) and IncFII(S), carrying antimicrobial resistance genes (ß-lactam, tetracycline, and aminoglycoside) and 166 virulence factor genes. The results of this study provide valuable insights for the surveillance and monitoring of ESBL-Sal in South Korean food chain.

Comparative analysis of antimicrobial resistance phenotype and genotype of Riemerella anatipestifer.

Riemerella anatipestifer is one of the important bacterial pathogens that threaten the waterfowl farming industry. In this study, 157 suspected R. anatipestifer strains were isolated from diseased ducks and geese from seven regions of China during 2019-2020, and identified using multiple polymerase chain reaction (PCR). Antimicrobial susceptibility tests and whole-genome sequence (WGS) analysis were then performed for comparative analysis of antimicrobial resistance phenotypes and genotypes. The results showed that these strains were susceptible to florfenicol, ceftriaxone, spectinomycin, sulfafurazole and cefepime, but resistant to kanamycin, amikacin, gentamicin, and streptomycin, exhibiting multiple antimicrobial resistance phenotypes. WGS analysis revealed a wide distribution of genotypes among the 157 strains with no apparent regional pattern. Through next-generation sequencing analysis of antimicrobial resistance genes, a total of 88 resistance genes were identified. Of them, 19 tetracycline resistance genes were most commonly found, followed by 15 efflux pump resistance genes, 11 glycopeptide resistance genes and seven macrolide resistance genes. The 157 R. anatipestifer strains contained 42-55 resistance genes each, with the strains carrying 47 different resistance genes being the most abundant. By comparing the antimicrobial resistance phenotype and genotype, it was observed that a high correlation between them for most antimicrobial resistance properties was detected, except for a difference in aminoglycoside resistance phenotype and genotype. In conclusion, 157 R. anatipestifer strains exhibited severe multiple antimicrobial resistance phenotypes and genotypes, emphasizing the need for improved antimicrobial usage guidelines. The wide distribution and diverse types of resistance genes among these strains provide a foundation for studying novel mechanisms of antimicrobial resistance.

Functional characterization of RhuB as a second TonB2-dependent hemin receptor in Riemerella anatipestifer CH-1.

In the previous study, it was shown that Riemerella anatipestifer (R. anatipestifer, RA), a pathogen in ducks and some other birds, encodes a hemin uptake system. The R. anatipestifer hemin uptake receptor RhuR is a TonB2-dependent hemin transporter. However, it remains unclear whether R. anatipestifer encodes additional TonB-dependent hemin transporters. Herein, we demonstrated that R. anatipestifer hemin uptake receptor B (RhuB) of R. anatipestifer CH-1 (RA CH-1) was negatively regulated by iron and mediated by the Fur protein, and knocking out rhuB damaged the ability of RA CH-1 to utilize iron from duck hemoglobin (Hb) but not that from duck serum. Moreover, the ability to use iron from Hb was restored by the expression rhuB in trans. Furthermore, the RhuB of RA CH-1 is a membrane protein, and recombinant RhuB could bind hemin at a 1:1 molar ratio in vitro. Compared to that of ΔtonB1ΔrhuR, the ability of ΔtonB1ΔrhuRΔrhuB to utilize hemin was impaired; meanwhile, compared to that of ΔtonB2ΔrhuR, the hemin utilization ability of ΔtonB2ΔrhuRΔrhuB was not affected, indicating that RhuB is a TonB2-dependent receptor. Compared to ΔrhuB, ΔrhuBΔrhuA did not affect hemin utilization. However, compared to ΔrhuA, ΔrhuBΔrhuA had reduced ability to utilize hemin, suggesting that RhuA relies on RhuB for its activity. Finally, the deletion of rhuB did not affect the virulence of RA CH-1. These results suggested that RhuB encodes a TonB2-dependent hemin receptor. The characterization of the second TonB-dependent receptor in R. anatipestifer enriches our understanding of the hemin uptake system of this bacterium.IMPORTANCEIron is essential for the survival of most bacteria, and hemin of hemoglobin can serve as an important iron source. In our previous studies, we showed that R. anatipestifer CH-1 encodes a TonB2-dependent hemin receptor RhuR, which is involved in hemin uptake. The deletion of rhuR did not abolish hemin utilization by RA CH-1. We hypothesized that additional hemin uptake systems exist in this bacterium. In this study, we identified the second TonB2-dependent hemin receptor RhuB in RA CH-1 through hemin utilization, protein localization, and hemin-binding experiments. The duck infection model showed that the deletion of rhuB did not affect the virulence of RA CH-1. This study is not only important for further understanding the hemin utilization mechanism of R. anatipestifer, but also for enriching the hemin uptake transporters of gram-negative bacteria.

Research Note: Antibiotic resistance and pathogenicity of geese-derived Riemerella anatipestifer isolated from eastern regions of Hebei Province, China.

Riemerella anatipestifer (R. anatipestifer) can cause serositis in multiple poultry species, resulting in significant losses. Although R. anatipestifer-caused infections in ducks have been well established, the literature about this disease in geese is rare. Here, we isolated and identified 56 strains of R. anatipestifer from the eastern regions of Hebei Province, China, and further determined their serotypes, antibiotic resistance, and pathogenicity. A total of 75 strains of causative bacteria were isolated from 70 sick geese with serositis. After Gram staining microscopy, PCR, and 16S rDNA sequence analysis, 56 isolates were identified as members of R. anatipestifer and 19 as Escherichia coli (E. coli). The results of serotyping showed that there were 4 serotypes prevalent in the isolate, including serotype 1 (37/56), serotype 2 (9/56), serotype 11 (8/56), and serotype 13 (2/56). The results of antibiotic susceptibility testing revealed that all 56 R. anatipestifer isolates showed varying degrees of multidrug resistance (MDR). A total of 10 antibiotic resistance genes (ARG) were determined in these isolates. Four isolates of different serotypes were selected for pathogenicity examination, and all were able to reproduce serositis-like symptoms in 15-day-old goslings, with neurological symptoms and a 100% mortality rate. Hemorrhagic congestion of the brain tissue, steatosis of the hepatocytes, and disorganization of some cardiac myofibers were observed in R. anatipestifer-infected geese. All these findings will contribute to our insights into the prevalence characteristics, antibiotic resistance profile, and pathogenicity of R. anatipestifer infection in geese in eastern Hebei Province and provide scientific guidance for the treatment and control of this disease.

Epidemiological investigation and ß-lactam antibiotic resistance of Riemerella anatipestifer isolates with waterfowl origination in Anhui Province, China.

Riemerella anatipestifer (R. anatipestifer) is a highly pathogenic and complex serotypes waterfowl pathogen with inherent resistance to multiple antibiotics. This study was aimed to investigate the antibiotic resistance characteristics and genomic features of R. anatipestifer isolates in Anhui Province, China in 2023. A total of 287 cases were analysed from duck farms and goose farms, and the R. anatipestifer isolates were subjected to drug resistance tests for 30 antimicrobials. Whole genome sequencing (WGS) and bioinformatics analysis were performed on the bacterial genomes, targeting the ß-lactam resistance genes. The results showed that a total of 74 isolates of R. anatipestifer were isolated from 287 cases, with a prevalence of 25.8%. The antimicrobial susceptibility testing (AST) revealed that all the 74 isolates were resistant to multiple drugs, ranging from 13 to 26 kinds of drugs. Notably, these isolates showed significant resistance to aminoglycosides and macrolides, which are also commonly used in clinical practices. Data revealed the presence of several ß-lactamase-related genes among the isolates, including a novel blaRASA-1 variant (16.2%), the class A extended-spectrum ß-lactamase blaRAA-1 (12.2%), and a blaOXA-209 variant (98.6%). Functional analysis of the variants blaRASA-1 and blaOXA-209 showed that the blaRASA-1 variant exhibited activity against various ß-lactam antibiotics while their occurrence in R. anatipestifer were not common. The blaOXA-209 variant, on the other hand, did not perform any ß-lactam antibiotic resistance. Furthermore, we observed that blaRAA-1 could undergo horizontal transmission among different bacteria via the insertion sequence IS982. In conclusion, this study delves into the high prevalence of R. anatipestifer infection in waterfowl in Anhui, China. The isolated strains exhibit severe drug resistance issues, closely associated with the prevalence of antibiotic resistance genes (ARG). Additionally, our research investigates the ß-lactam antibiotic resistance mechanism in R. anatipestifer.

Riemerella anatipestifer UvrC is required for iron utilization, biofilm formation and virulence.

UvrC is a subunit of excinuclease ABC, which mediates nucleotide excision repair (NER) in bacteria. Our previous studies showed that transposon Tn4531 insertion in the UvrC encoding gene Riean_1413 results in reduced biofilm formation by Riemerella anatipestifer strain CH3 and attenuates virulence of strain YZb1. In this study, whether R. anatipestifer UvrC has some biological functions other than NER was investigated. Firstly, the uvrC of R. anatipestifer strain Yb2 was in-frame deleted by homologous recombination, generating deletion mutant ΔuvrC, and its complemented strain cΔuvrC was constructed based on Escherichia coli - R. anatipestifer shuttle plasmid pRES. Compared to the wild-type (WT) R. anatipestifer strain Yb2, uvrC deleted mutant ΔuvrC significantly reduced biofilm formation, tolerance to H2O2- and HOCl-induced oxidative stress, iron utilization, and adhesion to and invasion of duck embryonic hepatocytes, but not its growth curve and proteolytic activity. In addition, animal experiments showed that the LD50 value of ΔuvrC in ducklings was about 13-fold higher than that of the WT, and the bacterial loads in ΔuvrC infected ducklings were significantly lower than those in Yb2-infected ducklings, indicating uvrC deletion in R. anatipestifer attenuated virulence. Taken together, the results of this study indicate that R. anatipestifer UvrC is required for iron utilization, biofilm formation, oxidative stress tolerance and virulence of strain Yb2, demonstrating multiple functions of UvrC.RESEARCH HIGHLIGHTSDeletion of uvrC in R. anatipestfer Yb2 significantly reduced its biofilm formation.uvrC deletion led to reduced tolerance to H2O2- and HOCl-induced oxidative stress.The iron utilization of uvrC deleted mutant was significantly reduced.The uvrC deletion in R. anatipestifer Yb2 attenuated its virulence.

Genome-wide association study reveals serovar-associated genetic loci in Riemerella anatipestifer.

BACKGROUND: The disease caused by Riemerella anatipestifer (R. anatipestifer, RA) results in large economic losses to the global duck industry every year. Serovar-related genomic variation, such as the O-antigen and capsular polysaccharide (CPS) gene clusters, has been widely used for serotyping in many gram-negative bacteria. RA has been classified into at least 21 serovars based on slide agglutination, but the molecular basis of serotyping is unknown. In this study, we performed a pan-genome-wide association study (Pan-GWAS) to identify the genetic loci associated with RA serovars. RESULTS: The results revealed a significant association between the putative CPS synthesis gene locus and the serological phenotype. Further characterization of the CPS gene clusters in 11 representative serovar strains indicated that they were highly diverse and serovar-specific. The CPS gene cluster contained the key genes wzx and wzy, which are involved in the Wzx/Wzy-dependent pathway of CPS synthesis. Similar CPS loci have been found in some other species within the family Weeksellaceae. We have also shown that deletion of the wzy gene in RA results in capsular defects and cross-agglutination. CONCLUSIONS: This study indicates that the CPS synthesis gene cluster of R. anatipestifer is a serotype-specific genetic locus. Importantly, our finding provides a new perspective for the systematic analysis of the genetic basis of the R anatipestifer serovars and a potential target for establishing a complete molecular serotyping scheme.

A novel bivalent inactivated vaccine for ducks against Riemerella anatipestifer based on serotype distribution in southern China.

Riemerella anatipestifer (RA) causes epizootic infectious polyserositis in ducks with high mortality and leads to huge economic losses worldwide. Bacterial resistance poses a challenge for the control of the disease, vaccines failed to provide ideal cross-protection. Thus, the preparation of vaccines based on popular serotypes is important. In this study, we collected 700 brain and liver tissues of dead ducks from 8 provinces in southern China from 2016 to 2022 and obtained 195 RA isolates with serotypes 1, 2, 7, and 10. Serotypes 1 and 2 were the most prevalent (82%). A novel bivalent inactivated vaccine WZX-XT5 containing propolis adjuvant was prepared, we chose XT5 (serotype 1) and WZX (serotype 2) as vaccine strains and evaluated WZX-XT5-induced immune response and protective efficacy in ducks. Results showed that the XT5 (LD50, 3.5 × 103 CFU) exhibited high virulence and provided better protection against RA compared with ZXP, DCR and LCF1 (LD50, 108 CFU). Notably, the dose of 109 CFU provided ideal protection compared with 108 CFU, propolis and oil emulsion adjuvants induced stronger protective efficacy compared with aluminum hydroxide adjuvant. Importantly, WZX-XT5 immunization induced high levels of RA-specific IgY, IFN-γ, IL-2, and IL-4 in serum and offered over 90% protection against RA with ultra-high lethal dose in ducks. Additionally, no clinical signs of RA infection or obvious pathological damage in tissues were observed in protected ducks. Overall, this study first reports the identification, serotyping and virulence of RA in ducks of southern China and the preparation of a novel bivalent inactivated vaccine, providing useful scientific information to prevent and control RA infection.

Functional characterization of a TerC family protein of Riemerella anatipestifer in manganese detoxification and virulence.

Manganese (Mn) is an essential element for bacteria, but the overload of manganese is toxic. In a previous study, we showed that the cation diffusion facilitator protein MetA and the resistance-nodulation-division efflux pump MetB are responsible for Mn efflux in the bacterial pathogen Riemerella anatipestifer CH-1. However, whether this bacterium encodes additional manganese efflux proteins is unclear. In this study, we show that R. anatipestifer CH-1 encodes a tellurium resistance C (TerC) family protein with low similarity to other characterized TerC family proteins. Compared to the wild type (WT), the terC mutant of R. anatipestifer CH-1 (∆terC) is sensitive to Mn(II) intoxication. The ability of TerC to export manganese is higher than that of MetB but lower than that of MetA. Consistently, terC deletion (∆terC) led to intracellular accumulation of Mn2+ under excess manganese conditions. Further study showed that ∆terC was more sensitive than the WT to the oxidant hypoclorite but not to hydrogen peroxide. Mutagenesis studies showed that the mutant at amino acid sites of Glu116 (E116), Asp122 (D122), Glu245 (E245) Asp248 (D248), and Asp254 (D254) may be involved in the ability of TerC to export manganese. The transcription of terC was upregulated under excess manganese and downregulated under iron-limited conditions. However, this was not dependent on the manganese metabolism regulator MetR. In contrast to a strain lacking the manganese efflux pump MetA or MetB, the terC mutant is attenuated in virulence in a duckling model of infection due to increased sensitivity to duck serum. Finally, comparative analysis showed that homologs of TerC are distributed across the bacterial kingdom, suggesting that TerC exerts a conserved manganese efflux function.IMPORTANCERiemerella anatipestifer is a notorious bacterial pathogen of ducks and other birds. In R. anatipestifer, the genes involved in manganese efflux have not been completely identified, although MetA and MetB have been identified as two manganese exporters. Additionally, the function of TerC family proteins in manganese efflux is controversial. Here, we demonstrated that a TerC family protein helps prevent Mn(II) intoxication in R. anatipestifer and that the ability of TerC to export manganese is intermediate compared to that of MetA and MetB. Sequence analysis and mutagenesis studies showed that the conserved key amino sites of TerC are Glu116, Asp122, Glu245, Asp248, and Asp254. The transcription of terC was regulated by manganese excess and iron limitation. Finally, we show that TerC plays a role in the virulence of R. anatipestifer due to the increased sensitivity to duck serum, rather than the increased sensitivity to manganese. Taken together, these results expand our understanding of manganese efflux and the pathogenic mechanisms of R. anatipestifer.

Lactobacillus plantarum supernatant inhibits growth of Riemerella anatipestifer and mediates intestinal antimicrobial defense in Muscovy ducks.

Riemerella anatipestifer (RA) is an important pathogen of waterfowl, with multiple serotypes and a lack of cross-protection between each serotype, which leads to the continued widespread in the world and causing significant economic losses to the duck industry. Thus, prevention and inhibition of RA infection are of great concern. Previous research has established that Lactobacillus plantarum supernatant (LPS) can prevents the pathogenic bacteria infection. However, LPS whether inhibits RA and underlying mechanisms have not yet been clarified. In this study, we investigated the direct and indirect effects of LPS-ZG7 against RA infection in Muscovy ducks. The results demonstrated that LPS-ZG7 prevented RA growth in the presence of pH-neutralized, and the inhibition was relatively stable and unaffected by heat, acid-base and ultraviolet light (UV). Following flow cytometry data found that LPS-ZG7 increased RA membrane permeability and leakage of intracellular molecules. And scanning electron microscopy revealed LPS-ZG7 damaged the RA membrane integrity and leading to RA death. Furthermore, quantitative real time polymerase chain reaction (qPCR) analysis represented that LPS-ZG7 upregulated mucosal tight junction proteins occludin, claudin-1, and Zo-1 in Muscovy ducks, and increasing mucosal transport channels SGLT-1, PepT1, AQP2, AQP3, and AQP10 in duodenum, jejunum, and colon, then decreased the intestinal permeability and intestinal barrier disruption which were caused from RA. From the data, it is apparent that LPS-ZG7 enhanced intestinal mucosal integrity by rising villus height, villus height-to-crypt depth ratio and lower crypt depth. LPS-ZG7 significantly decreased intestinal epithelia cells apoptosis caused by RA invasion, and enhanced intestinal permeability and contribute to barrier dysfunction, ultimately improving intestinal health of host, indirectly leading to reduce diarrhea rate and mortality caused by RA. Overall, this study strengthens the idea that LPS-ZG7 directly inhibited the RA growth by increased RA membrane permeability and damaged the RA membrane integrity, and then indirectly enhanced intestinal mucosal integrity, improved intestinal health of host and mediated intestinal antimicrobial defense.

Functional characterization of two TolC in the resistance to drugs and metals and in the virulence of Riemerella anatipestifer.

IMPORTANCE: Riemerella anatipestifer (RA) is a notorious duck pathogen, characterized by a multitude of serotypes that exhibit no cross-reaction with one another. Moreover, RA is resistant to various antibacterial agents. Consequently, understanding the mechanisms behind resistance and identifying potential targets for drug development have become pressing needs. In this study, we show that the two TolC proteins play a role in the resistance to different drugs and metals and in the virulence. The results suggest that TolCA has a wider range of efflux substrates than TolCB. Except for gentamicin, neither TolCA nor TolCB was involved in the efflux of the other tested antibiotics. Strikingly, TolCA but not TolCB enhanced the frequency of resistance-conferring mutations. Moreover, TolCA was involved in RA virulence. Given its conservation in RA, TolCA has potential as a drug target for the development of therapeutics against RA infections.

Calcium phosphate nanoparticles conjugated with outer membrane vesicle of Riemerella anatipestifer for vaccine development in ducklings.

Biodegradable calcium phosphate nanoparticles offer a viable substitute for traditional adjuvants such as aluminum in vaccine production. Calcium phosphate nanoparticle adjuvanted with outer membrane vesicle (OMV) of gram negative bacteria may induce efficient immune response in the host. The present study was carried out to evaluate the potential of a mucosal vaccine formulation of calcium phosphate (CAP) nanoparticle using OMV of Riemerella anatipestifer (RA) as antigen against New Duck disease in ducks. The work was initiated with isolation, identification of RA, followed by OMV production and extraction. The CAP-OMV nanoparticle was prepared and characterized. The efficacy of the vaccine formulation and toxicity were studied in ducks. The average OMV yield in terms of protein concentration was found to be 122.33 ± 3.48 mg per liter of BHI broth. In SDS-PAGE, isolated OMVs exhibited presence of 16 distinct protein bands with molecular weight ranging from 142.1 to 12.1 kDa. Seven protein bands of 74.1, 69.3, 55.5, 50.6, 45.6, 25.1 and 13.1 kDa were detected relatively more distinct. The major bands detected in our findings were 42 kDa, 37 kDa and 16 kDa that corresponds to OmpA, OmpH, P6 respectively. The mean zeta size (±SD) and potential of the nanoparticle were 246.20 ± 0.53 nm and -25.60 ± 5.97 respectively. In transmission electron microscopy (TEM), the nanoparticles exhibited an average diameter of 129.80 ± 11.10 nm and displayed spherical morphology. The median protective dose (PD50) of CAP-OMV nanoparticle was 1881.10 µg of protein. Group I ducks received 3762 µg of protein (entrapped protein in CAP-OMV nanoparticle) via intra nasal route and it showed the highest serum IgG and secretory IgA level than other immunized groups. All experimental ducks were challenged with 10 × LD50 on 35 days of post primary immunization. Group I showed 100 % survivability in the challenge study. No gross and biochemical indication of acute or chronic toxicity were recorded. In conclusion, our results suggest that CAP-OMV nanoparticle can be a safe and efficient mucosal vaccine delivery system for RA, eliciting strong immune response in the host.