The GE296_RS03820 and GE296_RS03830 genes are involved in capsular polysaccharide biosynthesis in Riemerella anatipestifer.

Riemerella anatipestifer is a pathogenic bacterium that causes duck serositis and meningitis, leading to significant harm to the duck industry. To escape from the host immune system, the meningitis-causing bacteria must survive and multiply in the bloodstream, relying on specific virulence factors such as capsules. Therefore, it is essential to study the genes involved in capsule biosynthesis in R. anatipestifer. In this study, we successfully constructed gene deletion mutants Δ3820 and Δ3830, targeting the GE296_RS03820 and GE296_RS03830 genes, respectively, using the RA-LZ01 strain as the parental strain. The growth kinetics analysis revealed that these two genes contribute to bacterial growth. Transmission and scanning electron microscopy (TEM and SEM) and silver staining showed that Δ3820 and Δ3830 produced the altered capsules and compounds of capsular polysaccharides (CPSs). Serum resistance test showed the mutants also exhibited reduced C3b deposition and decreased resistance serum killing. In vivo, Δ3820 and Δ3830 exhibited markedly declining capacity to cross the blood-brain barrier, compared to RA-LZ01. These findings indicate that the GE296_RS03820 and GE296_RS03830 genes are involved in CPSs biosynthesis and play a key role in the pathogenicity of R. anatipestifer. Furthermore, Δ3820 and Δ3830 mutants presented a tendency toward higher survival rates from RA-LZ01 challenge in vivo. Additionally, sera from ducklings immunized with the mutants showed cross-immunoreactivity with different serotypes of R. anatipestifer, including 1, 2, 7 and 10. Western blot and SDS-PAGE assays revealed that the altered CPSs of Δ3820 and Δ3830 resulted in the exposure of some conserved proteins playing the key role in the cross-immunoreactivity. Our study clearly demonstrated that the GE296_RS03820 and GE296_RS03830 genes are involved in CPS biosynthesis in R. anatipestifer and the capsule is a target for attenuation in vaccine development.

Iron efflux by IetA enhances ß-lactam aztreonam resistance and is linked to oxidative stress through cellular respiration in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer encodes an iron acquisition system, but whether it encodes the iron efflux pump and its role in antibiotic resistance are largely unknown. OBJECTIVES: To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance. METHODS: In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence. RESULTS: The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the ß-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes Ⅰ and Ⅱ. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT. CONCLUSION: Iron efflux is critical to alleviate oxidative stress damage and ß-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.

Occludin and collagen IV degradation mediated by the T9SS effector SspA contributes to blood-brain barrier damage in ducks during Riemerella anatipestifer infection.

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.

Isolation, identification, and proteomic analysis of outer membrane vesicles of Riemerella anatipestifer SX-1.

Riemerella anatipestifer, belonging to Weeksellaceae family Riemerella, is a bacterium that can infect ducks, geese, and turkeys, causing diseases known as duck infectious serositis, new duck disease, and duck septicemia. We collected diseased materials from ducks on a duck farm in China and then isolated and purified a strain of serotype 1 R. anatipestifer named SX-1. Animal experiments showed that SX-1 is a highly virulent strain with an LD50 value of 101 CFU/mL. The complete genome sequence was obtained. The complete genome sequence of R. anatipestifer SX-1 was 2,112,539 bp; 847 genes were involved in catalytic activity, and 445 genes were related to the cell membrane. The total length of the repetitive sequences was 8746 bp. Four CRISPR loci were predicted in R. anatipestifer strain SX-1, and 4 genomic islands were predicted. Concentration and ultra-high-speed centrifugation were used to extract the outer membrane vesicles of R. anatipestifer SX-1. The OMVs were extracted successfully. Particle size analysis revealed the size and abundance of particles: 147.4 nm, 94.9%; 293.6 nm, 1.1%; 327.2 nm, 1.1%; 397.2 nm, 0.3%; and 371.8 nm, 1.1%. The average size was 173.5 nm. Label-free proteomic technology was used to identify proteins in the outer membrane vesicles. ATCC 11845 served as the reference genome sequence, and 148 proteins were identified using proteomic analysis, which were classified into 5 categories based on their sources. Among them, 24 originated from cytoplasmic proteins, 4 from extracellular secreted proteins, 27 from outer membrane proteins, 10 from periplasmic proteins, and 83 from unknown sources. This study conducted a proteomic analysis of OMVs to provide a theoretical basis for the development of R. anatipestifer OMVs vaccines and adjuvants and lays the foundation for further research on the relationship between the pathogenicity of R. anatipestifer and OMVs.

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.

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.

Genomic analysis and experimental pathogenic characterization of Riemerella anatipestifer isolates from chickens in China.

Waterfowl have a high likelihood of being infected with Riemerella anatipestifer. Although the pathogen is found in domestic ducks, turkeys, geese, and wild birds, there is little information available about the consequences of infection during egg laying and hatching in chickens. Here, we present the first report of a novel sequence type of R. anatipestifer S63 isolated from chickens in China. On the basis of pan-genome analysis, we showed S63's genome occupies a distinct branch with other R. anatipestifer isolates from other hosts. Galleria mellonella larval tests indicated that S63 is less virulent than R. anatipestifer Ra36 isolated from ducks. Ducks and hens are susceptible to S63 infection. There is no mortality rate for chickens or ducks, but adult chickens experience neurological symptoms that reduce egg production and hatching rates. In chickens, S63 might be passed vertically from parents to offspring, resulting in "jelly-like" lifeless embryos. Using quantitative PCR, S63 was detected in the brain, liver, reproductive organs, and embryos. As far as we know, this is the first report of R. anatipestifer in hens, a disease that can reduce egg productivity, lower hatching rates, and produce jelly-like lifeless embryos, and the first report to raise the possibility that hens can be infected by roosters via semen.

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.

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.

Genome-based assessment of antimicrobial resistance reveals the lineage specificity of resistance and resistance gene profiles in Riemerella anatipestifer from China.

Riemerella anatipestifer (R. anatipestifer) is an important pathogen that causes severe systemic infections in domestic ducks, resulting in substantial economic losses for China's waterfowl industry. Controlling R. anatipestifer with antibiotics is extremely challenging due to its multidrug resistance. Notably, large-scale studies on antimicrobial resistance (AMR) and the corresponding genetic determinants in R. anatipestifer remain scarce. To solve this dilemma, more than 400 nonredundant R. anatipestifer isolates collected from 22 provinces in China between 1994 and 2021 were subjected to broth dilution antibiotic susceptibility assays, and their resistance-associated genetic determinants were characterized by whole-genome sequencing. While over 90% of the isolates was resistant to sulfamethoxazole, kanamycin, gentamicin, ofloxacin, norfloxacin, and trimethoprim, 88.48% of the isolates was resistant to the last-resort drug (tigecycline). Notably, R. anatipestifer resistance to oxacillin, norfloxacin, ofloxacin, and tetracycline was found to increase relatively over time. Genome-wide analysis revealed the alarmingly high prevalence of blaOXA-like (93.05%) and tet(X) (90.64%) genes and the uneven distribution of resistance genes among lineages. Overall, this study reveals a serious AMR situation regarding R. anatipestifer in China, with a high prevalence and high diversity of antimicrobial resistance genes, providing important data for the rational use of antibiotics in veterinary practice.IMPORTANCERiemerella anatipestifer (R. anatipestifer), an important waterfowl pathogen, has caused substantial economic losses worldwide, especially in China. Antimicrobial resistance (AMR) is a major challenge in controlling this pathogen. Although a few studies have reported antimicrobial resistance in R. anatipestifer, comprehensive data remain a gap. This study aims to address the lack of information on R. anatipestifer AMR and its genetic basis. By analyzing more than 400 isolates collected over two decades, this study reveals alarming levels of resistance to several antibiotics, including drugs of last resort. The study also revealed the lineage-specificity of resistance profiles and resistance gene profiles. Overall, this study provides new insights and updated data support for understanding AMR and its genetic determinants in R. anatipestifer.

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.

Promotion of Th1 and Th2 responses over Th17 in Riemerella anatipestifer stimulation in chicken splenocytes: Correlation of gga-miR-456-3p and gga-miR-16-5p with NOS2 and CCL5 expression.

Riemerella (R.) anatipestifer poses a significant threat to ducks, resulting in mortality rates ranging from 5-75%. This disease is highly infectious and economically consequential for domestic ducks. Although other avian species, such as chickens, also display susceptibility, the impact is comparatively less severe than in ducks. IL-17A has a pronounced correlation with R. anatipestifer infection in ducks, which is less in chickens. This study performed an in vitro transcriptome analysis using chicken splenic lymphocytes collected at 4-, 8-, and 24-hour intervals following R. anatipestifer stimulation. The primary objective was to discern the differentially expressed genes, with a specific focus on IL-17A and IL-17F expression. Moreover, an association between specific miRNAs with NOS2 and CCL5 was identified. The manifestation of riemerellosis in chickens was linked to heightened expression of Th1- and Th2-associated cells, while Th17 cells exhibited minimal involvement. This study elucidated the mechanism behind the absence of a Th17 immune response, shedding light on its role throughout disease progression. Additionally, through small RNA sequencing, we identified a connection between miRNAs, specifically miR-456-3p and miR-16-5p, and their respective target genes NOS2 and CCL5. These miRNAs are potential regulators of the inflammatory process during riemerellosis in chickens.

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.

Capsular polysaccharide determines the serotyping of Riemerella anatipestifer.

IMPORTANCE: Riemerella anatipestifer (R. anatipestifer) is one of the most important veterinary pathogens with at least 21 serotypes. However, the exact polysaccharide(s) that determine R. anatipestifer serotype is still unknown. This study has provided a preliminary exploration of the relationship between capsular polysaccharides and serotyping in R. anatipestifer and suggests possible directions for further investigation of the genetic basis of serotypes in this bacterium.

Effects of dietary nano-selenium supplementation on Riemerella anatipestifer vaccinated and challenged Pekin ducklings (Anas platyrhynchos).

Riemerella anatipestifer (RA) is a common disease causing economic losses to duck farms worldwide. Novel supplements are crucially needed to control this bacterium, enhance poultry performance, and produce synergistic effects with vaccines in stimulating the immune system. This study investigated the effect of nano-selenium (Nano-Se) on the vaccinated (VAC) and challenged (Ch) Pekin ducklings (Anas platyrhynchos) with RA. Five experimental groups (G1-G5) were included in this study: G1 was the control group, G2 was the RA-challenged group, G3 was the Nano-Se+Ch group, G4 was the VAC+Ch group, and G5 was the Nano-Se+VAC+Ch group. The Nano-Se (0.3 mg/kg diet) was supplemented for 5 weeks post-vaccination (PV). The ducklings were vaccinated subcutaneously with the RA vaccine at 7 days of age and challenged with RA at the 3rd week PV. Blood, pharyngeal swabs and tissue samples were collected at the 3rd week PV and at different times post-challenge (PC). The growth performance (weight gain and feed conversion ratio), clinical signs, gross lesions, mortality, bacterial shedding, haematological, immunological, and biochemical parameters, cytokines production, and histopathological lesion scores showed significant differences (P < 0.05) between the challenged (G2) group and the supplemented (G3 & G5) groups. G5 showed the highest (P < 0.05) growth performance, phagocytic activity, IgM and IgG, splenic interleukin-2 (IL-2), IL-10, and interferon-gamma (IFN-γ) gene expressions, and the lowest mortality, bacterial shedding, hepatic and renal damage, heterophil/lymphocyte ratio and lesion scores compared to the other groups. In conclusion, the supplementation of nano-selenium for five weeks in the diet can improve the growth performance, immune status, and cytokines production in ducklings vaccinated and challenged with RA.