UNASSIGNED: The increasing resistance of R. anatipestifer has posed a significant threat to the poultry industry in recent years. The tet gene is the primary determinant of tetracycline resistance in numerous bacteria, and the enzyme modification gene tet(X) is predominantly detected in tetracycline-resistant R. anatipestifer strains.
UNASSIGNED: In this study, we evaluated the susceptibility of both the standard strain and clinical isolates of R. anatipestifer to doxycycline. And the expression levels of tet(X), tet(A), and tet(O) genes were detected. To assess drug susceptibility, shuttle plasmids were constructed to transfer the tet(X) gene into the standard strain of R. anatipestifer followed by treatment with chlorogenic acid.
UNASSIGNED: The results revealed that the minimum inhibitory concentration of doxycycline for the standard strain was 0.25μg/mL, whereas it exceeded 8μg/mL for the clinical isolates. Furthermore, there was a significant upregulation observed in expression levels of tet(X), tet(A), and tet(O) genes among induced strains. Interestingly, when transferring the tet(X) gene into the standard strain, its sensitivity to doxycycline decreased; however, MIC values for chlorogenic acid remained consistent between both standard and drug-resistant strains of R. anatipestifer. Moreover, we made a surprising discovery that screening passage with chlorogenic acid resulted in increased sensitivity of R. anatipestifer to doxycycline. Further analysis demonstrated a reversal in expression trends among three differentially expressed genes within induced drug resistance group after intervention with chlorogenic acid. The main objective behind this study is to investigate both killing effect exerted by chlorogenic acid on drug-resistant R. anatipestifer as well as its regulatory impact on drug resistance genes. This will provide novel insights and theoretical basis towards development of chlorogenic acid as a promising drug for treatment and control of drug resistance in R. anatipestifer.

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 β-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 β-lactamase-related genes among the isolates, including a novel blaRASA-1 variant (16.2%), the class A extended-spectrum β-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 β-lactam antibiotics while their occurrence in R. anatipestifer were not common. The blaOXA-209 variant, on the other hand, did not perform any β-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 β-lactam antibiotic resistance mechanism in R. anatipestifer.

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.

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.

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.

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.

Riemerella anatipestifer (R. anatipestifer) is one of the common pathogens found in poultry flocks, resulting in serious economic losses for the poultry industry due to high mortality, reduced growth rate, poor feed conversion, increased condemnations, and high treatment costs. The aim of this study was to phenotypically characterize phylogenetic relationships and assess the presence of resistance gene strains of R. anatipestifer obtained from various poultry species in Poland. A total of 57 isolates of Riemerella were included in this study. A polymerase chain reaction (PCR) and matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) were used for identification of the strains. The phylogenetic relationship of the R. anatipestifer isolates was determined by analysing the rpoB gene sequence. The susceptibility to antibiotics was evaluated by minimum inhibitory concentration (MIC) in liquid media. All of the field strains of R. anatipestifer were grouped into one of two clades resulting from rpoB gene sequencing. High MIC50 and MIC90 values were obtained for gentamycin, amikacin, and colistin. Low MIC50 and MIC90 values were obtained for amoxicillin cefuroxime, cefoperazone, piperacillin, and trimethoprim/sulfamethoxazole. Among the resistance genes, tet(X) and ermF were identified most frequently. This is the first phenotypic characterization of R. anatipestifer strains obtained from poultry flocks in Poland.

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.

OBJECTIVE: 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.

UNASSIGNED: Ducks suffer a huge economic loss as a result of infections with Pasteurella multocida and Riemerella anatipestifer, which cause high morbidity and mortality. Because these pathogens induce similar clinical symptoms when coinfections occur, it is very difficult to differentiate between them based just on clinical signs. Hence, these major pathogens must be quickly and accurately detected.
UNASSIGNED: A total of 104 birds ranging from 2 days to 4 weeks old were collected from Egyptian farms, and the outcomes were compared statistically. Conventional cultural identification procedures and a direct multiplex polymerase chain reaction assay were utilized to recognize both pathogens in a single tube reaction simultaneously. Then, the obtained isolates were characterized phenotypically and genotypically.
UNASSIGNED: Clinical signs appear at 2-4 weeks of age with respiratory distress (dyspnea), white fluid feces, and stunting. The scrutinized data demonstrated a significantly higher detection rate by PCR directly compared to classical culture procedures. Pasteurella multocida was detected only by PCR. The disc diffusion technique against ten antibiotics showed absolute susceptibilities to amikacin, doxycycline, and florfenicol. High levels of beta-lactam resistance were observed. Riemerella anatipestifer isolates were screened for pathogenicity and plasmid-borne blaTEM genes. All six isolates harbored five virulence genes: aspC, RA46, m28, pstS, and Nlp/P60. Moreover, blaTEM was identified into four isolates and deposited to GenBank with accession numbers OP347083, OP347084, OP347085, and OP347086.
UNASSIGNED: These results suggest advanced PCR assays can be applied to the field for rapid and valuable diagnosis of two significant pathogens and focus on the worth of ducks in the propagation of transferable antibiotic resistance genes into the environment.