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.

The alleviating effect of Phillygenin on the regulation of respiratory microbiota and its metabolites in IBV-infected broilers by inhibiting the TLR7/MyD88/NF-κB axis.

Phillygenin (PHI) is an active ingredient derived from the leaf of Forsythia suspensa that has been found to alleviate inflammation and peroxidation response. Avian infectious bronchitis (IB) is a major threat to poultry industry viral respiratory tract disease that infected with infectious bronchitis virus (IBV). This study investigated the protection of PHI to CEK cell and broiler's tracheal injury triggered by avian infectious bronchitis virus (IBV). The results showed that IBV infection did not cause serious clinical symptoms and slowing-body weight in PHI-treated broilers. The expression of virus loads, pro-inflammation factors (IL-6, TNF-α, and IL-1ß) in CEK cell, and tracheas were decreased compared to the IBV group, exhibiting its potent anti-inflammation. Mechanistically, the study demonstrated that the inhibition of TLR7/MyD88/NF-κB pathway was mainly involved in the protection effect of PHI to inflammation injury. Interestingly, a higher abundance of Firmicutes and Lactobacillus in respiratory tract was observed in PHI-treated broilers than in the IBV group. Significant differences were observed between the IBV group and PHI-treated group in the Ferroptosis, Tryptophan metabolism, and Glutathione metabolism pathways. PHI exhibited potent protection effect on IBV infection and alleviated inflammation injury, mainly through inhibiting TLR7/MyD88/NF-κB pathway. The study encourages further development of PHI, paving the way to its clinical use as a new candidate drug to relieve IBV-induced respiratory symptoms.

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.

Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring.

This study aimed to develop a method for standardized broth microdilution antimicrobial susceptibility testing (AST) of Avibacterium (Av.) paragallinarum, the causative agent of infectious coryza in chickens. For this, a total of 83 Av. paragallinarum isolates and strains were collected from 15 countries. To select unrelated isolates for method validation steps, macrorestriction analyses were performed with 15 Av. paragallinarum. The visible growth of Av. paragallinarum was examined in six broth media and growth curves were compiled. In Veterinary Fastidious Medium and cation-adjusted Mueller-Hinton broth (CAMHB) + 1% chicken serum + 0.0025% NADH (CAMHB + CS + NADH), visible growth of all isolates was detected and both media allowed adequate bacterial growth. Due to the better readability of Av. paragallinarum growth in microtiter plates, CAMHB + CS + NADH was chosen for AST. Repetitions of MIC testing with five epidemiologically unrelated isolates using a panel of 24 antimicrobial agents resulted in high essential MIC agreements of 96%-100% after 48-h incubation at 35 ± 2°C. Hence, the remaining 78 Av. paragallinarum were tested and demonstrated easily readable MICs with the proposed method. Differences in MICs were detected between isolates from different continents, with isolates from Africa showing lower MICs compared to isolates from America and Europe, which more often showed elevated MICs of aminoglycosides, quinolones, tetracyclines, and/or trimethoprim/sulfamethoxazole. PCR analyses of isolates used for method development revealed that isolates with elevated MICs of tetracyclines harbored the tetracycline resistance gene tet(B) but none of the other tested resistance genes were detected. Therefore, whole-genome sequencing data from 62 Av. paragallinarum were analyzed and revealed the presence of sequences showing nucleotide sequence identity to the genes aph(6)-Id, aph(3″)-Ib, blaTEM-1B, catA2, sul2, tet(B), tet(H), and mcr-like. Overall, the proposed method using CAMHB + CS + NADH for susceptibility testing with 48-h incubation time at 35 ± 2°C in ambient air was shown to be suitable for Av. paragallinarum. Due to a variety of resistance genes detected, the development of clinical breakpoints is highly recommended. IMPORTANCE: Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.

Extended period of selection for antimicrobial resistance due to recirculation of persistent antimicrobials in broilers.

OBJECTIVES: Antimicrobials can select for antimicrobial-resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable, recirculation of subinhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens. METHODS: The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (unstable), doxycycline or enrofloxacin (stable). Antimicrobials were extracted from the faecal samples and were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism. RESULTS: Up to 37 days after treatment, doxycycline and enrofloxacin had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the amoxicillin treatment. The amoxicillin treatment showed a significant difference (P ≤ 0.01 and P ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic resistance and a significant difference (P ≤ 0.05 and P ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, enrofloxacin treatment resulted in a complete non-WT E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations. CONCLUSIONS: Based on our findings, we suggest that persistence of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.

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.

Exploring the predictive power of jejunal microbiome composition in clinical and subclinical necrotic enteritis caused by Clostridium perfringens: insights from a broiler chicken model.

BACKGROUND: Necrotic enteritis (NE) is a severe intestinal infection that affects both humans and poultry. It is caused by the bacterium Clostridium perfringens (CP), but the precise mechanisms underlying the disease pathogenesis remain elusive. This study aims to develop an NE broiler chicken model, explore the impact of the microbiome on NE pathogenesis, and study the virulence of CP isolates with different toxin gene combinations. METHODS: This study established an animal disease model for NE in broiler chickens. The methodology encompassed inducing abrupt protein changes and immunosuppression in the first experiment, and in the second, challenging chickens with CP isolates containing various toxin genes. NE was evaluated through gross and histopathological scoring of the jejunum. Subsequently, jejunal contents were collected from these birds for microbiome analysis via 16S rRNA amplicon sequencing, followed by sequence analysis to investigate microbial diversity and abundance, employing different bioinformatic approaches. RESULTS: Our findings reveal that CP infection, combined with an abrupt increase in dietary protein concentration and/or infection with the immunosuppressive variant infectious bursal disease virus (vIBDV), predisposed birds to NE development. We observed a significant decrease (p < 0.0001) in the abundance of Lactobacillus and Romboutsia genera in the jejunum, accompanied by a notable increase (p < 0.0001) in Clostridium and Escherichia. Jejunal microbial dysbiosis and severe NE lesions were particularly evident in birds infected with CP isolates containing cpa, netB, tpeL, and cpb2 toxin genes, compared to CP isolates with other toxin gene combinations. Notably, birds that did not develop clinical or subclinical NE following CP infection exhibited a significantly higher (p < 0.0001) level of Romboutsia. These findings shed light on the complex interplay between CP infection, the gut microbiome, and NE pathogenesis in broiler chickens. CONCLUSION: Our study establishes that dysbiosis within the jejunal microbiome serves as a reliable biomarker for detecting subclinical and clinical NE in broiler chicken models. Additionally, we identify the potential of the genera Romboutsia and Lactobacillus as promising candidates for probiotic development, offering effective alternatives to antibiotics in NE prevention and control.

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.

Accumulation of resistance genes in Salmonella Typhimurium transmitted between poultry and dairy farms increases the risk to public health.

Salmonella Typhimurium is a zoonotic pathogen that poses a major threat to public health. This generalist serotype can be found in many hosts and the environment where varying selection pressures may result in the accumulation of antimicrobial resistance determinants. However, the transmission of this serotype between food-producing hosts, specifically between poultry layer flocks and nearby dairy herds, was never demonstrated. We investigated an outbreak at a dairy in Israel to determine the role of nearby poultry houses to be sources of infection. The 2-month outbreak resulted in a 47% mortality rate among 15 calves born in that period. Routine treatment of fluid therapy, a nonsteroidal anti-inflammatory, and cefquinome was ineffective, and control was achieved by the introduction of vaccination of dry cows against Salmonella (Bovivac S, MSD Animal Health) and a strict colostrum regime. Whole genome sequencing and antimicrobial sensitivity tests were performed on S. Typhimurium strains isolated from the dairy (n = 4) and strains recovered from poultry layer farms (n = 10). We identified acquired antimicrobial-resistant genes, including the blaCTX-M-55 gene, conferring resistance to extended-spectrum cephalosporins, which was exclusive to dairy isolates. Genetic similarity with less than five single nucleotide polymorphism differences between dairy and poultry strains suggested a transmission link. This investigation highlights the severe impact of S. Typhimurium on dairy farms and the transmission risk from nearby poultry farms. The accumulation of potentially transferable genes conferring resistance to critically important antimicrobials underscores the increased public health risk associated with S. Typhimurium circulation between animal hosts.IMPORTANCESalmonella Typhimurium is one of the major causes of food-borne illness globally. Infections may result in severe invasive disease, in which antimicrobial treatment is warranted. Therefore, the emergence of multi-drug-resistant strains poses a significant challenge to successful treatment and is considered one of the major threats to global health. S. Typhimurium can be found in a variety of animal hosts and environments; however, its transmission between food-producing animals, specifically poultry layers flocks and dairy herds, was never studied. Here, we demonstrate the transmission of the pathogen from poultry to a nearby dairy farm. Alarmingly, the multi-drug-resistant strains collected during the outbreak in the dairy had acquired resistance to extended-spectrum cephalosporins, antibiotics critically important in treating Salmonellosis in humans. The findings of the study emphasize the increased risk to public health posed by zoonotic pathogens' circulation between animal hosts.

Gut microbiota intervention alleviates pulmonary inflammation in broilers exposed to fine particulate matter from broiler house.

The gut microbiota of poultry is influenced by a variety of factors, including feed, drinking water, airborne dust, and footpads, among others. Gut microbiota can affect the immune reaction and inflammation in the lungs. To investigate the effect of gut microbiota variation on lung inflammation induced by PM2.5 (fine particulate matter) in broilers, 36 Arbor Acres (AA) broilers were randomly assigned to three groups: control group (CON), PM2.5 exposure group (PM), and PM2.5 exposure plus oral antibiotics group (PMA). We used non-absorbable antibiotics (ABX: neomycin and amikacin) to modify the microbiota composition in the PMA group. The intervention was conducted from the 18th to the 28th day of age. Broilers in the PM and PMA groups were exposed to PM by a systemic exposure method from 21 to 28 days old, and the concentration of PM2.5 was controlled at 2 mg/m3. At 28 days old, the lung injury score, relative mRNA expression of inflammatory factors, T-cell differentiation, and dendritic cell function were significantly increased in the PM group compared to the CON group, and those of the PMA group were significantly decreased compared to the PM group. There were significant differences in both α and ß diversity of cecal microbiota among these three groups. Numerous bacterial genera showed significant differences in relative abundance among the three groups. In conclusion, gut microbiota could affect PM2.5-induced lung inflammation in broilers by adjusting the capacity of antigen-presenting cells to activate T-cell differentiation. IMPORTANCE: Gut microbes can influence the development of lung inflammation, and fine particulate matter collected from broiler houses can lead to lung inflammation in broilers. In this study, we explored the effect of gut microbes modified by intestinal non-absorbable antibiotics on particulate matter-induced lung inflammation. The results showed that modification in the composition of gut microbiota could alleviate lung inflammation by attenuating the ability of dendritic cells to stimulate T-cell differentiation, which provides a new way to protect lung health in poultry farms.

Evaluation of lyophilized bacteriophage cocktail efficiency against multidrug-resistant Salmonella in broiler chickens.

Currently, phage biocontrol is increasingly used as a green and natural technology for treating Salmonella and other infections, but phages exhibit instability and activity loss during storage. Therefore, in this study, the effects of lyophilization on the activity and stability of phage cocktails for the control of multidrug-resistant Salmonella in broiler chickens were determined. Eight serotypes of Salmonella were isolated and identified from broiler chicken farms, and bacteriophages against multidrug-resistant Salmonella enterica subsp. enterica serovar Kentucky, Salmonella enterica subsp. enterica serovar Typhimrium and Salmonella enterica subsp. enterica serovar Enteritidis were isolated. The bacteriophage cocktail was prepared and lyophilized, and it was subjected to in vitro and in vivo examinations. A reconstituted lyophilized bacteriophage cocktail was used for the oral treatment of chicks before and after challenge with multidrug-resistant S. Kentucky. The colonization of cecum by S. Kentucky was detected by using real-time PCR, and the serum levels of IgM, IgA and IL-4 and pathological changes in the different groups were detected. Three Caudovirales phages families were identified including Autographiviridae, Straboviridae and Drexlerviridae against multidrug-resistant S. Kentucky, S. Typhimrium and S. Enteritidis. The groups treated with the bacteriophage cocktail showed no clinical signs, no postmortem lesions, and a mortality rate of 0%, which improved the growth performance parameters. Additionally, the estimated serum levels of IgM, IgA and IL-4 were significantly greater in the bacteriophage cocktail-treated groups. Lyophilization effectively preserves the long-term storage stability of phages. Therefore, lyophilized bacteriophage cocktail therapy is a valuable approach for controlling multidrug-resistant Salmonella infections in broiler chickens.

Potential of ZnO nanoparticles for multi-drug resistant Escherichia coli having CRISPR-Cas from poultry market in Lahore.

BACKGROUND AND OBJECTIVES: Apart from known factors such as irrational use of antibiotics and horizontal gene transfer, it is now reported that clustered regularly interspaced short palindromic repeats (CRISPR) are also associated with increased antimicrobial resistance. Hence, it is critical to explore alternatives to antibiotics to control economic losses. Therefore, the present study aimed to determine not only the association of CRISPR-Cas system with antibiotic resistance but also the potential of Zinc Oxide nanoparticles (ZnO-NPs) for avian pathogenic Escherichia coli (APEC) isolated from poultry market Lahore. MATERIALS AND METHODS: Samples (n = 100) were collected from live bird markets of Lahore, and isolates were confirmed as Escherichia coli (E. coli) using the Remel One fast kit, and APEC was identified using PCR. The antibiotic resistance pattern in APEC was determined using the minimum inhibitory concentration (MIC), followed by genotypic confirmation of antibiotic-resistant genes using the PCR. The CRISPR-Cas system was also identified in multidrug-resistant (MDR) isolates, and its association with antibiotics was determined using qRT-PCR. The potential of ZnO-NPs was evaluated for multidrug-resistant (MDR) isolates by MIC. RESULTS: All isolates of APEC were resistant to nalidixic acid, whereas 95% were resistant to chloramphenicol and 89% were resistant to streptomycin. Nineteen MDR APEC were found in the present study and the CRISPR-Cas system was detected in all of these MDR isolates. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR isolates of APEC. ZnO-NPs inhibited the growth of resistant isolates. CONCLUSIONS: The findings showed the presence of the CRISPR-Cas system in MDR strains of APEC, along with the potential of ZnO-NPs for a possible solution to proceed. This highlights the importance of regulating antimicrobial resistance in poultry to reduce potential health consequences.

Occurrence, antibiotic resistance profiles and associated risk factors of Klebsiella pneumoniae in poultry farms in selected districts of Somalia Reginal State, Ethiopia.

BACKGROUND: Klebsiella pneumoniae is an opportunistic infection that causes production losses and death in the chicken industry. A cross-sectional study was conducted on exotic chicken breeds reared at the Jigjiga poultry farm from November 2022 to May 2023 to estimate the occurrence, associated risk factors, and antimicrobial susceptibility profiles of Klebsiella pneumoniae. The chickens were selected using systematic random sampling techniques. A total of 384 cloacal swabs were collected aseptically and transported to the laboratory for analysis. For statistical analysis, STATA® version 14.0 statistical software was used. RESULTS: From 384 examined faecal samples, 258 (67.2%) prevalences of Klebsiella pneumoniae were found. Furthermore, the association of the study's risk factors with the prevalence of Klebsiella pneumoniae was explored, and no statistically significant association was identified between sex and age. Nonetheless, relative prevalence at the age level was higher in chickens aged 12 months (67.6%) and Sasso breeds (90.0%). Similarly, male chickens and those raised for meat and egg production had a high prevalence rate of 72.5 and 75.8%, respectively. A total of 30 isolated Klebsiella pneumoniae colonies were tested in vitro for antibiotic sensitivity for six drugs, and it was shown that Klebsiella pneumoniae is moderately sensitive to Penicillin G (43.3%) while having higher resistance to Oxytetracycline (80.0%). CONCLUSIONS: The current findings revealed that the research area had the highest prevalence of Klebsiella pneumoniae, and the isolates were resistant to commonly used drugs in the study area. Thus, a long-term intervention plan, thorough research to determine a nationwide status, as well as further multi-drug resistance patterns and molecular characterization, were urged.

Comparative genomics of quinolone-resistant Escherichia coli from broilers and humans in Norway.

BACKGROUND: The usage of fluoroquinolones in Norwegian livestock production is very low, including in broiler production. Historically, quinolone-resistant Escherichia coli (QREC) isolated from Norwegian production animals rarely occur. However, with the introduction of a selective screening method for QREC in the Norwegian monitoring programme for antimicrobial resistance in the veterinary sector in 2014; 89.5% of broiler caecal samples and 70.7% of broiler meat samples were positive. This triggered the concern if there could be possible links between broiler and human reservoirs of QREC. We are addressing this by characterizing genomes of QREC from humans (healthy carriers and patients) and broiler isolates (meat and caecum). RESULTS: The most frequent mechanism for quinolone resistance in both broiler and human E. coli isolates were mutations in the chromosomally located gyrA and parC genes, although plasmid mediated quinolone resistance (PMQR) was also identified. There was some relatedness of the isolates within human and broiler groups, but little between these two groups. Further, some overlap was seen for isolates with the same sequence type isolated from broiler and humans, but overall, the SNP distance was high. CONCLUSION: Based on data from this study, QREC from broiler makes a limited contribution to the incidence of QREC in humans in Norway.

Survival of highly related ESBL- and pAmpC- producing Escherichia coli in broiler farms identified before and after cleaning and disinfection using cgMLST.

BACKGROUND: Broiler chickens are frequently colonized with Extended-Spectrum Beta-Lactamase- (ESBL-) and plasmid mediated AmpC Beta-Lactamase- (pAmpC-) producing Enterobacterales, and we are confronted with the potential spread of these resistant bacteria in the food chain, in the environment, and to humans. Research focused on identifying of transmission routes and investigating potential intervention measures against ESBL- and pAmpC- producing bacteria in the broiler production chain. However, few data are available on the effects of cleaning and disinfection (C&D) procedures in broiler stables on ESBL- and pAmpC- producing bacteria. RESULTS: We systematically investigated five broiler stables before and after C&D and identified potential ESBL- and pAmpC- colonization sites after C&D in the broiler stables, including the anteroom and the nearby surrounding environment of the broiler stables. Phenotypically resistant E. coli isolates grown on MacConkey agar with cefotaxime were further analyzed for their beta-lactam resistance genes and phylogenetic groups, as well as the relation of isolates from the investigated stables before and after C&D by whole genome sequencing. Survival of ESBL- and pAmpC- producing E. coli is highly likely at sites where C&D was not performed or where insufficient cleaning was performed prior to disinfection. For the first time, we showed highly related ESBL-/pAmpC- producing E. coli isolates detected before and after C&D in four of five broiler stables examined with cgMLST. Survival of resistant isolates in investigated broiler stables as well as transmission of resistant isolates from broiler stables to the anteroom and surrounding environment and between broiler farms was shown. In addition, enterococci (frequently utilized to detect fecal contamination and for C&D control) can be used as an indicator bacterium for the detection of ESBL-/pAmpC- E. coli after C&D. CONCLUSION: We conclude that C&D can reduce ESBL-/pAmpC- producing E. coli in conventional broiler stables, but complete ESBL- and pAmpC- elimination does not seem to be possible in practice as several factors influence the C&D outcome (e.g. broiler stable condition, ESBL-/pAmpC- status prior to C&D, C&D procedures used, and biosecurity measures on the farm). A multifactorial approach, combining various hygiene- and management measures, is needed to reduce ESBL-/pAmpC- E. coli in broiler farms.

In vivo overexpression of the avian interleukin-17 in a necrotic enteritis disease model modulates the expression of antimicrobial peptides in the small intestine of broilers.

In humans and mice, the induction of interleukin (IL)-17 expression enhances epithelial barrier integrity through the secretion of antimicrobial peptides (AMP), thereby improving antibacterial defense. However, it is unclear whether IL-17 has similar antibacterial effects in chickens by modulating the expression of AMPs, such as avian beta-defensins (also known as gallinacins) and cathelicidins. This study evaluated the in vivo effects of inoculating 20-day-old broiler chickens with two doses of a plasmid encoding chicken IL-17 (pCDNA3.1/rchIL-17-V5-HIS TOPO plasmid [pCDNA3.1-IL-17]; 5 or 10 µg/bird). On day 23 of age, all broilers, except those in the negative control group, were orally challenged with a virulent Clostridium perfringens strain for three days. To investigate IL-17-mediated effects against C. perfringens infection, the expression of avian beta-defensin 1 (avBD1), avBD2, avBD4, avBD6, cathelicidins, and inducible nitric oxide synthase (iNOS) genes were quantified, and gross necrotic enteritis (NE) lesion scores were assessed in the small intestine. The results showed that broilers receiving the higher dose of pCDNA3.1-IL-17 (10 µg) had significantly lower NE lesion scores compared to those receiving the lower dose (5 µg), the vector control, and the positive control groups. Furthermore, the expression of all avian beta-defensins and cathelicidin genes was detectable across all groups, regardless of treatment and time points. IL-17 treatment led to significantly higher expression of avBD1, avBD2, avBD4, avBD6, cathelicidin, and iNOS in the duodenum, jejunum, and ileum compared to control chickens. In C. perfringens-infected chickens, the expression of avBD1, avBD2, avBD4, cathelicidin, and iNOS in the ileum was significantly higher than in control chickens. Pre-treatment with the higher dose of pCDNA3.1-IL-17 (10 µg) in infected chickens was associated with reduced NE lesion severity and increased expression of avBD1, avBD2, cathelicidin, and iNOS in the ileum, but not avBD4 and avBD6. These findings provide new insights into the potential effect of IL-17 and reduction in NE lesion severity by modulating AMP expression which may be involved in mediating protective immunity against intestinal infection with C. perfringens.

Combined analysis of the microbiome, metabolome and transcriptome of silkie chickens in response to avian pathogenic E. coli (APEC).

Avian colibacillosis is a bacterial disease caused by avian pathogenic Escherichia coli (APEC) that results in great losses in the poultry industry every year. Individual Silkie chickens of the same breed that are given the same feed in the same feeding conditions have different levels of resistance or susceptibility to APEC. Differences in gut microbes, gut metabolites, and gene expression in the spleen of APEC-resistant and APEC-susceptible chickens were compared, and multiple omics associations were analyzed to explore the mechanism of resistance to APEC in Silkie chickens. Compared with those in the APEC-susceptible group, the APEC-resistant group showed significantly increased abundances of many gut microorganisms, including Bacillus, Thermoactinomyces, Arthrobacter, and Ureibacillus, which were positively correlated with norvaline, l-arginine, and valyl-glycine levels. Intestinal tryptophan, indole, and indole derivative-related differentially abundant metabolites played an active role in combatting APEC infection. In the spleen, "response to stimulus" was the most significantly enriched GO term, and "cytokine‒cytokine receptor interaction" was the most significantly enriched KEGG pathway. The arginine biosynthesis and PPAR signaling pathways were the KEGG pathways that were significantly enriched with differentially abundant metabolites and differentially expressed genes. This study provides new insight into the prevention and treatment of APEC infection in Silkie chickens and lays a foundation to study the mechanism of APEC infection in poultry.

Phenotypic, genotypic and proteomic variations between poor and robust colonizing Campylobacter jejuni strains.

Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 105 CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates.

The activity of hyaD contributed to the virulence of avian Pasteurella multocida.

Fowl cholera is an infectious disease that affects both poultry and wild birds, characterized by hemorrhagic and septicemic symptoms, caused by Pasteurella multocida (P. multocida), and leading to substantial economic losses in the poultry sector. The development of genetic engineering vaccines against avian P. multocida encountered early-stage challenges due to the limited availability of effective gene editing tools. Presently, NgAgoDM-enhanced homologous recombination stands as a potent technique for achieving efficient gene knockout in avian P. multocida. Hence, this study employed NgAgoDM-enhanced homologous recombination to target and knockout hyaE (239-359aa), hyaD, hexABC, and hexD, denoted as ΔhyaE (239-359aa), ΔhyaD, ΔhexABC, and ΔhexD, respectively. Additionally, we generated a hyaD recovery strain with two point mutations, designated as mhyaD. Thus, this study systematically examined the impact of capsular synthetic gene clusters on the pathogenicity of P. multocida. Moreover, the study demonstrated the critical role of hyaD activity in the virulence of avian P. multocida. This study offers novel insights for enhancing attenuated vaccines further.

Epidemiological and molecular investigations of Salmonella isolated from duck farms in southwest and around area of Shandong, China.

Salmonella is a zoonotic pathogen posing a serious risk to the farming industry and public health due to food animals serving as reservoirs for future contamination and spread of Salmonella. The present study is designed to monitor the contamination status of Salmonella in duck farms and the main control points during breeding. 160 strains of duck-derived Salmonella were isolated from the 736 samples (cloacal swabs, feces, water, feed, soil, air and dead duck embryos) collected in southwest Shandong Province and the province's surrounding area. The percentage of Salmonella-positive samples collected was 21.74 % (160/736), and the greatest prevalence from duck embryo samples (40.00 %, 36/90). These Salmonella were classified into 23 serotypes depending on their O and H antigens, in which S. Typhimurium (30.15 %), S. Kottbus (13.97 %) and S. Enteritidis (10.29 %) were the prevailing serotypes. Subsequently, the molecular subtyping was done. Clustered regularly interspaced short palindromic repeats (CRISPR) analysis showed that 41 strains of S. Typhimurium and 14 strains of S. Enteritidis were classified into 13 and 3 genotypes, respectively. 19 S. Kottbus isolates from different sources featured ST1546, ST198, ST321, and ST1690 by multilocus sequence typing (MLST) analysis, among which ST1546 belongs to S. Kottbus was a new ST. The minimum spanning tree analysis based on the two CRISPR loci and seven MLST loci from all S. Typhimurium, S. Enteritidis and S. Kottbus isolates revealed that duck embryos, feed and water were key control points to the spread of Salmonella along the breeding chain. Meanwhile, the emergence of S. Kottbus in duck flocks was considered a potential public health hazard.