Serological survey of avian metapneumovirus in vaccinated and unvaccinated broiler chickens in Hong Kong.

In chickens, avian metapneumovirus (aMPV) causes the swollen head syndrome, a respiratory disease often associated with a reduction in egg production. The virus' epidemiology in East and Southeast Asia is poorly understood. An aMPV serological survey was conducted on broiler chicken farms of Hong Kong SAR to assess the seroprevalence of aMPV in unvaccinated batches and the serological status of vaccinated batches. Blood samples were collected from 53-93-day-old chickens in 24 chicken farms of Hong Kong SAR and sera were tested for aMPV antibodies by ELISA. Seroprevalence in aMPV unvaccinated birds was 80.6% (95% confidence interval (CI): 78.9-82.2) with a high variation between batches. Batch-level seroprevalence was not significantly different between birds hatched during the rainy season (74.3%, 95% CI: 64.0-84.5) and the ones hatched during the dry season (88.7%, 95% CI: 80.1-97.3, p = 0.5). The high seroprevalence and high antibody titers that are reported in this study indicate repeated exposure of broiler chickens to aMPV in Hong Kong SAR poultry farms. Based on these results, we recommend improving the surveillance of respiratory pathogens and applying appropriate prophylactic measures against aMPV such as vaccination.

Typhlitis induced by Histomonas meleagridis affects relative but not the absolute Escherichia coli counts and invasion in the gut in turkeys.

Unlike in chickens, dynamics of the gut microbiome in turkeys is limitedly understood and no data were yet published in context of pathological changes following experimental infection. Thus, the impact of Histomonas meleagridis-associated inflammatory changes in the caecal microbiome, especially the Escherichia coli population and their caecal wall invasion in turkeys was investigated. Birds experimentally inoculated with attenuated and/or virulent H. meleagridis and non-inoculated negative controls were divided based on the severity of macroscopic caecal lesions. The high throughput amplicon sequencing of 16SrRNA showed that the species richness and diversity of microbial community significantly decreased in severely affected caeca. The relative abundances of operational taxonomic units belonging to Anaerotignum lactatifermentans, E. coli, and Faecalibacterium prausnitzii were higher and paralleled with a decreased abundances of those belonging to Alistipes putredinis, Streptococcus alactolyticus, Lactobacillus salivarius and Lactobacillus reuteri in birds with the highest lesion scores. Although the relative abundance of E. coli was higher, the absolute count was not affected by the severity of pathological lesions. Immunohistochemistry showed that E. coli was only present in the luminal content of caecum and did not penetrate even severely inflamed and necrotized caecal wall. Overall, it was demonstrated that the fundamental shift in caecal microbiota of turkeys infected with H. meleagridis was attributed to the pathology induced by the parasite, which only led to relative but not absolute changes in E. coli population. Furthermore, E. coli cells did not show tendency to penetrate the caecal tissue even when the intestinal mucosal barriers were severely compromised.

Aerosol is the optimal route of respiratory tract infection to induce pathological lesions of colibacillosis by a lux-tagged avian pathogenic Escherichia coli in chickens.

Pathogenesis of colibacillosis caused by avian pathogenic Escherichia coli (APEC) in poultry is unclear and experimental studies reveal substantial inconsistency. In this study, the impact of three infection routes differing in the site of deposition of inoculum in the respiratory tract, were investigated. Two-weeks-old chickens were infected with a lux-tagged APEC strain via aerosol, intranasally or intratracheally, and sequentially sampled along with uninfected birds. At 1 and 3 days post infection (dpi), liver or spleen to body-weight ratios in all infected groups were significantly higher than in negative control, while at 7 dpi, such differences were significant in both organs in the aerosol-infected group. The infection-strain colonized tracheas and lungs in infected birds at 1 dpi and persisted until 7 dpi. Among infected groups, in lungs, bacterial load at 1 dpi was significantly lower in intranasally-inoculated birds. Histology revealed that, independent of infection route, lesions were mostly seen in the lower respiratory organs (lungs and air sacs) characterized by bronchitis/pneumonia and airsacculitis. Birds infected via aerosol showed the highest mean lesion score in lungs while intranasal application caused the mildest pathological changes, and difference between the two groups was significant at 1 dpi. In spleen, heterophilic infiltrations were prominent in affected birds. Interestingly, tracheas were pathologically unaffected. Altogether, the results demonstrated the importance of infection route, with aerosol being the most suitable to induce pathological lesions of colibacillosis without predisposing factors. Furthermore, the lux-tagged APEC strain was discriminated from native isolates enabling exact differentiation and enumeration.RESEARCH HIGHLIGHTS Lux-tagged APEC strain was used for infection to differentiate from native E. coli.Pathologically, lungs, air sacs and spleen but not trachea were affected.The route of infection strongly impacts the pathological outcome with APEC.The infection with APEC via aerosol caused the most severe lesions in chickens.

Bacterial Infection in Chicken Embryos and Consequences of Yolk Sac Constitution for Embryo Survival.

Bacterial infections in chicken eggs often cause mortality of embryos and clinical consequences in chicks but the pathological mechanism is unclear. We investigated the pathological changes and bacterial growth kinetics in dead and live embryos following infection with 2 Escherichia coli strains with a different clinical background and with 1 Salmonella Enteritidis strain. In 2 experiments, 12-day-old embryos were infected via the allantoic sac with 100 µl of 1 to 5 × 102 CFU/ml of one of the bacteria. In experiment 1, only dead embryos were sampled until 4 days postinfection (dpi), and surviving embryos were sampled at 5 dpi. In experiment 2, sampling was performed in dead and killed embryos sequentially at 1, 2, 3, and 4 dpi. The bacteria showed varying pathogenicity in embryos. The yolk sacs of dead embryos showed congestion, inflammation, damaged blood vessels, and abnormal endodermal epithelial cells. Such lesions were absent in the yolk sacs of negative control embryos and in those of embryos that survived infection. The livers and hearts of dead embryos showed congestion and lysed erythrocytes with no morphological changes in hepatocytes or myocardial cells. All bacteria multiplied rapidly in the yolks of infected embryos, although this did not predict survival. However, the livers of dead embryos contained significantly higher bacterial loads than the livers of the embryos that survived infection. The results provide evidence that lesions in the yolk sac, which have been neglected to date, coincide with embryonic mortality, underlining the importance of healthy yolk sacs for embryo survival.

Escherichia coli isolates from femoral bone marrow of broilers exhibit diverse pheno- and genotypic characteristics that do not correlate with macroscopic lesions of bacterial chondronecrosis with osteomyelitis.

The pheno- and genotypic relatedness among Escherichia coli isolates from broilers with and without macroscopic lesions of the femoral head were investigated. In total, 219 isolates obtained from the bone marrow were characterized by serotyping, antimicrobial resistance (AMR) profiles, phylogenetic grouping, detection of virulence-associated genes (VAGs) and pulsed-field gel electrophoresis (PFGE). Serotyping revealed that 48.4% of the isolates were assigned to one of the three serotypes (O78:K80: 21.0%, O2:K1: 18.7%, O1:K1: 8.7%). Substantial phenotypic variation was also noticed in AMR testing as most of the birds harboured E. coli isolates with different AMR profiles, which is of high clinical relevance. The majority of isolates could be classified into phylogenetic groups D (54.3%) and B2 (25.6%), followed by A (11.4%) and B1 (8.7%). Virulotyping showed that the highest number of isolates contained genes iucD (86.8%) and iss (84.9%), whereas papC (16.0%) and astA (12.3%) were present in least number of isolates. PFGE resulted in 58 different profiles from 200 typeable isolates. No correlation was found between specific serotypes, AMR profiles, phylogenetic groups, PFGE types or VAG profiles of E. coli and the occurrence of bacterial chondronecrosis with osteomyelitis, contradicting the hypothesis of a specific bacterial pheno- or genotype being involved in the disease.

High genetic diversity among extraintestinal Escherichia coli isolates in pullets and layers revealed by a longitudinal study.

BACKGROUND: Various information about the genetic diversity of Escherichia coli isolates from chickens are available but a detailed epidemiological investigation based upon isolates obtained from interrelated pullet and layer flocks is still missing. Therefore, in the course of a longitudinal epidemiological study on pullets and layers, 144 E. coli isolates from chickens with or without pathological lesions of the reproductive tract were serotyped and genotyped with pulsed-field gel electrophoresis (PFGE). These isolates were collected during rearing, peak and at the end of production. The actual study is the first of its kind so as to elucidate genetic relatedness among extraintestinal E. coli isolated from chickens with varying pathological conditions in interrelated layer farms/flocks at different stages of rearing. RESULTS: Serotyping revealed that 63.19 % of the isolates could not be assigned to any of the three serotypes tested whereas 30.55 % of the isolates belonged to serotype O1:K1, 4.86 % to O2:K1 and 1.38 % to O78:K80. After macrorestriction digest with XbaI, 91.66 % of the isolates were typeable resulting in 96 distinct PFGE profiles. Among them, five PFGE types included isolates collected from diseased chickens as well as from birds without pathological lesions. This finding shows that pathogenicity of E. coli in layers seems to be largely influenced by concurrent susceptibility factors. Furthermore, in six out of eight cases where two isolates were collected from each of eight birds, different PFGE types were found in the same or different organs of the same bird. The existence of predominant or persistent E. coli genotypes was only observed in two cases. CONCLUSIONS: It is concluded that extraintestinal E. coli genotypes and serotypes in pullets and layers are heterogenous and also do not maintain a single clonality within the same bird. The facts that E. coli strains did not show any definite clonal population structure based on geographical region, age of the host and pathological lesions should have relevance in further epidemiological studies and control strategies.

Pathogenesis of Gallibacterium anatis in a natural infection model fulfils Koch's postulates: 1. Folliculitis and drop in egg production are the predominant effects in specific pathogen free layers.

Pathogenicity of Gallibacterium anatis was studied in specific pathogen free layers in a controlled environment, applying the intranasal route for experimental infection. At 30 weeks, 37 hens were infected with 0.4 ml of 1.53 × 10(8) colony-forming units/ml suspension of G. anatis strain 07990 whereas equal numbers of hens were left uninfected for control. Following experimental infection, clinical signs and the number and weight of the eggs were recorded daily until 5 weeks post infection. Three birds from each group were killed at 3, 7, 10, 28 and 38 days post infection (d.p.i.) for necropsy and sampling for bacteriological and histopathological examinations. Additionally, necropsy examination was performed on all remaining birds at 38 d.p.i. G. anatis infection was found to have an immediate and severe effect on egg production, showing early and persistent colonization in respiratory and reproductive organs as well as in the gut of infected layers. In birds killed at various time points, G. anatis infection caused focal necrosis in the liver (1/37), folliculitis (2/37), pericarditis (3/37), haemorrhagic follicles (2/37), ruptured follicles (20/37), yolk in the body cavity (2/37) and egg peritonitis (1/37). The inflammation of the ovaries could be further confirmed by histopathological examination. Recovery of G. anatis from yolk at 10 d.p.i. indicates the potential of vertical transmission. Altogether, lesions reflect typical findings of G. anatis infection reported in natural cases. Thus, for the first time, lesions and the consecutive disease caused by G. anatis infection have been reproduced experimentally in a natural infection model.

Pathogenesis of Gallibacterium anatis in a natural infection model fulfils Koch's postulates: 2. Epididymitis and decreased semen quality are the predominant effects in specific pathogen free cockerels.

Pathogenesis of Gallibacterium anatis was investigated in specific pathogen free cockerels. Birds aged 35 weeks were infected intranasally with G. anatis whereas negative controls were left uninfected. Following infection, necropsy, bacteriological and histopathological investigations were performed in birds killed at 3, 7, 10, 28 and 38 days post infection (d.p.i.). Additionally, semen samples were collected twice a week until 5 weeks post infection for quality assessment. No clinical signs and gross pathological lesions were seen throughout the experiment. Bacteriological investigation revealed that G. anatis colonized the upper respiratory tract at 3 d.p.i. and could be isolated from testis and epididymis at 7 d.p.i. onwards. Bacterial persistence was found in the respiratory tract, gut and testis until the termination of the study at 38 d.p.i. Furthermore, G. anatis was isolated from semen arguing for the possibility of vertical transmission. Histopathological examination showed infiltration of mononuclear cells in epididymal tissue, indicating an inflammation. Density, total motility, progressive motility and membrane integrity of sperms were significantly decreased in infected birds as compared with control chickens. Along with these findings, an increase in spermatozoa with morphological defects was observed at different time points. In conclusion, the present study provides novel data on the impact of a G. anatis infection in cockerels in a natural infection model, thus helping to elucidate bacterial distribution, pathological lesions as well as influences on semen quality.

Association Between Escherichia coli Load in the Gut and Body Weight Gain in Broiler Chickens: A Systematic Review and Meta-Analysis.

Escherichia coli is one of the first commensal bacteria to colonize the chicken gut, where it predominates at an early stage of broiler chick life. Escherichia coli can potentially cause colibacillosis in chickens, spreading to extraintestinal systemic organs, which results in high economic losses in poultry industry, as well as a potential risk to public health. Many studies conducted to investigate the effectiveness of natural products as alternatives to antibiotics and to enhance the production performance in broiler chickens have assessed E. coli load in the chicken gut, but it is still unknown how the E. coli count is linked to broiler growth performance. A systematic search of published research articles, including key terms of interest such as broiler chickens, growth performance, and E. coli count, was conducted using two main databases (PubMed and the Web of Science). A random effects metaregression model was built to evaluate the association between E. coli count and weight gain in untreated groups of broilers (negative controls) from eligible studies. Of 2108 articles in the initial screening, 60 were included in the final meta-analysis. After data extraction, records from the ileum and cecum at 21, 35, and 42 days of age were considered for the meta-analysis. The meta-analysis showed that the average E. coli count in both the ileum and cecum at 21 days of age was positively associated with the average weight gain in the studied broiler chickens, while no statistically significant associations were found at 35 and 42 days of age. In conclusion, the positive association between E. coli load and body weight gain in young broiler chickens may be attributed to the relative dominance of E. coli in the gut of this age group when the microbial population is less diverse. The dynamic association between the production performance and the load of E. coli that has dubious pathogenic potential suggests the importance of careful assessment of commensal E. coli to develop strategies to enhance production, particularly in young broiler chickens.

A systematic review and meta-analysis on the efficacy of vaccination against colibacillosis in broiler production.

Colibacillosis, a disease caused by Escherichia coli in broiler chickens has serious implications on food safety, security, and economic sustainability. Antibiotics are required for treating the disease, while vaccination and biosecurity are used for its prevention. This systematic review and meta-analysis, conducted under the COST Action CA18217-European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), aimed to assess the efficacy of E. coli vaccination in broiler production and provide evidence-based recommendations. A comprehensive search of bibliographic databases, including, PubMed, CAB Abstracts, Web of Science and Agricola, yielded 2,722 articles. Following a defined protocol, 39 studies were selected for data extraction. Most of the studies were experimental infection trials, with only three field studies identified, underscoring the need for more field-based research. The selected studies reported various types of vaccines, including killed (n = 5), subunit (n = 8), outer membrane vesicles/protein-based (n = 4), live/live-attenuated (n = 16), and CpG oligodeoxynucleotides (ODN) (n = 6) vaccines. The risk of bias assessment revealed that a significant proportion of studies reporting mortality (92.3%) or feed conversion ratio (94.8%) as outcomes, had "unclear" regarding bias. The meta-analysis, focused on live-attenuated and CpG ODN vaccines, demonstrated a significant trend favoring both vaccination types in reducing mortality. However, the review also highlighted the challenges in reproducing colibacillosis in experimental setups, due to considerable variation in challenge models involving different routes of infection, predisposing factors, and challenge doses. This highlights the need for standardizing the challenge model to facilitate comparisons between studies and ensure consistent evaluation of vaccine candidates. While progress has been made in the development of E. coli vaccines for broilers, further research is needed to address concerns such as limited heterologous protection, practicability for application, evaluation of efficacy in field conditions and adoption of novel approaches.

Prevalence and characterization of antimicrobial-resistant Escherichia coli in chicken meat from wet markets in Hong Kong.

Given the close contact between animals, animal products, and consumers in wet markets, fresh meat products are considered a potential source and disseminator of antimicrobial-resistant (AMR) bacteria near the end of the food chain. This cross-sectional study was conducted to estimate the prevalence of select AMR-E. coli in fresh chicken meat collected from wet markets in Hong Kong and to determine target genes associated with the observed resistance phenotypes. Following a stratified random sampling design, 180 fresh half-chickens were purchased from 29 wet markets across Hong Kong in 2022 and immediately processed. After incubation, selective isolation was performed for extended-spectrum ß-lactamase producing (ESBL), carbapenem-resistant (CRE), and colistin-resistant (CSR) E. coli. The bacterial isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Disc Diffusion was used to determine the susceptibility of ESBL- and CRE-E. coli isolates. The broth microdilution method was used to determine the minimum inhibitory concentration of CSR-E. coli. Targeted resistance genes were then detected by PCR. The prevalence of ESBL-E. coli and CSR-E. coli were estimated at 88.8% (95% CI: 83.4-93.1%) and 6.7% (95% CI: 3.5-11.4%), respectively. No CRE-E. coli isolate was detected. The blaCTX-M-1 gene was the most common ß-lactamase group in isolated E. coli (80%), followed by blaTEM (63.7%); no blaSHV gene was detected. Forty-five percent of the isolates had blaTEM and blaCTX-M-1 simultaneously. The mcr-1 gene was detected in all 12 CSR isolates. Of 180 meat samples, 59 were from Mainland China, and 121 were locally sourced. There was no statistically significant difference in the prevalence of ESBL- and CSR-E. coli between the two sources. Our findings can be used to inform food safety risk assessments and set the stage for adopting targeted control and mitigation measures tailored to the local wet markets.

A comprehensive study of colisepticaemia progression in layer chickens applying novel tools elucidates pathogenesis and transmission of Escherichia coli into eggs.

Colisepticaemia caused by avian pathogenic Escherichia coli (APEC) is a challenging disease due to its high economic importance in poultry, dubious pathogenesis and potential link with zoonosis and food safety. The existing in vitro studies can't define hallmark traits of APEC isolates, suggesting a paradigm shift towards host response to understand pathogenesis. This study investigated the comprehensive pathological and microbial progression of colisepticaemia, and transmission of E. coli into eggs using novel tools. In total 48 hens were allocated into three groups and were inoculated intratracheally with ilux2-E. coli PA14/17480/5-/ovary (bioluminescent strain), E. coli PA14/17480/5-/ovary or phosphate buffered saline. Infection with both strains led to typical clinical signs and lesions of colibacillosis as in field outbreaks. Based on lung histopathology, colisepticaemia progression was divided into four disease stages as: stage I (1-3 days post infection (dpi)), stage II (6 dpi), stage III (9 dpi) and stage IV (16 dpi) that were histologically characterized by predominance of heterophils, mixed cells, pyogranuloma, and convalescence, respectively. As disease progressed, bacterial colonization in host organs also decreased, revealed by the quantification of bacterial bioluminescence, bacteriology, and quantitative immunohistochemistry. Furthermore, immunofluorescence, immunohistochemistry, and bacteria re-isolation showed that E. coli colonized the reproductive tract of infected hens and reached to egg yolk and albumen. In conclusion, the study provides novel insights into the pathogenesis of colisepticemia by characterizing microbial and pathological changes at different disease stages, and of the bacteria transmission to table eggs, which have serious consequences on poultry health and food safety.

Assessing pathogenicity of Gallibacterium anatis in a natural infection model: the respiratory and reproductive tracts of chickens are targets for bacterial colonization.

Two separate bird trials were performed to establish a reliable route of infection for Gallibacterium anatis in chickens, comparing intranasal (i.n.) and intravenous (i.v.) applications. Additionally, three mutually divergent isolates from three geographical locations, as shown by MALDI-TOF-MS and partial rpoB gene sequence analysis, were compared. In the first trial, birds were infected with one of the selected isolates by the i.v. or i.n. route. Subsequently, birds were killed 3, 12 and 24 h post infection following i.v. infection while at 3, 7 and 10 days post infection (dpi) in the case of i.n. infection along with birds of the control group. As a result, i.n. infection showed prominent and consistent bacterial tissue distribution in different organs persisting until 10 dpi, which was a striking contrast to the i.v. infection route. Likewise, histopathology revealed mild to severe tracheal lesions following i.n. infection. The second trial was set up to confirm both the achieved results and the robustness of i.n. infection but with an extended observation period, until 28 dpi In agreement with the preceding trial, identical results for bacteriological and histopathological examinations were obtained with persistency of bacteria until 28 dpi Comparing the three different isolates from Mexico, China and Austria, the Mexican isolate showed a somewhat higher pathogenicity than the other strains. Consequently, pathogenesis of G. anatis strains was studied in chickens elucidating i.n. infection as the most reliable route characterized by a long-lasting bacteraemia, targeting the respiratory and reproductive tract.

The genetic network underlying the evolution of pathogenicity in avian Escherichia coli.

Introduction: Colibacillosis is a worldwide prevalent disease in poultry production linked to Escherichia coli strains that belong to the avian pathogenic E. coli (APEC) pathotype. While many virulence factors have been linked to APEC isolates, no single gene or set of genes has been found to be exclusively associated with the pathotype. Moreover, a comprehensive description of the biological processes linked to APEC pathogenicity is currently lacking. Methods: In this study, we compiled a dataset of 2015 high-quality avian E. coli genomes from pathogenic and commensal isolates, based on publications from 2000 to 2021. We then conducted a genome-wide association study (GWAS) and integrated candidate gene identification with available protein-protein interaction data to decipher the genetic network underlying the biological processes connected to APEC pathogenicity. Results: Our GWAS identified variations in gene content for 13 genes and SNPs in 3 different genes associated with APEC isolates, suggesting both gene-level and SNP-level variations contribute to APEC pathogenicity. Integrating protein-protein interaction data, we found that 15 of these genes clustered in the same genetic network, suggesting the pathogenicity of APEC might be due to the interplay of different regulated pathways. We also found novel candidate genes including an uncharacterized multi-pass membrane protein (yciC) and the outer membrane porin (ompD) as linked to APEC isolates. Discussion: Our findings suggest that convergent pathways related to nutrient uptake from host cells and defense from host immune system play a major role in APEC pathogenicity. In addition, the dataset curated in this study represents a comprehensive historical genomic collection of avian E. coli isolates and constitutes a valuable resource for their comparative genomics investigations.

Trend of Antimicrobial Use in Food-Producing Animals from 2018 to 2020 in Nepal.

Antimicrobial resistance is a global public health problem and is primarily driven by the widespread overuse of antibiotics. However, antimicrobial use data in animals are not readily available due to the absence of a national database in many developing countries, including Nepal. This study was conducted to estimate the quantities of antimicrobials available in Nepal as an indicator of their use in food-producing animals between 2018 and 2020. Data were collected through surveys targeting major stakeholders: (i) the Department of Drug Administration (DDA), the Government of Nepal (GoN) for the authorized antimicrobials for veterinary use in Nepal, (ii) veterinary pharmaceuticals for antimicrobials produced in Nepal, (iii) the DDA and Veterinary Importers Association for antimicrobials bought by veterinary drug importers, and (iv) the Department of Customs, GoN, for antibiotics sourced through customs. Data showed that in the 3 years, a total of 96 trade names, comprising 35 genera of antibiotics representing 10 classes, were either produced or imported in Nepal. In total, 91,088 kg, 47,694 kg, and 45,671 kg of active ingredients of antimicrobials were available in 2018, 2019, and 2020, respectively. None of the antibiotics were intended for growth promotion, but were primarily for therapeutic purposes. Oxytetracycline, tilmicosin, and sulfadimidine were among the most-used antibiotics in Nepal in 2020. Oxytetracycline was primarily intended for parenteral application, whereas tilmicosin was solely for oral use. Sulfadimidine was available for oral use, except for a small proportion for injection purposes. Aminoglycosides, fluroquinolones, nitrofurans, sulfonamides, and tetracyclines were mostly produced locally, whereas cephalosporins, macrolides and "other" classes of antimicrobials were imported. Amphenicols and penicillins were exclusively imported and nitrofurans were produced locally only. In general, except for tetracyclines, the volume of antimicrobials produced locally and/or imported in 2020 was lower than that in 2018, which corresponded to a decreasing trend in total antimicrobials available. Furthermore, the subsequent years have seen a decrease in the use of critically important antibiotics, particularly class I antibiotics. Finally, this study has firstly established a benchmark for future monitoring of antimicrobial usage in food-producing animals in Nepal. These data are useful for risk analysis, planning, interpreting resistance surveillance data, and evaluating the effectiveness of prudent use, efforts, and mitigation strategies.

Aerosol delivered irradiated Escherichia coli confers serotype-independent protection and prevents colibacillosis in young chickens.

Escherichia coli causes colibacillosis in chickens, which has severe economic and public health consequences. For the first time, we investigated the efficacy of gamma-irradiated E. coli to prevent colibacillosis in chickens considering different strains and application routes. Electron microscopy, alamarBlue assay and matrix assisted laser desorption/ionization time-of- flight mass spectrometry showed that the cellular structure, metabolic activity and protein profiles of irradiated and non-treated E. coli PA14/17480/5-ovary (serotype O1:K1) were similar. Subsequently, three animal trials were performed using the irradiated E. coli and clinical signs, pathological lesions and bacterial colonization in systemic organs were assessed. In the first animal trial, the irradiated E. coli PA14/17480/5-ovary administered at 7 and 21 days of age via aerosol and oculonasal routes, respectively, prevented the occurrence of lesions and systemic bacterial spread after homologous challenge, as efficient as live infection or formalin-killed cells. In the second trial, a single aerosol application of the same irradiated strain in one-day old chickens was efficacious against challenges with a homologous or a heterologous strain (undefined serotype). The aerosol application elicited better protection as compared to oculonasal route. Finally, in the third trial, efficacy against E. coli PA15/19103-3 (serotype O78:K80) was shown. Additionally, previous results of homologous protection were reconfirmed. The irradiated PA15/19103-3 strain, which also showed lower metabolic activity, was less preferred even for the homologous protection, underlining the importance of the vaccine strain. In all the trials, the irradiated E. coli did not provoke antibody response indicating the importance of innate or cell mediated immunity for protection. In conclusion, this proof-of-concept study showed that the non-adjuvanted single aerosol application of irradiated "killed but metabolically active" E. coli provided promising results to prevent colibacillosis in chickens at an early stage of life. The findings open new avenues for vaccine production with E. coli in chickens using irradiation technology.

Aerosol vaccination of chicken pullets with irradiated avian pathogenic Escherichia coli induces a local immunostimulatory effect.

The present study investigated the expression of cytokines and cellular changes in chickens following vaccination with irradiated avian pathogenic Escherichia coli (APEC) and/or challenge. Four groups of 11-week-old pullets, each consisting of 16 birds were kept separately in isolators before they were sham inoculated (N), challenged only (C), vaccinated (V) or vaccinated and challenged (V+C). Vaccination was performed using irradiated APEC applied via aerosol. For challenge, the homologous strain was administered intratracheally. Birds were sacrificed on 3, 7, 14 and 21 days post challenge (dpc) to examine lesions, organ to body weight ratios and bacterial colonization. Lung and spleen were sampled for investigating gene expression of cytokines mediating inflammation by RT-qPCR and changes in the phenotype of subsets of mononuclear cells by flow cytometry. After re-stimulation of immune cells by co-cultivation with the pathogen, APEC-specific IFN-γ producing cells were determined. Challenged only birds showed more severe pathological and histopathological lesions, a higher probability of bacterial re-isolation and higher organ to body weight ratios compared to vaccinated and challenged birds. In the lung, an upregulation of IL-1ß and IL-6 following vaccination and/or challenge at 3 dpc was observed, whereas in the spleen IL-1ß was elevated. Changes were observed in macrophages and TCR-γδ+ cells within 7 dpc in spleen and lung of challenged birds. Furthermore, an increase of CD4+ cells in spleen and a rise of Bu-1+ cells in lung were present in vaccinated and challenged birds at 3 dpc. APEC re-stimulated lung and spleen mononuclear cells from only challenged pullets showed a significant increase of IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. Vaccinated and challenged chickens responded with a significant increase of IFN-γ+CD8α+ T cells in the lung and IFN-γ+TCR-γδ+ cells in the spleen. Re-stimulation of lung mononuclear cells from vaccinated birds resulted in a significant increase of both IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. In conclusion, vaccination with irradiated APEC caused enhanced pro-inflammatory response as well as the production of APEC-specific IFN-γ-producing γδ and CD8α T cells, which underlines the immunostimulatory effect of the vaccine in the lung. Hence, our study provides insights into the underlying immune mechanisms that account for the defense against APEC.

Quality Assessment of Day-Old Chickens on the Broiler Farms of Hong Kong.

The present study aimed to investigate the quality of newly hatched broiler chicks delivered to Hong Kong (imported or local), and to develop tailored recommendations to improve their management. During 2019-2021, 70 batches (34 imported from mainland China and 36 local) of one/three day old chicks on 11 broiler farms were studied. From each batch, 23 or 24 chicks (1647 in total) were assessed for abnormalities in appearance, navel, crop filling, dehydration, body weight, and length uniformity. Chicks were sacrificed, and yolk sac residues in three day old chicks were measured. Maternally derived antibody levels against Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), and avian reovirus (ARV) were measured in all chicks using an enzyme-linked immunosorbent assay (ELISA). The proportion of abnormal navel in most batches is high (median: 59%), ranging between 0 and 100%. The average length of chicks within batches ranges between 16.3 and 20.7 cm, and their average weights are 31-38.5 g upon delivery to the farms. On average, imported batches have a higher body weight and length than their local counterparts. The average yolk-free weight varies between 45 and 55 g, which is significantly lower in local batches (33-43 g). The mean antibody titers against NDV and ARV are higher in imported batches than in the local ones. In contrast, the mean antibody titer against IBDV is significantly lower in the imported batches. Concerning the overall lower quality of local chicks compared to the imported batches, establishing a well-managed broiler breeder farm and a hatchery in Hong Kong is highly recommended to enhance the health and productivity of the local broiler chicken industry.

Production of interferon gamma and interleukin 17A in chicken T-cell subpopulations hallmarks the stimulation with live, irradiated and killed avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis with different clinical manifestations. The disease is associated with compromised animal welfare and results in substantial economic losses in poultry production worldwide. So far, immunological mechanisms of protection against colibacillosis are not comprehensively resolved. Therefore, the present study aimed to use an ex vivo model applying chicken mononuclear cells stimulated by live and inactivated APEC. For this purpose, an 8-color flow cytometry panel was set up to target viable chicken immune cells including CD45+, CD8α+, CD4+, TCR-γδ+, Bu-1+ cells and monocytes/macrophages along with the cytokines interferon gamma (IFN-γ) or interleukin 17A (IL-17A). The 8-color flow cytometry panel was applied to investigate the effect of live and two different types of inactivated APEC (formalin-killed APEC and irradiated APEC) on the cellular immune response. For that, mononuclear cells from spleen, lung and blood of 10-week-old specific pathogen-free layer birds were isolated and stimulated with live, irradiated or killed APEC. Intracellular cytokine staining and RT-qPCR assays were applied for the detection of IFN-γ and IL-17A protein level, as well as at mRNA level for spleenocytes. Ex vivo stimulation of isolated splenocytes, lung and peripheral blood mononuclear cells (PBMCs) from chickens with live, irradiated or killed APEC showed an increasing number of IFN-γ and IL-17A producing cells at protein and mRNA level. Phenotyping of the cells from blood and organs revealed that IFN-γ and IL-17A were mainly produced by CD8α+, TCR-γδ+ T cells as well as CD4+ T cells following stimulation with APEC. Expression level of cytokines were very similar following stimulation with live and irradiated APEC but lower when killed APEC were applied. Consequently, in the present study, an ex vivo model using mononuclear cells of chickens was applied to investigate the cellular immune response against APEC. The results suggest the relevance of IFN-γ and IL-17A production in different immune cells following APEC infection in chickens which needs to be further investigated in APEC primed birds.

Identification of Chicken Transglutaminase 1 and In Situ Localization of Transglutaminase Activity in Avian Skin and Esophagus.

Transglutaminase 1 (TGM1) is a membrane-anchored enzyme that cross-links proteins during terminal differentiation of epidermal and esophageal keratinocytes in mammals. The current genome assembly of the chicken, which is a major model for avian skin biology, does not include an annotated region corresponding to TGM1. To close this gap of knowledge about the genetic control of avian cornification, we analyzed RNA-sequencing reads from organotypic chicken skin and identified TGM1 mRNA. By RT-PCR, we demonstrated that TGM1 is expressed in the skin and esophagus of chickens. The cysteine-rich sequence motif required for palmitoylation and membrane anchorage is conserved in the chicken TGM1 protein, and differentiated chicken keratinocytes display membrane-associated transglutaminase activity. Expression of TGM1 and prominent transglutaminase activity in the esophageal epithelium was also demonstrated in the zebra finch. Altogether, the results of this study indicate that TGM1 is conserved among birds and suggest that chicken keratinocytes may be a useful model for the study of TGM1 in non-mammalian cornification.