Mohamed Kamal Abdelhamid

Broiler birds as compared to layers are more susceptible to avian colibacillosis.An aerosol challenge using an ilux2-APEC effectively evaluates disease pathogenesis.The ilux2-APEC persistently colonized the bursa of Fabricius after aerosol exposure.

Avian pathogenic Escherichia coli (APEC) infections in poultry adversely affect health and production, with public health implications. This study assessed the potential of bioluminescence imaging for real-time, noninvasive tracking of microbial progression in 12-day-old chicken embryos inoculated with an APEC strain or its derivatives integrated either with luxABCDE or ilux2 operon. Eggs were imaged daily for bioluminescence detection, with dead embryos sampled immediately and survivors killed at 5 days post inoculation (dpi). The eggs were opened, and egg contents were imaged for bioluminescence. Yolks were sampled for E. coli isolation and quantification. Results showed lethality rates of 100%, 93.3% and 80% in embryos inoculated with native strain, luxABCDE or ilux2, respectively. Bioluminescence analysis showed increased bioluminescence signal strength over time preceding embryo death. Surviving embryos exhibited a sequential reduction in signal strength. A strong positive correlation was found between bioluminescence signal intensity in ovo and ex ovo, with ilux2-APEC infected eggs showing a higher luminoscore than luxABCDE-APEC. The E. coli load in yolks of APEC-inoculated eggs showed a positive trend over time. Overall, bioluminescence imaging of ilux2 operon labelled bacteria enabled more efficient real-time detection and monitoring of E. coli in ovo. Multiple imaging sessions on the same embryo throughout the experiment allow precise monitoring of infection progression without sequential culling. This offers a controlled platform for evaluating antimicrobial treatments' efficacy in an in ovo model that closely resembles in vivo environment in chickens. It also bears the potential to study other pathogens' infection patterns, especially those pose risks to public health.

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.

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.

Avian infectious bronchitis is one of the most common viral infections in chickens affecting all ages. The tropism of infectious bronchitis virus (IBV) strains became broader and more variable posing major implications for the effective control of IBV infection. In this study, two IBV viruses representing classic and variant strains were inoculated intranasally into day-old SPF chicks (10⁵ EID₅₀/0.2 ml/bird). Clinical signs were observed for 15 days post-infection (DPI). Five chicks from each group were euthanized at 2, 4, 6, 8, 10, 12, and 15 DPI for histopathology and virus antigen detection by IHC and quantitative rRT-PCR. Results revealed that both classic and variant IBV strains induced mild clinical signs with no mortalities and fewer various histopathological lesions in infected SPF chickens. Although the viruses were detected by rRT-PCR up to 12 DPI, the affected tissues showed regeneration after 10 DPI with IHC revealing no IBV antigen. In summary, no differences were found in the behaviour of both IBV isolates in chickens. The broad tissue tropism for both IBV strains as indicated by viral antigen detection in various organs with no clinical or gross lesion suggest that the main cause of death in IBV infection under field conditions occurs as a result of complication with secondary infections rather single IBV infection. Due to positive immunostaining in the bursa, it is thought that IBV infection has immunosuppressive consequences, hence further study is required to validate this impact.

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-1β and IL-6 following vaccination and/or challenge at 3 dpc was observed, whereas in the spleen IL-1β 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.

Pathogenesis of colibacillosis caused by avian pathogenic Escherichia coli (APEC) in poultry is not clear and experimental studies reveal substantial inconsistency. In this study, the impact of three infection routes that differ with respect to 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 routes, 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 the difference 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.

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, Streptococcusalactolyticus, 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.

Histomonosis in chickens often appears together with colibacillosis in the field. Thus, we have experimentally investigated consequences of the co-infection of birds with Histomonas meleagridis and avian pathogenic Escherichia coli (APEC) on the pathology, host microbiota and bacterial translocation from the gut. Commercial chicken layers were infected via oral and cloacal routes with lux-tagged APEC with or without H. meleagridis whereas negative controls were left uninfected. Except one bird, which died due to colibacillosis, no clinical signs were recorded in birds infected with bioluminescence lux gene tagged E. coli. In co-infected birds, depression and ruffled feathers were observed in 4 birds and average body weight gain significantly decreased. Typhlitis caused by H. meleagridis was present only in co-infected birds, which also had pronounced microscopic lesions in systemic organs such as liver, heart and spleen. The 16S rRNA gene amplicon sequencing showed that in co-infected birds, corresponding to the severity of cecal lesions, microbial species richness and diversity in caeca greatly decreased and the abundance of the Escherichia group, Helicobacter and Bacteroides was relatively higher with a reduction of commensals. Most of the shared Amplicon Sequencing Variants between cecum and blood in co-infected birds belonged to Pseudomonas, Staphylococcus, and members of Enterobacteriaceae while those assigned as Lactobacillus and members of Ruminococcaceae and Lachnospiraceae were found mainly in negative controls. In infected birds, E. coli in the cecal lumen penetrated into deeper layers, a phenomenon noticed with higher incidence in the dead and co-infected birds. Furthermore, numbers of lux-tagged E. coli in caeca were significantly higher at every sampling date in co-infected birds. Altogether, infection of layers with H. meleagridis and E. coli resulted in more severe pathological changes, dramatic shift in the cecal mucosa-associated microbiota, higher tissue colonization of pathogenic bacteria such as avian pathogenic E. coli in the gut and increased penetration of E. coli from the cecal lumen toward peritoneum. This study provides novel insights into the parasite-bacteria interaction in vivo highlighting the role of H. meleagridis to support E. coli in the pathogenesis of colibacillosis in chickens.