Hanem El-Sharkawy

Fungal infections have a key effect on the commercial poultry production and welfare. Infections caused by fungi and their food contaminants are zoonotic and influence food safety. Eggs and cooked meats remain major public health concerns. Therefore, this research is aimed at examining the pathology and understanding the epidemiology of fungal infection in layer chicken flocks. The study was carried out on twenty-layer flocks from Kafrelsheikh Governorate, Egypt, from January 2019 to December 2020. In total, 600 samples were collected from 100 healthy and diseased layer chickens from different organs (skin, liver, lung, kidney, spleen, and ovary). In this work, we present the clinical and pathological characteristics of some fungal pathogens (Aspergillus spp. and Fusarium spp.) in layer chicken flocks, as they are responsible for reducing the egg production. In total, 19 fungal strains were isolated from individual chickens, and these were analysed to determine the fungal species. The total proportion of fungal infections at the farm level was (3/20) 15%. The main clinical signs were emaciation and mortalities that reached 4.34 % ± 0.84 . We report the first isolation of Aspergillus piperis and Fusarium species from the ovary of poultry, which is the main reason for egg retention and multiple numerous nodules of occasional caseating centers in layer ovaries. The histopathological findings of Aspergillus infection are indicated by the presence of branched hyphae that tend to be numerous and progressive. Furthermore, we found spherules with multiple endospores of Fusarium spp. in the ovaries. Morphological and molecular identification and analysis were performed to confirm the etiological agents.

Salmonella is a major cause of foodborne outbreaks. It causes gastroenteritis in humans and animals. This micro-organism causes severe illness in chickens and has a major impact on chicken productivity and the poultry industry. This study aimed to address the prevalence of Salmonella infection in broiler chicken farms in Kafrelsheikh, Gharbia, and Menofeya provinces in Egypt during 20202022. This work also aimed to evaluate the genetic characterization and antibiotic resistance of the isolated Salmonella strains. Clinical signs and mortalities were observed and recorded. In total, 832 samples were collected from 52 broiler flocks, including 26 from both one-week-old and 6-week-old chicken farms from different organs (liver, intestinal content, spleen, and gallbladder). The prevalence of Salmonella infections was reported in the study region to be 36.54. Of the 26 one-week-old farms surveyed, 11 (42.31) and 8/26 (30.77) of the six-week-old broiler chicken farms had Salmonella infections. Recovered isolates were serotyped as 9 (47.37) S. enteritidis O 1,9,12, ad monophasic H: g, m: -, 6 (31.58.) S. shangani 2, (10.53) S. gueuletapee 1, (5.26) S. II (salamae), and 1 (5.26) untypable. The results showed that Salmonella infection was predominant in one-week-old chicks compared to infection in six-week-old and uninfected flocks. All Salmonella isolates were resistant to ampicillin and erythromycin, while all isolates were sensitive to ciprofloxacin, chloramphenicol, and levofloxacin. The isolates also contained 10.53 (2/19) streptomycin, 10.53 (2/21) gentamicin, 15.79 (3/19) doxycycline, and 26.32 (5/19) lincomycin and colistin. The phenotypically resistant Salmonella samples against ampicillin, erythromycin, and macrolide harbored bla_TEM, bla_SHV, ermB, ereA, mphA, and ermB, respectively. This baseline data on Salmonella spp. prevalence, serotyping, and antibiotic profiles are combined to define the antimicrobial resistance to this endemic disease. Elucidation of the mechanisms underlying this drug resistance should be of general importance in understanding both the treatment and prevention of Salmonella infection in this part of Egypt.

This work aimed to characterize S. aureus isolates from the eyes of healthy and clinically affected equines in the Kafrelsheikh Governorate, Egypt. A total of 110 animals were examined for the presence of S. aureus, which was isolated from 33 animals with ophthalmic lesions and 77 healthy animals. We also investigated the antimicrobial resistance profile, oxacillin resistance mechanism, and the major virulence factors implicated in many studies of the ocular pathology of pathogenic S. aureus. The association between S. aureus eye infections and potential risk factors was also investigated. The frequency of S. aureus isolates from clinically affected equine eyes was significantly higher than in clinically healthy equids. A significant association was found between the frequency of S. aureus isolation from clinically affected equine eyes and risk factors including age and season but not with sex or breed factors. Antimicrobial resistance to common antibiotics used to treat equine eyes was also tested. Overall, the isolates showed the highest sensitivity to sulfamethoxazole (100%) and the highest resistance to cephalosporin (90.67%) and oxacillin (90.48%). PCR was used to demonstrate that mecA was present in 100% of oxacillin- and β-lactam-resistant S. aureus strains. The virulence factor genes Spa (x region), nuc, and hlg were identified in 62.5%, 100%, and 56%, of isolates, respectively, from clinically affected equines eyes. The severity of the eye lesions increased in the presence of γ-toxin-positive S. aureus. The phylogenetic tree of the Spa (x region) gene indicated a relationship with human reference strains isolated from Egypt as well as isolates from equines in Iran and Japan. This study provides insight into the prevalence, potential risk factors, clinical pictures, zoonotic potential, antimicrobial resistance, and β-lactam resistance mechanism of S. aureus strains that cause eye infection in equines from Egypt.

Enteropathogenic (EPEC) and Enterohemorrhagic (EHEC) Escherichia coli are considered emerging zoonotic pathogens of worldwide distribution. The pathogenicity of the bacteria is conferred by multiple virulence determinants, including the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (T3SS) and effector proteins, including the multifunctional secreted effector protein (EspF). EspF sequences differ between EPEC and EHEC serotypes in terms of the number and residues of SH3-binding polyproline-rich repeats and N-terminal localization sequence. The aim of this study was to discover additional cellular interactions of EspF that may play important roles in E. coli colonization using the Yeast two-hybrid screening system (Y2H). Y2H screening identified the anaphase-promoting complex inhibitor Mitotic Arrest-Deficient 2 Like 2 (MAD2L2) as a host protein that interacts with EspF. Using LUMIER assays, MAD2L2 was shown to interact with EspF variants from EHEC O157:H7 and O26:H11 as well as EPEC O127:H6. MAD2L2 is targeted by the non-homologous Shigella effector protein invasion plasmid antigen B (IpaB) to halt the cell cycle and limit epithelial cell turnover. Therefore, we postulate that interactions between EspF and MAD2L2 serve a similar function in promoting EPEC and EHEC colonization, since cellular turnover is a key method for bacteria removal from the epithelium. Future work should investigate the biological importance of this interaction that could promote the colonization of EPEC and EHEC E. coli in the host.

Diagnosis and treatment of ocular fungal infection in equine seems very challenging for owners and clinicians. The present study aimed to identify and characterize fungal species isolated from the eyes of clinically healthy and diseased equines (N = 100) from Dakahlia Governorate, Egypt. The work also involved morphological and molecular characterization of the major fungal species. In addition, correlations between the occurrence of isolated fungi and some of the potential risk factors were also investigated. Interestingly, the prevalence rate of ocular mycosis in all examined equines in the study was 28% and there were major clinical signs associated with ocular fungal infection. Moreover, the identified fungal species included Aspergillus flavus, A. fumigatus, A. niger, Penicillium spp., Mucor spp., and Alternari spp. with a corresponding prevalence rate of 63.9%, 27.8%, 15.3%, 18.1%, 13.9%, and 4.2%, respectively, in healthy equine eyes, while their prevalence in diseased equine eyes was 57.1%, 32.1%, 21.4%, 7.1%, 3.6%, and 0%. Furthermore, a statistical significant association (p < 0.05) was found between the frequency of isolation of A. fumigatus and Penicillium and several risk factors (breed, sex, and ground type), while the remaining risk factors and occurrence of fungi were not statistically correlated. A subset of the Aspergillus species samples positive by polymerase chain reaction (PCR) were sequenced and their phylogenetic analysis identified three species of Aspergillus. Taken together, our study provides novel data related to the occurrence of ocular mycosis in equine in Egypt. Given the zoonotic potential of some identified fungi, our data may be helpful for implementation of novel diagnostic and therapeutic strategies for combating this sight-threatening infection in equine.

Avipoxviruses (APVs) are among the most complex viruses that infect a wide range of birds’ species. The infection by APVs is often associated with breathing and swallowing difficulties, reduced growth, decreased egg production, and high mortalities in domestic poultry. In the present study, 200 cutaneous nodular samples were collected from different avian species (chicken, pigeon, turkey, and canary) suspected to be infected with APVs from Dakahlia Governorate, Egypt. Pooled samples (n = 40) were prepared and inoculated in embryonated chicken eggs (ECEs). APVs were then identified by polymerase chain reaction (PCR) and sequence analysis of the APV P4b gene. Furthermore, the forty strains of APVs were screened for the presence of reticuloendotheliosis virus (REV)-5′LTR in their genomes. Interestingly, the phylogenic tree of the APV P4b gene was separated into 2 clades: clade 1, in which our fowlpox virus (FWPV), turkeypox virus (TKPV), and canarypox virus (CNPV) isolates were grouped, along with reference FWPVs and TKPVs retrieved from GenBank, whereas, in clade2, the pigeonpox virus (PGPV) isolate was grouped with PGPVs retrieved from GenBank. Likewise, REV-5′LTR was amplified from 30 strains isolated from chicken, turkey, and canary, while PGPV strains were free from REV-5′LTR integration. To the best of our knowledge, this study involved the detection and characterization of REV-5′LTR insertions in the APVs field isolates in Egypt for the first time. Given the above information, further future research seems recommended to understand the impact of the resulting REV-5′LTR insertions on the pathogenesis, virulence, and inadequate vaccine protection against APVs.

Chicken Salmonella enterica serovars are enteric bacteria associated with massive public health risks and economic losses. There is a widespread antimicrobial resistance among S. enterica serotypes, and innovative solutions to antibiotic resistance are needed. We aimed to use probiotics to reduce antibiotic resistance and identify the major probiotic players that modify the early interactions between S. enterica and host cells. One-day-old cobb broiler chicks were challenged with S. typhimurium after oral inoculation with different probiotic strains for 3 days. The adherence of different probiotic strains to Caco-2 intestinal epithelial cells was studied in vitro. Lactobacillus (Lacticaseibacillus) casei ATTC334 and Bifidobacterium breve JCM1192 strains attached to Caco-2 cells stronger than B. infantis BL2416. L. casei ATTC334 and B. breve JCM1192 reduced S. typhimurium recovery from the cecal tonsils by competitive exclusion mechanism. Although B. infantis BL2416 bound poorly to Caco-2 epithelial cells, it reduced S. typhimurium recovery and increased IFN-γ and TNF-α production. L. casei ATTC334, B. breve JCM1192 and B. infantis BL2416 improved body weight gain and the food conversion rate in S. typhimurium-infected broilers. B. longum Ncc2785 neither attached to epithelial cells nor induced IFN-γ and TNF-α release and consequently did not prevent S. typhimurium colonization in broiler chickens. In conclusion, probiotics prevented the intestinal colonization of S. typhimurium in infected chickens by competitive exclusion or cytokine production mechanisms.

Avian influenza virus (AIV) remains one of the enzootic zoonotic diseases that challenges the poultry industry in Egypt. In the present study, a total of 500 tissue samples were collected from 100 chicken farms (broilers and layers) suspected to be infected with AIV through the period from 2009 to 2019 from Dakahlia governorate, Egypt. These samples were pooled in 100 working samples and screened for AIV then the positive samples were subjected to histopathological examination combined with real time-polymerase chain reaction (RRT-PCR). RRT-PCR positive samples were also subjected to conventional reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of H5 AIV and some of these resulting positive samples were sequenced for detection of the molecular nature of the studied virus. Interestingly, the histopathological examination revealed necrotic liver with leukocytic infiltration with degenerative changes with necrotic pancreatitis, edema, and intense lymphoid depletion of splenic tissue and hyperplastic tracheal epithelium. Likewise, edema and congested sub mucosal blood vessels and intense bronchial necrosis with hyalinized wall vascular wall and heterophils infiltration were reported. Pneumonic areas with intense leukocytic aggregation mainly and vasculitis of the pulmonary blood vessels were also detected in lung. Collectively, these significant pathological changes in examined tissues cohered with AIV infection. Regarding the molecular characterization, 66 samples were positive for AIV by RRT-PCR and 52 of them were positive for H5 AIV by RT-PCR. The phylogenetic analysis revealed that the H5 viruses identified in this study were aligned with other Egyptian H5N1 AIVs in the Egyptian sub clade 2.2.1, while some of the identified strains were aligned with other Egyptian H5N8 strains in the new Egyptian sub clade 2.3.4.4. Taken together, our present findings emphasize the wide spread of AIV in Egypt and the importance of developing an efficient surveillance and periodical screening program for controlling such disease of public health concern.

Inflammation is critical for infection control and acts as an arsenal defense mechanism against invading microbes through activation of the host immune system. It works via its inflammasome components to sense the dangerous invading microorganism and send messages to the immune system to destroy them. To date, the function of bovine macrophage inflammasome and its relationship with actin has not been identified. This study aimed to investigate the activation of bovine inflammasome by phase one flagellin from Salmonella typhimurium and its interaction with actin. Bovine monocyte-derived macrophages were prepared and challenged with S. typhimurium SL1344 phase one flagellin. The results demonstrated the relationship between the flagellin-based activation of inflammasome and actin rearrangement. The flagellin-based activation of inflammasome promoted the activation and co-localization of F-actin and the inflammasome complex. Actin was remodeled to different degrees according to the stage of inflammasome activation. The actin redistribution varied from polymerization to filopodia, while at the stage of pyroptotic cell death, actin was broken down and interacted with activated inflammasome complexes. In conclusion, flagellin-dependent inflammasome activation and actin localization to the inflammasome at the stage of pyroptotic cell death may be of importance for appropriate immune responses, pending further studies to explore the exact cross-linking between the inflammasome complex and actin.

Bifidobacterium longum 105-A produces markedly high amounts of capsular polysaccharides (CPS) and exopolysaccharides (EPS) that should play distinct roles in bacterial–host interactions. To identify the biological function of B. longum 105-A CPS/EPS, we carried out an informatics survey of the genome and identified the EPS-encoding genetic locus of B. longum 105-A that is responsible for the production of CPS/EPS. The role of CPS/EPS in the adaptation to gut tract environment and bacteria-gut cell interactions was investigated using the ΔcpsD mutant. A putative B. longum 105-A CPS/EPS gene cluster was shown to consist of 24 putative genes encoding a priming glycosyltransferase (cpsD), 7 glycosyltransferases, 4 CPS/EPS synthesis machinery proteins, and 3 dTDP-L-rhamnose synthesis enzymes. These enzymes should form a complex system that is involved in the biogenesis of CPS and/or EPS. To confirm this, we constructed a knockout mutant (ΔcpsD) by a double cross-over homologous recombination. Compared to wild-type, the ∆cpsD mutant showed a similar growth rate. However, it showed quicker sedimentation and formation of cell clusters in liquid culture. EPS was secreted by the ∆cpsD mutant, but had altered monosaccharide composition and molecular weight. Comparison of the morphology of B. longum 105-A wild-type and ∆cpsD by negative staining in light and electron microscopy revealed that the formation of fimbriae is drastically enhanced in the ∆cpsD mutant while the B. longum 105-A wild-type was coated by a thick capsule. The fimbriae expression in the ∆cpsD was closely associated with the disappearance of the CPS layer. The wild-type showed low pH tolerance, adaptation, and bile salt tolerance, but the ∆cpsD mutant had lost this survivability in gastric and duodenal environments. The ∆cpsD mutant was extensively able to bind to the human colon carcinoma Caco-2 cell line and was phagocytosed by murine macrophage RAW 264.7, whereas the wild-type did not bind to epithelial cells and totally resisted internalization by macrophages. Our results suggest that CPS/EPS production and fimbriae formation are negatively correlated and play key roles in the survival, attachment, and colonization of B. longum 105-A in the gut.