Mastitis is a significant disease affecting dairy cattle farms in Egypt. The current study aimed to investigate the prevalence and major bacterial pathogens causing subclinical mastitis (SCM) in three bovine dairy herds, with a history of SCM, at three Governorates in North Upper Egypt. The antimicrobial resistance profiles and specific virulence-associated genes causing bovine SCM were investigated. One thousand sixty-quarter milk samples (QMS) were collected aseptically from 270 apparently healthy cows in three farms and examined. The total prevalence of SCM was 46% and 44.8% based on California Mastitis Test (CMT) and Somatic Cell Count (SCC), respectively. Bacteriological examination of CMT positive quarters revealed that the prevalence of bacterial isolation in subclinically mastitic quarters was 90.4% (26 and 64.3% had single and mixed isolates, respectively). The most frequent bacterial isolates were E. coli (49.8%), Staphylococcus aureus (44.9%), streptococci (44.1%) and non-aureus staphylococci (NAS) (37.1%). Antimicrobial susceptibility testing of isolates revealed a high degree of resistance to the most commonly used antimicrobial compound in human and veterinary medicine. Implementation of PCR revealed the presence of mecA and blaZ genes in 60% and 46.7% of S. aureus isolates and in 26.7% and 53.3% of NAS, respectively. Meanwhile 73.3% of streptococci isolates harbored aph(3’)-IIIa gene conferring resistance to aminoglycosides and cfb gene. All E. coli isolates harbored tetA gene conferring resistance to tetracycline and sul1 gene conferring resistance to sulfonamides. The fimH and tsh genes were found in 80% and 60%, respectively. A significant association between the phenotypes and genotypes of AMR in different bacteria was recorded. The presence of a high prevalence of SCM in dairy animals impacts milk production and milk quality. The coexistence of pathogenic bacteria in milk is alarming, threatens human health and has a public health significance. Herd health improvement interventions are required to protect human health and society.

Although normally regarded harmless commensals, enterococci may cause a range of different infections in humans, including urinary tract infections, sepsis, and endocarditis. The acquisition of vancomycin resistance by enterococci (VRE) has seriously affected the treatment and infection control of these organisms. VRE are frequently resistant to all antibiotics that are effective treatment for vancomycin-susceptible enterococci, which leaves clinicians treating VRE infections with limited therapeutic options. With VRE emerging as a global threat to public health, we aimed to isolate, identify enterococci species from tilapia and their resistance to van-mediated glycopeptide (vanA and vanC) as well as the presence of enterococcal surface protein (esp) using conventional and molecular methods. The cultural, biochemical (Vitek 2 system) and PCR results revealed eight Enterococcus isolates from the 80 fish samples (10%) to be further identified as E. faecalis (6/8, 75%) and E gallinarum (2/8, 25%). Intraperitoneal injection of healthy Nile tilapia with the eight Enterococcus isolates caused significant morbidity (70%) within 3 days and 100% mortality at 6 days post injection with general signs of septicemia. All of the eight Enterococcus isolates were found to be resistant to tetracycline. The 6/6 E. faecalis isolates were susceptible for penicillin, nitrofurantoin, gentamicin, and streptomycin. On the other hand 5/6 were susceptible for ampicillin, vancomycin, chloramphenicol and ciprofloxacin. The two isolates of E. gallinarum were sensitive to rifampicin and ciprofloxacin and resistant to vancomycin, chloramphenicol and erythromycin. Molecular characterization proved that they all presented the prototypic vanC element. On the whole, one of the two vancomycin resistance gene was present in 3/8 of the enterococci isolates, while the esp virulence gene was present in 1/8 of the enterococci isolates. The results in this study emphasise the potential role that aquatic environments are correlated to proximity to anthropogenic activities in determining the antimicrobial resistance patterns of Enterococcus spp. recovered from fish in the river Nile in Giza, Elmounib, Egypt as a continuation of our larger study on the reservoirs of antibiotic resistance in the environment.

Pasteurella (P.) multocida and Mannheimia (M.) haemolytica are the most two common pathogenic bacterial agents causing pneumonia in calves. Both bacteria are associated with significant economic losses in the cattle industry due to high morbidity and mortality rates, especially in the case of severe infections. The objectives of the present study were to perform serotyping and genotyping, as well as characterization of the virulence-associated genes in 48 bacterial isolates; 33 P. multocida and 15 M. haemolytica. All strains were isolated from pneumonic cattle calves showing respiratory manifestations such as fever, nasal discharges, and rapid breathing in North Upper Egypt governorates (Beni-Suef and El-Fayoum). PCR was applied as a confirmatory test using a specific universal gene, kmt1, and rpt2 for P. multocida and M. haemolytica, respectively. The results show that 29 (87.9%) P. multocida and 15 (100%) M. haemolytica isolates were positive for the corresponding universal gene. The results of serotyping indicate that 86.2% of P. multocida isolates belonged to serotype B:2, while 13.8% were untyped. Meanwhile, 60% and 40% of M. haemolytica isolates belonged to serotype 2 and serotype 1, respectively. Investigation of virulence-associated genes showed that all the tested P. multocida isolates harbored nanB, omp87, and toxA genes. Four M. haemolytica isolates harbored both gcp and lktC genes and of these, three isolates harbored the ssa gene. Sequencing of toxA gene of P. multocida and lktC gene of M. haemolytica in the current strains indicated a great homology with strains uploaded in gene banks from different hosts and localities worldwide.

In this study, 6150 animals (3600 dairy cattle and 2550 buffaloes) in different Egyptian governorates were tested by single intradermal cervical tuberculin test using PPD-B, 72 cattle (2%) and 26 buffaloes (1%) reacted positively. After slaughtering the PM examination revealed 49 (68.1%) of the slaughtered cattle showed visible lesions (VL); comparing with 17 (65.4%) in buffaloes, while 23 (31.9%) showed non visible lesions (NVL) comparing with 9 (64.6%) in buffaloes. The bacteriological examination of processed samples from the 72 slaughtered cattlerevealed 44 M. bovis isolates (61.1%); 40 (81.6%) were from 49 VL and 4 (17.4%) were from 23 NVL, compared to 15 (57.7%) Mycobacterial isolates from buffaloes; 10 (38.5%) were M .bovis, all were from the 17 VL (58.8%), and 5 isolates (19.2%) were MOTT, 3 (17.6%) were from the 17 VL and 2 (22.2%) were from the 9 NVL. The ESAT6-CFP10 mixture using ELISA of the tuberculin positive animals sera showed VL could detect 83.7% and 70.6% in cattle and buffaloes, respectively; compared to 89.8% and 76.5% in cattle and buffaloes, respectively using PPD-B. On the other hand, the sera collected from tuberculin positive animals with NVL, the antigen mixture revealed 17.4% and 22.2%in cattle and buffaloes, respectively; compared to 21.7% in cattle and 33.3% in buffaloes with the PPD-B. The PCR assay using Oligonucleotide primer that amplifies a 350bp fragment in RD7 region of M. bovis confirmed the cultural and biochemical identification of M. bovis isolates (PCR-positive) and MOTT (PCR-negative

| Pseudomonas aeruginosa is an opportunistic environmental pathogen causing serious problems in poultry farms. Our study investigated the prevalence of P. aeruginosa in 80 examined kidney samples of broiler chicken where 6 P. aeruginosa isolates (7.5%) were isolated. The extended spectrum β-lactamases (ESβLs) production was detected phenotypically in P. aeruginosa isolates using modified CLSI ESβLs confirmatory test and 66.7% of isolates were ESβLs producers. PCR was conducted on all isolates for detection for bla TEM and exoS genes that were found in 66.7 and 100% of isolates, respectively. Antibacterial activity of carvacrol oil was tested against all P. aeruginosa isolates at concentrations of 800, 400, 200, 100 and 50 µl/ml. Concentrations of 800, 400 and 200µl/ml showed complete growth inhibition while 100 and 50 µl/ml concentrations inhibited the growth of 66.7% and 33.3% of isolates, respectively. Real time-PCR (RT-PCR) was conducted on the non-inhibited P. aeruginosa isolates at 100 µl/ml concentration after treatment with carvacrol oil for detection of the possible effect on bla TEM and exoS genes expression and the results indicated mild reduction of both genes expression after treatment (0.6-0.8 folds). It was concluded that carvacrol oil could be used as alternatives for synthetic antimicrobial drugs.