Prevalence and Molecular Characterization of Mycoplasma Species, Pasteurella multocida, and Staphylococcus aureus Isolated from Calves with Respiratory Manifestations.

Bovine respiratory disease (BRD) is a complex syndrome associated with high mortality in young calves and causes severe economic losses in the cattle industry worldwide. The current study investigated the prevalence and molecular characterization of common bacterial pathogens associated with respiratory symptoms in young calves from Sadat City, one of the largest industrial cities in Menoufiya Governorate, Egypt. In between December 2020 and March 2021, 200 mixed-breed young calves of 6-12 months were examined clinically. Of them, sixty (30%) calves showed signs of respiratory manifestations, such as coughing, serous to mucopurulent nasal discharges, fever, and abnormal lung sound. Deep nasal (Nasopharyngeal) swabs were collected from the affected calves for bacteriological investigation. Phenotypic characterization and identification revealed Mycoplasma bovis, Mycoplasma bovigenitalium, Pasteurella multocida, and Staphylococcus aureus in 8.33%, 5%, 5%, and 5% of the tested samples, respectively. The PCR technique using species-specific primer sets successfully amplified the target bacterial DNA in all culture-positive samples, confirming the identity of the isolated bacterial species. Partial gene sequencing of 16S rRNA gene of M. bovigenitalium, P. multocida, and S. aureus, and mb-mp 81 gene of M. bovis revealed high nucleotide similarity and genetic relationship with respective bacterial species reported from Egypt and around the world, suggesting transmission of these bacterial species between animal host species and localities. Our study highlights the four important bacterial strains associated with respiratory disorders in calves and suggests the possible spread of these bacterial pathogens across animal species and different geographic locations. Further studies using WGS and a large number of isolates are required to investigate the realistic lineage of Egyptian isolates and globally.

Clinical, histopathological, and molecular characterization of Mycoplasma species in sheep and goats in Egypt.

BACKGROUND AND AIM: Mycoplasma infection in small ruminants is a serious problem in sheep and goat herds around the world. It is responsible for high economic losses and decreased animal productivity. This study aimed to highlight the clinical, histopathological, minimum inhibitory concentration (MIC), and molecular characterization of Mycoplasma species in sheep and goats in Menoufiya Governorate, Egypt. MATERIALS AND METHODS: A total of 234 samples were collected; 104 samples were collected from pneumonic lung tissues from the abattoir, in addition, 10 and 20 samples collected from apparently and diseased sheep, respectively, and 40 and 60 samples were collected from apparently and diseased goats, respectively, which were subjected to isolation onto pleuropneumonia-like organism medium. Polymerase chain reaction (PCR), histopathological examination, and determination of the MIC were also performed. RESULTS: Of 104 samples of lung tissues showing pneumonic lesions, 56 (53.84%) were positive for Mycoplasma isolation. The positive isolation of Mycoplasma from 10 and 20 samples from apparently and diseased sheep was 30% and 40%, respectively as well as the positive isolation of Mycoplasma was 17% and 56.66% out of 40 and 60 apparently healthy and diseased field goat's cases, respectively. All the diseased sheep and goats showed respiratory manifestations, including cough, bilateral nasal discharge, conjunctivitis, and systemic reaction. Evaluation of the MIC for Mycoplasma ovipneumoniae revealed that lincospectin and tylosin were the most effective antibiotics at 2.5 mg/mL. Histopathological examination of affected lung tissue showed extensive hemorrhagic pneumonia with extensive alveolar hemorrhage. The PCR technique proved to be a rapid, specific, and sensitive method for the detection of M. ovipneumoniae and Mycoplasma arginini at 390 and 326 bp, respectively. CONCLUSION: M. ovipneumoniae and M. arginini were the most prevalent species associated with respiratory infections in sheep and goats in the study area. Further studies are needed to investigate the role of these species in dissemination of the disease within herds of small ruminants.

Prevalence, Antibiogram and Genetic Characterization of Listeria monocytogenes from Food Products in Egypt.

World Health Organization classified Listeria monocytogenes as a major notable foodborne pathogen associated with high mortality and hospitalization. The study reports the prevalence, antibiogram, virulence determination and genetic characterization of L. monocytogenes from different food products. A total of 250 food samples, fifty samples each from raw milk, ice cream, minced meat, fish fillet and sausage were collected from the Menoufiya governorate in Egypt. L. monocytogenes was detected in 17 (6.8%) of the tested food samples including minced meat (14%), fish fillet (8%), sausage (6%) and raw milk (6%). The antimicrobial susceptibility assay of 17 L. monocytogenes isolates against seventeen antibiotics belonging to eight antibiotics classes revealed a high susceptibility to norfloxacin (82.3%), amoxicillin-clavulanic acid (76.4%), cefotaxime (70.5%), erythromycin (64.6%), amoxicillin (64.6%), gentamicin (58.7%) and vancomycin (58.7%). While, high resistance was observed against oxytetracycline (76.4%), trimethoprim-sulfamethoxazole (76.4%), chloramphenicol (70.5%), doxycycline (64.6%), levofloxacin (41.2%) and azithromycin (41.2%). Of note, all L. monocytogenes isolates were multidrug-resistant. The multiplex PCR successfully amplified L. monocytogenes in all tested isolates. Screening of the five virulence-related genes revealed the hlyA and iap as the most prevalent genes followed by actA gene, however, the inlA and prfA genes were not detected in any of the studied isolates. The partial 16S rRNA gene sequencing of three L. monocytogenes isolates showed a high nucleotide similarity (99.1-99.8%) between the study isolates and various global clones, and phylogenetic analysis clustered these L. monocytogenes strains with other Listeria species including L. welshimeri, L. seeligeri and L. innocua. This study demonstrates the impact of L. monocytogenes as a major contaminant of various food products and suggests more attention to the awareness and hygienic measures in the food industry.

Chronic incurable mastitis in sheep: prevalence, identification of predisposing factors, and genotyping of fungal causative species using PCR-RFLP.

Mycotic mastitis is a neglected problem type of incurable chronic mastitis in sheep flock of many countries which associated with wide economic burden. In the current study, a total of 600 ewes at Menofia governorate, Egypt, were subjected to clinical and molecular examination using PCR-RFLP to estimate the prevalence of chronic mycotic mastitis and identify the causative agent. A structured questionnaire is distributed to shepherds in the study area to identify the risky behavioral practices being followed and lead to increase the prevalence of mycotic mastitis cases. The results showed that out of 600 ewes examined, 150 showed clinical signs of mastitis (25%). A total of 25 ewes with clinical mastitis did not respond to antibiotic treatment for long time and suffered from mycotic mastitis (16.7%, CI 11.1-23.6%). A total of 31 fungal isolates were identified: 14 yeast spp., Candida albicans, Candida parapasilosis, Candida rugosa, and Saccharomyces spp. and 17 mold spp., Alternaria spp., and Fusarium spp. Results showed also the widespread of risky practices among shepherds which could be responsible for the increase the prevalence of mycotic mastitis among ewes in the study area including presenting of decayed food to sheep, uncontrolled usage of antibiotics for mastitis treatment, lack of usage of antiseptics, and keeping of chronic infected animals in flocks for breeding. In conclusions, using of specific ITS1 and ITS4 primer sets with PCR-RFLP technique provided a suitable method for rapid identification and genotyping of Candida spp., Scaccharomyces, Alternaria, and Fusarium vertolliodes isolated from chronic mastitis in sheep. Furthermore, this study is considered up to our knowledge one of scarce estimates available on mycotic mastitis in sheep flocks in Egypt. Mycotic mastitis existed at higher prevalence estimates in the study area and educational campaigns to shepherds are much required to increase their awareness on the threat of risky of behaviors responsible for spread of the disease among their animals.

Prevalence and Characterization of Coagulase Positive Staphylococci from Food Products and Human Specimens in Egypt.

Methicillin-resistant Staphylococcus aureus (MRSA) strains have veterinary and public health importance as they are responsible for a wide range of difficult to treat infections and food poisoning. Two hundred samples (50 samples each of minced meat, beef luncheon, Karish cheese, and human samples (pus swab from open wounds)) were cultured, and MRSA strains were identified using disk diffusion tests and mecA gene-based PCR. A total of 35% (70/200) of the examined samples were confirmed as coagulase-positive S. aureus in minced meat (46%), beef luncheon (44%), Karish cheese (44%), and human samples (22%). The MRSA strains showed resistance to amoxicillin (91.4%), penicillin (97.1%), cefoxitin (85.7%), cephradine (82.9%), tetracycline (57.2%), and erythromycin (52.8%). More than half of the tested S. aureus isolates harbored the mecA gene. The sequence analysis of the mecA gene from the minced meat, Karish cheese, and human samples revealed high genetic similarities between the S. aureus isolates from these sources. In conclusion, our findings indicate a risk for the transmission of the mecA gene of S. aureus across the food chain between humans and animal food products. Further studies should focus on finding additional epidemiological aspects of the MRSA strains in food chain.

Antibiogram and phylogenetic diversity of enterotoxigenic Staphylococcus aureus strains from milk products and public health implications.

Food poisoning caused by Staphylococcus aureus (S. aureus) toxins is considered one of the foremost public health threat that usually occurs through the ingestion of raw milk contaminated with staphylococcal enterotoxins. The current study spotlights on the prevalence, antibiogram and genetic diversity of S. aureus enterotoxin genes. One hundred and fifty of raw milk (90) and ice cream (60) samples were randomly collected from local markets from Sadat city, Egypt. S. aureus was recovered from 44% of raw milk and 20% of ice cream samples. The identification for the obtained S. aureus isolates was confirmed through targeting the nuc gene. Antibiogram pattern of 32 S. aureus isolates showed high resistance to Cefoxitin, Sulpha/Trimethoprim, Tetracycline, Norfloxacin, Penicillin and Cephradine. However, high susceptibility to Gentamycin and Vancomycin were observed. Multiplex PCR was a competent practise for the recognition of Staphylococcus enterotoxin (SE) genes (SEA, SEB and SED). The phylogenetic analysis of the SED gene of enterotoxigenic S. aureus strains showed identical similarity with 100% to each other and high similarity with other international isolates in GenBank from different localities and sources. The frequency of enterotoxigenic S. aureus strains in milk products could have serious hazardous effects on humans. These results suggested possible strains transmission between different geographical areas through the food and milk product trades.

Synergistic and Antagonistic Effects of Metal Nanoparticles in Combination with Antibiotics Against Some Reference Strains of Pathogenic Microorganisms.

BACKGROUND AND AIM: Nanosized inorganic antibacterial materials have received increasing attention in recent years. The present study aimed to determine the antimicrobial activity of silver (Ag) and zinc oxide (ZnO) nanoparticles alone and in combination with antibiotics against reference strains of pathogenic microorganisms as Staphylococcus aureus (Staph. aureus), Salmonella enterica subsp. Bukuru, Escherichia coli (E.coli) and Candida albicans ( C. albicans). METHODS: The antimicrobial effect of metal-nanoparticles (AgNPs and ZnONPS) and in combination with antibiotics was studied using the normal disc-diffusion method. RESULTS: Both AgNPs and ZnONPs had increased antibacterial activity with an increase in their concentration against Gram-positive bacterium (Staph. aureus), Gram-negative bacteria (E. coli and Salmonella spp) and no effect on C. albicans. The synergistic effect of antibiotics (azithromycin, cefotaxime, cefuroxime, fosfomycin and chloramphenicol) against E. coli was significantly increased in the presence of AgNPs compared to antibiotic only. However, all antibiotics had a synergistic effect in the presence of AgNps against Salmonella spp. On the other hand, the antibacterial action of AgNPs with oxacillin and neomycin antibiotics against Staph. aureus was significantly decreased in comparison with antibiotics only. The synergistic effect of antibiotics (azithromycin, oxacillin, cefotaxime, cefuroxime, fosfomycin and oxytetracycline) against E. coli was significantly increased in presence of ZnONPs compared to antibiotic only and also the synergistic effect of antibiotics (azithromycin, cefotaxime, cefuroxime, fosfomycin, chloramphenicol and oxytetracycline) against Staph. aureus was significantly increased in the presence of ZnONPs compared to antibiotics only. On the other hand, most antibiotics had an antagonistic effect in presence of ZnONps against Salmonella spp. CONCLUSION: AgNPs and ZnONPs demonstrate a good synergistic effect with antibiotics and this may open the door for a future combination therapy against pathogenic bacteria.

Genetic Diversity Among Candida albicans Isolated from Humans and Cattle with Respiratory Distress in Egypt.

As human populaces develop, they are progressively squeezed into higher living densities. The same is true for horticulture and animals expected to bolster these communities. Despite the high potential for zoonotic transmission, connections among humans and cattle have been understudied; however, Candida albicans remains the most important medical mycosis. The genesis of the mycobiome can vary, and interactions between humans and cattle are progressively being perceived as a key interface for disease transmission. αINT1 is a unique gene from Candida albicans; hence, it has been used for detection as well as intraspecific and interspecific phylogenetic analysis of C. albicans collected from human patients and cattle with pulmonary distress in urban-rural populations. A total of 1,921 specimens were examined by direct microscopy and culture to recover yeast associated with human infection. Identification was performed by micromorphology using an API 20C AUX system. The fungal species identified in bovine nasal specimens were Alternaria species (15%), Penicillium species, and C. albicans (6.7%). Other fungal species, such as Aspergillus niger, Torulopsis species, Mucor species (5%), Aspergillus flavus, Fusarium species, Trichosporon species (3.3%), C. rugosa, C. tropical, and Saccharomyces species (1.7%), were also isolated. In human sputum specimens, C. albicans (20%) and C. parapsilosis (2.7%) were the only reported yeast species in our samples. The four identified C. albicans species (two human and two cattle) were subjected to αINT1 gene sequence analysis, which confirmed major phylogenetic relationships among human and cattle isolates. This finding highlights the public health importance of bovines as a potential source for C. albicans zoonotic transmission to humans in an urban-rural community. Additionally, the close relationship between circulating C. albicans strains recorded in Egypt and the United States indicates the possible cross-species transmission of C. albicans between imported foreign and native cattle breeds.

Substantial Virulence Genes among Brucella melitensis Field Strains Isolated from Cattle in Egypt.

BACKGROUND AND OBJECTIVE: The economic losses due to brucellosis as well as its potential public health in human worldwide encourage more researches to find novel pathways for effective control methods of the disease. The objective of this study was to investigate the most prevalent Brucella strains obtained from cattle and their virulence genes. MATERIALS AND METHODS: Three hundred small-holders cows in Menoufia governorate, Egypt, were screened for brucellosis using rose bengal test (RBT) and confirmed by complement fixation test (CFT). Milk samples and supra-mammary lymph nodes of serologically positive cows were collected for bacteriological isolation and identification. The obtained isolates were genotyped using PCR and their virulence genes (omp25, omp31, manA, manB, virB and znuA) were screened. RESULTS: The prevalence rate of bovine brucellosis was 15 (5%), 11 (3.6%) and 7 (2.33%) by RBT, CFT and bacteriological examination, respectively. The seven isolates were identified and genotyped as Brucella melitensis biotype3. Furthermore, the molecular detection of substantial virulence genes revealed that manA, manB, omp25 and omp31 genes were detected in all tested B. melitensis strains. Meanwhile, the virB genes were detected in 4 strains and the znuA genes were detected in 3 strains among the isolated B. melitensis strains. CONCLUSION: It was concluded that B. melitensis biotype3 was the pre-dominant Brucella spp. as well as omp25, omp31, manA and manB were the most common related-virulence genes which assumed to play a worthy function in the pathogenesis of brucellosis.