Development of LC-MS/MS analytical method for the rapid determination of Diquat in water and beverages.

This study aimed to develop simple, fast, and sensitive methods for the determination of diquat (DQ) in various matrices such as water and beverages. For water, direct injection was tested first, however, the sensitivity of the incurred samples were too low and couldn't possibly achieve the targeted limit of quantification. Hence, dilution with "weaker" injection solvents were tested, and the final conditions involved the dilution of water with acetonitrile (0.4 % ammonium hydroxide) which increased the sensitivity by more than ten times. Nevertheless, the beverages samples needed further treatment to achieve acceptable spiked recovery. The final conditions involved extraction using the aforementioned solvent, followed by heating and partitioning. Both of the methods satisfied the validation requirements, with an average recovery ranging from 85.9 to115 % and associated relative standard deviation (RSD %) within the range 3-8. Further applications on real samples were done to test the levels of contamination.

Seroprevalence of Toxoplasma gondii, Neospora caninum and Trichinella spp. in Pigs from Cairo, Egypt.

Pork production is a niche economy in Egypt, and pigs are typically raised as backyard animals with no sanitary control, potentially exposing them to various pathogens. Commercially available ELISAs were used to detect specific antibodies to the food-borne zoonotic parasites Toxoplasma gondii and Trichinella spp., as well as to Neospora caninum, in serum samples of pigs slaughtered at Egypt's only licensed pig abattoir, the El-Bassatin abattoir in Cairo. Among the tested sera (n = 332), seroreactivity for T. gondii was 45.8% (95% confidence interval: 40.4-51.3), N. caninum was 28.0% (95% CI: 23.3-33.2), and Trichinella spp. was 1.2% (95% CI: 0.4-3.3). Mixed infection was only detected for T. gondii and N. caninum (18.7%; 95% CI: 14.7-23.4). The seroprevalence of T. gondii was significantly higher (p = 0.0003) in animals collected from southern Cairo (15 May city slum) than in eastern Cairo (Ezbet El Nakhl slum). Seroprevalence for N. caninum was higher in western (Manshiyat Naser slum; p = 0.0003) and southern Cairo (15 May city slum; p = 0.0003) than in that of eastern Cairo (Ezbet El Nakhl slum; p = 0.0003). Moreover, female pigs exhibited a higher rate of N. caninum antibodies than male ones (p < 0.0001). This study provides the first seroprevalence data for N. caninum in pigs in Egypt, and updates the prevalence of the zoonotic parasites Trichinella spp. and T. gondii.

Multidrug-Resistant Enteropathogenic Escherichia coli Isolated from Diarrhoeic Calves, Milk, and Workers in Dairy Farms: A Potential Public Health Risk.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhoeagenic diseases in humans and cattle worldwide. The emergence of multidrug-resistant (MDR) EPEC from cattle sources is a public health concern. A total of 240 samples (75 diarrhoeic calves, 150 milk samples, and 15 workers) were examined for prevalence of EPEC in three dairy farms in Egypt. Antimicrobial resistance (AMR) traits were determined by antibiogram and polymerase chain reaction (PCR) detection of ß-lactamase-encoding genes, plasmid-mediated quinolone resistance genes, and carbapenemase-encoding genes. The genetic relatedness of the isolates was assessed using repetitive extragenic palindromic sequence-based PCR (REP-PCR). EPEC isolates were detected in 22.7% (17/75) of diarrhoeic calves, 5.3% (8/150) of milk samples, and 20% (3/15) of worker samples. The detected serovars were O26 (5%), O111 (3.3%), O124 (1.6%), O126 (0.8%), and O55 (0.8%). AMR-EPEC (harbouring any AMR gene) was detected in 9.2% of samples. Among isolates, blaTEM was the most detected gene (39.3%), followed by blaSHV (32.1%) and blaCTX-M-1 (25%). The qnrA, qnrB, and qnrS genes were detected in 21.4%, 10.7%, and 7.1% of isolates, respectively. The blaVIM gene was detected in 14.3% of isolates. All EPEC (100%) isolates were MDR. High resistance rates were reported for ampicillin (100%), tetracycline (89.3%), cefazolin (71%), and ciprofloxacin (64.3%). Three O26 isolates and two O111 isolates showed the highest multiple-antibiotic resistance (MAR) indices (0.85-0.92); these isolates harboured blaSHV-12 and blaCTX-M-15 genes, respectively. REP-PCR genotyping showed high genetic diversity of EPEC, although isolates belonging to the same serotype or farm were clustered together. Two worker isolates (O111 and O26) showed high genetic similarity (80-95%) with diarrhoeic calf isolates of matched serotypes/farms. This may highlight potential inter-species transmission within the farm. This study highlights the potential high risk of cattle (especially diarrhoeic calves) as disseminators of MDR-EPEC and/or their AMR genes in the study area. Prohibition of non-prescribed use of antibiotics in dairy farms in Egypt is strongly warranted.

Validation of real-time polymerase chain reaction versus conventional polymerase chain reaction for diagnosis of brucellosis in cattle sera.

BACKGROUND AND AIM: Different polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera. MATERIALS AND METHODS: One hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard. RESULTS: TaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%. CONCLUSION: A cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.