In the presented study, we developed a nanogold lateral glow immunoassay-based technique (LFI-COVID-19 antigen test) for the detection of SARS-CoV-2 nucleocapsid proteins; the developed LFI-COVID-19 Ag test has been tested for limit of detection (LOD), cross-reactivity and interfering substances, and performance. It was found that the performance of the developed LFI-COVID-19 antigen test when it was evaluated by RT-qPCR indicated 95, 98, and 97% for sensitivity, specificity and accuracy, respectively. This complies with the WHO guidelines. It was concluded that the developed LFI-COVID-19 antigen test is a point of care and an alternative approach to current laboratory methods, especially RT-qPCR. It provides an easy, rapid (within 20 min), and on-site diagnostic tool for COVID-19 infection, and it is a cheap test if it is manufactured on a large scale for commercial use.
Objective: This work was conducted for the development of a 5-combi lateral flow immunochro¬matographic kit (LFK) for rapid and simultaneous identification of the common bacterial causes of bovine mastitis. The following pathogens are the identification targets of this kit: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus agalactiae, and Streptococcus pyogenes in milk samples from suspected bovine mastitis cases. The conventional microbiological identification of these agents is not only time-consuming and requires a fully equipped laboratory but also requires experienced personnel. Materials and Methods: Rabbit polyclonal antibodies (PAbs) specific to the antigenic components of the selected pathogens were prepared, and the pathogen-specific IgG was separated, purified, and conjugated with nanogold that was laid on the conjugate pad. Guinea pig PAbs specific to the microbial antigens of the selected pathogens were prepared, and their IgG content was separated, purified, and used as a capture antibody in the test (T) line on the nitrocellulose (NC) strips. Goat anti-rabbit IgG antibodies were used to capture the rabbit antibodies in the control (C) line of NC strips. The kit was held in a device comprising five strip-holding channels for the above-mentioned five bacterial species antigens. The developed LFK was evaluated, and its sensitivity and specificity were determined. Results: The developed kits were applied for the examination of bovine milk samples from suspected mastitis cases, and the average sensitivity, specificity, and accuracy of 5-combi LFK for the detection of the five selected bacterial species compared to bacteriological examination (gold standard test) were 93.90%, 80.83%, and 90.53%, respectively. The minimal microbial count that gave positive results using the developed LFK was 103 colony forming unit/ml. Treatment of the milk samples with an application buffer and its pre-incubation in trypticase soy broth for 6 h at 37°C before testing significantly increased the sensitivity of the prepared LFK. The developed kit proved simple and convenient, and the results could be obtained in less than 10 min.
A total of 3608 quarter's milk samples were aseptically collected form 902 Friesian cows, of which 68 (7.54%) suffered from clinical mastitis and 120 (13.3%) proved to suffer from subclinical form of mastitis. Bacteriological examination of the milk samples revealed the isolation of the following pathogens from clinical and subclinical mastitis cases, respectively; E. coli (29.41% and 29.2%), S. aureus (19.12% and 25%), Str. agalactiae (16.2% and 16.6%), Str. dysgalactiae (14.7% and 20%), Str. uberis (7.3% and 2.5%), K. pneumoniae (4.4% and 3.3%), Str. fecalis (4.4% and 0%), Str. pyogenes (2.9% and 0%) and Pseudomonas aeruginosa (1.5% and 3.3%). Among the recovered E. coli serotypes, the serotype O111:K58 (B4) was the most prevalent (38.2%). An inactivated polyvalent autogenous mastitis vaccine was prepared from the most encountered bacterial pathogens, namely, E. coli, S. aureus, Str. agalactiae and Str. dysgalactiae. The selected strains for vaccine preparation were inactivated with a predetermined minimal lethal dose of gamma radiation (4krad kGy/min) and the bacterial inactivation was assured using bacteriological examination. Two experiments were done; in experiment No.1, the immunizing efficacy of the prepared vaccine was evaluated in two groups of apparently normal lactating cows (4 cows /group). The first group was unvaccinated and kept as a control group all over the study while the animals in the second group were immunized with the prepared vaccine. Vaccinated cows received two vaccinal doses (5ml) at two week interval. The vaccine was injected subcutaneously in the brachiocephalic muscle. In experiment No. 2, the therapeutic potential of the prepared vaccine was tested in four groups of lactating cows suffering from subclinical mastitis. The first group was left untreated. The second group was treated only with antibiotics (neomycin and penicillin) and the third group was immunized with the prepared vaccine alone. The fourth group was treated simultaneously both with the antibiotics and the prepared vaccine. In both experiments blood and milk samples from cows in all groups were collected before and at 2 weeks intervals post immunization and/or antibiotic treatment. Vaccinated cows in the two experiments did not develop clinical mastitis during the whole observation period that extends up to 20 weeks post treatment. The total immunoglobulin concentration (g/dl) measured four weeks post immunization was significantly higher in the serum and milk whey of immunized cows as compared to the non-immunized cows (P<0.01). The highest ELISA titers against E. coli (O111), S. aureus, Str. agalactiae and Str. dysgalactiae were recorded 4 weeks post immunization in the serum and milk whey of immunized cows as compared to the non-immunized group. In cows suffering from subclinical mastitis the vaccination and /or antibiotic treatment induced significant decrease in the somatic cell counts (P<0.01). Also, significant reduction of the CFU/ml of the examined milk samples (P<0.01) and spontaneous recovery were recorded...
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