Polymeric nanocarrier-based adjuvants to enhance a locally produced mucosal coryza vaccine in chicken.

Infectious coryza (IC) is an acute upper respiratory disease of chicken caused by Avibacterium (A.) paragallinarum. This disease results in an increased culling rate in meat chicken and a marked decrease in egg production (10% to more than 40%) in laying and breeding hens. Vaccines were first used against IC and effectively controlled the disease. Nanotechnology provides an excellent way to develop a new generation of vaccines. NPs have been widely used in vaccine design as adjuvants and antigen delivery vehicles and as antibacterial agents; thus, they can be used as inactivators for bacterial culture. In this research, the antibacterial effects of several nanoparticles (NPs), such as silicon dioxide with chitosan (SiO2-CS), oleoyl-chitosan (O.CS), silicon dioxide (SiO2), and iron oxide (Fe3O4), on A. paragallinarum were studied. Additionally, different A. paragallinarum vaccines were made using the same nanomaterials at a concentration of 400 µg/ml to help control infectious coryza disease in chicken. A concentration of 400 µg/ml of all the NPs tested was the best concentration for the inactivation of A. paragallinarum. Additionally, this study showed that the infectious coryza vaccine adjuvanted with SiO2 NPs had the highest immune response, followed by the infectious coryza vaccine adjuvanted with Fe3O4 NPs, the infectious coryza vaccine adjuvanted with SiO2-CS NPs, and the infectious coryza vaccine adjuvanted with O.CS NPs in comparison with the infectious coryza vaccine adjuvanted with liquid paraffin (a commercial vaccine).

Development of a lateral flow device for rapid simultaneous multiple detections of some common bacterial causes of bovine mastitis.

Objective: This work was conducted for the development of a 5-combi lateral flow immunochromatographic 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.

Development, preparation, and evaluation of a novel dotted lateral flow immunochromatographic kit for rapid diagnosis of dermatophytosis.

Dermatophytosis is a widely spread contagious zoonotic disease, affecting both man (tinea) and animals (ringworm). This disease is caused by a group of closely related keratinophilic fungi known collectively as the dermatophytes group. Although the wide distribution of dermatophytosis cases throughout the whole world and its adverse clinical effect on human health, economical effect on productive animals, and pet animal welfare, there is no rapid accurate diagnostic tool for such disease. The current conducted study tries to accomplish the difficult equation by achieving an accurate, sensitive, specific, user-friendly, rapid, robust, device-less, deliverable to end-users, and economic cost for the development and production of diagnostic kits. Through the development of a rapid diagnostic kit based on immunochromatographic assay with three major affordable reproducible production stages; preliminary stage, developmental and standardization stage, and evaluation stage. Obtaining dermatophytes-specific polyclonal antibodies against criteria-based selected dermatophytes strains associating proper gold nanoparticle preparation, characterization, and conjugation, with proper loading of the different bio-reactants on the efficiently laminated and fabricated lateral flow strips were the main challenge and control points through the whole process. Also, as a result of examining 100 animal samples using the new kit, the κ coefficients of the kit with the direct microscopy while the kit with the culture were 0.44 and 0.76, respectively. Therefore, the newly designated and developed kit showed a very promising competitive diagnostic result within 5-7 min through easy-to-be-performed three steps.

Development of Lateral Flow Immunochromatographic Test for Rapid Detection of SARS-CoV-2 Virus Antigens in Clinical Specimens.

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.

Preparation and evaluation of a lateral flow immunochromatographic nanogold diagnostic kit for brucellosis in sheep.

Background and Aim: Brucellosis is a zoonotic disease with a worldwide distribution. It has a serious impact on the health of humans and animals, along with a negative impact on the economy. This study aimed to prepare and evaluate the diagnostic performance of a lateral flow immunochromatographic test (LFIT) nanogold diagnostic kit for detecting brucellosis in sheep. Materials and Methods: A rapidly developed LFIT, in which lipopolysaccharide conjugates with nanogold molecules, was placed on the conjugate pad. One hundred ovine serum samples were tested to detect Brucella antibodies (Ab) using the prepared lateral flow immunochromatography assay (LFA) kit and Rose Bengal test. The evaluation of specificity, sensitivity, and accuracy for LFIT and Rose Bengal plate test was conducted using the P04310-10 IDEXX brucellosis ovine/caprine Ab enzyme-linked immunosorbent assay (ELISA) test (gold standard). Results: The lower amount of Brucella Ab in the ovine serum samples was detected and was 1.58 S/P ratio ELISA titer/100 µL using LFIT and with Rose Bengal to detect 1.86 S/P ratio ELISA. The results showed that the developed LFIT had high specificity with no cross-reactivity with other tested bacteria. The calculated sensitivity, specificity, and accuracy of LFIT and Rose Bengal test using the P04310-10 IDEXX brucellosis ovine/caprine Ab ELISA test (gold standard) were 74% and 89%, 81% and 59%, and 76.9% and 66%, respectively. Conclusion: The present results showed interesting results implying that the LFIA strip test could be used as a substantial diagnostic tool for field screening ovine Brucella as an essential step in the control of brucellosis. However, further studies for the validation of the present findings are necessary.

Preparation of ELISA and Lateral Flow Kits for rapid Diagnosis of Mycoplasma gallisepticum in Poultry.

Avian mycoplasmas were mainly the cause of poultry industry economic losses; reduced meat and egg production and increases the antibiotic treatment cost. Mycoplasma gallisepticum (MG) infection is designated as infectious sinusitis of turkeys and chronic respiratory disease of chickens (gasping, depression, semi closed eyes, infraorbital sinuses edema and decrease in egg production). This study aimed to prepare, evaluate and Compare in-house ELISA kits and lateral flow assay (LFA) from a local strain of MG with commercial ELISA kits and PCR consequently. A total of 54 samples (27 tracheal swabs, 10 trachea and 17 lung) and 50 serum samples collected from birds suffering from chronic respiratory disease were tested by prepared in-house ELISA, commercial ELISA kits, PCR and LFA; a high correlation coefficient between in-house ELISA using whole antigen or sonicated antigen and commercial kit was recorded. Lateral Flow assay (LFA) performance indicate a low sensitivity (77.5%) but maintain a high specificity (92%) compared to PCR. The in-house ELISA kits and LFA prepared could be used as a fast diagnostic technique for detection of MG in Egypt. According to the available knowledge the prepared LFA for diagnosis of MG infection in chickens was developed for the first time in Egypt.

Preparation and evaluation of Salmonella Enteritidis antigen conjugated with nanogold for screening of poultry flocks.

AIM: The present work aimed to develop lateral flow immunochromatographic strip (ICS) test for detection of Salmonella Enteritidis (SE) specific antibodies in chicken sera. MATERIALS AND METHODS: A rapid lateral flow immunochromatographic test (LFIT) has been developed, in which SE Group D antigen labeled with the gold chloride molecules laid on the conjugate pad. Staphylococcus aureus protein A was used as capture antibody at the test line (T) of a nitrocellulose (NC) membrane and anti-SE antigen-specific rabbit antibodies were used as capture antibody at the control line (C) of the NC strip in the lateral flow layout device. RESULTS: Using the developed LFIT, the minimal amount of SE-specific antibodies that can be detected in chicken serum sample was 1427 enzyme-linked immunosorbent assay (ELISA) unit/100 µl that was equal to 0.1 µg (Ab)/100 µl sample. 100 suspected serum samples collected from a poultry flock were tested with the prepared SE-LFIT kits and the locally prepared stained Salmonella antigen, and the results were compared with those obtained from examination of these samples with Salmonella Group D antibody ELISA kit as the gold standard test. The sensitivity, specificity, and accuracy of the prepared SE-LFIT antigen kits were 94.4%, 90%, and 94%, respectively, while those obtained with stained Salmonella antigen were 88.8%, 90%, and 89%, respectively. CONCLUSION: The developed test is a simple field rapid test of high sensitivity, specificity, and accuracy that can improve and facilitates rapid field surveillance of salmonellosis among chickens.