Synthesis of Antibodies-Conjugated Fluorescent Dye-Doped Silica Nanoparticles for a Rapid Single Step Detection of Campylobacter jejuni

The preparation of antibodies-conjugated fluorescent dye-doped silica nanoparticles (FDS-NPs) was developed to detect Campylobacter jejuni cells under a fluorescence microscope. The particles prepared by sol-gel microemulsion techniques have a round shape with an average size of 43 ± 4 nm. They were highly photo stable and could emit strong orange fluorescent for 60 min. Both amine- and carboxyl-functionalized properties were evident from FTIR and FT Raman spectra. The FDS-NPs conjugated with antibodies against C. jejuni were well dispersed in PBS solution at 20 mM of NaCl. The conjugation with monoclonal antibodies against C. jejuni was successful. The direct observation of the antibodies-conjugated FDS-NPs- that bounds C. jejuni with Petroff Hausser counting chamber at 40x was clear. The different focus lengths clearly separated bound and unbound FDS-NPs under the microscope. We successfully synthesis the bio-conjugated dye doped silica nanoparticles for C. jejuni that are easy to use and giving clear detection in due time

Characterization of Amino-Functionalized Ferromagnetic Nanoparticles with Glutaraldehyde Cross-linking

Characterization of amino-functionalised magnetic nanoparticles (FMNs) modified surfaces with glutaraldehyde cross linker, which enables the attachment to a specific antibody against Salmonella was examined using FTIR spectroscopy. The magnetism property of the particles before and after attachment to the target cells was studied. Synchrotron FTIR spectroscopy technique was employed to investigate the chemistry of the crosslinking reaction to amino-functionalized ferromagnetic nanoparticles. Results indicated that the bonding pattern imine bond (C=N) and amide bond (-CONH-) were detected. The finding indicated the attachment of aldehyde group (CHO) to amino region of the particle and the other free end to link to the antibodies against Salmonella.  The bonding allowed the antibodies to bear reactive sites to catch the target Salmonella in food samples. The FMNs demonstrated hysteresis characteristics that could be changed due to cell attachment that caused reduction of loop coercive force (H). Results showed evidence of glutaraldehyde crosslinking that could be also used as quality control for immobilization of antibodies to the particles. Magnetism properties (the coercive force H) and FTIR characteristics could be further used for signal of attachment of cells to the FMNs as well

Reducing Risk of Campylobacteriosis from Poultry: A Mini Review

The worldwide annual cost of campylobacteriosis is at least several billion dollars. Risk analysis is being used to reduce the magnitude of the problem and to support regulations and voluntary actions that are successful in that the number of cases of illness is decreasing. The new regulations in the U.S. have resulted in commercial products with fewer Campylobacter. During the last 16 years there has been significant progress in New Zealand because of new regulations that have resulted in reduced numbers of Campylobacter on marketed products. While some progress has been made in reducing cross contamination, it remains an important issue. Food safety education on the general principles of food hygiene and food handling as well as applications of hazard analysis and critical control point (HACCP) principles in food safety management are recommended to address the challenges associated with cross contamination. Economic analysis of campylobacteriosis and the poultry meat industry shows that there are significant benefits of addressing the challenges associated with Campylobacter in poultry products. Freezing has been shown to be an excellent cost-effective method to reduce the number of viable Campylobacter and the number of cases of campylobacteriosis

Distribution and Genetic Profiles of Campylobacter in Commercial Broiler Production from Breeder to Slaughter in Thailand

Poultry and poultry products are commonly considered as the major vehicle of Campylobacter infection in humans worldwide. To reduce the number of human cases, the epidemiology of Campylobacter in poultry must be better understood. Therefore, the objective of the present study was to determine the distribution and genetic relatedness of Campylobacter in the Thai chicken production industry. During June to October 2012, entire broiler production processes (i.e., breeder flock, hatchery, broiler farm and slaughterhouse) of five broiler production chains were investigated chronologically. Representative isolates of C. jejuni from each production stage were characterized by flaA SVR sequencing and multilocus sequence typing (MLST). Amongst 311 selected isolates, 29 flaA SVR alleles and 17 sequence types (STs) were identified. The common clonal complexes (CCs) found in this study were CC-45, CC-353, CC-354 and CC-574. C. jejuni isolated from breeders were distantly related to those isolated from broilers and chicken carcasses, while C. jejuni isolates from the slaughterhouse environment and meat products were similar to those isolated from broiler flocks. Genotypic identification of C. jejuni in slaughterhouses indicated that broilers were the main source of Campylobacter contamination of chicken meat during processing. To effectively reduce Campylobacter in poultry meat products, control and prevention strategies should be aimed at both farm and slaughterhouse levels

Model of inactivation of Campylobacter jejuni in poultry scalding

The purpose of this study was to develop a model of inactivation of Campylobacter jejuni in industrial scalding of chickens. Models can be used as a guide for broiler slaughterhouse operations for reducing levels of C. jejuni contamination on broiler carcasses. Mean concentrations of C. jeuni in terms of colony farming units (CFU) in scald tank water and in carcass rinse solution after scalding were 2.90±0.07 and 3.86±0.11 LogCFU/ml, respectively. Scald tank water temperature, flow rate, pH, and total solids in scalding process water were 54.15±0.2°C, 172.0±8.4 L/min, 8.0±0.01, and 2,565±114.3 mg/L, respectively. Inactivation models were developed by using literature data for inactivating kinetics, of Campylobacter and the Arrhenius equation. Results of the inactivation models of scalding process indicated that high temperature and short time (less than 2 minutes) of scalding process were effective in reducing the number of viable cells. The model fits the experimental data well and the values of the estimated parameters provide insight for this process. The model can be used for process design and potential process modifications

Validation of a Rapid Visual Screening of Campylobacter jejuni in Chicken Using Antibody-Conjugated Fluorescent Dye-Doped Silica Nanoparticle Reporters

The objective of this work was to validate the antibody-conjugated fluorescent dye-doped silica nanoparticle- (FDS-NP-) based assay for the rapid detection of Campylobacter jejuni in chicken samples using ISO16140:2003 single-laboratory validation guidelines. This assay reversed the paradigm of microbial testing. The noncultured FDS-NPs increased the fluorescent light signal of the test, not the cell numbers, and significantly reduced the detection time. Validation results showed that relative detection level (LOD) of the assay was 103 cfu/ml. The antibody-conjugated FDS-NPs were evaluated for the detection of C. jejuni in 140 chicken samples collected from slaughterhouses (50 carcass rinse water, 60 rectal swabs, and 30 viscera contents) against ISO 10272 reference method with duplex PCR confirmation. The relative accuracy (AC), relative specificity (SP), and relative sensitivity (SE) were 95.67%, 100%, and 94.87%, respectively. The inclusivity test of C. jejuni strains was 100% positive. The exclusivity test demonstrated no cross-reactivity with 32 strains of non-Campylobacter strains. The FDS-NP assay was very fast and specific, with time to result of 30 min compared with the 2–5 days required by the reference method. The noncultured FDS-NPs demonstrated comparable performance to the cultured reference method to detect C. jejuni in poultry samples. It is applicable for effective screening of poultry product category

Rubpy Dye-Doped Silica Nanoparticles as Signal Reporter in a Dot Fluorescence Immunoassay Strip

This paper describes an application of Rubpy dye-doped silica nanoparticles (RSNPs) as signal reporter in a dot fluorescence immunoassay strip for rapid screening of Vibrio cholera O1 (VCO1). These nanoparticles have a spherical shape with an average diameter of 45 nm. They appear luminescent orange when excited with a 312 nm UV lamp. Based on the sandwich immunoassay principle, a test strip was made of a nitrocellulose membrane dotted with monoclonal antibodies against VCO1 as analyte capture molecules. After introducing a test sample, followed by polyclonal rabbit anti-VCO1 antibody conjugated RSNPs as detection reporters and one washing step, the presence or absence of the target bacteria could be identified under UV light by naked eyes. A positive sample would signal a bright orange dot on the strip. The proposed assay had a detection limit of 4.3×103 cfu/mL and was successfully applied as a rapid screening test for VCO1 in food samples with high sensitivity, specificity, and accuracy

Recent Trends in Rapid Environmental Monitoring of Pathogens and Toxicants: Potential of Nanoparticle-Based Biosensor and Applications

Of global concern, environmental pollution adversely affects human health and socioeconomic development. The presence of environmental contaminants, especially bacterial, viral, and parasitic pathogens and their toxins as well as chemical substances, poses serious public health concerns. Nanoparticle-based biosensors are considered as potential tools for rapid, specific, and highly sensitive detection of the analyte of interest (both biotic and abiotic contaminants). In particular, there are several limitations of conventional detection methods for water-borne pathogens due to low concentrations and interference with various enzymatic inhibitors in the environmental samples. The increase of cells to detection levels requires long incubation time. This review describes current state of biosensor nanotechnology, the advantage over conventional detection methods, and the challenges due to testing of environmental samples. The major approach is to use nanoparticles as signal reporter to increase output rather than spending time to increase cell concentrations. Trends in future development of novel detection devices and their advantages over other environmental monitoring methodologies are also discussed

Synchrotron FTIR Light Reveals Signal Changes of Biofunctionalized Magnetic Nanoparticle Attachment on Salmonella sp.

The objective of this research was to develop new technology for possible noncontact, nondestructive, and culture-independent rapid detection of Salmonella using ferromagnetic nanoparticles. Light signal changes of particles, cells, and their reaction stages were investigated. Amino-functionalized ferromagnetic nanoparticles (amino-FMNs) were synthesized and modified by glutaraldehyde to crosslink the attachment of specific antibodies to the particles. The nanoparticle complex was used to capture, concentrate, and isolate Salmonella in a culture broth. Signal changes of the four stages of the nanoparticles-amino-glutaraldehyde-antibodies-Salmonella cell attachments were tracked with sensitive Synchrotron FTIR spectroscopy (SR-FTIR). The unique peaks from these four steps were identified. Results can be applied to develop a new test method or a new test/universal reader for rapid, nondestructive, and culture-independent detection of Salmonella in food products using IR spectroscopy at wave numbers 1454 cm-1, 1542 cm-1, and 1414 cm-1, respectively