Rapid and simple detection of foot-and-mouth disease virus: Evaluation of a cartridge-based molecular detection system for use in basic laboratories.

Highly contagious transboundary animal diseases such as foot-and-mouth disease (FMD) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen-side. Here, we report on a study to evaluate a fully automated cartridge-based real-time RT-PCR diagnostic system (Enigma MiniLab® ) for the detection of FMD virus (FMDV). The modular system integrates both nucleic acid extraction and downstream real-time RT-PCR (rRT-PCR). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV, and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter-laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge-based automated real-time RT-PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world.

Transfer of the coccidiostats monensin and lasalocid from feed at cross-contamination levels to whole egg, egg white and egg yolk.

Recent legislation has addressed the unavoidable carry-over of coccidiostats and histomonostats in feed, which may lead to the presence of residues of these compounds in eggs. In this study, laying hens received cross-contaminated feed at a ratio of 2.5%, 5% and 10% of the therapeutic dose of monensin and lasalocid for broilers. The eggs were collected during the treatment and depletion period and were analysed using liquid chromatography-tandem mass spectrometry. The different egg matrices were separated and analysed during the plateau phase. High lasalocid concentrations, which exceeded the maximum residue level, and low monensin concentrations were found in whole egg. Plateau levels were reached at days 7-9 for lasalocid and at days 3-5 for monensin. For lasalocid, the highest concentrations were measured in egg yolk; residue concentrations in egg white were very low.

Transfer of flubendazole and tylosin from feed at cross-contamination levels to various poultry matrices.

Residues of veterinary drugs and feed additives used extensively in animal husbandry are sometimes found in edible matrices. In this study, broilers received experimental feed, containing either flubendazole or tylosin, at cross-contamination levels of 2.5%, 5%, and 10% of the therapeutic dose to determine the transfer ratio of these molecules from feed to poultry matrices. Breast and thigh muscle and liver samples were collected during treatment and depletion periods and then analyzed using liquid chromatography-tandem mass spectrometry. The parent molecule flubendazole and its 2 major metabolites were quantified. After 3 to 5 d, a plateau phase was reached, and a few days after withdrawal of the experimental feed, a depletion of residues was noted. Significant difference between both muscle types was noted for flubendazole. Strong metabolization of flubendazole in the liver was seen. For tylosin, no residue concentrations above the limit of quantification could be detected in muscle. None of the residue concentrations for either molecule exceeded the corresponding maximum residue limits.

Transfer of flubendazole and tylosin at cross contamination levels in the feed to egg matrices and distribution between egg yolk and egg white.

Chemical residues may be present in eggs from laying hens' exposure to drugs or contaminants. These residues may pose risks to human health. In this study, laying hens received experimental feed containing flubendazole or tylosin at cross contamination levels of 2.5, 5, and 10% of the therapeutic dose. Eggs were collected daily and analysis of the whole egg, egg white, and egg yolk was performed using liquid chromatography tandem mass spectrometry. Highest concentrations of the parent molecule flubendazole, as well as the hydrolyzed and the reduced metabolite, were detected in egg yolk. Residue concentrations of the parent molecule were higher compared with those of the metabolites in all egg matrices. No tylosin residue concentrations were detected above the limit of quantification for all concentration groups and in all egg matrices. Neither molecule exceeded the set maximum residue limits.

Residues of sulfadiazine and doxycycline in egg matrices due to cross-contamination in the feed of laying hens and the possible correlation with physicochemical, pharmacokinetic and physiological parameters.

In the poultry industry, the widespread use of veterinary drugs such as antimicrobial compounds may lead to the presence of residues in whole eggs, egg white and egg yolk. During this study, laying hens received experimental feed containing sulfadiazine or doxycycline at cross-contamination levels of 2.5%, 5% and 10% of the therapeutic concentration. Since the therapeutic dose is 250 mg kg(-1) for both substances, cross-contamination concentrations in the feed of 6.25, 12.5 and 25 mg kg(-1) were expected. Whole egg, egg white and egg yolk samples were collected during the treatment and depletion period and were analysed via liquid chromatography-tandem mass spectrometry. For both drugs, a plateau phase was reached within 3-5 days and residue concentrations were detected in all egg matrices. For the 10% cross-contamination group, residual sulfadiazine concentrations of 208, 299 and 60 µg kg(-1) and residual doxycycline concentrations of 455, 332, 206 µg kg(-1) were detected in whole egg, egg white and egg yolk on day 13 of the treatment period, respectively. Both sulfadiazine and doxycycline had higher concentrations in egg white than in egg yolk, but the egg white-egg yolk ratio was higher for sulfadiazine than for doxycycline. As neither drug is allowed in Belgium for use in laying hens, residues may pose food safety concerns.

Residues of sulfadiazine and doxycycline in broiler liver and muscle tissue due to cross-contamination of feed.

Veterinary drugs, such as antimicrobial compounds, are widely used in poultry and may lead to the presence of residues in matrices of animal origin, such as muscle and liver tissue. In this study, broilers received an experimental feed containing sulfadiazine or doxycycline at cross-contamination levels of 2.5, 5 and 10% of the therapeutic dose in feed. Breast and thigh muscle and liver samples were collected during treatment and depletion period and analysed via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Concentrations reached a plateau phase 3-5 days after the start of experimental feeding. A rapid depletion of residues was noted after withdrawal of the experimental feed. No significant differences in measured concentrations were observed between the various muscle types. Residue concentrations for some experimental groups; the 10% group of sulfadiazine and the 5 and 10% group of doxycycline, however, exceeded their corresponding maximum residue limits (MRLs).

Accumulation of advanced glycation end products in canine atherosclerosis.

Atherosclerosis is an uncommon lesion in animals and particularly in dogs. Prominent atherosclerotic lesions of the coronary arteries are described in three dogs. These comprised an expansion of the tunica media by the accumulation of foam cells and/or cholesterol crystals, with subsequent narrowing of the vascular lumen. Immunohistochemical analysis revealed the accumulation of advanced glycation end products (AGEs) in foam cells, macrophages and lymphocytes. As in man, these findings suggest a possible role of AGEs in the development of canine atherosclerosis.