Deposition and depletion of five anticoccidials in eggs

Anticoccidials are compounds that are widely used as feed additives to prevent and treat coccidiosis. They are licensed for use in a prescribed concentration and during a certain time interval for broilers and pullets but not for laying hens. It was shown in the past that carry-over at the feeding mill is found to be the main reason for the presence of residues in eggs. An animal experiment was set up to investigate the effect of carry-over at the feeding mill on the presence of residues of anticoccidials in eggs. For the compounds diclazuril, robenidine, halofuginone and nicarbazin in combination with narasin, two concentration levels were tested: the maximum allowed concentration for broilers (100%) and a concentration corresponding to 5% carry-over during feed preparation. Also dimetridazole was included in the experiment but only at one concentration level. Eggs were sampled during treatment (14 days) and for a period of 30 days after withdrawal of the anticoccidial-containing feed. Residues were determined, and deposition and depletion curves were generated. Analyses were performed by ELISA and LC-MS/MS. For all compounds, substantial residues could be found in the 5% groups, which points out the risk of carry-over at the feeding mill. The distribution of the residues between egg yolk and white was determined by analyzing both fractions.

Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (sORNA-soaked M incognita eggs Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH2)-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s. which recovered within 24 h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48 h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-en-I transcript which encodes an RNAi-inhibiting siRNA exonuclease We observe a marked up-regulation of MI-en-I transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RN/NI-based reverse genetic screens in plant parasitic nematodes. (C) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

The bird GPS - long-range navigation in migrants

Nowadays few people consider finding their way in unfamiliar areas a problem as a GPS (Global Positioning System) combined with some simple map software can easily tell you how to get from A to B. Although this opportunity has only become available during the last decade, recent experiments show that long-distance migrating animals had already solved this problem. Even after displacement over thousands of kilometres to previously unknown areas, experienced but not first time migrant birds quickly adjust their course toward their destination, proving the existence of an experience-based GPS in these birds. Determining latitude is a relatively simple task, even for humans, whereas longitude poses much larger problems. Birds and other animals however have found a way to achieve this, although we do not yet know how. Possible ways of determining longitude includes using celestial cues in combination with an internal clock, geomagnetic cues such as magnetic intensity or perhaps even olfactory cues. Presently, there is not enough evidence to rule out any of these, and years of studying birds in a laboratory setting have yielded partly contradictory results. We suggest that a concerted effort, where the study of animals in a natural setting goes hand-in-hand with lab-based study, may be necessary to fully understand the mechanism underlying the long-distance navigation system of birds. As such, researchers must remain receptive to alternative interpretations and bear in mind that animal navigation may not necessarily be similar to the human system, and that we know from many years of investigation of long-distance navigation in birds that at least some birds do have a GPS-but we are uncertain how it works.

Survey of the anticoccidial feed additive nicarbazin (as dinitrocarbanilide residues) in poultry and eggs

A survey was carried out on the occurrence of dinitrocarbanilide (DNC), the marker residue for nicarbazin, in poultry produced in Ireland during 2002-2004. Liver (n = 736) and breast muscle samples (n = 342) were tested. DNC residues were found in 40 and 26% of liver and breast muscle samples at levels greater than 12.5 and 5 mu g kg(-1), respectively. DNC residues were found at >200 mu g kg(-1) in 12 and 0% of liver and muscle samples, respectively. Samples of breast muscle (n = 217) imported from 11 countries were also tested for DNC residues. A lower incidence of DNC residues (6%) was found in imported breast muscle. Egg samples (n = 546) were tested and DNC residues were found in nine samples, with levels ranging between 14 and 122 mu g kg(-1). Analysis of poultry, carried out as part of official food inspection in the period 2004-2006, indicated a reduction in the number of broiler liver samples containing DNC at >200 mu g kg(-1), to approximately 7%. Low levels of DNC residues continue to be found in

An all-in-one dry chemistry immunoassay for the screening of coccidiostat nicarbazin in poultry eggs and liver

An automated immunoassay for the detection of nicarbazin residues in poultry eggs and liver was developed. The assay was based on a novel all-in-one dry chemistry concept and time-resolved fluorometry. The analyte specific antibody was immobilized into a single microtiter well and covered with an insulation layer, on top of which the label was dried in a small volume. The extracted sample was added automatically to the dry microtiter well, and the result was available within 18 min. Due to the rapidity and simplicity, the quantitative immunoassay could also be used as a high throughput screening method. The analytical limit of detection for the assay was calculated as 0.1 ng mL(-1) (n = 12) and the functional limit of detection as 3.2 ng g(-1) for egg (n = 6) and 11.3 ng g(-1) for liver (n = 6) samples. The sample recovery varied from 97.3 to 115.6%. Typically, the intra-assay variations were less than 10%, and interassay variations ranged between 8.1 and 13.6%.

Development and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS-MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid-liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H](+) ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 mug kg (-1) in liver and 5, 15 and 50 mug kg (-1) in eggs. The new analytical limits, CCalpha and CCbeta were calculated for liver and were 35.4 and 43.6 mug kg (-1), respectively. (C) 2003 Elsevier Science B.V. All rights reserved.

Development and validation of a method for the confirmation of nicarbazin in chicken liver and eggs using LC-electrospray MS-MS according to the revised EU criteria for veterinary drug residue analysis

A method is described for the quantitative confirmation of 4,4'-dinitrocarbanilide (DNC), the marker residue for nicarbazin in chicken liver and eggs. The method is based on LC coupled to negative ion electrospray MS-MS of tissue extracts prepared by liquid-liquid extraction. The [M-H](-) ion at m/z 301 is monitored along with two transition ions at m/z 137 and 107 for DNC and the [M-H](-) ion at m/z 309 for the internal standard, d(8)-DNC. The method has been validated according to the new EU criteria for the analysis of veterinary drug residues at 100, 200 and 300 mug kg(-1) in liver and at 10, 30 and 100 mug kg(-1) in eggs. Difficulties concerning the application of the new analytical limits, namely the decision limit (CC) and the detection capability (CC) to the determination of DNC in both liver and eggs are discussed.

Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4'-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 µg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 µg/kg, respectively.