Development of a rapid screening test for veterinary sedatives and the beta-blocker carazolol in porcine kidney by ELISA

Sedatives and tranquillisers are frequently used to reduce stress during the transportation of food producing animals. The most widely used classes of sedatives include the butyrophenone azaperone, the phenothiazines acepromazine, propionylpromazine, chlorpromazine and the beta-blocker, carazolol. For regulatory control purposes, tolerances for azaperone and carazolol have been set by the European Union as 100 and 25 mug kg(-1), respectively. Furthermore, the use of the phenothiazines is prohibited and therefore has a zero tolerance. A method for the detection of residues of five tranquillisers and one beta-blocker using a single ELISA plate has been developed. Kidney samples (2.5 g) were extracted with dichloromethane and applied to a competitive enzyme immunoassay using three polyclonal antibodies raised in rabbits against azaperol, propionylpromazine and carazolol conjugates. In sample matrix, the azaperol antibody cross-reacted 28.0% with azaperone and the propionylpromazine antibody cross-reacted 24.9% with acepromazine and 11.7% with chlorpromazine. In the ELISA, the detection capabilities of the six sedatives, azaperol, azaperone, carazolol, acepromazine, chlorpromazine, and propionylpromazine are 5, 15, 5, 5, 20 and 5 mug kg(-1), respectively. The proposed method is a sensitive and rapid multi-residue technique that offers a cost effective alternative to current published procedures, without any concession on the ability to detect sedative misuse.

Comparison of porcine urine and bile as matrices to screen for the residues of two sulfonamides using a semi-automated enzyme immunoassay.

Porcine urine enzyme immunoassays for sulfamethazine and sulfadiazine have previously been employed as screening tests to predict the concentrations of the drugs in the corresponding tissues (kidneys), If a urine was found positive (> 800 ng ml(-1)) the corresponding kidney was then analysed by an enzyme immunoassay and, if found positive, a confirmatory analysis by HPLC was performed. Urine was chosen as the screening matrix since sulfonamides are mainly eliminated through this body fluid, However, after obtaining a number of false positive predictions, an investigation was carried out to assess the possibility of using an alternative body fluid which would act as a superior indicator of the presence of sulfonamides in porcine kidney, An initial study indicated that serum, plasma and bile could all be used as screening matrices. From these, bile was chosen as the preferred sample matrix and an extensive study followed to compare the efficiencies of sulfonamide positive bile and urine at predicting sulphonamide positive kidneys, Bile was found to be 17 times more efficient than urine at predicting a sulfamethazine positive kidney and 11 times more efficient at predicting a sulfadiazine positive kidney, With this enhanced performance of the initial screening test, the need for the costly and time consuming kidney enzyme immunoassay, prior to HPLC analysis, was eliminated

Determination of carazolol residues in porcine tissue by radioreceptor assay

A radioreceptor assay was developed for the determination of the p-blocker carazolol in porcine muscle and kidney. The method involves a simple alkaline extraction procedure using diethyl ether followed by a competitive assay between carazolol residues and [H-3]-dihydroalprenolol ([H-3]-DHA) using solubilised beta2-adrenoceptors isolated from a transfected cell line. The limit of detection (LOD) was determined using 20 reference blank samples of pig kidney and pig muscle. LODs for muscle (0.93 mug kg(-1)) and kidney (1.47 mug kg(-1)) were well below their respective European community maximum residue limits, (MRLs 5 and 25 mug kg(-1), respectively). The assay was used to investigate if carazolol residues persisted in pig tissues for up to 30 h post-intramuscular injection at the recommended dose rate (10 mug carazolol/kg body weight). The highest mean +/- S.D. concentrations were detected at 1 h post-injection in kidney (10.84 +/- 1.3 mug kg(-1)) and muscle (3.59 +/- 0.2 mug kg(-1)) which were less than the respective MRLs. It is concluded that this method offers a robust and rapid alternative to other methods for the screening of carazolol residues in pig meat. (C) 2002 Elsevier Science B.V. All rights reserved.

Multi sulfonamide screening in porcine muscle using a surface plasmon resonance biosensor

A binding protein displaying broad-spectrum cross-reactivity within the sulfonamide group was used in conjunction with a sulfonamide specific sensor chip and a surface plasmon resonance biosensor to develop a rapid broad spectrum screening assay for sulfonamides in porcine muscle. Results for 40 samples were available in just over 5 h after the completion of a simple sample preparation protocol. Twenty sulfonamide compounds were detected. Acetylated metabolites were not recognised by the binding protein. Limit of detection (mean-three times standard deviation value when n = 20) was calculated to be 16.9 ng g(-1) in tissue samples. Intra-assay precision (n = 10) was calculated at 4.3 %CV for a sample spiked at 50 ng g(-1) with sulfamethazine, 3.6 %CV for a sample spiked at 100 ng g(-1) with sulfamethazine, 7.2 %CV for a sample spiked at 50 ng g(-1) with sulfadiazine and 3.1 %CV for a sample spiked at 100 ng g-1 with sulfadiazine. Inter-assay precision (n = 3) was calculated at 9.7 %CV for a sample spiked at 50 ng g-1 with sulfamethazine, 3.8 %CV for a sample spiked at 100 ng g(-1) with sulfamethazine, 3.5 %CV for a sample spiked at 50 ng g(-1) with sulfadiazine and 2.8 %CV for a sample spiked at 100 ng g(-1) with sulfadiazine. (C) 2004 Elsevier B.V. All rights reserved.

Evaluation of Microcollimated Pars Plana External Beam Radiation in the Porcine Eye

Purpose: To evaluate the clinical and histological side effects of a prototype stereotactic radiotherapy system delivering microcollimated external beam radiation through pars plana in porcine eyes.

Methods: Five Yucatan mini-swine (10 eyes) were randomized to five treatment groups. Eight eyes were dosed with X-ray radiation on Day 1, and two eyes served as untreated controls. Treated eyes received doses up to 60 Gy to the retina and up to 130 Gy to the sclera using single or overlapping beams. The treatment beams were highly collimated such that the diameter was approximately 2.5 mm on the sclera and 3 mm on the retinal surface. Fundus photography, fluorescein angiography (FA), and spectral domain optical coherence tomography (SD-OCT) were obtained on days 7, 30, 60, and 110. Images were examined by a masked grader and evaluated for abnormalities. Animals were sacrificed on day 111 and gross and histopathological analysis was conducted.

Results: Histological and gross changes to eye structures including conjunctiva and lens were minimal at all doses. Fundus, FA, and SD-OCT of the targeted region failed to disclose any abnormality in the control or 21 Gy treated animals. In the 42 and 60 Gy animals, hypopigmented spots were noted after treatment on clinical exam, and corresponding hyperfluorescent staining was seen in late frames. No evidence of choroidal hypoperfusion was seen. The histological specimens from the 60 Gy animals showed photoreceptor loss and displacement of cone nuclei.

Conclusion: Transcleral stereotactic radiation dosing in porcine eyes can be accomplished with no significant adverse events as doses less than 42 Gy.

Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE

Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO₂-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO₂-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO₂-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO₂-DDE (10μM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO₂-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO₂-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO₂-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO₂-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathione-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO₂-DDE induced endocrine disruption in Leydig cells.

Prédiction de la digestibilité des acides aminés des ingrédients utilisés en alimentation porcine : approche par méta-analyse

En alimentation animale, l’utilisation adéquate des aliments nécessite une connaissance précise des valeurs nutritionnelles de leurs composantes, dont celle en acides aminés. Cependant, ces valeurs nutritionnelles dépendent de la teneur en acides aminés essentiels (AAE) totaux et de la digestibilité iléale standardisée (DIS) de ces AAE. Cette dernière varie en fonction de plusieurs facteurs, dont l’origine botanique des graines, les conditions de culture des récoltes, le stockage des aliments, les traitements physico-chimiques appliqués aux grains, les facteurs antinutritionnels (FANs) et les techniques expérimentales utilisées pour le dosage et l’estimation de la digestibilité des AAE. Une approche par méta-analyse a permis d’établir des modèles de prédiction de la valeur nutritionnelle en AAE des ingrédients à partir de leur composition chimique en considérant la protéine brute (PB), les AAE totaux, la teneur en fibre (Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF) et Fibre Brute (FB)) et les FANs comme les inhibiteurs de la trypsine. En se référant à l’analyse graphique et statistique, les données ont été réparties en 4 groupes : 1) les tourteaux (tourteau de soja, colza/canola et coton); 2) les légumineuses (féveroles, lupins, pois et soja); 3) les céréales (blé, orge, avoine, sorgho et maïs); 4) les drêches de distilleries (blé et maïs). Ainsi, un modèle général ajusté en fonction du type d’ingrédients a été généré et les facteurs de variation de la digestibilité en AAE ont été identifiés. Pour les tourteaux, la DIS des AAE est réduite par un accroissement de la teneur en NDF, tandis que la DIS des AAE de la féverole, pois et lupin est principalement influencée par la teneur en PB et en FANs. Concernant les graines de soja la DIS des AAE est réduite par une hausse de la teneur en fibre (FB et ADF). Enfin pour les céréales et les sous- produit de céréales telles que les drêches, la PB et les fibres (ADF ou NDF) étaient les meilleurs nutriments pour prédire la DIS des AAE. Ces résultats démontrent que la DIS des AAE peut être prédite avec précision à partir de la composition chimique pour la plupart des ingrédients.

Gastric supply manipulation to modulate ghrelin production and enhance vascularization to the cardia: proof of the concept in a porcine model.

Introduction. Selective embolization of the left-gastric artery (LGA) reduces levels of ghrelin and achieves significant short-term weight loss. However, embolization of the LGA would prevent the performance of bariatric procedures because the high-risk leakage area (gastroesophageal junction [GEJ]) would be devascularized. Aim. To assess an alternative vascular approach to the modulation of ghrelin levels and generate a blood flow manipulation, consequently increasing the vascular supply to the GEJ. Materials and methods. A total of 6 pigs underwent a laparoscopic clipping of the left gastroepiploic artery. Preoperative and postoperative CT angiographies were performed. Ghrelin levels were assessed perioperatively and then once per week for 3 weeks. Reactive oxygen species (ROS; expressed as ROS/mg of dry weight [DW]), mitochondria respiratory rate, and capillary lactates were assessed before and 1 hour after clipping (T0 and T1) and after 3 weeks of survival (T2), on seromuscular biopsies. A celiac trunk angiography was performed at 3 weeks. Results. Mean (±standard deviation) ghrelin levels were significantly reduced 1 hour after clipping (1902 ± 307.8 pg/mL vs 1084 ± 680.0; P = .04) and at 3 weeks (954.5 ± 473.2 pg/mL; P = .01). Mean ROS levels were statistically significantly decreased at the cardia at T2 when compared with T0 (0.018 ± 0.006 mg/DW vs 0.02957 ± 0.0096 mg/DW; P = .01) and T1 (0.0376 ± 0.008mg/DW; P = .007). Capillary lactates were significantly decreased after 3 weeks, and the mitochondria respiratory rate remained constant over time at the cardia and pylorus, showing significant regional differences. Conclusions. Manipulation of the gastric flow targeting the gastroepiploic arcade induces ghrelin reduction. An endovascular approach is currently under evaluation.

Characterization of in vivo vertebral growth modulation by shape memory alloy staples on a porcine model for the correction of the scoliosis

La recherche de nouvelles voies de correction de la scoliose idiopathique a une longue histoire. Le traitement conventionnel de la scoliose idiopathique est présenté par le port du corset ou par la correction opératoire de la déformation. Depuis leur introduction, les deux méthodes ont prouvé leur efficacité. Cependant, malgré des caractéristiques positives évidentes, ces méthodes peuvent causer un nombre important d'effets indésirables sur la santé du patient. Les techniques sans fusion pour le traitement de la scoliose semblent être une alternative perspective de traitement traditionnel, car ils apportent moins de risques et des complications chirurgicales que les méthodes conventionnelles avec la conservation de la mobilité du disque intravertébral. Cependant, l'utilisation de techniques mentionnées exige une connaissance profonde de la modulation de croissance vertébrale. L'objectif principal de la présente étude est d'estimer le potentiel d'agrafes à l’AMF de moduler la croissance des vertèbres porcines en mesurant la croissance osseuse sur la plaque de croissance de vertèbres instrumentées en comparaison avec le groupe contrôle. La méthode est basée sur la loi de Hueter-Volkmann. Nous avons choisi NiTi agrafes à l’AMF pour notre étude et les porcs de race Landrace comme un animal expérimental. Les agrafes ont été insérés sur 5 niveaux thoracique de T6 à T11. En outre, les radiographies ont été prises toutes les 2 semaines. La présence d'agrafes en alliage à mémoire de forme a produit la création de courbes scoliotiques significatives dans 4 de 6 animaux chargés et le ralentissement considérable de la croissance osseuse (jusqu'à 35,4%) comparativement aux groupes contrôle et sham. L'étude a démontré in vivo le potentiel d'agrafes en alliage à mémoire de formes de moduler la croissance des vertèbres en créant des courbes scoliotiques sur les radiographies et en ralentissant le taux de croissance sur les plaques de croissance instrumenté. La position précise de l'agrafe est essentielle pour la modulation de croissance osseuse et le développement de la scoliose expérimentale.

The effects of administration of a nuclear factor kappa-B inhibitor on pulmonary endothelial dysfunction after cardiopulmonary bypass: impact on oxygenation and hemodynamics and development of therapeutic and preventive modalities in a porcine model

Introduction: La circulation extracorporelle (CEC) peut entraîner une dysfonction endothéliale pulmonaire et l’hypertension pulmonaire. Le SN50 agit au niveau de la signalisation cellulaire pour prévenir ces réactions à la CEC et pourrait renverser la dysfonction endothéliale pulmonaire post-CEC sans effets néfastes sur l’hémodynamie. Méthodes: Quatre groups de porcs ont reçu un parmi quatre traîtements avant de subir 90 minutes de CEC et 60 minutes de reperfusion: (1) milrinone nébulisé; (2) sildenafil nébulisé; (3) placebo nébulisé; et (4) SN-50 intraveineux. Un monitoring hémodynamique invasif a été utilisé. La réactivité vasculaire des artères pulmonaires de deuxième ordre a été évaluée face à l’acétylcholine et la bradykinine. Résultats: Le sildénafil produit une augmentation significative de la pression de l’artère pulmonaire (PAP) moyenne à 60 minutes de reperfusion par rapport au début de la chirurgie. Les relaxations dépendantes de l’endothélium face à la bradykinine étaient meilleurs dans les groupes milrinone et SN-50 et surtout dans le groupe sildénafil par rapport au groupe placébo. Le SN-50 produisait de moins bonnes relaxations dépendantes de l’endothélium face à l’acétylcholine que les autres traitements incluant placébo. Conclusion: Le sildénafil prévient mieux la dysfonction endothéliale pulmonaire que les autres traitements. Les bénéfices du SN-50 sont possiblement sous-estimés vu que la dose n’a pas pu être ajustée à la durée de CEC. Le sildenafil inhalé mérite une étude plus importante chez l’humain et le SN-50 dans un model de CEC animal.

In vivo effect of deoxynivalenol (DON) naturally contaminated feed on porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Deoxynivalenol (DON), also known as vomitoxin, is the most prevalent type B trichothecene mycotoxin worldwide. Pigs show a great sensitivity to DON, and because of the high proportion of grains in their diets, they are frequently exposed to this mycotoxin. The objective of this study was to determine the impact of DON naturally contaminated feed on porcine reproductive and respiratory syndrome virus (PRRSV) infection, the most important porcine viral pathogen in swine. Experimental infections were performed with 30 animals. Piglets were randomly divided into three groups of 10 animals based on DON content of diets (0, 2.5 and 3.5 mg/kg DON). All experimental groups were further divided into subgroups of 6 pigs and were inoculated with PRRSV. The remaining pigs (control) were sham-inoculated with PBS. Pigs were daily monitored for temperature, weight and clinical signs for 21 days. Blood samples were collected and tested for PRRSV RNA and for virus specific antibodies. Results of PRRSV infection showed that ingestion of diet highly contaminated with DON greatly increases the effect of PRRSV infection on weight gain, lung lesions and mortality, without increasing significantly viral replication, for which the tendency is rather directed toward a decrease of replication. These results suggest that PRRSV infection could exacerbate anorectic effect of DON, when ingested in large doses. Results also demonstrate a DON negative effect on PRRSV-specific humoral responses. This study demonstrate that high concentrations of DON naturally contaminated feed decreased the immune response against PRRSV and influenced the course of PRRSV infection in pigs.