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The European Union has implemented breeding programmes to increase scrapie resistance in sheep. A similar approach can be applied also in goats since the K222 allele provides a level of resistance equivalent to that of ARR in sheep. The European Food Safety Authority stated that breeding for resistance could be offered as an option for Member States to control classical scrapie in goats. We assessed the impact of different breeding strategies on PRNP genotype frequencies using a mathematical model that describes in detail the evolution of K222 in two goat breeds, Chamois Coloured and Saanen. Different patterns of age structure and replacement rate were modelled as factors affecting response to selection. Breeding for scrapie resistance can be implemented in goats, even though the initial K222 frequencies in these breeds are not particularly favourable and the rate at which the resistant animals increase, both breeding and slaughtered for meat production, is slow. If the goal is not to achieve the fixation of resistance allele, it is advisable to carry out selection only until a desired frequency of K222-carriers has been attained. Nucleus selection vs. selection on the overall populations is less expensive but takes longer to reach the desired output. The programme performed on the two goat breeds serves as a model of the response the selection could have in other breeds that show different initial frequencies and population structure. In this respect, the model has a general applicability.
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Cruzamento , Resistência à Doença/genética , Doenças das Cabras/genética , Proteínas Priônicas/genética , Scrapie/genética , Seleção Genética , Animais , Genótipo , Cabras/genética , Itália , Modelos Genéticos , Proteínas Priônicas/metabolismoRESUMO
The presence of lysine (K) at codon 222 has been associated with resistance to classical scrapie in goats, but few scrapie cases have been identified in 222Q/K animals. To investigate the contribution of the 222K variant to PrPres formation in natural and experimental Q/K scrapie cases, we applied an immunoblotting method based on the use of two different monoclonal antibodies, F99/97.6.1 and SAF84, chosen for their different affinities to 222K and 222Q PrP variants. Our finding that PrPres seems to be formed nearly totally by the 222Q variant provides evidence that the 222K PrP variant confers resistance to conversion to PrPres formation and reinforces the view that this mutation has a protective role against classical scrapie in goats.
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Doenças das Cabras/metabolismo , Lisina/metabolismo , Proteínas PrPSc/metabolismo , Scrapie/metabolismo , Motivos de Aminoácidos , Animais , Códon/genética , Códon/metabolismo , Genótipo , Cabras , Lisina/genética , Peptídeo Hidrolases/metabolismo , Proteínas PrPSc/química , Proteínas PrPSc/genética , Scrapie/genéticaRESUMO
In accordance with U.S. FDA Foods Program Regulatory Science Steering Committee guidelines, with this study, we optimized and validated a commercial real-time PCR method for the detection of low amounts of lupin in four classes of food matrices: chocolate cookies, ragù, Olivier salad, and barley and rice flour. DNA extracted from blank (true negative) samples artificially contaminated with lupin (Lupinus albus) flour at 1000 ppm underwent dilutions with the DNA extracted from the true negative samples up to 0.5 ppm. The limit of detection for real-time PCR was 0.5 ppm in the complex matrices (range, Ct 26-34), making this a specific, robust, and rapid method for lupin allergen detection and labeling. Our validation data support the suitability of this commercially available real-time PCR method for this purpose.
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SOD1 gene is associated with progressive motor neuron degeneration in the familiar forms of amyotrophic lateral sclerosis. Although studies on mutant human SOD1 transgenic rodent models have provided important insights into disease pathogenesis, they have not led to the discovery of early biomarkers or effective therapies in human disease. The recent generation of a transgenic swine model expressing the human pathological hSOD1G93A gene, which recapitulates the course of human disease, represents an interesting tool for the identification of early disease mechanisms and diagnostic biomarkers. Here, we analyze the activation state of CNS cells in transgenic pigs during the disease course and investigate whether changes in neuronal and glial cell activation state can be reflected by the amount of extracellular vesicles they release in biological fluids. To assess the activation state of neural cells, we performed a biochemical characterization of neurons and glial cells in the spinal cords of hSOD1G93A pigs during the disease course. Quantification of EVs of CNS cell origin was performed in cerebrospinal fluid and plasma of transgenic pigs at different disease stages by Western blot and peptide microarray analyses. We report an early activation of oligodendrocytes in hSOD1G93A transgenic tissue followed by astrocyte and microglia activation, especially in animals with motor symptoms. At late asymptomatic stage, EV production from astrocytes and microglia is increased in the cerebrospinal fluid, but not in the plasma, of transgenic pigs reflecting donor cell activation in the spinal cord. Estimation of EV production by biochemical analyses is corroborated by direct quantification of neuron- and microglia-derived EVs in the cerebrospinal fluid by a Membrane Sensing Peptide enabled on-chip analysis that provides fast results and low sample consumption. Collectively, our data indicate that alteration in astrocytic EV production precedes the onset of disease symptoms in the hSODG93A swine model, mirroring donor cell activation in the spinal cord, and suggest that EV measurements from the cells first activated in the ALS pig model, i.e. OPCs, may further improve early disease detection.
Assuntos
Esclerose Lateral Amiotrófica , Vesículas Extracelulares , Camundongos , Animais , Humanos , Suínos , Superóxido Dismutase-1/genética , Neurônios Motores/metabolismo , Superóxido Dismutase/genética , Camundongos Transgênicos , Esclerose Lateral Amiotrófica/patologia , Medula Espinal/patologia , Neuroglia/patologia , Biomarcadores/metabolismo , Peptídeos/metabolismo , Modelos Animais de DoençasRESUMO
In human medicine, p53 immunohistochemistry (IHC) is a common method that is used for the identification of tumors with TP53 mutations. In veterinary medicine, several studies have performed IHC for p53 in canine tumors, but it is not known how well it actually predicts the mutation. The aim of this study was to estimate the accuracy of the IHC method for p53 (clone PAb240) using a lab-developed NGS panel to analyze TP53 mutations in a subset of malignant tumors in dogs. A total of 176 tumors were analyzed with IHC and then 41 were subjected to NGS analysis; among them, 15 were IHC positive and 26 were negative, and 16 out of 41 (39%) were found to be inadequate for NGS analysis. Excluding the non-evaluable cases at NGS, of the remaining eight IHC-positive cases, six were mutants and two were wild-type. Among the 17 IHC-negative cases, 13 were wild type, and 4 were mutants. The sensitivity was 60%, specificity was 86.7%, and the accuracy was 76%. These results suggest that when using IHC for p53 with this specific antibody to predict mutation, up to 25% wrong predictions can be expected.
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Complete animal welfare evaluation in intensive farming is challenging. With this study, we investigate new biomarkers for animal physical and mental health by comparing plasma expression of biochemical indicators in dairy cows reared in three different systems: (A) semi-intensive free-stall, (B) non-intensive tie-stall, and (C) intensive free-stall. Additionally, protein levels of mature brain-derived neurotrophic factor (mBDNF) and its precursor form (proBDNF) and indoleamine 2,3-dioxygenase (IDO1) specific activity were evaluated in brain samples collected from 12 cattle culled between 73 and 138 months of age. Alterations in plasma lipid composition and in the kynurenine pathway of tryptophan metabolism were observed in the tie-stall-reared animals. The total plasma BDNF concentration was higher in tie-stall group compared to the two free-housing groups. Brain analysis of the tie-stall animals revealed a different mBDNF/proBDNF ratio, with a higher level of proBDNF (p < 0.001). Our data are similar to previous studies on animal models of depression, which reported that inhibition of the conversion of proBDNF in its mature form and/or elevated peripheral kynurenine pathway activation may underlie cerebral biochemical changes and induce depressive-like state behavior in animals.
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A sudden increase in the incidence of scrapie in Italy in 1997 was subsequently linked to the use of a potentially infected vaccine against contagious agalactia. The relative risk for the exposed farms ranged between 6 and 40. The aim of this study was to assess the long-term impact of exposure to the potentially scrapie-contaminated vaccine on the Italian classical scrapie epidemic. We carried out a retrospective cohort study, fitting mixed-effects Poisson regression models, dividing national geographic areas into exposure categories on the basis of the vaccine circulation levels. We took into account the sensitivity of the surveillance system applied in the different areas. The population attributable fraction (PAF) was used to assess the impact on the total population of farms associated with the effect of circulation of the vaccine. The provinces where the vaccine was more often sold were noted to have a higher level of disease when compared to those provinces where the vaccine was sold less often (incidence rate ratio [IRR]: 2.7; 95% confidence interval [CI]: 1.1-6.5). The population attributable fraction was high (68.4%). Standardization techniques allowed to account for the potential of geographical variability in the sensitivity of the Italian surveillance system. Although the number of the directly exposed farms was limited, an important long-term impact of the vaccine circulation could be quantified in terms of secondary outbreaks likely due to the exchange of animals from directly exposed flocks.
Assuntos
Vacinas Bacterianas/efeitos adversos , Contaminação de Medicamentos , Epidemias/veterinária , Doenças das Cabras/epidemiologia , Mycoplasma agalactiae/imunologia , Scrapie/epidemiologia , Animais , Estudos de Coortes , Doenças das Cabras/patologia , Doenças das Cabras/transmissão , Cabras , Incidência , Itália/epidemiologia , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/prevenção & controle , Estudos Retrospectivos , Scrapie/patologia , Scrapie/transmissão , OvinosRESUMO
BACKGROUND: Processed animal products (PAPs) could be a great alternative to common protein supplements and represent a good example of recycling and valorization of by-products. Due to the reintroduction of certain types of PAPs in feed, a deeper knowledge of these heterogeneous matrices is needed. Thus, the aim of this study is to evaluate the levels of essential elements and inorganic contaminants in 55 PAPs considered as potential alternatives to common protein supplements. METHODS: PAPs samples were analysed for essential (cobalt, nickel, chromium, copper, zinc, iron and manganese) and non-essential elements (arsenic, cadmium, lead and mercury) by Inductively Coupled Plasma Mass Spectrometer (ICP-MS), Graphite Furnace Atomization Atomic Absorption Spectrometer (GF-AAS) and dual cell Direct Mercury Analyzer spectrometer (DMA-80). RESULTS: Essential elements were found with the following decreasing order iron>zinc>copper>manganese>chromium>nickel>cobalt (mg kg-1). Only one sample was found non-compliant to lead concentration according to the European Union Regulation while negligible values of others non-essential elements were found. CONCLUSIONS: This study suggests that PAPs could be a useful supplement for animal diet due to their natural content of essential elements. A careful monitoring of chemical elements should be required and eventually guidelines have to be drafted for a correct use of PAPs to ensure a safe and sustainable feed production.
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Mercúrio , Metais Pesados , Oligoelementos , Animais , Metais Pesados/análise , Manganês/análise , Cobre/análise , Níquel/análise , Zinco/análise , Cromo/análise , Cádmio/análise , Mercúrio/análise , Cobalto , Chumbo/análise , Ferro , Oligoelementos/análiseRESUMO
Food safety laboratories rely on validated methods that detect hidden allergens in food to ensure the safety and health of allergic consumers. Here we present test results for the validation and accreditation of a real-time PCR assay for the detection of peanut traces in food products. The method was tested on five classes of food matrices: bakery and pastry products, meats, ready-to-eat and dairy products, and grains and milling products. Blank samples were spiked starting with the peanut samples (Arachis hypogaea) at a concentration of 1000 ppm. Serial dilutions were then prepared with the DNA extracted from the blank samples to a final concentration of 0.5 ppm. The limit of detection in grains and milling products, ready-to-eat, meats, bakery and pastry products was 0.5 ppm (range, Ct 27-34) and 2.5 ppm in dairy products (range, Ct 25-34). In order to determine the exclusivity parameter of the method, the ragù matrix was contaminated with Prunus dulcis (almonds), Glycine max (soy), Sinapis alba (mustard), Apium graveolens (celery), Allium cepa (onion), Pisum sativum (peas), Daucus carota (carrots), and Theobroma cacao (cocoa); no cross-reactions were observed. The method was rated satisfactory for sensitivity (98%), specificity (100%), robustness, and repeatability and it was fully validated and accredited.
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The illicit use of dexamethasone and other glucocorticoids for cattle fattening in livestock production has been widely described; evidence for illegal treatments can be obtained by direct or indirect detection. In our previous study, we applied two-dimensional electrophoresis (2DE) to identify plasma protein markers of dexamethasone administration in veal calves. Comparison of 2DE maps obtained from blood samples before and after treatment showed the disappearance of two protein spots identified as serum paraoxonase/arylesterase 1 precursor (PON1). In the present study, we validated PON1 as a marker by analysing a larger number of samples treated with dexamethasone for illicit use. Analysis of samples from experimental treatment with other glucocorticoids, androgens and oestrogens confirmed that their influence on PON1 could be excluded. The specificity of the PON1 protein marker was verified on expected negative field samples to exclude interfering factors. However, there is poor statistical evidence to support a significant association between the outcome of PON1 and the considered variables. The results on field samples were compared with histological examination of the thymus as a biomarker of corticosteroid treatment monitored in the Italian histological plan for the control of growth promoters in animals. Two suspect cases were identified from two Piedmont farms where other animals had tested positive at histological examination. In conclusion, the absence of PON1 in the plasma of veal calves can indirectly reveal illicit dexamethasone treatment in individual animals and so identify suspect farms for further investigation. It is effective in a period ranging from 3 to about 10 days from illicit treatment, covering a time span that goes beyond the limits of official chemical controls and preceding histological controls on the thymus of slaughtered animals. PON1 detection in plasma can be coupled with other tests to identify illegal dexamethasone use on veal calf farms.
Assuntos
Glucocorticoides , Carne Vermelha , Animais , Arildialquilfosfatase , Biomarcadores , Hidrolases de Éster Carboxílico , Bovinos , DexametasonaRESUMO
European sea bass (Dicentrarchus labrax L.) is one of the most economically important fish species in the Mediterranean Sea area. Despite strict requirements regarding indications of production method (wild/farmed), incorrect labelling of sea bass is a practice still frequently detected. The aim of this study was to evaluate the capabilities of two techniques, Near-InfraRed (NIR) spectroscopy and mass spectrometry, to discriminate sea bass according to the production method. Two categories were discriminated based on the docosahexaenoic and arachidonic fatty acid ratio by using a Direct Sample Analysis (DSA) system integrated with a time-of-flight (TOF) mass spectrometer. The cut-off value of 3.42, of fatty acid ratio, was able to discriminate between the two types of fish with sensitivity and specificity of 100%. It was possible to classify fish production by using multivariate analysis with portable NIR. The results achieved by the developed validation models suggest that this approach is able to distinguish the two product categories with high sensitivity (100%) and specificity (90%). The results obtained from this study highlight the potential application of two easy, fast, and accurate screening methods to detect fraud in commercial sea bass production.
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The description of the pattern of livestock movements between herds provides essential information for both improving risk-based surveillance and to understand the likely spread of infectious diseases. This study provides a description of the temporal pattern of pig movements recorded in Italy on a 4-year period (2013-2016). Data, provided by the National Livestock registry, were described by social network analysis and the application of a walk-trap algorithm for community detection. Our results show a highly populated community located in Northern Italy, which is the focal point of the Italian industrial pig production and as a general pattern an overall decline of medium and backyard farms and an increase in the number of large farms, in agreement with the trend observed by other EU pig-producing countries. A seasonal pattern of all the parameters evaluated, including the number of active nodes in both the intensive and smaller production systems, emerged: that is characterized by a higher number of movements in spring and autumn, linked with the breeding and production cycle as pigs moved from the growing to the finishing phase and with periods of increased slaughtering at Christmas and Easter. The same pattern was found when restricting the analysis to imported pig batches. Outbreaks occurring during these periods would have a greater impact on the spread of infectious diseases; therefore, targeted surveillance may be appropriate. Finally, potential super-spreader nodes have been identified and represent 0.47% of the total number of pig holdings (n = 477). Those nodes are present during the whole study period with a similar ranking in their potential of being super-spreaders. Most of them were in Northern Italy, but super-spreaders with high mean out-degree centrality were also located in other Regions. Seasonality, communities and super-spreaders should be considered when planning surveillance activity and when applying disease control strategies.
Assuntos
Criação de Animais Domésticos , Doenças dos Suínos , Animais , Itália/epidemiologia , Estações do Ano , Suínos , Doenças dos Suínos/epidemiologia , Meios de TransporteRESUMO
BACKGROUND: During the last decade, active surveillance for transmissible spongiform encephalopathies in small ruminants has been intensive in Europe. In many countries this has led to the detection of cases of atypical scrapie which, unlike classical scrapie, might not be contagious. EU legislation requires, that following detection of a scrapie case, control measures including further testing take place in affected flocks, including the culling of genotype susceptible to classical scrapie. This might result in the detection of additional cases. The aim of this study was to investigate the occurrence of additional cases in flocks affected by atypical scrapie using surveillance data collected in Europe in order to ascertain whether atypical scrapie, is contagious. RESULTS: Questionnaires were used to collect, at national level, the results of active surveillance and testing associated with flock outbreaks in 12 European countries. The mean prevalence of atypical scrapie was 5.5 (5.0-6.0) cases per ten thousand in abattoir surveillance and 8.1 (7.3-9.0) cases per ten thousand in fallen stock. By using meta-analysis, on 11 out of the 12 countries, we found that the probability of detecting additional cases of atypical scrapie in positive flocks was similar to the probability observed in animals slaughtered for human consumption (odds ratio, OR = 1.07, CI95%: 0.70-1.63) or among fallen stock (OR = 0.78, CI95%: 0.51-1.2). In contrast, when comparing the two scrapie types, the probability of detecting additional cases in classical scrapie positive flocks was significantly higher than the probability of detecting additional cases in atypical scrapie positive flocks (OR = 32.4, CI95%: 20.7-50.7). CONCLUSIONS: These results suggest that atypical scrapie is not contagious or has a very low transmissibility under natural conditions compared with classical scrapie. Furthermore this study stressed the importance of standardised data collection to make good use of the analyses undertaken by European countries in their efforts to control atypical and classical scrapie.
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Scrapie/epidemiologia , Scrapie/transmissão , Animais , Europa (Continente)/epidemiologia , Vigilância da População , Prevalência , Ovinos , Inquéritos e QuestionáriosRESUMO
OBJECTIVE: To evaluate the effects of 2 constant rate infusions of hydroxyethyl starch (HES) 130/0.4 on plasma colloid osmotic pressure (COP) in hypoalbuminemic dogs. DESIGN: Prospective, randomized clinical trial. ANIMALS: A total of 24 client-owned dogs. INTERVENTIONS: Hypoalbuminemic euvolemic dogs (albumin < 20 g/L [<2 g/dL]) with normal perfusion parameters requiring IV fluid therapy were enrolled. In addition to crystalloid, HES 130/0.4 was administered as a constant rate infusion over 24 hours at 1 mL/kg/h (group 1, n = 15) or at 2 mL/kg/h (group 2, n = 9), in order to support plasma COP. Before infusion, a blood sample was collected to perform CBC, serum electrophoresis, and serologic tests for some infective diseases. Plasma COP, albumin concentration, PCV, and total plasma protein concentration were evaluated serially at baseline (T0) and then at 6, 12, and 24 hours after the start of infusion, and a multilevel model was performed for these parameters to detect statistically significant differences between the 2 groups. MEASUREMENT AND MAIN RESULTS: Twenty-four dogs were included. No statistically significant differences in COP were found between the 2 groups; however, a high level of variability has been identified within the single individual. Among the other laboratory analyses, PCV was significantly decreased in group 1 at T12 and T24 compared with T0 (P < 0.001) and total plasma protein concentration was significantly increased in group 2 at T12 and T24 compared with T0 (P < 0.008). CONCLUSION: No significant effect on plasma COP was found following infusion with HES 130/0.4 at doses of 1 mL/kg/h and 2 mL/kg/h for 24 hours to hypoalbuminemic dogs. The administered concomitant dose of crystalloids, underlying disease, and small sample size were all potential confounding factors.
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Doenças do Cão/terapia , Derivados de Hidroxietil Amido/farmacologia , Hipoalbuminemia/veterinária , Pressão Osmótica/efeitos dos fármacos , Animais , Coloides , Soluções Cristaloides , Cães , Hidratação/veterinária , Derivados de Hidroxietil Amido/administração & dosagem , Hipoalbuminemia/terapia , Masculino , Plasma , Substitutos do Plasma/administração & dosagem , Estudos ProspectivosRESUMO
In the context of mRNA biomarker profiling, formalin fixed paraffin embedded (FFPE) samples represent an interesting source for retrospective analysis. However, the implementation in routine analysis of FFPE samples, following legislation demands for validated and accredited methods, often requires critical revision and optimization. In the frame of an official control program the validation study of a molecular test for detection of sex steroids administration in calves, based on quantification of progesterone-Receptor mRNA in bulbo-urethral gland samples, was performed: analyses were made on FFPE tissues sections routinary used for histological investigations and compared with RNAlater tissue preservation. To overcome the limitations of original assays several modifications were tested. Obtained results confirmed how Progesterone-Receptor assay represent a useful tool to study suspected cases of sex steroid illicit administration in veal calves, complementary to histological and/or immune histochemical investigation, overcoming the limitation of field studies, where optimal pre-analytical condition cannot always be guarantee.
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Reação em Cadeia da Polimerase em Tempo Real/métodos , Carne Vermelha , Animais , Biomarcadores/análise , Bovinos , Feminino , Formaldeído/química , Perfilação da Expressão Gênica , Humanos , Inclusão em Parafina , RNA Mensageiro/genética , Receptores de Progesterona/genética , Estudos Retrospectivos , Esteroides/farmacologia , Fixação de TecidosRESUMO
Scrapie is a fatal neurodegenerative disease of sheep and goats belonging to the group of Transmissible Spongiform Encephalopathy or prion diseases. The EU has adopted mandatory measures for scrapie surveillance to safeguard public and animal health because it is highly contagious and might decimate all genetic susceptible animals in affected flocks. Definite diagnosis of scrapie relies on the detection of the pathological prion protein in brain tissues and there are still no blood biomarkers available for making diagnosis in living animals that can be used for the screening of sheep in scrapie-affected flocks. Neurofilament light (NfL) protein, a valid biomarker for neuronal and axonal damages, can now be easily measured in blood by the ultra-sensitive single molecule array (Simoa) technology. Recent work reported that serum NfL is increased in neurodegenerative diseases, including human prion diseases, but no data are available for scrapie or other animal prion diseases. Here, we found that the median serum NfL concentration in scrapie animals (56.2, IQR 42.2-84.8, n = 9) was more than 15 times higher (p = 0.00084) than that found in control samples (3.4, IQR 3.0-26.3, n = 11). Moreover, serum NfL concentration in scrapie sheep with clinical signs (n = 2; 75.3, 15.7 pg/ml) did not significantly (p = 0.541; t-test) differ from scrapie animals without clinical signs (n = 7; 61.0, 10.7 pg/ml). The receiver operating characteristic (ROC) curve analysis estimated the cut-off value of 31 pg/ml serum NfL for distinguishing scrapie-infected sheep from controls. The application of this cut-off value gives an accuracy of the test of 95% (percent error of 5.23%). These data indicate that the Simoa test for serum NfL might be a useful screening method for detecting preclinical scrapie in living sheep. Finally, the preliminary data reported here need confirmation in large and more structured studies.
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Técnicas de Diagnóstico Molecular/veterinária , Proteínas de Neurofilamentos/sangue , Scrapie/diagnóstico , Animais , Biomarcadores/sangue , Técnicas de Diagnóstico Molecular/normas , Scrapie/sangue , Sensibilidade e Especificidade , OvinosRESUMO
We compared results of a serum immunofluorescence assay (IFA) and lymph node quantitative PCR (qPCR) in dogs classified as exposed, infected, or sick because of leishmaniasis. We also determined how IFA or qPCR results changed in response to treatment and reflected different clinical and clinicopathologic improvement of dogs. We included 108 dogs in our retrospective study: 12 exposed, 25 infected, and 71 sick, as classified according to Canine Leishmaniasis Working Group standards. Between-group comparison showed higher IFA values ( p < 0.01) for sick dogs; qPCR values were higher for sick than infected dogs ( p < 0.01). A novel clinical and clinicopathologic score was created and applied to 50 sick dogs. Using this score, 41 were reclassified as partially recovered (PR) within 3 mo, and 37 as totally recovered (TR) 3-6 mo after presentation. Statistically significant differences in IFA values were found between the sick and TR dogs ( p < 0.01), but not between sick and PR dogs ( p = 0.98). During follow-up, qPCR revealed a progressive decrease in parasite load, with a statistically significant difference in sick versus PR ( p < 0.01), sick versus TR ( p < 0.01), and PR versus TR ( p < 0.01) dogs. A decrease of 1 point in the clinical score corresponded to 1.3 Leishmania/µL qPCR decrease ( p < 0.01) and decrease of 1:42 in IFA ( p < 0.01). Our findings confirm that the clinical status of dogs affected by leishmaniasis is closely related to parasite load and antibody level, both before and after treatment.
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Antiprotozoários/uso terapêutico , Doenças do Cão/parasitologia , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Leishmania infantum , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Doenças do Cão/tratamento farmacológico , Cães , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Estudos RetrospectivosRESUMO
Member States of the EU are required to monitor the use of pharmacologically active substances in food-producing animals. There is evidence, however, that the target-based approach currently applied in official monitoring plans might under-estimate the real incidence of growth promoter abuse in livestock. As demonstrated for sex hormones, the association of effect-oriented biological screening with chemical confirmatory techniques could be the best strategy in revealing the abuse of veterinary drugs. Here we demonstrate the reliability of a cell-based assay to screen calf urine samples for synthetic glucocorticoids. The validation included the most widely used synthetic drugs (flumethasone, dexamethasone, betamethasone, methylprednisolone and prednisolone) and was developed according to the Commission Decision 2002/657/EC, thus including the verification of cut-off level, the ß error, the specificity, ruggedness and stability. The study was carried out using prednisolone as representative substance at 5 ng mL-1 concentration. All blank and spiked urine fulfilled the EU criteria, moreover the method resulted in being specific and sound, and the analytes in urine were stable for at least 30 days. The assay results indicated its suitability for a qualitative analysis of calf urine samples. This method enabled the detection of low doses of synthetic glucocorticoids (GCs) in matrix (<2 ng mL-1 for flumethasone, dexamethasone, betamethasone; < 4 ng mL-1 for methylprednisolone; 5 ng mL-1 for prednisolone), with the possibility of detecting new or unknown molecules and cumulative effects of low-level mixtures with glucocorticoid bioactivity.
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Bioensaio , Glucocorticoides/urina , Animais , Bovinos , Cromatografia Líquida , Glucocorticoides/síntese química , Glucocorticoides/metabolismo , Luciferases/genética , Luciferases/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The bovine spongiform encephalopathy epidemic is thought to have occurred as a consequence of feeding prion-infected material to cattle. To avoid the risk of bovine spongiform encephalopathy diffusion, the European Commission (Directive 2003/126/EC) established an official method to detect the presence of animal-derived constituents in feedstuffs, using microscopic examination. This method allows easy identification of bone fragments among other animal constituents. The analysis is based on morphological conformation of the fragments and their characterization (mainly of the shape of lacunae) to discriminate among mammalian, poultry, and fish tissues. The aim of this study was to assess the performances of nine European laboratories through a ring trial of the official microscopic method, and to calculate accuracy and reproducibility of the method. In general the reproducibility of the microscopic method was very good (kappa overall = 0.83), with a high sensitivity for all laboratories. Concerning the analysis on the different animal-derived constituents, the results show values of sensitivity with large variability between fish and poultry or mammal. It was generally more difficult to discriminate between mammalian and poultry tissues than fish tissue.