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.

Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed

Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D(®)) and a more cost-efficient and transportable planar imaging detector (MAGPIX(®)), hence demonstrating adequate transferability.

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely. E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Copper-transporting P-Type ATPase, ATP7A, confers multidrug resistance and its expression is related to resistance to SN-38 in clinical colon cancer

We and others have shown that the copper transporters ATP7A and ATP7B play a role in cellular resistance to cisdiaminedichloroplatinum (II) (CDDP).  In this study, we found that ATP7A transfection of Chinese hamster ovary  cells (CHOK1) and fibroblasts isolated from Menkes disease patients  enhanced resistance not only to CDDP but also to various anticancer drugs, such as vincristine, paclitaxel, 7-ethyl-10- hydroxy-camptothecin (SN-38),  etoposide, doxorubicin, mitoxantron, and 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11). ATP7A preferentially localized
doxorubicin fluorescence to the Golgi apparatus in contrast to the more intense nuclear staining of doxorubicin in the parental cells. Brefeldin A   partially and monensin completely altered the distribution of doxorubicin to the nuclei in the ATP7A-expressing cells. ATP7A expression also enhanced the efflux rates of doxorubicin and SN-38 from cells and increased the uptake of SN-38 in membrane vesicles. These findings strongly suggested that   ATP7A confers multidrug resistance to the cells by compartmentalizing drugs in the Golgi apparatus and by enhancing efflux of these drugs, and the trans-Golgi network has an important role of ATP7A-related drug resistance. ATP7A was expressed in 8 of 34 (23.5%) clinical colon cancer specimens but not in the adjacent normal epithelium. Using the histoculture drug response assay that is useful for the prediction of drug sensitivity of clinical cancers, ATP7A-expressing colon cancer cells were significantly more  resistant to SN-38 than ATP7Anegative cells. Thus, ATP7A confers  resistance to various anticancer agents on cancer cells and might be a good index of drug resistance in clinical colon cancers.

Characterization of proADAMTS5 processing by proprotein convertases.

ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in arthritis, is synthesized as a zymogen, proADAMTS5. We report a detailed characterization of the propeptide excision mechanism and demonstrate that it is a major regulatory step with unusual characteristics. Using furin-deficient cells and a furin inhibitor, we found that proADAMTS5 was processed by proprotein convertases, specifically furin and PC7, but not PC6B. Mutagenesis of three sites containing basic residues within the ADAMTS5 propeptide (RRR⁴⁶, RRR⁶⁹ and RRRRR²⁶¹) suggested that proADAMTS5 processing occurs after ^Arg261. That furin processing was essential for ADAMTS5 activity was illustrated using the known ADAMTS5 substrate aggrecan, as well as a new substrate, versican, an important regulatory proteoglycan during mammalian development. When compared to other ADAMTS proteases, proADAMTS5 processing has several distinct features. In contrast to ADAMTS1, whose furin processing products were clearly present intracellularly, cleaved ADAMTS5 propeptide and mature ADAMTS5 were found exclusively in the conditioned medium. Despite attempts to enhance detection of intracellular proADAMTS5 processing, such as by immunoprecipitation of total ADAMTS5, overexpression of furin, and secretion blockade by monensin, neither processed ADAMTS5 propeptide nor the mature enzyme were found intracellularly, which was strongly suggestive of extracellular processing. Extracellular ADAMTS5 processing was further supported by activation of proADAMTS5 added exogenously to HEK293 cells stably expressing furin. Unlike proADAMTS9, which is processed by furin at the cell-surface, to which it is bound, ADAMTS5 does not bind the cell-surface. Thus, the propeptide processing mechanism of ADAMTS5 has several points of distinction from those of other ADAMTS proteases, which may have considerable significance in the context of osteoarthritis.

Produção de metano entérico em pastagens tropicais

Na presente revisão, buscou-se apresentar os principais impactos ambientais causados pela pecuária, sobretudo, em relação às emissões de gases efeito estufa (GEE). Além disso, buscou-se apresentar possíveis formas de mitigar essas externalidades. A criação de bovinos, no Brasil, acontece de forma extensiva, muitas vezes em áreas com pastagem degradada e, portanto, de baixa produtividade. Isso possibilita à atividade uma oportunidade de redução do impacto causado ao meio ambiente, uma vez que ações tomadas, no sentido de melhorar o rendimento animal, devem resultar em um menor consumo de recursos naturais (terra e água) e maior eficiência do sistema digestivo animal. Os principais problemas apontados pelos pesquisadores, no que tange à pecuária extensiva, são o metano emitido pela fermentação entérica dos ruminantes, o óxido nitroso emitido pelos dejetos dos animais em pastejo e o dióxido de carbono trocado pelo solo e vegetação. Muitos fatores influenciam a produção de CH4 entérico dos ruminantes, inclusive o tipo de carboidrato fermentado, o sistema digestivo do animal, a quantidade e o tipo de alimentos consumidos. Diante do exposto, pesquisadores têm desenvolvido tecnologias para reduzir a emissão de metano, através da melhoria das práticas de manejo alimentar, manipulação ruminal, por meio de suplementação com monensina, lipídios, ácidos orgânicos e compostos de plantas. Outras estratégias de redução de metano que foram investigadas são: defaunação e vacinas, que buscam inibir micro-organismos metanogênicos e a metanogênese. Assim, a busca por sistemas de produção eficientes tem sido uma das perspectivas da pecuária mundial para reduzir a emissão de poluentes e intensificar a produção animal.

Fermentação ruminal e produção de metano em bovinos alimentados com feno de capim-tifton 85 e concentrado com aditivos

Objetivou-se avaliar o efeito do uso de monensina, complexo de leveduras, ácidos graxos poliinsaturados e aminoácidos no consumo de matéria seca e nutrientes, na estimativa da digestibilidade ruminal, nos parâmetros de fermentação ruminal (pH, concentração de nitrogênio amoniacal e de ácidos graxos de cadeia curta), na população de protozoários e na produção de metano. Foram utilizados seis bovinos e com peso corporal de 530 ± 15 kg, recebendo complexo de leveduras, ácidos graxos poliinsaturados e aminoácidos (5 g/dia); monensina (5 g/dia); caulim (5 g/dia), usado como controle adicionado à dieta composta de feno de capim-tifton 85 (Cynodon spp.); e concentrado, na relação 80:20. O delineamento experimental adotado para análise do consumo e da digestibilidade foi o de blocos completos casualizados e, para análise dos parâmetros ruminais e da produção de metano, o de parcelas subdivididas. O consumo foi influenciado pelo uso de monensina na dieta, mas não diferiu entre os aditivos. As digestibilidades da matéria seca e dos nutrientes não foram influenciadas pelo fornecimento dos aditivos. A relação acetato:propionato nos animais alimentados com a dieta com monensina foi menor que naqueles que receberam o complexo de leveduras e ácidos graxos poliinsaturados e aminoácidos, diminuindo a perda de energia na forma de metano. O pH e a concentração de nitrogênio amoniacal foram adequados para o crescimento bacteriano. A concentração de metano não é alterada pelo uso dos aditivos testados.

Molecular Identification and Characterization of Ileal and Cecal Fungus Communities in Broilers Given Probiotics, Specific Essential Oil Blends, and Under Mixed Eimeria Infection

Broiler digestive tract fungal communities have gained far less scrutiny than that given corresponding bacterial communities. Attention given poultry-associated fungi have focused primarily on feed-associated toxin-producers, yeast, and yeast products. The current project focused on the use of pyrosequencing and denaturing gradient gel electrophoresis (DGGE) to identify and monitor broiler digestive fungal communities. Eight different treatments were included. Four controls were an Uninfected-Unmedicated Control, an Unmedicated-Infected Control, the antibiotic bacitracin methylene disalicylate plus the ionophore monensin as Positive Control, and the ionophore monensin alone as a Negative Control. Four treatments were two probiotics (BC-30 and Calsporin) and two specific essential oil blends (Crina Poultry Plus and Crina Poultry AF). All chickens except the Unmedicated-Uninfected Control were given, at 15 days of age, a standard oral Eimeria inoculum of sporulated oocysts. Ileal and cecal digesta were collected at pre-Eimeria infection at 14 days of age and at 7 days post-Eimeria infection at 22 days of age. Extracted cecal DNA was analyzed by pyrosequencing to examine the impact of diet supplements and Eimeria infection on individual constituents in the fungal community, while DGGE was used to compare more qualitative changes in ileal and cecal communities. Pyrosequencing identified three phyla, seven classes, eight orders, 13 families, 17 genera, and 23 fungal species. Ileal and cecal DGGE patterns showed fungal communities were clustered mainly into pre- and post-infection patterns. Post-infection Unmedicated-Uninfected patterns were clustered with pre-infection groups demonstrating a strong effect of Eimeria infection on digestive fungal populations. These combined techniques offered added versatility towards unraveling the effects of enteropathogen infection and performance enhancing feed additives on broiler digestive microflora.

Efeito da monensina sódica sobre a produção e composição do leite, a produção de mozzarela e o escore de condição corporal de búfalas Murrah

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Características da carcaça e qualidade da carne de ovinos terminados em pastagens cultivadas

The objective of this study was to evaluate the quality of housing and the physical and chemical characteristics of meat from sheep raised on pasture Brachiaria brizantha and Panicum maximum. The experiment was conducted in the physical area of the Study Group on Forage (GEFOR), located in the Academic Unit Specialized in Agricultural Sciences - Federal University of Rio Grande do Norte - UFRN in Macaíba, RN, Brazil. We used 32 lambs SPRD, obtained from herds in the state, with liveweight (LW) of 24.5 kg were assigned randomly to four treatments consisting of tropical grasses, two cultivars of Brachiaria brizantha, Marandu and Piatã, and two of Panicum maximum, Aruana and Massai. The experimental area was 2.88 ha, divided into 4 paddocks of 0.72 ha, where each picket consisted of a farm and was divided into six plots of 0.12 ha, where the animals remained under rotational grazing. The period of adaptation to the pickets was seven days. At the beginning of the experiment the animals were weighed, identified with plastic earrings and necklaces colored according to the treatment, and treated against. The lambs were loose in the paddock at 8 am and collected at 16 hours, which returned to collective pens. During the time of grazing animals had free access to mineral supplement with monensin Ovinofós ® and water. Before entering the paddocks of pasture were sampled to characterize the chemical composition. Every seven days occurred at weighing, with fasting, to monitor the weight development. Cultivars Marandu, Aruana, Piatã and Massai were grazed for 133, 129, 143 and 142 days, respectively, until the lambs reach slaughter weight. Arriving at 32 kg lambs were evaluated subjectively for body condition score by, passed through fasting period, diet and water for 16 hours were slaughtered. Measurements were made in the inner and outer casings in addition to subjective evaluations regarding muscling, finish and quantity of pelvic-renal fat, then each was divided longitudinally into two half-carcases and cuts were made in the commercial left half, and after heavy calculated their income. Between the 12th and 13th thoracic vertebrae, was performed a cut to expose the cross section of the Longissimus dorsi, which was drawn on the rib eye area (REA) in transparent film. Fat thickness and extent of AOL GR were determined using a caliper. A tissue composition was determined by dissection of the legs. Analyzes were performed physical (color, cooking loss and shear force) and chemical composition of meat (moisture, ash, protein and lipids) in Longissimus dorsi muscle. Grazing tropical grass Brachiaria brizantha cvs. Marandu and Piatã and Panicum maximum cvs. Aruana and Massai can be used for lambs SRPD in the rainy season, because not alter the physico-chemical and chemical composition of meat

Avaliação da monensina sódica em vacas leiteiras

Utilizaram-se quinze vacas da raça Holandesa, submetidas a três tratamentos: T1-controle, T2-monensina sódica 300 mg/animal/dia, T3-monensina sódica 225 mg/animal/dia. A produção de leite, consumo de ração e o peso dos animais foram mensurados, juntamente com a avaliação da condição corporal Após o período experimental de 112 dias, evidenciou-se aumento na produção de leite de 13,5% e 10% (T2) em relação a TI e T3 respectivamente. Houve redução de consumo de ração de 4,5% (T2) e 3% (T3) em relação ao controle.

Performance, carcass characteristics and gain cost of feedlot cattle fed a high level of concentrate and different feed additives

The objective of this study was to evaluate the effects of feeding cattle with isoprotein and isoenergetic diets, with and without the addition of polyclonal antibody preparation (PAP), yeasts (YST) or monensin sodium (MON) on performance, carcass characteristics and gain cost in feedlot. Ninety-five 20-month old bullocks (323.3±21.8 kg) were distributed in 25 pens. The completely randomized experimental design had a 2 × 2 + 1 factorial arrangement and the treatments were replicated 5 times. There was no effect of MON for DMI throughout the feedlot period; however, MON reduced the dry matter intake (DMI) in g/kg of BW in the first 28 days when compared with the other treatments. The gain cost decreased with MON addition in relation to the other treatments. Inclusion of YST decreased average daily gain (ADG), final body weight, hot carcass weight, carcass weight, gain to feed ratio and DMI in g/kg body weight, worsening feed conversion and increasing the gain cost in the feeding periods. Inclusion of PAP increased ADG and decreased the gain cost, besides improving feed conversion. For MON and PAP, a difference was found for kidney-pelvic fat and kidney-pelvic fat per 100 kg of hot carcass weight. For MON and YST, there was a difference in ADG, feed conversion, gain cost and carcass yield and kidney-pelvic fat per 100 kg of hot carcass. Treatment YST worsened performance in relation to the non-supplemented treatments. Feeding PAP to animals did not influence performance and carcass characteristics of bullocks in feedlot negatively. Thus, PAP shows potential to substitute MON in cattle feeding using isoprotein and isoenergetic diets.

Parâmetros de fermentação ruminal em bovinos alimentados com grãos de milho ou sorgo de alta umidade ensilados

Os parâmetros de fermentação ruminal de dietas contendo silagem de sorgo úmido em substituição à de milho úmido foram estudados em 12 fêmeas bovinas, com peso médio de 584 kg. O delineamento foi inteiramente casualizado com três tratamentos: substituição do milho úmido pelo sorgo úmido ensilado, nos níveis de 0, 50 e 100%. As dietas continham grão úmido de milho ou de sorgo ensilados, soja extrusada, uréia, feno de aveia (Avena sativa sp.), suplemento mineral e monensina. Adicionalmente, foi avaliada a degradabilidade in situ da matéria seca e da fibra em detergente neutro do feno de aveia. Não houve diferença sobre produção total de ácidos graxos voláteis (AGVs) no rúmen, porcentagem molar dos ácidos acético, propiônico e butírico, relação acético/propiônico, pH ruminal, concentração de N-NH3 no rúmen, fluxo e volume de líquidos do rúmen, nos diferentes tratamentos. A degradabilidade da matéria seca e da fibra em detergente neutro do feno não apresentou diferenças. Não se constatou melhora nos parâmetros de fermentação ruminal com a associação dos grãos.

Effects of feeding a multivalent polyclonal antibody preparation on feedlot performance, carcass characteristics, rumenitis, and blood gas profile in Bos indicus biotype yearling bulls

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of adding polyclonal antibody preparations on ruminal fermentation patterns and digestibility of cows fed different energy sources

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)