Performance of RT-PCR-ELISA for the detection of peste des petits ruminants virus

A highly sensitive and specific reverse transcription polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR-ELISA) was developed for the objective detection of nucleoprotein (N) gene of peste des petits ruminants (PPR) virus from field outbreaks or experimentally infected sheep. Two primers (IndF and Np4) and one probe (Sp3) available or designed for the amplification/probing of the 'N' gene of PPR virus, were chosen for labeling and use in RT-PCR-ELISA based on highest analytical sensitivity of detection of infective virus or N-gene containing recombinant plasmid, higher nucleotide homology at the primer binding sites of the 'N' gene sequences available and the ability to amplify PPR viral genome from different sources of samples. RT-PCR was performed with unlabeled IndF and Np4 digoxigenin labeled primers followed by a microplate hybridization probe reaction with biotin labeled Sp3 probe. RT-PCR-ELISA was found to be 10-fold more sensitive than the conventional RT-PCR followed by agarose gel based detection of PCR product. Based on the Mean (mean +/- 3S.D.) optical density (OD) values of 47 RT-PCR negative samples, OD values above 0.306 were considered positive in RT-PCR-ELISA. A total of 82 oculo-nasal swabs and tissue samples from suspected PPR cases were analyzed by RT-PCR and RT-PCR-ELISA, which revealed 54.87 and 58.54% positivity, respectively. From an experimentally infected sheep, both RT-PCR and RT-PCR-ELISA could detect the virus from 6 days post-infection up to 9 days in oculo-nasal swabs. On post-mortem, PPR viral genome was detected in spleen, lymph node, lung, heart and liver. The correlation co-efficient between RT-PCR-ELISA OD values and either TCID50 of virus or molecules of DNA was 0.622 and 0.657, respectively. The advantages of RT-PCR-ELISA over the conventional agarose gel based detection of RT-PCR products are discussed. (c) 2006 Elsevier B.V. All rights reserved.

Determination of digesta flow entering the omasal canal of dairy cows using different marker systems

Four studies were conducted to compare the effect of four indigestible markers (LiCoEDTA, Yb-acetate, Cr-mordanted straw and indigestible neutral-detergent fibre (INDF)) and three marker systems on the flow of digesta entering the omasal canal of lactating dairy cows. Samples of digesta aspirated from the omasal canal were pooled and separated using filtration and high-speed centrifugation into three fractions defined as the liquid phase, small particulate and large particulate matter. Co was primarily associated with the liquid phase, Yb was concentrated in small particulate matter, whilst Cr and INDF were associated with large particles. Digesta flow was calculated based on single markers or using the reconstitution system based on combinations of two (Co + Yb, Co + Cr and Co + INDF) or three markers (Co + Yb + Cr and Co + Yb + INDF). Use of single markers resulted in large differences between estimates of organic matter (OM) flow entering the omasal canal suggesting that samples were not representative of true digesta. Digesta appeared to consist of at least three phases that tended to separate during sampling. OM was concentrated in particulate matter, whilst the liquid phase consisted mainly of volatile fatty acids and inorganic matter. Yb was intimately associated with nitrogenous compounds, whereas Cr and INDF were concentrated in fibrous material. Current data indicated that marker systems based on Yb in combination with Cr or INDF are required for the accurate determination of OM, N and neutral-detergent fibre flow. In cases where the flow of water-soluble nutrients entering the omasal canal is also required, the marker system should also include Co.

Inovações e desafios na avaliação de alimentos na nutrição de ruminantes

A avaliação de alimentos é um dos principais pontos a serem observados no setor de nutrição animal, a qual diz respeito à utilização de métodos para descrever alimentos ao seu público e à sua capacidade de sustentar diferentes tipos e níveis de desempenho animal. Atualmente, as pesquisas têm buscado relacionar o conteúdo de nutrientes dos alimentos com seu aproveitamento digestivo e metabólico. A nutrição animal é uma ciência bastante dinâmica, sempre lança mão de novas tecnologias e estratégias para melhorar o aproveitamento dos alimentos e busca novas metodologias para respondem às demandas emergenciais.. Dessa forma, o estabelecimento de estratégias apropriadas para obtenção de respostas é essencial para que o sucesso na pesquisa seja atingido. Assim, esta revisão irá abordar e discutir as principais técnicas utilizadas na avaliação de alimentos aplicadas à nutrição de ruminantes.

Estimation of fecal production, digesta flow and digestibility in dairy heifers with different indicator

Objetivou-se com este trabalho avaliar a produção fecal por meio do indicador interno, fibra em detergente neutro indigestível (FDNi), e externos, cromo complexado com ácido etilenodiaminotetracético (Cr-EDTA ) e o cloreto de itérbio (YbCl3), além de estimar o fluxo duodenal da matéria seca e os coeficientes de digestibilidades aparentes total, ruminal e pós-ruminal, de diferentes nutrientes. Foram utilizadas oito novilhas mestiças Holandês/Zebu, distribuídas em duplo quadrado latino 4 x 4. Os indicadores Cr-EDTA, YbCl3 e o FDNi não estimaram produção fecal de forma eficiente (p < 0,05), obtendo resultado de 1,64; 1,71 e 2,71 kg dia-1, respectivamente, quando comparado à coleta total de fezes, que obteve resultado de 1,39 kg dia-1. Os valores estimados de fluxo de matéria seca, tanto para a metodologia de único, quanto para de duplo indicador, podem ser considerados biologicamente aceitáveis. Contudo, o valor obtido pela associação Cr-EDTA/YbCl3, utilizada na forma de duplo indicador, foi o mais confiável, pela melhor recuperação dos indicadores externos (Cr-EDTA e YbCl3), que obtiveram médias de 89 e 85%, respectivamente, em comparação ao interno (FDNi), que obteve média 67%. Os coeficientes de digestibilidade ruminal e pós ruminal, estimados pela associação Cr-EDTA/YbCl3, foram considerados melhores, em consequência do valor de fluxo de matéria seca estimado por esta associação.

Digestibilidade e degradabilidade de rações à base de milho desintegrado com palha e sabugo em diferentes graus de moagem

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

Consumo e digestibilidade total e parcial de dietas utilizando farelo de girassol e três fontes de energia em novilhos confinados

Avaliaram-se o consumo alimentar, a digestibilidade parcial e total e o balanço de nitrogênio, em novilhos confinados. Foram utilizados três novilhos de corte cruzados, canulados no rúmen e no duodeno, distribuídos em dois delineamentos em quadrado latino 3 x 3. As dietas experimentais foram constituídas de 60% de silagem de milho, tendo como fonte de proteína o farelo de girassol e, como fonte de energia, o milho (MI). O milho foi substituído parcialmente pela casca de soja (CS) ou pelo farelo de gérmen de milho (FGM). Quatro indicadores internos (lignina e FDA, FDN e lignina indigestíveis) foram submetidos a 144 horas de digestão in vitro, para estimativa da digestibilidade parcial e total das dietas. Houve maior ingestão dos componentes fibrosos na dieta CS, porém não foi observado efeito sobre o consumo de matéria seca. A lignina subestimou significativamente a digestibilidade. A utilização da FDAi, FDNi e lignina-i para estimar a digestibilidade total é viável, no entanto, a FDAi e lignina-i não estimaram adequadamente as digestibilidades parciais. Houve efeito significativo na digestibilidade ruminal da FDA, com valores mais elevados para CS e semelhantes para FGM, em relação à dieta MI. A digestibilidade total da FDA foi maior na dieta CS, porém, as digestibilidades dos demais componentes não foram afetadas pelas diferentes fontes energéticas. Não houve diferença significativa para a digestibilidade da energia e nos valores obtidos de NDT, com média de 61,5%. A casca de soja e o farelo de gérmen de milho, em substituição parcial do milho, mostraram-se fontes alternativas satisfatórias para a inclusão na dieta de bovinos.

Comparison of indigestible markers from in situ and in vivo incubation to predict apparent digestibility in hay- and corn-fed horses

Foram utilizados quatro cavalos castrados sem raça definida pelo delineamento em blocos casualizados. Objetivou-se viabilizar os indicadores internos indigestíveis, FDNi e FDAi, obtidas pelas técnicas in situ nos bovinos e in vivo nos equinos por meio do saco de náilon móvel (SNM). Os tratamentos consistiram de digestibilidade determinada por método direto com a coleta total de fezes (CT) e indireto pelo uso de FDNi e FDAi obtidos pelas técnicas in situ (IS) na cavidade ruminal de bovinos e in vivo (IV) nos equinos por meio do SNM. As FDNi-IV e FDAi-IV promoveram a melhor taxa de recuperação (TR) do indicador, igualando-se ao grupo CT, em 91,50%, enquanto a técnica in situ resultou nas piores taxas, na média 86,50%, para ambos os indicadores indigestíveis (p < 0,05). As estimativas dos CD dos nutrientes foram adequadamente preditos pela FDAi-IV, para equinos alimentados com feno, no qual os valores observados foram de 46,41; 48,16; 47,92 e 45,51% para MS, MO, FDN e EB, respectivamente. Concluiu-se que o SNM em equinos pode ser usado para obter a FDAi em equinos alimentados exclusivamente com feno de coast-cross e para cavalos consumindo dieta mista, as FDNi e FDAi foram eleitas para predizer CD de nutrientes.

Diferentes procedimentos na determinação de indicadores internos para estimativa de produção fecal e fluxo duodenal de matéria seca em bovinos

Times of in situ incubation (144 and 288h) for determination of internal markers IADF and INDF and the effects of differents procedures (wash or not the nylon bag every 72h incubation) was evaluated in samples of diet, duodenal digesta and cattle feces. The duodenal flow dry matter and fecal production utilizing the internal markers to compare with the external marker chromium oxide there was estimated. The animals were fed with sorgum silage, concentrate or urea. In this experiment, a latin square design was used, in a factorial scheme (two times of incubation × two processing nylon bag). No was observed effect of the incubation time or processing in the internal markers INDF and IADF concentration and the in situ incubation after 144h is adequate to reproduce the indigestible markers fraction in samples. For fecal production estimation, the external marker chromium oxide presented similar result (1.26 kg day-1) as the total fecal collection (1.49 kg day-1). Both the internal markers overestimate the duodenal flow dry matter when compared with the external marker chromium oxide.

Indicadores na estimativa do fluxo de digesta em bovinos de corte

single and double marker methods were compared in seven Nellore steers with average body weight 250 kg, and previously canullated in the rumen and duodenum. The animals were fitted with continuous-infusion pump that administered CoEDTA solutions intraruminally. In addition, a capsule containing Cr 2O 3, and other, containing external n-alkanes C 32, C 36 were inserted into the rumen of each steer. Internal markers indigestible neutral-detergent fiber and acid-detergent fiber (iNDF and iADF after 144 h in situ incubation) and internal n-alkanes C 31, C 33, C 35 were components of the diet. Steers were fed with palisade grass (Brachiaria brizantha cv. marandu) in two age of regrowth, 30 and 60 days. The duodenal flow experimental design was a factorial 2×2×12 while omasal experimental design was a factorial 2×2×4. Markers iNDF, CoEDTA and the combination Co+iNDF were efficacious to estimate duodenal dry matter flow whereas iNDF, iADF and the Co+iADF combination were all efficacious to predict omasal dry matter flow. In conclusion, the double marker method for estimation of omasal and duodenal dry matter flow was the most appropriate considering the ruminal fiber digestibility.

Development of equations to estimate microbial contamination in ruminal incubation residues of forage produced under tropical conditions using 15N as a label1

The objective of this study was to use 15N to label microbial cells to allow development of equations for estimating the microbial contamination in ruminal in situ incubation residues of forage produced under tropical conditions. A total of 24 tropical forages were ruminal incubated in 3 steers at 3 separate times. To determine microbial contamination of the incubated residues, ruminal bacteria were labeled with 15N by continuous intraruminal infusion 60 h before the first incubation and continued until the last day of incubation. Ruminal digesta was collected for the isolation of bacteria before the first infusion of 15N on adaptation period and after the infusion of 15N on collection period. To determine the microbial contamination of CP fractions, restricted models were compared with the full model using the model identity test. A value of the corrected fraction A was estimated from the corresponding noncorrected fraction by this equation: Corrected A fraction (ACPC) = 1.99286 + 0.98256 × A fraction without correction (ACPWC). The corrected fraction B was estimated from the corresponding noncorrected fraction and from CP, NDF, neutral detergent insoluble protein (NDIP), and indigestible NDF (iNDF) using the equation corrected B fraction (BCPC) = -17.2181 - 0.0344 × fraction B without correction (BCPWC) + 0.65433 × CP + 1.03787 × NDF + 2.66010 × NDIP - 0.85979 × iNDF. The corrected degradation rate of B fraction (kd)was estimated using the equation corrected degradation rate of B fraction (kdCPC) = 0.04667 + 0.35139 × degradation rate of B fraction without correction (kdCPWC) + 0.0020 × CP - 0.00055839 × NDF - 0.00336 × NDIP + 0.00075089 × iNDF. This equation was obtained to estimate the contamination using CP of the feeds: %C = 79.21 × (1 - e-0.0555t) × e-0.0874CP. It was concluded that A and B fractions and kd of CP could be highly biased by microbial CP contamination, and therefore these corrected values could be obtained mathematically, replacing the use of microbial markers. The percentage of contamination and the corrected apparent degradability of CP could be obtained from values of CP and time of incubation for each feed, which could reduce cost and labor involved when using 15N. © 2013 American Society of Animal Science. All rights reserved.

Estimativa do fluxo de nutrientes pelo trato digestivo de bovinos de corte alimentados com Brachiaria brizantha (Hochst A. Rich) Stapf. cv. Marandu em duas idades de rebrota

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Indicadores na estimativa do fluxo de nutrientes no duodeno, produção fecal, consumo de concentrado e volumoso por bovinos

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

Fontes protéicas em dietas à base de cana-de-açúcar para novilhas leiteiras

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Consumo alimentar residual e produção de metano entérico de bovinos em confinamento e pastagem

Pós-graduação em Zootecnia - FCAV

Avaliação das características físico-químicas e microbiológicas dos componentes fibrosos obtidos da moagem úmida de grãos de milho

Fibers are non-digestible materials by human organism, and they are insoluble in specific conditions. The corn fiber has been used as an alternative to enrich the food products for human consumption, in order to add nutritious value to this co-product. This study aimed at determining the chemical and fractions components of corn fiber, extracted by semi-wet milling in laboratory (LabF), and by semi-humid milling in industrial process (IndF); and microbiological analyses were perform in the last fraction. The fiber composition differed significantly among these two products as to moisture, ash, lipids, proteins, insoluble dietary fiber, neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose and lignin contents. The sample IndF showed microbiological parameters in compliance with the specifications for consumption. The investigated corn fiber may effectively contribute to increase the fiber intake in the diet. As the industrially processed fiber (IndF) showed compliant microbiological parameters with the specifications for food, this component might be incorporated into the food products formulation.