Determining anthelmintic residues in goat milk in Brazil / Determinação de resíduos anti-helmínticos no leite caprino no Brasil

Anthelmintics are used to combat nematodes. The misuse of anthelmintics can raise the cost of milk production. The objective of this research was to determine the presence of anthelmintics in goat milk. Twenty goats were used, divided into four groups of five animals: I- animals treated with an ivermectin-based anthelmintic; II- animals treated with moxidectin; III- animals treated with levamisole hydrochloride; and IV: animals treated with albendazole. Milk samples were collected individually: before, and 1, 2, 3, 15 and 21 days after administration of the anthelmintics. Determination of anthelmintic residues was performed by liquid chromatography-mass spectrometry (LC-MS). According to the results, there was an exponential effect (P<0.05) for ivermectin and moxidectin. Moxidectin was the anthelmintic that left a residue in the milk for the longest time, up to 21 days. However, with all the anthelmintics researched, residues were below the maximum limit recommended by the inspecting agencies.(AU)

Anthelmintics são usados ​​para combater nematodes. O mau uso de anti-helmínticos pode aumentar o custo da produção de leite. O objetivo desta pesquisa foi determinar a presença de anti-helmínticos no leite de cabra. Foram utilizados vinte cabras, divididos em quatro grupos de cinco animais: I - animais tratados com um antihelmíntico à base de ivermectina; II - animais tratados com moxidectina; III- animais tratados com cloridrato de levamisol; E IV: animais tratados com albendazole. Amostras de leite foram coletadas individualmente: antes, e 1, 2, 3, 15 e 21 dias após a administração dos anti-helmínticos. A determinação dos resíduos antihelmínticos foi realizada por cromatografia líquida-espectrometria de massa (LC-MS). De acordo com os resultados, houve um efeito exponencial (P <0.05) para ivermectina e moxidectina. A moxidectina foi o anti-helmíntico que deixou um resíduo no leite por mais tempo, até 21 dias. Contudo, com todos os anthelmintics pesquisados, os resíduos estavam abaixo do limite máximo recomendado pelas agências de inspeção.(AU)

Traceability of animal protein byproducts in ruminants by multivariate analysis of isotope ratio mass spectrometry to prevent transmission of prion diseases

Background: Ruminant feed containing animal byproduct proteins (ABPs) is prohibited in many countries due to its risk of transmitting prion diseases (PD). In most cases the entire herd is sacrificed, which causes great harm to the producer countries by preventing their exportation of ruminant derived-products. Methods: We used stable isotope ratio mass spectrometry (IRMS) of carbon (13C/12C) and nitrogen (15N/14N) to trace the animal protein in the blood of 15 buffaloes (Bubalus bubalis) divided into three experimental groups: 1 - received only vegetable protein (VP) during 117 days; 2 - received animal and vegetable protein (AVP); and 3 - received animal and vegetable protein with animal protein subsequently removed (AVPR). Groups 2 and 3 received diets containing 13.7% bovine meat and bone meal (MBM) added to a vegetable diet (from days 21-117 in the AVP group and until day 47 in the AVPR group, when MBM was removed). Results: On the 36th day, differences were detectable in the feeding profile (p 0.01) among the three experimental groups, which remained for a further 49 days (85th day). The AVPR group showed isotopic rate reversibility on the 110th day by presenting values similar to those in the control group (VP) (p> 0.05), indicating that it took 63 days to eliminate MBM in this group. Total atoms exchange (> 95%) of 13C and 15N was observed through incorporation of the diet into the AVP and AVPR groups. Conclusions: IRMS is an accurate and sensitive technique for tracing the feeding profile of ruminants through blood analysis, thus enabling investigation of ABP use.(AU)

Determining anthelmintic residues in goat milk in Brazil / Determinação de resíduos anti-helmínticos no leite caprino no Brasil

Anthelmintics are used to combat nematodes. The misuse of anthelmintics can raise the cost of milk production. The objective of this research was to determine the presence of anthelmintics in goat milk. Twenty goats were used, divided into four groups of five animals: I- animals treated with an ivermectin-based anthelmintic; II- animals treated with moxidectin; III- animals treated with levamisole hydrochloride; and IV: animals treated with albendazole. Milk samples were collected individually: before, and 1, 2, 3, 15 and 21 days after administration of the anthelmintics. Determination of anthelmintic residues was performed by liquid chromatography-mass spectrometry (LC-MS). According to the results, there was an exponential effect (P<0.05) for ivermectin and moxidectin. Moxidectin was the anthelmintic that left a residue in the milk for the longest time, up to 21 days. However, with all the anthelmintics researched, residues were below the maximum limit recommended by the inspecting agencies.

Anthelmintics são usados ​​para combater nematodes. O mau uso de anti-helmínticos pode aumentar o custo da produção de leite. O objetivo desta pesquisa foi determinar a presença de anti-helmínticos no leite de cabra. Foram utilizados vinte cabras, divididos em quatro grupos de cinco animais: I - animais tratados com um antihelmíntico à base de ivermectina; II - animais tratados com moxidectina; III- animais tratados com cloridrato de levamisol; E IV: animais tratados com albendazole. Amostras de leite foram coletadas individualmente: antes, e 1, 2, 3, 15 e 21 dias após a administração dos anti-helmínticos. A determinação dos resíduos antihelmínticos foi realizada por cromatografia líquida-espectrometria de massa (LC-MS). De acordo com os resultados, houve um efeito exponencial (P <0.05) para ivermectina e moxidectina. A moxidectina foi o anti-helmíntico que deixou um resíduo no leite por mais tempo, até 21 dias. Contudo, com todos os anthelmintics pesquisados, os resíduos estavam abaixo do limite máximo recomendado pelas agências de inspeção.

Identificação por espectrometria de massa MALDI-TOF de Salmonella spp. e Escherichia coli isolados de carcaças bovinas / MALDI-TOF mass spectrometry identification of Salmonella spp. and Escherichia coli isolated from bovine carcasses

O objetivo deste trabalho foi introduzir a técnica de espectrometria de massa com fonte de ionização e dessorção a laser assistida por matriz e analisador de tempo-de-voo (MALDI-TOF) para incrementar o método tradicional microbiológico na detecção de Salmonella spp. e Escherichia coli em carcaças bovinas. Foram avaliadas 270 amostras de 90 carcaças de bovinos. Para isolamento de Salmonella spp. e E. coli, foram utilizadas, respectivamente, as metodologias descritas na ISO 6579:2002 e no Compendium of Methods for the Microbiological Examination of Foods. As análises por MALDI-TOF foram realizadas a partir de isolados cultivados em ágar nutriente ou em caldo triptona de soja, provenientes das amostras com características bioquímicas positivas (n=7), inconclusivas (n=4) e negativas (n=85) para Salmonella spp. e bioquímicas positivas (n=37) e negativas (n=85) para E. coli. Os perfis de massas foram adquiridos com o espectrômetro de massas MALDI-TOF Autoflex III SmartBeam e os espectros brutos foram processados usando o programa MALDI Biotyper (Bruker Daltonics). De acordo com a identificação preliminar, com base na morfologia das colônias e nas reações bioquímicas, sete isolados foram considerados positivos para Salmonella spp. Através do MALDI Biotyper, esses sete isolados foram classificados como pertencentes ao gênero Salmonella e, além disso, identificados como S. enterica. Quatro isolados que apresentaram características fenotípicas não usuais e resultados inconclusivos nos testes bioquímicos para Salmonella foram identificados como pertencentes aos gêneros Citrobacter e Proteus após análise por MALDI. Para E. coli, 37 amostras foram positivas pelos testes bioquímicos da espécie, o que foi confirmado por MALDI Biotyper. A metodologia MALDI-TOF permitiu a rápida confirmação da identidade de Salmonella spp. e E. coli, podendo ser utilizada para detecção desses microrganismos em isolados bacterianos de carcaças bovinas.(AU)

The aim of this study was to introduce matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry to improve the traditional microbiological method for the detection of Salmonella spp. and Escherichia coli in beef carcasses. Two hundred seventy samples from 90 beef carcasses were evaluated. The methodologies described in ISO 6579:2002 and in the Compendium of Methods for the Microbiological Examination of Foods were used for Salmonella spp. and E. coli isolation, respectively. MALDI-TOF analysis were performed on tryptone soya broth suspension isolates or directly from nutrient agar colonies, from the positive, inconclusive or negative biochemically tested samples for Salmonella and E. coli. Mass profiles were acquired on an Autoflex III SmartBeam MALDI-TOF mass spectrometer and the raw spectra were processed using the MALDI Biotyper software (Bruker Daltonics). According to the preliminary identification based on colony morphology and the biochemical reactions, seven isolates were positive for Salmonella spp. Through MALDI Biotyper these seven isolates were also classified as belonging to the genus Salmonella and further identified as S. enterica. Four isolates showing unusual phenotypic characteristics and inconclusive results in biochemical tests for Salmonella were identified as belonging to Citrobacter and Proteus genera after MALDI analysis. Regarding Escherichia coli, 37 were positive for species biochemical testing which MALDI Biotyper confirmed. MALDI-TOF methodology allowed rapid Salmonella spp. and E. coli identity confirmation and may be used to detect these microrganisms within bacterial isolates from beef carcasses.(AU)

Identificação por espectrometria de massa MALDI-TOF de Salmonella spp. e Escherichia coli isolados de carcaças bovinas / MALDI-TOF mass spectrometry identification of Salmonella spp. and Escherichia coli isolated from bovine carcasses

O objetivo deste trabalho foi introduzir a técnica de espectrometria de massa com fonte de ionização e dessorção a laser assistida por matriz e analisador de tempo-de-voo (MALDI-TOF) para incrementar o método tradicional microbiológico na detecção de Salmonella spp. e Escherichia coli em carcaças bovinas. Foram avaliadas 270 amostras de 90 carcaças de bovinos. Para isolamento de Salmonella spp. e E. coli, foram utilizadas, respectivamente, as metodologias descritas na ISO 6579:2002 e no Compendium of Methods for the Microbiological Examination of Foods. As análises por MALDI-TOF foram realizadas a partir de isolados cultivados em ágar nutriente ou em caldo triptona de soja, provenientes das amostras com características bioquímicas positivas (n=7), inconclusivas (n=4) e negativas (n=85) para Salmonella spp. e bioquímicas positivas (n=37) e negativas (n=85) para E. coli. Os perfis de massas foram adquiridos com o espectrômetro de massas MALDI-TOF Autoflex III SmartBeam e os espectros brutos foram processados usando o programa MALDI Biotyper (Bruker Daltonics). De acordo com a identificação preliminar, com base na morfologia das colônias e nas reações bioquímicas, sete isolados foram considerados positivos para Salmonella spp. Através do MALDI Biotyper, esses sete isolados foram classificados como pertencentes ao gênero Salmonella e, além disso, identificados como S. enterica. Quatro isolados que apresentaram características fenotípicas não usuais e resultados inconclusivos nos testes bioquímicos para Salmonella foram identificados como pertencentes aos gêneros Citrobacter e Proteus após análise por MALDI. Para E. coli, 37 amostras foram positivas pelos testes bioquímicos da espécie, o que foi confirmado por MALDI Biotyper. A metodologia MALDI-TOF permitiu a rápida confirmação da identidade de Salmonella spp. e E. coli, podendo ser utilizada para detecção desses microrganismos em isolados bacterianos de carcaças bovinas.(AU)

The aim of this study was to introduce matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry to improve the traditional microbiological method for the detection of Salmonella spp. and Escherichia coli in beef carcasses. Two hundred seventy samples from 90 beef carcasses were evaluated. The methodologies described in ISO 6579:2002 and in the Compendium of Methods for the Microbiological Examination of Foods were used for Salmonella spp. and E. coli isolation, respectively. MALDI-TOF analysis were performed on tryptone soya broth suspension isolates or directly from nutrient agar colonies, from the positive, inconclusive or negative biochemically tested samples for Salmonella and E. coli. Mass profiles were acquired on an Autoflex III SmartBeam MALDI-TOF mass spectrometer and the raw spectra were processed using the MALDI Biotyper software (Bruker Daltonics). According to the preliminary identification based on colony morphology and the biochemical reactions, seven isolates were positive for Salmonella spp. Through MALDI Biotyper these seven isolates were also classified as belonging to the genus Salmonella and further identified as S. enterica. Four isolates showing unusual phenotypic characteristics and inconclusive results in biochemical tests for Salmonella were identified as belonging to Citrobacter and Proteus genera after MALDI analysis. Regarding Escherichia coli, 37 were positive for species biochemical testing which MALDI Biotyper confirmed. MALDI-TOF methodology allowed rapid Salmonella spp. and E. coli identity confirmation and may be used to detect these microrganisms within bacterial isolates from beef carcasses.(AU)

Degenerative axonopathy associated with copper deficiency in pigs / Axonopatia degenerativa associada com deficiência de cobre em suínos

The epidemiological, clinic and morphological (pathological and ultrastructural) aspects of four outbreaks of copper deficiency affecting 21- to 90-day-old pigs in the Northeast region of Brazil are reported. Clinical signs began with paraparesis and ataxia and progressed to flaccid or spastic paralysis of the pelvic and thoracic limbs, followed by sternal and/or lateral recumbence. In addition, some animals showed dog-sitting position and intention tremors. The clinical manifestation period was 5-20 days. Significant gross lesions were not observed; however, microscopically, symmetrical degeneration of the white matter with ballooned myelin sheaths containing occasional macrophages was observed, mainly in the spinal cord. Two pigs presented with necrosis ad loss of Purkinje cells and ectopic Purkinje cells in the granular layer and cerebellar white matter. A ultrastructural analysis showed different degrees of damage of myelinated axons in the spinal segments, including an absence of the axoplasm structures with only axonal residues remaining. The myelin sheaths were degenerated and often collapsed into the space previously occupied by the axon. These results suggest that myelin degeneration is secondary to the axonal lesion. Finally, the concentration of copper in the liver was determined using atomic absorption spectrophotometry and was found to be low (ranging from 2.2 to 10.8 ppm). In conclusion, in the Brazilian semiarid region, Cu deficiency occurs in 21 to 90-day-old pigs that ingested different types of waste in their food.(AU)

São relatados os achados epidemiológicos, clínicos e morfológicos (patológicos e ultraestruturais) de quatro surtos de deficiência de cobre em suínos afetados entre 21 e 90 dias de idade na região Nordeste do Brasil. Os sinais clínicos iniciaram com paraparesia e ataxia, que progrediu a paralisia flácida ou espástica dos membros pélvicos e torácicos, seguido de decúbito esternal e/ou lateral. Além disso, alguns animais apresentaram posição de cão sentado e tremores de intenção. O período de manifestação clínica variou de 5-20 dias. Não foram observadas lesões macroscópicas significativas; no entanto, microscopicamente, foi observada degeneração simétrica da substância branca com fragmentação das bainhas de mielina, contendo ocasionais macrófagos, principalmente na medula espinal. Dois suínos apresentaram necrose e perda de células de Purkinje e células de Purkinje ectópicos na camada granular da substância branca cerebelar. A análise ultraestrutural mostrou diferentes graus de lesões em axônios mielinizados em segmentos da medula espinhal, incluindo o desaparecimento de estruturas do axoplasma, restando apenas restos axonais. A bainha de mielina encontrava-se degenerada e muitas vezes, colapsada dentro do espaço previamente ocupado pelo axônio. Esses resultados sugerem que a degeneração da mielina é secundária à lesão axonal. Finalmente, a concentração do cobre no fígado foi determinada usando espectrometria de absorção atômica e revelou baixos valores (variando de 2,2-10,8ppm). Conclui-se que na região semiárida do Brasil ocorre deficiência de cobre em suínos de 21 a 90 dias de idade alimentados com diferentes tipos de resíduos.(AU)

Degenerative axonopathy associated with copper deficiency in pigs / Axonopatia degenerativa associada com deficiência de cobre em suínos

The epidemiological, clinic and morphological (pathological and ultrastructural) aspects of four outbreaks of copper deficiency affecting 21- to 90-day-old pigs in the Northeast region of Brazil are reported. Clinical signs began with paraparesis and ataxia and progressed to flaccid or spastic paralysis of the pelvic and thoracic limbs, followed by sternal and/or lateral recumbence. In addition, some animals showed dog-sitting position and intention tremors. The clinical manifestation period was 5-20 days. Significant gross lesions were not observed; however, microscopically, symmetrical degeneration of the white matter with ballooned myelin sheaths containing occasional macrophages was observed, mainly in the spinal cord. Two pigs presented with necrosis ad loss of Purkinje cells and ectopic Purkinje cells in the granular layer and cerebellar white matter. A ultrastructural analysis showed different degrees of damage of myelinated axons in the spinal segments, including an absence of the axoplasm structures with only axonal residues remaining. The myelin sheaths were degenerated and often collapsed into the space previously occupied by the axon. These results suggest that myelin degeneration is secondary to the axonal lesion. Finally, the concentration of copper in the liver was determined using atomic absorption spectrophotometry and was found to be low (ranging from 2.2 to 10.8 ppm). In conclusion, in the Brazilian semiarid region, Cu deficiency occurs in 21 to 90-day-old pigs that ingested different types of waste in their food.(AU)

São relatados os achados epidemiológicos, clínicos e morfológicos (patológicos e ultraestruturais) de quatro surtos de deficiência de cobre em suínos afetados entre 21 e 90 dias de idade na região Nordeste do Brasil. Os sinais clínicos iniciaram com paraparesia e ataxia, que progrediu a paralisia flácida ou espástica dos membros pélvicos e torácicos, seguido de decúbito esternal e/ou lateral. Além disso, alguns animais apresentaram posição de cão sentado e tremores de intenção. O período de manifestação clínica variou de 5-20 dias. Não foram observadas lesões macroscópicas significativas; no entanto, microscopicamente, foi observada degeneração simétrica da substância branca com fragmentação das bainhas de mielina, contendo ocasionais macrófagos, principalmente na medula espinal. Dois suínos apresentaram necrose e perda de células de Purkinje e células de Purkinje ectópicos na camada granular da substância branca cerebelar. A análise ultraestrutural mostrou diferentes graus de lesões em axônios mielinizados em segmentos da medula espinhal, incluindo o desaparecimento de estruturas do axoplasma, restando apenas restos axonais. A bainha de mielina encontrava-se degenerada e muitas vezes, colapsada dentro do espaço previamente ocupado pelo axônio. Esses resultados sugerem que a degeneração da mielina é secundária à lesão axonal. Finalmente, a concentração do cobre no fígado foi determinada usando espectrometria de absorção atômica e revelou baixos valores (variando de 2,2-10,8ppm). Conclui-se que na região semiárida do Brasil ocorre deficiência de cobre em suínos de 21 a 90 dias de idade alimentados com diferentes tipos de resíduos.(AU)

Detecting animal by-product intake using stable isotope ratio mass spectrometry (IRMS).

Sheep are used in many countries as food and for manufacturing bioproducts. However, when these animals consume animal by-products (ABP), which is widely prohibited, there is a risk of transmitting scrapie - a fatal prion disease in human beings. Therefore, it is essential to develop sensitive methods to detect previous ABP intake to select safe animals for producing biopharmaceuticals. We used stable isotope ratio mass spectrometry (IRMS) for 13C and 15N to trace animal proteins in the serum of three groups of sheep: 1 - received only vegetable protein (VP) for 89 days; 2 - received animal and vegetable protein (AVP); and 3 - received animal and vegetable protein with animal protein subsequently removed (AVPR). Groups 2 and 3 received diets with 30% bovine meat and bone meal (MBM) added to a vegetable diet (from days 16-89 in the AVP group and until day 49 in the AVPR group, when MBM was removed). The AVPR group showed 15N equilibrium 5 days after MBM removal (54th day). Conversely, 15N equilibrium in the AVP group occurred 22 days later (76th day). The half-life differed between these groups by 3.55 days. In the AVPR group, 15N elimination required 53 days, which was similar to this isotope's incorporation time. Turnover was determined based on natural 15N signatures. IRMS followed by turnover calculations was used to evaluate the time period for the incorporation and elimination of animal protein in sheep serum. The δ13C and δ15N values were used to track animal protein in the diet. This method is biologically and economically relevant for the veterinary field because it can track protein over time or make a point assessment of animal feed with high sensitivity and resolution, providing a low-cost analysis coupled with fast detection. Isotopic profiles could be measured throughout the experimental period, demonstrating the potential to use the method for traceability and certification assessments.

Detection of Perkinsus marinus in the oyster Crassostrea rhizophorae in southern Bahia by proteomic analysis / Detecção de Perkinsus marinus na ostra Crassostrea rhizophorae do sul da Bahia por análise proteômica

This study reports the presence of the pathogen Perkinsus marinus, notifiable to the World Organization for Animal Health (Office International des Èpizooties = OIE) in the oyster Crassostrea rhizophorae in southern Bahia via proteomic analysis. We analyzed Crassostrea brasiliana from a long-line cultivation system and C. rhizophorae from an adjacent mangrove in Porto do Campo, Camamu Bay, Bahia, Brazil. The collections (n = 100) were performed in October 2012. In the laboratory, the oysters were measured and opened to remove the meat, which was steeped in dry ice. For extraction of proteins, adaptation of a protocol used for mussels was used, after which separation in the first dimension was taken by isoelectric focusing (IEF). The peptides were transferred to a Mass Spectrometer. The obtained spectra were analyzed with the ProteinLynx Global Server 4.2 software tool and also by MASCOT (Matrix Science) and compared to the databases of the SWISSPROT and NCBI, respectively. The identification was evidenced by beta-tubulin, Perkinsus marinus ATCC 50983 and protein homology code in the database NCBI = gi | 294889481. This is the first record of P. marinus in Bahia and the fourth in Brazil.(AU)

Este estudo relata a presença do patógeno Perkinsus marinus, de notificação obrigatória à Organização Internacional de Epizootias (OIE) na ostra Crassostrea rhizophorae no sul da Bahia, via análise proteômica. Foram analisadas as ostras Crassostrea brasiliana de um cultivo em espinhel e C. rhizophorae de um manguezal adjacente, na localidade de Porto do Campo, Baía de Camamu, Bahia. As coletas (n = 100) foram efetuadas em outubro de 2012. Em laboratório, as ostras foram medidas e abertas para a retirada da carne, que foi macerada em gelo seco. Para a extração das proteínas, foi adotada a adaptação de um protocolo utilizado para mexilhões, após o que foi realizada a separação na primeira dimensão, por focalização isoelétrica (IEF). Os peptídeos foram transferidos para um Espectrômetro de Massas. Os espectros obtidos foram analisados no software ProteinLynx Global Server 4.2 e também pela ferramenta MASCOT (Matrix Science) e comparados com os bancos de dados do SWISSPROT e do NCBI, respectivamente. A identificação foi evidenciada por meio da beta-tubulina, homologia Perkinsus marinus ATCC 50983 e código da proteína no banco de dados NCBI = gi|294889481. Este é o primeiro registro de P. marinus na Bahia e o quarto no Brasil.(AU)

Detection of Perkinsus marinus in the oyster Crassostrea rhizophorae in southern Bahia by proteomic analysis / Detecção de Perkinsus marinus na ostra Crassostrea rhizophorae do sul da Bahia por análise proteômica

This study reports the presence of the pathogen Perkinsus marinus, notifiable to the World Organization for Animal Health (Office International des Èpizooties = OIE) in the oyster Crassostrea rhizophorae in southern Bahia via proteomic analysis. We analyzed Crassostrea brasiliana from a long-line cultivation system and C. rhizophorae from an adjacent mangrove in Porto do Campo, Camamu Bay, Bahia, Brazil. The collections (n = 100) were performed in October 2012. In the laboratory, the oysters were measured and opened to remove the meat, which was steeped in dry ice. For extraction of proteins, adaptation of a protocol used for mussels was used, after which separation in the first dimension was taken by isoelectric focusing (IEF). The peptides were transferred to a Mass Spectrometer. The obtained spectra were analyzed with the ProteinLynx Global Server 4.2 software tool and also by MASCOT (Matrix Science) and compared to the databases of the SWISSPROT and NCBI, respectively. The identification was evidenced by beta-tubulin, Perkinsus marinus ATCC 50983 and protein homology code in the database NCBI = gi / 294889481. This is the first record of P. marinus in Bahia and the fourth in Brazil.

Este estudo relata a presença do patógeno Perkinsus marinus, de notificação obrigatória à Organização Internacional de Epizootias (OIE) na ostra Crassostrea rhizophorae no sul da Bahia, via análise proteômica. Foram analisadas as ostras Crassostrea brasiliana de um cultivo em espinhel e C. rhizophorae de um manguezal adjacente, na localidade de Porto do Campo, Baía de Camamu, Bahia. As coletas (n = 100) foram efetuadas em outubro de 2012. Em laboratório, as ostras foram medidas e abertas para a retirada da carne, que foi macerada em gelo seco. Para a extração das proteínas, foi adotada a adaptação de um protocolo utilizado para mexilhões, após o que foi realizada a separação na primeira dimensão, por focalização isoelétrica (IEF). Os peptídeos foram transferidos para um Espectrômetro de Massas. Os espectros obtidos foram analisados no software ProteinLynx Global Server 4.2 e também pela ferramenta MASCOT (Matrix Science) e comparados com os bancos de dados do SWISSPROT e do NCBI, respectivamente. A identificação foi evidenciada por meio da beta-tubulina, homologia Perkinsus marinus ATCC 50983 e código da proteína no banco de dados NCBI = gi/294889481. Este é o primeiro registro de P. marinusna Bahia e o quarto no Brasil.

Detection of Perkinsus marinus in the oyster Crassostrea rhizophorae in southern Bahia by proteomic analysis / Detecção de Perkinsus marinus na ostra Crassostrea rhizophorae do sul da Bahia por análise proteômica

This study reports the presence of the pathogen Perkinsus marinus, notifiable to the World Organization for Animal Health (Office International des Èpizooties = OIE) in the oyster Crassostrea rhizophorae in southern Bahia via proteomic analysis. We analyzed Crassostrea brasiliana from a long-line cultivation system and C. rhizophorae from an adjacent mangrove in Porto do Campo, Camamu Bay, Bahia, Brazil. The collections (n = 100) were performed in October 2012. In the laboratory, the oysters were measured and opened to remove the meat, which was steeped in dry ice. For extraction of proteins, adaptation of a protocol used for mussels was used, after which separation in the first dimension was taken by isoelectric focusing (IEF). The peptides were transferred to a Mass Spectrometer. The obtained spectra were analyzed with the ProteinLynx Global Server 4.2 software tool and also by MASCOT (Matrix Science) and compared to the databases of the SWISSPROT and NCBI, respectively. The identification was evidenced by beta-tubulin, Perkinsus marinus ATCC 50983 and protein homology code in the database NCBI = gi / 294889481. This is the first record of P. marinus in Bahia and the fourth in Brazil.(AU)

Este estudo relata a presença do patógeno Perkinsus marinus, de notificação obrigatória à Organização Internacional de Epizootias (OIE) na ostra Crassostrea rhizophorae no sul da Bahia, via análise proteômica. Foram analisadas as ostras Crassostrea brasiliana de um cultivo em espinhel e C. rhizophorae de um manguezal adjacente, na localidade de Porto do Campo, Baía de Camamu, Bahia. As coletas (n = 100) foram efetuadas em outubro de 2012. Em laboratório, as ostras foram medidas e abertas para a retirada da carne, que foi macerada em gelo seco. Para a extração das proteínas, foi adotada a adaptação de um protocolo utilizado para mexilhões, após o que foi realizada a separação na primeira dimensão, por focalização isoelétrica (IEF). Os peptídeos foram transferidos para um Espectrômetro de Massas. Os espectros obtidos foram analisados no software ProteinLynx Global Server 4.2 e também pela ferramenta MASCOT (Matrix Science) e comparados com os bancos de dados do SWISSPROT e do NCBI, respectivamente. A identificação foi evidenciada por meio da beta-tubulina, homologia Perkinsus marinus ATCC 50983 e código da proteína no banco de dados NCBI = gi/294889481. Este é o primeiro registro de P. marinusna Bahia e o quarto no Brasil.(AU)

Resistencia antimicrobiana y epidemiología molecular de aislamientos de Staphylococcus pseudintermedius de muestras clínicas de caninos / Antimicrobial resistance and molecular epidemiology of Staphylococcus pseudintermedius strains isolated from dog clinical samples

Se estudiaron 28 aislamientos obtenidos de muestras clínicas de perros e identificados por espectrometría de masas (MALDI-TOF) como Staphylococcus pseudintermedius; el objetivo fue evaluar la sensibilidad a los antimicrobianos por el método de difusión y establecer la relación clonal entre aislamientos por electroforesis en campo pulsado (PFGE). La resistencia a meticilina se evaluó mediante PCR por amplificación del gen mecA y se observó en 3/28 aislamientos (10,7 %). Quince aislamientos (53,6 %) presentaron resistencia a alguno de los antibióticos ensayados y 11 de ellos (39,3 %) presentaron resistencia múltiple (resistencia a 3 o más familias de antibióticos). Once aislamientos (39,3 %) presentaron resistencia a eritromicina, debido a la presencia de metilasa ribosomal ermB, y no se detectó resistencia inducible a clindamicina. Por PFGE se pudieron diferenciar 27 tipos clonales, lo cual demuestra gran diversidad clonal. Se destaca el hallazgo de aislamientos de S. pseudintermedius multirresistentes como una eventual problemática a considerar en el diagnóstico veterinario de laboratorio, el tratamiento de las infecciones caninas y el ámbito de la salud pública.

Twenty-eight strains isolated from dog clinical samples identified as Staphylococcus pseudintermedius by mass spectrometry (MALDI-TOF) were studied to assess antimicrobial susceptibility by the diffusion method and clonal relationship by pulsed field gel electrophoresis (PFGE). Methicillin resistance (3/28 isolates; 10,7 %) was evaluated by mecA PCR. Fifteen strains (53.6 %) were resistant to at least one of the antibiotics tested, and eleven of them (39.3 %) showed multiple resistance (3 or more antimicrobial families). Eleven isolates (39.3 %) were resistant to erythromycin due to the presence of ribosomal methylase ermB, whereas clindamycin inducible resistance was not detected. Twenty-seven (27) clonal types were differentiated by PFGE, suggesting high clonal diversity. We emphasize that the finding of multiresistant S. psedintermedius strains is an emerging problem to be considered in veterinary diagnostic laboratory treatment of canine infections and in public health settings.

Eletroforese bidimensional e espectrometria de massa como ferramentas proteômicas aplicadas à definição de marcadores proteicos associados à eficiência reprodutiva de caprinos / Two-dimensional electrophoresis and mass spectrometry as proteomic tools applied to the definition of protein markers associated with reproductive efficiency goats

O crescimento e desenvolvimento do rebanho caprino no Nordeste são observados com o aumento na produção da pecuária do Brasil. Esse aumento é reflexo, a princípio, das maiores exigências do mercado consumidor por produtos de melhor qualidade obtidos a partir de rebanhos de alto padrão zootécnico. As pesquisas atuais ilustram a necessidade de dispor de biomarcadores que auxiliem a indicação do potencial reprodutivo dos animais, uma vez que isso não pode ser expresso apenas com o exame andrológico. A avaliação da expressão das proteínas, tomando-as como biomarcadores, é análise potencial uma vez que estas, dentre os constituintes do plasma seminal, são encontradas em maior quantidade na forma de complexos associados, desempenhando papel crucial em todos os processos relacionados à capacidade fecundante dos espermatozoides. Essa análise é realizada por métodos de separação e detecção simultânea de proteínas utilizando técnicas como a eletroforese bidimensional (2DE) ou cromatografias, acoplados a métodos cada vez mais eficientes e sensíveis de identificação e quantificação de níveis de expressão de proteínas por espectrometria de massas. O objetivo desta revisão é abordar sobre a técnica de eletroforese bidimensional associada à espectrometria de massa como ferramenta na análise da expressão de proteínas dentro do campo da proteômica.

The growth and development of goat herds in the northeast are being observed due to the increase in livestock production in Brazil. This increase reflects demands of the consumer market for high quality product, which are obtained from flocks of high standard zootechnics. Current research illustrates the need for biomarkers that indicate an animal ́s reproductive potential, since this cannot be expressed solely with andrologic evaluation. For this reason, the expression of the proteins as biomarkers is a potential analysis. The proteins from the seminal plasma constituents are found in larger amounts in the form of associated complexes, playing a crucial role in all processes related to the fertilizing capacity of sperm. This analysis is performed by separation methods and simultaneous detection of proteins using techniques such as two-dimensional electrophoresis (2DE) or chromatography. These techniques are coupled with methods to identify and quantify expression levels of proteins by mass spectrometry that are increasingly efficient and sensitive. The aim of this review was to discuss the technique of two-dimensional electrophoresis combined with mass spectrometry as a tool in the analysis of protein expression within the field of proteomics.

Eletroforese bidimensional e espectrometria de massa como ferramentas proteômicas aplicadas à definição de marcadores proteicos associados à eficiência reprodutiva de caprinos / Two-dimensional electrophoresis and mass spectrometry as proteomic tools applied to the definition of protein markers associated with reproductive efficiency goats

O crescimento e desenvolvimento do rebanho caprino no Nordeste são observados com o aumento na produção da pecuária do Brasil. Esse aumento é reflexo, a princípio, das maiores exigências do mercado consumidor por produtos de melhor qualidade obtidos a partir de rebanhos de alto padrão zootécnico. As pesquisas atuais ilustram a necessidade de dispor de biomarcadores que auxiliem a indicação do potencial reprodutivo dos animais, uma vez que isso não pode ser expresso apenas com o exame andrológico. A avaliação da expressão das proteínas, tomando-as como biomarcadores, é análise potencial uma vez que estas, dentre os constituintes do plasma seminal, são encontradas em maior quantidade na forma de complexos associados, desempenhando papel crucial em todos os processos relacionados à capacidade fecundante dos espermatozoides. Essa análise é realizada por métodos de separação e detecção simultânea de proteínas utilizando técnicas como a eletroforese bidimensional (2DE) ou cromatografias, acoplados a métodos cada vez mais eficientes e sensíveis de identificação e quantificação de níveis de expressão de proteínas por espectrometria de massas. O objetivo desta revisão é abordar sobre a técnica de eletroforese bidimensional associada à espectrometria de massa como ferramenta na análise da expressão de proteínas dentro do campo da proteômica.(AU)

The growth and development of goat herds in the northeast are being observed due to the increase in livestock production in Brazil. This increase reflects demands of the consumer market for high quality product, which are obtained from flocks of high standard zootechnics. Current research illustrates the need for biomarkers that indicate an animal ́s reproductive potential, since this cannot be expressed solely with andrologic evaluation. For this reason, the expression of the proteins as biomarkers is a potential analysis. The proteins from the seminal plasma constituents are found in larger amounts in the form of associated complexes, playing a crucial role in all processes related to the fertilizing capacity of sperm. This analysis is performed by separation methods and simultaneous detection of proteins using techniques such as two-dimensional electrophoresis (2DE) or chromatography. These techniques are coupled with methods to identify and quantify expression levels of proteins by mass spectrometry that are increasingly efficient and sensitive. The aim of this review was to discuss the technique of two-dimensional electrophoresis combined with mass spectrometry as a tool in the analysis of protein expression within the field of proteomics.(AU)

Produção e composição do leite de vacas com mastite causada por Corynebacterium spp / Milk yield and composition of cows with subclinical mastitis caused by Corynebacterium spp

A mastite é a inflamação da glândula mamária cuja causa mais comum são as infecções bacterianas. É considerada a doença de maiores prejuízos e prevalência em vacas leiteiras. A forma subclínica da mastite é o tipo mais predominante de infecção intramamária (IIM), sendo Corynebacterium spp. um dos agentes mais frequentemente isolados. Portanto, os objetivos gerais deste estudo foram: 1) avaliar o efeito de IIMs subclínicas causadas por Corynebacterium spp. sobre a composição e a produção de leite de quartos mamários; 2) determinar o efeito da IIM causada por Corynebacterium spp. sobre o teor de gordura, proteína bruta, lactose, caseína, extrato seco total, extrato seco desengordurado do leite e contagem de células somáticas (CCS); 3) avaliar a técnica de Espectrometria de massas por Ionização e Dessorção à Laser Assistida por Matriz Tempo de Voo (MALDI/TOF-MS) para a identificação das espécies de Corynebacterium isoladas de quartos mamários. Foram utilizados 21 rebanhos leiteiros (n=1.242 animais), nos quais foram selecionadas vacas em lactação previamente identificadas com IIM causada por Corynebacterium spp. (n=285). [...] Houve equivalência de resultados de 95% entre as duas técnicas de identificação utilizadas. Em conclusão, IIMs causadas por Corynebacterium spp. não apresentam efeito sobre a produção e composição de leite, porém apresentam aumento significativo da CCS. Adicionalmente, a técnica de MALDI/TOF-MS com o uso da extração em tubo por ácido fórmico/acetonitrila foi aplicada com 91,57% de sensibilidade de identificação das espécies de Corynebacterium nos casos de mastite bovina subclínica, quando comparado aos resultados do sequenciamento de genes 16S rRNA

Mastitis is an inflammation of the mammary glands which bacteria are the most common cause of infections. It is considered a disease with higher prevalence and losses in dairy cows. The subclinical form of mastitis is the most prevalent type of intramammary infection (IMI), and Corynebacterium spp., one of the agents most frequently isolated. Therefore, the main objectives of this study were: 1) to evaluate the effect of subclinical IMI caused by Corynebacterium spp. on milk composition and yield of mammary quarters; 2) to determine the effect of IMI caused by Corynebacterium spp. on the concentration of fat, protein, lactose, casein, total solids, non fat dry milk and somatic cell count (SCC); 3) to evaluate the technique of Matrix-Assisted Laser Desorption Ionization - Time-of-Flight - Mass Spectrometry (MALDI/TOF-MS) for species identification of Corynebacterium isolated from mammary glands. Twenty-one dairy herds were used (n = 1242 animals) on which lactating cows were selected previously identified with IMI caused by Corynebacterium spp. (n = 285). [...]There were equivalence results of 95% between the two identification techniques used. In conclusion, IMI caused by Corynebacterium spp. have no effect on milk yield and composition, but exhibit a significant increase in CCS. Additionally, the technique of MALDI/TOF-MS using the tube-extraction by formic acid/acetonitrile was applied with 91.57% sensitivity for identifying the species of Corynebacterium on cases of bovine mastitis subclinical when compared to the results of genes sequencing 16S rRNA

Determination of trace elements in bovine semen samples by inductively coupled plasma mass spectrometry and data mining techniques for identification of bovine class.

The reproductive performance of cattle may be influenced by several factors, but mineral imbalances are crucial in terms of direct effects on reproduction. Several studies have shown that elements such as calcium, copper, iron, magnesium, selenium, and zinc are essential for reproduction and can prevent oxidative stress. However, toxic elements such as lead, nickel, and arsenic can have adverse effects on reproduction. In this paper, we applied a simple and fast method of multi-element analysis to bovine semen samples from Zebu and European classes used in reproduction programs and artificial insemination. Samples were analyzed by inductively coupled plasma spectrometry (ICP-MS) using aqueous medium calibration and the samples were diluted in a proportion of 1:50 in a solution containing 0.01% (vol/vol) Triton X-100 and 0.5% (vol/vol) nitric acid. Rhodium, iridium, and yttrium were used as the internal standards for ICP-MS analysis. To develop a reliable method of tracing the class of bovine semen, we used data mining techniques that make it possible to classify unknown samples after checking the differentiation of known-class samples. Based on the determination of 15 elements in 41 samples of bovine semen, 3 machine-learning tools for classification were applied to determine cattle class. Our results demonstrate the potential of support vector machine (SVM), multilayer perceptron (MLP), and random forest (RF) chemometric tools to identify cattle class. Moreover, the selection tools made it possible to reduce the number of chemical elements needed from 15 to just 8.

Iodine-129 in animal thyroids from Argentina.

(129)I and (127)I concentrations in animal thyroids coming from several regions of Argentina were determined by accelerator mass spectrometry (AMS) and gas chromatography (GC), respectively. The measured (129)I/(127)I ratios, ranging from 3 × 10(-12) to 4 × 10(-10), are significantly lower than those typical for areas in the northern hemisphere (10(-10)-10(-7)). The (129)I concentrations show a clear dependence with latitude and season, which can be understood considering tropospheric circulation patterns, possible (129)I sources and regional precipitation rates.

Identification of immunodominant antigens of Mycobacterium bovis by expression library immunization.

This study combines two methodologies - vector expression of a genomic library and proteomics - to identify immunogenic proteins of Mycobacterium bovis. Immunization of BALB/c mice with a plasmid DNA pool from the library, containing approximately 8000 clones, induced a humoral response that facilitated the detection of 12 antigenic proteins by Western blotting. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry identified four proteins (Cpn60-1, HSP70, EF-Tu, and AdoHcyase). Such genomic immunization offers the possibility of in vivo screening of potential candidate M. bovis antigens.

Contribution and perspectives of the impact of proteomics in study of placentas and embryonic and fetal membranes / Contribuição e perspectivas do impacto da proteômica em estudo de placentas e membranas embrionárias e fetais

Proteomic approaches have revolutionized the analysis of biological samples by allowing the identification of non-characterized protein expression and its role on protein interactions and post-translational modifications during embryonic development or as a consequence of pathological states. This new frontier of knowledge is, nowadays, an essential contribution to the elucidation of biological problems, by providing information that cannot be obtained by other target driven methods. This review highlights the new perspectives of proteomics studies based on bidimensional eletrophoresis (2D-PAGE) associated to mass spectrometry (MS) analytical methods of placenta, embryonic membranes and embryo evaluation, with emphasis on comparisons between normal and diseased tissues consequence of reproductive biotechnology manipulation.

Abordagens proteômicas revolucionaram a análise de amostras biológicas por permitir a identificação de padrões ainda não caracterizados de expressão de proteínas e/ou suas interações e modificações pós-traducionais em diferentes tecidos, como a placenta ou em embriões e fetos em diferentes estágios de desenvolvimento. Esta nova fronteira de conhecimento se constitui atualmente numa ferramenta fundamental para a elucidação de problemas biológicos, por fornecer informações que não podem ser obtidas por métodos mais focalizados, ou com alvos específicos. Essa revisão salienta as novas perspectivas geradas pela contribuição da proteômica baseada na eletroforese bidimensional (2D-PAGE) associada à espectrometria de massas (MS) para o estudo da placenta, das membranas embrionárias e do embrião, com ênfase nas comparações entre condições normais e aqueles provenientes de estados patológicos ou conseqüentes ao uso de biotecnologias reprodutivas.

Withdrawal time of four pharmaceutical formulations of enrofloxacin in poultry according to different maximum residues limits.

To ensure delivery of safe animal products to consumers, the withdrawal time (WDT) of drugs must be respected. Property differences among pharmaceutical formulations, for the same drugs, can lead to differences in the WDTs estimation. The WDTs of four commercial formulations of enrofloxacin (ENRO) in broiler chickens, considering MRLs established by different countries, were studied. Two hundred-thirty-four broiler chicks were allotted among four groups; the formulations were orally administered daily with 10 mg/kg bw. After treatment, six chickens of each group and two controls were slaughtered daily until day 9 post-treatment. Samples of muscle and liver were collected, and analyzed using HPLC-MS-MS. The WDTs among formulations of ENRO showed differences of 24 and 48 h. Based on the European Community and Chile MRLs of 100 microg/kg (muscle) and 200 microg/kg (liver), the WDTs did not exceed 5 days. When Japan MRL was considered (10 microg/kg(,)), the WDTs increased up to 8 days. These results indicate that for WDTs determination, the differences among pharmaceutical formulations of a drug must be considered as well as the MRLs.