MS/MS analysis of four scorpion venoms from Colombia: a descriptive approach

Background: Scorpions are widely known for the neurotoxic effects of their venoms, which contain peptides affecting ionic channels. Although Colombia is recognized for its scorpion diversity, only a few studies are available describing the venom content. Methods: In this descriptive study, we analyzed the MS/MS sequence, electrophoretic and chromatographic profile linked to a bioinformatics analysis of the scorpions Chactas reticulatus (Chactidae), Opisthacanthus elatus (Hormuridae), Centruroides edwardsii (Buthidae) and Tityus asthenes (Buthidae) from Colombia. Results: Each scorpion showed a specific electrophoretic and chromatographic profile. The electrophoretic profiles indicate the presence of high molecular mass compounds in all venoms, with a predominance of low molecular mass compounds in the Buthidae species. Chromatographic profiles showed a similar pattern as the electrophoretic profiles. From the MS/MS analysis of the chromatographic collected fractions, we obtained internal peptide sequences corresponding to proteins reported in scorpions from the respective family of the analyzed samples. Some of these proteins correspond to neurotoxins affecting ionic channels, antimicrobial peptides and metalloproteinase-like fragments. In the venom of Tityus asthenes, the MSn analysis allowed the detection of two toxins affecting sodium channels covering 50% and 84% of the sequence respectively, showing 100% sequence similarity. Two sequences from Tityus asthenes showed sequence similarity with a phospholipase from Opisthacanthus cayaporum indicating the presence of this type of toxin in this species for the first time. One sequence matching a hypothetical secreted protein from Hottentotta judaicus was found in three of the studied venoms. We found that this protein is common in the Buthidae family whereas it has been reported in other families - such as Scorpionidae - and may be part of the evolutionary puzzle of venoms in these arachnids. Conclusion: Buthidae venoms from Colombia can be considered an important source of peptides similar to toxins affecting ionic channels. An interesting predicted antimicrobial peptide was detected in three of the analyzed venoms.(AU)

MS/MS analysis of four scorpion venoms from Colombia: a descriptive approach

Background: Scorpions are widely known for the neurotoxic effects of their venoms, which contain peptides affecting ionic channels. Although Colombia is recognized for its scorpion diversity, only a few studies are available describing the venom content. Methods: In this descriptive study, we analyzed the MS/MS sequence, electrophoretic and chromatographic profile linked to a bioinformatics analysis of the scorpions Chactas reticulatus (Chactidae), Opisthacanthus elatus (Hormuridae), Centruroides edwardsii (Buthidae) and Tityus asthenes (Buthidae) from Colombia. Results: Each scorpion showed a specific electrophoretic and chromatographic profile. The electrophoretic profiles indicate the presence of high molecular mass compounds in all venoms, with a predominance of low molecular mass compounds in the Buthidae species. Chromatographic profiles showed a similar pattern as the electrophoretic profiles. From the MS/MS analysis of the chromatographic collected fractions, we obtained internal peptide sequences corresponding to proteins reported in scorpions from the respective family of the analyzed samples. Some of these proteins correspond to neurotoxins affecting ionic channels, antimicrobial peptides and metalloproteinase-like fragments. In the venom of Tityus asthenes, the MSn analysis allowed the detection of two toxins affecting sodium channels covering 50% and 84% of the sequence respectively, showing 100% sequence similarity. Two sequences from Tityus asthenes showed sequence similarity with a phospholipase from Opisthacanthus cayaporum indicating the presence of this type of toxin in this species for the first time. One sequence matching a hypothetical secreted protein from Hottentotta judaicus was found in three of the studied venoms. We found that this protein is common in the Buthidae family whereas it has been reported in other families - such as Scorpionidae - and may be part of the evolutionary puzzle of venoms in these arachnids. Conclusion: Buthidae venoms from Colombia can be considered an important source of peptides similar to toxins affecting ionic channels. An interesting predicted antimicrobial peptide was detected in three of the analyzed venoms.(AU)

Electrophoretic analysis (sds-page) of canine urinary proteins according to the stage of chronic kidney disease / [Análise eletroforética (SDS-PAGE) de proteínas urinárias de cães de acordo com o estágio da doença renal crônica]

Glomerular proteinuria is characterized by the loss of high-molecular-weight proteins (HMWPs), while tubulointerstitial proteinuria is characterized by the loss of low-molecular-weight proteins (LMWPs). The objective was to assess the molecular weight of urinary proteins (MWUP) in dogs with naturally acquired CKD and determine the proportion of HMWPs and LMWPs according to CKD stage. Twenty-eight dogs with CKD were recruited and divided into 4 groups based on serum creatinine (Cr) levels (group1: Cr<1,4, n=8; group2: 1,45,0, n=5). The control group consisted of 5 healthy dogs. The MWUP was determined by SDS-PAGE. The urinary protein-to-creatinine ratio (UP/C) was used to quantitatively assess proteinuria. The electrophoresis pattern revealed a proportionally greater loss of HMWPthan of LMWP in all groups with CKD and an increased loss of LMWP in group 4 (P<0.05). These results suggest a predominance of glomerular injuries throughout all stages of CKD in these dogs and an increase in tubulointerstitial injury towards the end-stage of the disease. The results of the present study support the recommendation of SDS-PAGE as an effective technique for the qualitative assessment of proteinuria, as well as a method for assessing the severity and location of renal injury.(AU)

A proteinúria glomerular é caracterizada pela perda de proteínas de alto peso molecular (PAPM), enquanto a proteinúria tubulointersticial se caracteriza pela perda de proteínas de baixo peso molecular (PBPM). O objetivo do trabalho foi determinar o peso molecular das proteínas urinárias (PMPU) de cães com DRC naturalmente adquirida e a proporção de PAPM e PBPM de acordo com o estágio da DRC. Foram utilizados 28 cães com DRC, divididos em quatro grupos, de acordo com o nível sérico de creatinina (cr) (grupo 1: cr<1,4, n=8; grupo 2: 1,45,0, n=5). O grupo controle era composto por cinco cães saudáveis. O PMPU foi determinado por SDS-PAGE. A relação proteína:creatinina urinária (RPCU) foi utilizada como um método quantitativo de proteinúria. A eletroforese revelou uma perda proporcionalmente maior de PAPM, quando comparada às PBPM, em todos os grupos de DRC, bem como uma perda crescente de PBPM no grupo 4 (P<0,05). Esses resultados sugerem uma predominância de lesão glomerular em todos os estágios de DRC nesses cães e uma progressão crescente na lesão túbulo-intersticial no estágio terminal da doença. Os resultados deste estudo reafirmam a recomendação de que a eletroforese de proteínas urinárias é uma técnica qualitativa efetiva de avaliação da proteinúria, bem como um método que permite avaliar a extensão e a localização da lesão renal.(AU)

Electrophoretic analysis (sds-page) of canine urinary proteins according to the stage of chronic kidney disease / [Análise eletroforética (SDS-PAGE) de proteínas urinárias de cães de acordo com o estágio da doença renal crônica]

Glomerular proteinuria is characterized by the loss of high-molecular-weight proteins (HMWPs), while tubulointerstitial proteinuria is characterized by the loss of low-molecular-weight proteins (LMWPs). The objective was to assess the molecular weight of urinary proteins (MWUP) in dogs with naturally acquired CKD and determine the proportion of HMWPs and LMWPs according to CKD stage. Twenty-eight dogs with CKD were recruited and divided into 4 groups based on serum creatinine (Cr) levels (group1: Cr<1,4, n=8; group2: 1,45,0, n=5). The control group consisted of 5 healthy dogs. The MWUP was determined by SDS-PAGE. The urinary protein-to-creatinine ratio (UP/C) was used to quantitatively assess proteinuria. The electrophoresis pattern revealed a proportionally greater loss of HMWPthan of LMWP in all groups with CKD and an increased loss of LMWP in group 4 (P<0.05). These results suggest a predominance of glomerular injuries throughout all stages of CKD in these dogs and an increase in tubulointerstitial injury towards the end-stage of the disease. The results of the present study support the recommendation of SDS-PAGE as an effective technique for the qualitative assessment of proteinuria, as well as a method for assessing the severity and location of renal injury.(AU)

A proteinúria glomerular é caracterizada pela perda de proteínas de alto peso molecular (PAPM), enquanto a proteinúria tubulointersticial se caracteriza pela perda de proteínas de baixo peso molecular (PBPM). O objetivo do trabalho foi determinar o peso molecular das proteínas urinárias (PMPU) de cães com DRC naturalmente adquirida e a proporção de PAPM e PBPM de acordo com o estágio da DRC. Foram utilizados 28 cães com DRC, divididos em quatro grupos, de acordo com o nível sérico de creatinina (cr) (grupo 1: cr<1,4, n=8; grupo 2: 1,45,0, n=5). O grupo controle era composto por cinco cães saudáveis. O PMPU foi determinado por SDS-PAGE. A relação proteína:creatinina urinária (RPCU) foi utilizada como um método quantitativo de proteinúria. A eletroforese revelou uma perda proporcionalmente maior de PAPM, quando comparada às PBPM, em todos os grupos de DRC, bem como uma perda crescente de PBPM no grupo 4 (P<0,05). Esses resultados sugerem uma predominância de lesão glomerular em todos os estágios de DRC nesses cães e uma progressão crescente na lesão túbulo-intersticial no estágio terminal da doença. Os resultados deste estudo reafirmam a recomendação de que a eletroforese de proteínas urinárias é uma técnica qualitativa efetiva de avaliação da proteinúria, bem como um método que permite avaliar a extensão e a localização da lesão renal.(AU)

Concentrations of acute-phase proteins and immunoglobulins in serum and synovial fluid in clinically healthy heifers and steers / Concentração de proteínas de fase aguda e de imunoglobulinas no soro e no líquido sinovial de bovinos clinicamente saudáveis

The aim of the study was to determine the concentration pattern of intra-articular acute phase proteins (APPs) and immunoglobulins in healthy crossbred cattle. Synovial fluid (SF) samples were collected from the radiocarpal joint of 25 heifers and 25 steers. Concentrations of APPs were measured by SDS-PAGE. The results were submitted to analysis of variance using the SAS statistical program, and means were compared by the Student-Newman-Keuls test (P<0.05). Thirty-seven proteins with molecular weights ranging from 7 to 37kDa were identified in SF of all animals. Eight were nominally identified with immunoglobulin A (IgA) and G (IgG), ceruloplasmin (Cp), transferrin (Tf), albumin (Ab), α1-antitripsin (AAT), α1-acid glycoprotein (AGP), and haptoglobin (Hp). The α1-antitripsin was only identified in the Sf of the heifers. The SF values of Cp, Hp, AGP and IgA were significantly higher in heifers than in steers. In sera, 34 proteins with molecular weights between 7 and 244kDa were identified in heifers and steers. Similar proteins were nominally identified in the sera, however the α1-antitrypsin was identified only in SF. The serum values Tf, AGP and IgG were significantly higher in heifers compared with steers. In conclusion, the physiological acute-phase proteins concentrations in synovial fluid of healthy ruminants can be useful in the interpretation of samples from animals with joint diseases. The SF electrophoretic profile of healthy ruminants differs depending on gender. Similar proteins were nominally identified in the sera, but only the SF of α1-antitrypsin.(AU)

O objetivo do estudo foi determinar o padrão de concentração de proteínas de fase aguda e de imunoglobulinas intra-articulares (APPs) em bovinos mestiços saudáveis. As amostras de fluido sinovial (SF) foram coletadas da articulação radiocárpica de 25 novilhas e 25 novilhos. As concentrações de APPs foram mensuradas por SDS-PAGE. Os resultados foram submetidos à análise de variância usando o programa estatístico SAS, e os meios foram comparados pelo teste Student-Newman-Keuls (P<0,05). Trinta e sete proteínas com pesos moleculares variando de 7 a 37kDa foram identificadas no SF de todos os animais. Oito foram nominalmente identificadas como imunoglobulina A (IgA) e G (IgG), ceruloplasmina (Cp), transferrina (Tf), albumina (Ab), a1-antitripsina (AAT), glicoproteína a1-ácido (AGP) e haptoglobina (Hp). A α1-antitripsina foi identificada apenas no SF das novilhas. Os valores de Cp, Hp, AGP e IgA no SF foram significativamente maiores em novilhas do que em novilhos. No soro, 34 proteínas com pesos moleculares entre 7 e 244kDa foram identificadas nas novilhas e novilhos. Proteínas similares foram identificadas nos soros, mas apenas o SF das novilhas apresentou a α1-antitripsina. Os valores séricos de Tf, AGP e IgG foram significativamente maiores em novilhas em relação aos novilhos. Conclui-se que a mensuração das concentrações das proteínas da fase aguda no líquido sinovial de animais saudáveis pode ser útil na avaliação de amostras oriundas de bovinos com afecções articulares. O perfil eletroforético do SF de ruminantes saudáveis difere em função do gênero e as diferenças devem ser levadas em consideração na interpretação dos achados.(AU)

Concentrations of acute-phase proteins and immunoglobulins in serum and synovial fluid in clinically healthy heifers and steers / Concentração de proteínas de fase aguda e de imunoglobulinas no soro e no líquido sinovial de bovinos clinicamente saudáveis

The aim of the study was to determine the concentration pattern of intra-articular acute phase proteins (APPs) and immunoglobulins in healthy crossbred cattle. Synovial fluid (SF) samples were collected from the radiocarpal joint of 25 heifers and 25 steers. Concentrations of APPs were measured by SDS-PAGE. The results were submitted to analysis of variance using the SAS statistical program, and means were compared by the Student-Newman-Keuls test (P<0.05). Thirty-seven proteins with molecular weights ranging from 7 to 37kDa were identified in SF of all animals. Eight were nominally identified with immunoglobulin A (IgA) and G (IgG), ceruloplasmin (Cp), transferrin (Tf), albumin (Ab), α1-antitripsin (AAT), α1-acid glycoprotein (AGP), and haptoglobin (Hp). The α1-antitripsin was only identified in the Sf of the heifers. The SF values of Cp, Hp, AGP and IgA were significantly higher in heifers than in steers. In sera, 34 proteins with molecular weights between 7 and 244kDa were identified in heifers and steers. Similar proteins were nominally identified in the sera, however the α1-antitrypsin was identified only in SF. The serum values Tf, AGP and IgG were significantly higher in heifers compared with steers. In conclusion, the physiological acute-phase proteins concentrations in synovial fluid of healthy ruminants can be useful in the interpretation of samples from animals with joint diseases. The SF electrophoretic profile of healthy ruminants differs depending on gender. Similar proteins were nominally identified in the sera, but only the SF of α1-antitrypsin.(AU)

O objetivo do estudo foi determinar o padrão de concentração de proteínas de fase aguda e de imunoglobulinas intra-articulares (APPs) em bovinos mestiços saudáveis. As amostras de fluido sinovial (SF) foram coletadas da articulação radiocárpica de 25 novilhas e 25 novilhos. As concentrações de APPs foram mensuradas por SDS-PAGE. Os resultados foram submetidos à análise de variância usando o programa estatístico SAS, e os meios foram comparados pelo teste Student-Newman-Keuls (P<0,05). Trinta e sete proteínas com pesos moleculares variando de 7 a 37kDa foram identificadas no SF de todos os animais. Oito foram nominalmente identificadas como imunoglobulina A (IgA) e G (IgG), ceruloplasmina (Cp), transferrina (Tf), albumina (Ab), a1-antitripsina (AAT), glicoproteína a1-ácido (AGP) e haptoglobina (Hp). A α1-antitripsina foi identificada apenas no SF das novilhas. Os valores de Cp, Hp, AGP e IgA no SF foram significativamente maiores em novilhas do que em novilhos. No soro, 34 proteínas com pesos moleculares entre 7 e 244kDa foram identificadas nas novilhas e novilhos. Proteínas similares foram identificadas nos soros, mas apenas o SF das novilhas apresentou a α1-antitripsina. Os valores séricos de Tf, AGP e IgG foram significativamente maiores em novilhas em relação aos novilhos. Conclui-se que a mensuração das concentrações das proteínas da fase aguda no líquido sinovial de animais saudáveis pode ser útil na avaliação de amostras oriundas de bovinos com afecções articulares. O perfil eletroforético do SF de ruminantes saudáveis difere em função do gênero e as diferenças devem ser levadas em consideração na interpretação dos achados.(AU)

Influência da mastite nas frações eletroforéticas do colostro de vacas da raça Holandesa / Influence of mastitis on electrophoretic fractions of colostrum from Holstein cows

O objetivo desse estudo foi o de avaliar as frações proteicas em secreções colostrais de vacas acometidas por mastite clínica imediatamente após o parto. Para tanto, foram utilizadas 30 vacas da raça Holandesa distribuídas em três grupos, a saber: Grupo I (GI)- 10 vacas pluríparas sadias, Grupo II (GII) 10 vacas pluríparas que pariram com mastite assintomática e Grupo III (GIII) 10 vacas pluríparas que pariram com mastite clínica. Foram avaliadas as concentrações de imunoglobulina a (IgA), lactoferrina (LF), albumina, imunoglobulina G (IgG), ß-lactoglobulina (ß-Lg) e α-lactoalbumina (α-La) por meio da eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE).Observou-se que a IgG, LF e a albumina variaram entre as glândulas com mastite assintomática e clínica quando comparadas às glândulas sadias, e que a presença de um único microrganismo é capaz de promover alterações no proteinograma, com ou sem manifestações clínicas na glândula mamária.(AU)

The aim of this study was to evaluate the protein fractions in colostral secretions of cows affected by mastitis immediately after calving. Therefore, 30 Holstein cows were divided into three groups: Group I (GI) composed of ten multiparous cows calving without mastitis; Group II (GII) composed of ten multiparous cows calving with subclinical mastitis, and Group III (GIII) composed of ten multiparous cows calving with mastitis. The concentration of immunoglobulin A (IgA), lactoferrin (LF), albumin, immunoglobulin G (IgG), ß-lactoglobulin (ß-Lg) and α-lactoalbumin (α-La) was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the IgG, LF and albumin vary among glands of subclinical and clinical mastitis and healthy and that the presence of a bacteria in the mammary gland was the key role for changing of the pattern of serum protein source.(AU)

Influência da mastite nas frações eletroforéticas do colostro de vacas da raça Holandesa / Influence of mastitis on electrophoretic fractions of colostrum from Holstein cows

O objetivo desse estudo foi o de avaliar as frações proteicas em secreções colostrais de vacas acometidas por mastite clínica imediatamente após o parto. Para tanto, foram utilizadas 30 vacas da raça Holandesa distribuídas em três grupos, a saber: Grupo I (GI)- 10 vacas pluríparas sadias, Grupo II (GII) 10 vacas pluríparas que pariram com mastite assintomática e Grupo III (GIII) 10 vacas pluríparas que pariram com mastite clínica. Foram avaliadas as concentrações de imunoglobulina a (IgA), lactoferrina (LF), albumina, imunoglobulina G (IgG), ß-lactoglobulina (ß-Lg) e α-lactoalbumina (α-La) por meio da eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE).Observou-se que a IgG, LF e a albumina variaram entre as glândulas com mastite assintomática e clínica quando comparadas às glândulas sadias, e que a presença de um único microrganismo é capaz de promover alterações no proteinograma, com ou sem manifestações clínicas na glândula mamária.(AU)

The aim of this study was to evaluate the protein fractions in colostral secretions of cows affected by mastitis immediately after calving. Therefore, 30 Holstein cows were divided into three groups: Group I (GI) composed of ten multiparous cows calving without mastitis; Group II (GII) composed of ten multiparous cows calving with subclinical mastitis, and Group III (GIII) composed of ten multiparous cows calving with mastitis. The concentration of immunoglobulin A (IgA), lactoferrin (LF), albumin, immunoglobulin G (IgG), ß-lactoglobulin (ß-Lg) and α-lactoalbumin (α-La) was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the IgG, LF and albumin vary among glands of subclinical and clinical mastitis and healthy and that the presence of a bacteria in the mammary gland was the key role for changing of the pattern of serum protein source.(AU)

Cambios en el patrón electroforético de proteínas plasmáticas de tilapia (Oreochromis niloticus) (Linnaeus, 1759) macho y hembra, por radiacion gamma / Changes in the electrophoretic pattern of plasma proteins of male and female tilapia (Oreochromis niloticus) (Linnaeus, 1759), caused by gamma radiation / Alterações tegumentares e no eletroferograma de proteinas plasmáticas de tilápias (Oreochromis niloticus ) (Linnaeus, 1759) macho e fêmea, provocadas por radiação gama

Este estudio tuvo como objetivo evaluar la respuesta de las proteínas del plasma de la sangre de tilapia macho y hembra, a dosis de 20Gy, expuestos a la radiación gamma. Se observó un patrón diferencial entre los sexos. La reducción e incluso la eliminación de ciertas bandas fueram detectadas por electroforesis en gel de agarosa.(AU)

This study aimed to evaluate the response of plasma proteins from blood of male and female tilapia at a dose of 20Gy, exposed to gamma radiation. A differential pattern between the sexes was observed. A reduction and even elimination of certain bands were detected by electrophoresis in agarose gel.(AU)

O presente trabalho teve por objetivo avaliar a resposta de proteínas plasmáticas de sangue de tilápias macho e fêmea, à dose de 20Gy expostas à radiação gama. Observou-se um padrão diferencial entre os sexos, bem como diminuição e até mesmo supressão de determinadas bandas detectadas por eletroforese em gel de agarose.(AU)

Cambios en el patrón electroforético de proteínas plasmáticas de tilapia (Oreochromis niloticus) (Linnaeus, 1759) macho y hembra, por radiacion gamma / Changes in the electrophoretic pattern of plasma proteins of male and female tilapia (Oreochromis niloticus) (Linnaeus, 1759), caused by gamma radiation / Alterações tegumentares e no eletroferograma de proteinas plasmáticas de tilápias (Oreochromis niloticus ) (Linnaeus, 1759) macho e fêmea, provocadas por radiação gama

Este estudio tuvo como objetivo evaluar la respuesta de las proteínas del plasma de la sangre de tilapia macho y hembra, a dosis de 20Gy, expuestos a la radiación gamma. Se observó un patrón diferencial entre los sexos. La reducción e incluso la eliminación de ciertas bandas fueram detectadas por electroforesis en gel de agarosa.

This study aimed to evaluate the response of plasma proteins from blood of male and female tilapia at a dose of 20Gy, exposed to gamma radiation. A differential pattern between the sexes was observed. A reduction and even elimination of certain bands were detected by electrophoresis in agarose gel.

O presente trabalho teve por objetivo avaliar a resposta de proteínas plasmáticas de sangue de tilápias macho e fêmea, à dose de 20Gy expostas à radiação gama. Observou-se um padrão diferencial entre os sexos, bem como diminuição e até mesmo supressão de determinadas bandas detectadas por eletroforese em gel de agarose.

Physiological performance and electrophoretic pattern of isoenzymes in Phaseolus vulgaris Lam. seeds treated with essential oil of Moringa oleifera Lam / Desempenho fisiológico e padrão eletroforético de isoenzimas em sementes de Phaseolus vulgaris Lam. tratadas com óleo essencial de Moringa oleifera Lam

In order to investigate the effect of different doses of essential oil of Moringa oleifera (EOM) on physiological potential and the expression of isoenzymes in bean seeds, an experiment was conducted with two cultivars (Colibri and Campos Gerais) and seven treatments (bactericidal, fungicide and the doses 0, 0.1, 0.2, 0.4 and 0.8% EOM). Germination, first count of germination, germination speed index (GSI), emergence and emergence rate index (ERI) were evaluated in addition to the enzymes esterase (EST), alcohol dehydrogenase (ADH), superoxide dismutase (SOD) and isocitrate lyase (ICL) by electrophoresis. In an experiment conducted in the laboratory, the dose containing 0.4% EOM promoted germination and higher averages for first germination in seeds and GSI in the Colibri cultivar. However, the highest dose used (0.8%) was responsible for the worst physiological performance by the cultivar. For Campos Gerais, increasing doses of essential oil stimulated germination, first count of germination and GSI. In the field, higher doses of essential oil gave higher germination percentages for the Colibri cultivar, not influencing the seeds of Campos Gerais. The ERI was not altered by the treatments with OEM and changes in the molecular forms of the isoenzymes were observed, with low expression values of bands for EST, ICL and ADH-related vigor, the germination of both cultivars, as well as increased expression of SOD, which is associated with reductions in germination.(AU)

A fim de investigar o efeito de diferentes doses de óleo essencial de Moringa oleifera (OEM) no potencial fisiológico e expressão de isoenzimas em sementes de feijão, conduziu-se um experimento com duas cultivares (Campos Gerais e Colibri) e sete tratamentos (bactericida, fungicida e as doses 0, 0.1, 0.2, 0.4 e 0.8% de óleo essencial). Foram avaliados a germinação, primeira contagem de germinação, índice de velocidade de germinação (IVG), emergência e índice de velocidade de emergência (IVE), além das enzimas esterase (EST), álcool desidrogenase (ADH), superóxido dismutase (SOD) e isocitrato liase (ICL) por meio de eletroforese. Em experimento conduzido em laboratório, a dose contendo 0.4% de OEM promoveu incremento na germinação e maiores médias para primeira contagem de germinação e IVG nas sementes da cultivar Colibri. No entanto, a maior dose utilizada (0.8%) foi responsável pelo pior desempenho fisiológico para a cultivar. Para Campos Gerais, as doses crescentes de óleo essencial estimularam a germinação, a primeira contagem de germinação e IVG. À campo, as doses de óleo essencial conferiram maior porcentagem de emergência para a cultivar Colibri, não influenciando as sementes da cultivar Campos Gerais. O IVE não sofreu alteração e modificações nas formas moleculares das isoenzimas foram observadas, estando os baixos valores de expressão de bandas para EST, ICL e ADH, relacionadas ao maior vigor e germinação das sementes de ambas as cultivares, bem como, maior expressão de SOD, associada à redução na germinação.(AU)

BIODIVERSIDADE FENOTÍPICA E GENOTÍPICA DE Salmonella spp. ISOLADAS DE ALIMENTOS ENVOLVIDOS EM SURTOS DE DOENÇAS TRANSMITIDAS POR ALIMENTOS (DTA) EM MINAS GERAIS/BRASIL

Salmonella spp. é um dos principais patógenos causadores de Doenças Transmitidas por Alimentos (DTA) no mundo. Neste estudo, analisamos 70 amostras de Salmonella spp., isoladas de alimentos implicados em casos de salmonelose humana em Minas Gerais/Brasil, de 2003 a 2017, em relação à sorotipagem bacteriana, à susceptibilidade a 16 antimicrobianos (oito classes terapêuticas), à presença dos genes de virulência avrA, flgL, flgK, hilA, invA, iroB, lfpA, sefA, sipA, sipB, sipD, sivH, sopB, sopE, spvB e spvC e do gene PT4 e ao polimorfismo genético, por meio das sequências de elementos repetitivos REP-PCR (Repetitive Extragenic Palindromic). Treze sorotipos foram identificados neste estudo. As frequências de S. Enteritidis e S. Typhimurium foram estatisticamente maiores (p 0,05) que os demais sorotipos, ambas correspondendo a 74,2%. Produtos de origem animal ou com algum ingrediente de origem animal representaram 90% dos alimentos envolvidos nos surtos de salmonelose. S. Enteritidis apresentou maior associação (p 0,05) com os produtos de origem animal. Cerca de 94,3% das amostras de Salmonella spp. demonstraram ser resistentes a pelo menos um dos 16 antimicrobianos avaliados. Percentual estatisticamente significativo (p 0,05) de resistência antimicrobiana foi identificado para nitrofurantoína (71,4%), ácido nalidíxico (68,6%) e ciprofloxacina (40%), principalmente nas amostras de S. Enteritidis. O fenótipo de resistência a múltiplos antimicrobianos variou de 10 a 12,8%, com predominância no sorotipo S. Typhimuirum. A presença dos genes de virulência oscilou de 75,7 a 100% nas 70 amostras de Salmonella ssp.. Maior associação entre os genes de virulência e os sorotipos, após decomposição das variáveis pela Análise de Componentes Principais, foi observada em S. Enteritidis. O fagotipo PT4 foi observado em 90% das amostras de S. Enteritidis. Na análise de diversidade genética, S. Enteritidis (n = 40) subdividiu-se em três clusters que compartilharam mais de 80% de similaridade e 14 padrões eletroforéticos (ID = 0,80), um deles composto por 17 amostras (42,5%) indistinguíveis procedentes de diferentes surtos de salmonelose. O agrupamento formado por S. Typhimurium (n=12) apresentou dois clusters e seis perfis distintos (ID=0,74). O grupo Outros sorotipos, composto por vários sorotipos, caracterizou-se por três clusters e 11 perfis com impressão digital única (ID=0,89). Neste experimento, REP-PCR apresentou reprodutibilidade, tipabilidade e potencial discriminatório. Conclui-se que as amostras de Salmonella ssp., isoladas de alimentos envolvidos em surtos de DTA em Minas Gerais/Brasil, de 2003 a 2017, apresentam padrão sorológico similar àquele reportado 9 pelos principais centros de Saúde Pública mundial. Caracterizam-se por elevada prevalência de resistência às classes terapêuticas quinolona e nitrofurano, demonstram significativo potencial patogênico, em especial o sorotipo S. Enteritidis e, embora algumas amostras de Salmonella spp. possuam um genoma relativamente variável, é comum uma elevada similaridade genética entre as mesmas, em alguns casos, com impressões digitais idênticas. Por meio deste experimento foi possível também observar a circulação de perfis genéticos únicos, constantes e predominantes, em várias mesorregiões estaduais e em períodos distintos, corroborando, dessa forma, a hipótese de circulação clonal de alguns sorotipos no Estado de Minas Gerais/Brasil.

Salmonella spp. is one of the main pathogens that cause foodborne diseases (DTA) in the world. In this study, we analyzed 70 samples of Salmonella spp., isolated from foods implicated in cases of human salmonellosis in Minas Gerais State / Brazil, to 2003 from 2017, as for bacterial serotyping, antimicrobial susceptibility to 16 antimicrobials (eight therapeutic classes), presence of the virulence genes avrA, flgL, flgK, hilA, invA, iroB, lfpA, sefA, sipA, sipB, sipD, sivH, sopB, sopE, spvB e spvC and the PT4 gene and genetic polymorphism, using the sequences of repetitive elements REP-PCR (Repetitive Extragenic Palindromic). Thirteen serotypes were identified in this study. The frequencies of S. Enteritidis and S. Typhimurium were significantly higher (p 0.05) than the other serotypes, both corresponding to 74.2% of those. Products of animal origin or with any ingredient of animal origin accounted for 90% of those involved in salmonellosis outbreaks. S. Enteritidis showed a greater association (p 0.05) with products of animal origin. About 94.3% of the samples of Salmonella spp. demonstrated to be resistant to at least one of the 16 evaluated antimicrobials. Statistically significant percentage (p 0.05) of antimicrobial resistance was identified for nitrofurantoin (71.4%), nalidixic acid (68.6%) and ciprofloxacin (40%), mainly in the samples of S. Enteritidis. The phenotype of resistance to multiple antimicrobidans varied from 10 to 12.8%, with predominance in the S. Typhimuirum serotype. The presence of virulence genes ranged from 75.7% to 100% in the 70 samples of Salmonella ssp.. Greater association between virulence genes and serotypes, after decomposition of the variables by Principal Component Analysis, was observed in S. Enteritidis. The PT4 phagotype was observed in 90% of S. Enteritidis samples. In the analysis of genetic diversity, S. Enteritidis (n = 40) was subdivided into three clusters that shared more than 80% similarity and 14 electrophoretic patterns (ID = 0.80), the largest consisting of 17 samples (42.5%) indistinguishable originated from different outbreaks of salmonellosis. The cluster formed by S. Typhimurium (n = 12) had two clusters and six distinct profiles (ID = 0.74). The group Other serotypes, composed of several serotypes, was characterized by three clusters and 11 profiles with a unique fingerprint (ID = 0.89). In this experiment, REP-PCR showed reproducibility, typability and discriminatory potential. We concluded that the samples of Salmonella ssp., isolated from food in outbreaks of DTA in Minas Gerais/Brazil, from 2003 to 2017, feature a serological pattern similar to that reported by the main centers of Public Health worldwide, are characterized by a high prevalence of resistance to therapeutic classes 11 quinolone and nitrofuran, demonstrate significant pathogenic potential, especially the serotype S. Enteritidis and, although some samples of Samonella spp. have a relatively variable genome, a high genetic similarity between them is common, in some cases, with identical fingerprints. According to this experimente, was also possible to observe the circulation of unique, constant and predominant genetic profiles, in several State mesoregions and in different periods, thus corroborating the hypothesis of clonal circulation of some serotypes in the Minas Gerais/Brasil.

Proteomics to investigate bulls semen freezability / Proteômica para investigar a congelabilidade do semen bovino

The present study was undertaken the protein composition in 2D-electrophoretic pattern (2DE) of the seminal plasma (SP) can interfere in the semen bull freezability, and if we can use that for predicting semen bull freezability. Samples were obtained of 20 bulls (different breeds) with a minimum of 3 years history semen production in commercial semen collection Center. All animals ranged between 2 - 7 years of age. The semen freezability was calculated by # of thawed and approved ejaculates / # of ejaculates submitted to cryopreservation (after semen evaluation and approved to submitted to freeze). The bulls were divided in 3 groups: HIGH (=>80% ejaculates approved); MEDIUM (>60% and <79% ejaculates approved); LOW (=<59% ejaculates approved); the pattern and criteria were the same used in theroutine of the commercial semen Center. 68 gels were carried through by 2DE of SP samples indicated 225 detected spots with protein different amount (VION) comparing. Comparing bull´s semen freezability and VION of each spot found difference among 2 spots from High and Low, even considering just spots with % of detection frequency bigger than 75%. The taurine bulls demonstrated more homogeneous profile when comparing with zebu bulls, considering number and frequency of appearance of spots. The results showed that proteomics can be a useful tool to predict the semen freezability, but we´ll need to study better the interactions between sperm membrane, seminal plasma and extender to comprehend better which proteome phenotype interfere positive or negatively in the semen freezability.(AU)

O objetivo do presente estudo foi avaliar se a composição proteica de plasma seminal, verificada por eletroforese bidimensional (2DE), pode interferir na congelabilidade do sêmen de touros, e se existe a possibilidade de predizer tal característica em touros doadores de sêmen. Amostras de 20 touros (diferentes raças) com mínimo de três anos de histórico de produção em Central de Coleta de sêmen foram coletadas. Todos os touros tinham de 2 a 7 anos de idade. A congelabilidade do sêmen foi calculada por no de ejaculados aprovados pós-descongelação / no de ejaculados submetidos à congelação (depois do crivo do padrão de qualidade da empresa para aprovar o ejaculado para ser submetido a congelação). Os touros foram divididos em três grupos: ALTA (=>80% aproveitamento de ejaculados encaminhados à congelação); MÉDIA (>60 a 79% ejaculados aprovados) e BAIXA (=<59% ejaculados aprovados). Sessenta e oito corridas de géis 2DE foram avaliadas nas amostras de plasma seminal, que detectaram 225 spots com quantidade de proteína diferente entre as mesmas (VION). Pela estatística utilizada, comparando-se a congelabilidade do sêmen com quantidade de proteína detectada de cada spot (VION), contatouse que 2 spots apresentaram-se com quantidade significativa de proteína diferente entre os dois grupos de congelabilidade do sêmen, considerando spots que foram detectados em mais de 75% das amostras corridas. Os touros taurinos apresentaram perfil proteico mais homogêneo quando comparado com os zebuínos, considerando número e frequência de spots. Tais resultados demonstram um potencial para uso da proteômica como ferramenta preditiva da congelabilidade do sêmen de touros, porém com necessidade de maiores estudos, principalmente para melhor compreensão de interações proteicas entre membrana espermática, plasma seminal e diluidores de sêmen utilizados, já que podem interferir no fenótipo proteico avaliado e na congelabilidade do sêmen, seja de forma positiva ou negativa.(AU)

Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen / Desenvolvimento de ELISA indireto para detectar anticorpos específicos contra Corynebacterium pseudotuberculosis em ovinos utilizando o supernadante de cultura da cepa T1 como antígeno

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI) broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN-gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep. (AU)

Corynebacterium pseudotuberculosis é o agente etiológico da linfadenite caseosa (LC) uma doença crônica que afeta ovinos e caprinos caracterizada pela formação de granulomas em linfonodos. A LC causa perdas econômicas significativas em criações de pequenos ruminantes. Este trabalho teve como objetivo testar antígenos secretados da cepa T1 da bactéria em um sistema de ELISA indireto para detecção de anticorpos específicos contra C. pseudotuberculosis. O perfil eletroforético do antígeno foi analisado, bem como o padrão de reconhecimento por soros de animais infectados. Os resultados do ELISA foram comparados com ensaio de multiplex PCR e com teste de indução de produção específica de IFN-gama. O ELISA foi capaz de discriminar animais positivos de animais negativos, com sensibilidade de 89% e especificidade de 99%, usando o isolamento microbiológico como padrão ouro. Quando o ensaio foi comparado com o multiplex PCR e a produção específica de IFN-gama, somente seis resultados discrepantes foram encontrados em trinta e duas amostras. Pode-se concluir que o ensaio de ELISA desenvolvido pode ser utilizado com alto grau de confiança para o diagnóstico da LC em ovinos. (AU)

A hematologic and electrophoretic study in puppies vaccinated against canine distemper virus and canine parvovirus

Background: Canine distemper is a contagious multisystemic viral disease that affects canines and others carnivores. Canine parvovirus infection is one of the most important viral diseases in young dogs. Side effects of vaccine generally include fever, lethargy and local inflammation. Complementary exams are important to evaluate the strenght of immungenic stimulation. This study was aimed at evaluating hematological and electrophoretic alterations in puppies after inoculation of live attenuated vaccine against canine distemper virus and canine parvovirus. Materials, Methods & Results: Five non-breeding newborn dogs of the same litter were used. Animals received three subcutaneous injection of 1mL (at days 0, 21 and 42). Blood was collected at day 0 (day of vaccination) and for three times for each dose: at days 7, 14 and 21 (first dose); at days 28, 35 and 42 (second dose); and at days 49, 56 and 63 (third dose). Blood containing ethylenediaminetetraacetic acid as anticoagulant was used for hematological evaluation. The total serum protein were determined by the biuret method, using commercial reagent, according to fabricant instructions. Serum was used for protein fractionation by using cellulose acetate strip electrophoresis. A decrease in platelet count was observed at days 7 and 28 post-vaccination. Lymphocyte number increased 88.4%, as well as the level of the protein fractions alpha-1 globulin (68%) and alpha-2 globulin (41.4%) at day 7. Moreover, a 5-fold increase in the fibrinogem concentration and in the number of eosinophils was observed at day 14. Thereafter, the platelet count decreased by 27.3% and the number of monocytes increased 5-fold at day 28. Discussion: Mild to moderate thrombocytopenia is often observed in dogs 3-5 days post-vaccination with live attenuated vaccines, mainly those against CDV and CPV. Besides the platelet damage caused by the CDV per se, infected animals showed secondary immune-mediated thrombocytopenia and decreased platelet production due to direct viral megakaryocyte infection. The increase in alpha-1 globulin may be related to the augment in the synthesis of alpha-1 antitrypsin, the main protein of the alpha-1 globulin region, in response to the vaccine-induced acute inflammatory process. The alpha-2 globulin region includes haptoglobin, alpha-2 macroglobulin and ceruloplasmin, and the increase observed in this fraction suggested that both haptoglobin and ceruloplasmin levels were augmented, following acute inflammatory response pattern. Fibrinogen is a soluble plasma glycoprotein that is converted by thrombin into fibrin during blood clotting. Despite the increase in fibrinogen concentration be the best indicator of inflammation in large animals, the hyperfibrinogenemia observed suggests that the inflammatory process was adequate to stimulate synthesis of this acute phase protein (P < 0,05). Absence of lymphocytosis observed at days 49, 56 and 63 associated to the progressive increase of the gamma globulin fraction, although not statistically significant, suggested an augment of B lymphocytes. The eosinophilia was observed in highlighting the presence of inflammation. Moreover, an increase in monocyte count indicating the presence of subacute or chronic inflammation after the second dose of the vaccine.

Isoflavone and protein content in soybeans grains submitted to flooding at different stages of development

The stress imposed on plants by soil flooding constitutes a major barrier to growth and productivity. The identification of soybean varieties that produce higher levels of isoflavones, is necessary as soybeans have been used as human food to reduce risks of chronic diseases. Thus, this study was conducted with the objective of quantifying proteins and isoflavones in soybean cultivars subjected to flooding at various stages of development. The cultivars 'BRS267', 'BRS257' and 'BRS213' were subjected to 15 days of flooding, starting at the stages V6 and V8 and 11 days under stress starting at the stage R4. The proteins in the grain were extracted and quantified and analyzed by SDS-PAGE electrophoresis. Isoflavones were extracted, separated and quantified on HPLC. The electrophoretic analysis of the three cultivars under study revealed the same pattern of banding relative to the total protein regardless of the treatment. However, it was noted that flooding led to an increase in the total contents of isoflavones in the BRS 267 plants flooded in stage R4, remaining constant in other cultivars.

O estresse imposto às plantas pelo alagamento do solo constitui uma barreira para o crescimento e produtividade destas. A identificação de cultivares de soja que possam produzir maior teor de isoflavonas torna-se necessária à medida que a soja vem sendo utilizada na alimentação humana e para redução dos riscos de doenças crônicas. Dessa forma, o presente estudo foi realizado com o objetivo de quantificar isoflavonas e proteínas em cultivares de soja submetidas ao alagamento em diferentes estádios de desenvolvimento. As cultivares 'BRS 267', 'BRS 257' e 'BRS 213' foram submetidas a 15 dias de alagamento, nos estádios V6 e V8 e 11 dias sob estresse, no estádio R4. As proteínas no grão foram extraídas e quantificadas e analisadas as eletroforeses em SDS-PAGE. Os isoflavonóides foram extraídos, separados e quantificados por HPLC. A análise por eletroforese das três cultivares revelou o mesmo padrão de bandas relativas às proteínas totais, independente do tratamento utilizado. O alagamento promoveu aumento no teor total de isoflavonas nas plantas alagadas no estádio R4 da cultivar 'BRS 267', permanecendo constante nas demais cultivares.

Isoflavone and protein content in soybeans grains submitted to flooding at different stages of development

The stress imposed on plants by soil flooding constitutes a major barrier to growth and productivity. The identification of soybean varieties that produce higher levels of isoflavones, is necessary as soybeans have been used as human food to reduce risks of chronic diseases. Thus, this study was conducted with the objective of quantifying proteins and isoflavones in soybean cultivars subjected to flooding at various stages of development. The cultivars 'BRS267', 'BRS257' and 'BRS213' were subjected to 15 days of flooding, starting at the stages V6 and V8 and 11 days under stress starting at the stage R4. The proteins in the grain were extracted and quantified and analyzed by SDS-PAGE electrophoresis. Isoflavones were extracted, separated and quantified on HPLC. The electrophoretic analysis of the three cultivars under study revealed the same pattern of banding relative to the total protein regardless of the treatment. However, it was noted that flooding led to an increase in the total contents of isoflavones in the BRS 267 plants flooded in stage R4, remaining constant in other cultivars.

O estresse imposto às plantas pelo alagamento do solo constitui uma barreira para o crescimento e produtividade destas. A identificação de cultivares de soja que possam produzir maior teor de isoflavonas torna-se necessária à medida que a soja vem sendo utilizada na alimentação humana e para redução dos riscos de doenças crônicas. Dessa forma, o presente estudo foi realizado com o objetivo de quantificar isoflavonas e proteínas em cultivares de soja submetidas ao alagamento em diferentes estádios de desenvolvimento. As cultivares 'BRS 267', 'BRS 257' e 'BRS 213' foram submetidas a 15 dias de alagamento, nos estádios V6 e V8 e 11 dias sob estresse, no estádio R4. As proteínas no grão foram extraídas e quantificadas e analisadas as eletroforeses em SDS-PAGE. Os isoflavonóides foram extraídos, separados e quantificados por HPLC. A análise por eletroforese das três cultivares revelou o mesmo padrão de bandas relativas às proteínas totais, independente do tratamento utilizado. O alagamento promoveu aumento no teor total de isoflavonas nas plantas alagadas no estádio R4 da cultivar 'BRS 267', permanecendo constante nas demais cultivares.

DETECÇÃO E ISOLAMENTO DE ROTAVÍRUS CAPRINO DO GRUPO A

ABSTRACT Rotavirus is the main cause of diarrhea in young animals of several species. They are classified as a genus within the virus family Reoviridae. Rotaviruses have a segmented genome made up of a double-stranded RNA. It is divided into 11 segments and surrounded by a triple protein capsid. In this study, Rotavirus was detected by polyacrylamide gel electrophoresis (PAGE) in feces samples of 80-day-old goats during a diarrhea outbreak in a milk-producing herd. The samples electrophoretic pattern, called cap01/2007, was characterized as Group A rotavirus. A 20% fecal suspension in buffer TRIS 0.1M pH 7.3 was filtered and treated with crystalline trypsin in a 5 mg/mL concentration for viral isolation. Caprine sample Cap01/2007 was inoculated in MA-104 cells (Rhesuss kidneys) with a 48-hour growth. Two treatments (T1 and T2) were tested. In T1, maintenance medium without trypsin was added (Dulbecco-MEM) after adsorption (buffer at 37º C for 1h) the inoculum was maintained. In treatment T2, the previous procedure without the inoculum was performed. Cytopathic effect was observed in the first passage for both treatments (T1 and T2) with 48h inoculation, and viral isolation was then confirmed by PAGE. Migration design remained unchanged after 3 successive passages. This is the first report of isolation of caprine rotavirus in Brazil.

RESUMO Rotavírus é a principal causa de diarreia em animais jovens de várias espécies. Os rotavirus estão classificados na família Reoviridae e contêm um genoma formado por 11 segmentos de RNA de cadeia dupla, envolvidos em três camadas proteicas. Neste estudo, foi detectado rotavírus pela eletroforese em gel de poliacrilamida (PAGE) em amostras de fezes de caprinos, com 80 dias de idade, durante surto de diarreia em um rebanho leiteiro. O perfil eletroforético das amostras, denominadas cap01/2007 e cap02/2007, foi caracterizado no PAGE como sendo de rotavírus do grupo A. Para o isolamento viral, suspensão fecal a 20% em tampão tris 0,1M pH 7,3 foi filtrada e tratada com tripsina cristalina na concentração de 5mg/mL. A amostra caprina cap01/2007 foi inoculada em cultura de células MA104 (Rim de Macaco Rhesus). Foram testados dois tratamentos (T1 e T2): após adsorção (estufa 37º C por uma hora) adicionou-se meio de manutenção (Dulbecco-MEM) sem tripsina, mantendo o inóculo (T1). No tratamento 2 (T2), foi realizado o mesmo pro cedimento anterior, porém o inóculo foi dispensado. O efeito citopático foi observado na primeira passagem para ambos os tratamentos (T1 e T2) com 48 horas de inoculação e o isolamento viral foi confirmado no PAGE. O perfil de migração manteve-se inalterado após três passagens sucessivas. Este é o primeiro relato de isolamento de rotavírus em caprinos no Brasil.

HEMATOLOGIA, BIOQUÍMICA E ELETROFORESE DE PROTEÍNAS SÉRICAS DE TAMANDUÁS-BANDEIRAS (Myrmecophaga tridactyla, LINNAEUS, 1758) EM CATIVEIRO

O tamanduá-bandeira (Myrmecophaga tridactyla), mamífero silvestre pertencente à superordem dos Xenarthras, é originário das Américas Central e do Sul. Atualmente, está classificado como espécie vulnerável, devido principalmente ao desmatamento, queimadas e atropelamento. A ausência de valores de referência para exames laboratoriais dificulta a avaliação desses animais, especialmente devido ao aumento na casuística de atendimento dessa espécie em instituições veterinárias. Dada à importância de tais dados, objetivou-se com este estudo estabelecer parâmetros hematológicos, bioquímicos e eletroforéticos para tamanduás-bandeiras de cativeiro do cerrado, bem como, saber quais os efeitos de estocagem nas amostras hematológicas nessa espécie em duas concentrações distintas de anticoagulante. Para isso, foram utilizados 12 animais de cativeiro clinicamente saudáveis. O sangue foi colhido da jugular e armazenado em tubos apropriados para cada tipo de exame. Os hemogramas foram realizados logo após as colheitas e em outros dois diferentes momentos para verificação dos efeitos de estocagem (M0 análise imediata, M2 24 horas pós-colheita, M3 48 horas pós-colheita). As análises bioquímicas foram executadas uma semana após as colheitas. A eletroforese foi feita no suporte de gel de agarose. A média e o desvio padrão das variáveis hematológicas das amostras processadas logo após a colheita foram: hemácias (2,07 x106/µL ± 0,40); volume globular (38,08 % ± 5,93); hemoglobina (11,33 g/dL ± 2,15); VCM (186,52 fL ± 21,72); CHCM (29,68 g/dL ± 2,56); HCM (55,08 pcg ± 5,94); leucócitos totais (8.142/µL ± 2.441); neutrófilos (5.913/µL ± 2.168); linfócitos (1.460/µL ± 740); eosinófilos (522/µL ± 385); monócitos (247/µL ± 176); plaquetas (123.458/µL ± 31.362) e proteínas plasmáticas totais (6,23 g/dL ± 0,49). As médias e o desvio padrão correspondentes às análises bioquímicas foram: albumina (3,29 g/dL ± 0,33); ALT (15,49 UI/L ± 7,98); amilase (1037,92 UI/L ± 149,04); AST (21,12 UI/L ± 7,50); colesterol total (62,79 mg/dL ± 20,08); colesterol HDL (14,73 mg/dL ± 4,98); colesterol LDL (26,60 mg/dL ± 11,05); colesterol VLDL (2,14 mg/dL ± 1,06); CK (111,61 UI/L ± 70,16); creatinina (1,05 mg/dL ± 0,37); ferro (194,64 µg/dL ± 81,17); GGT (65,18 UI/L ± 54,57); glicose (103,71 mg/dL ± 29,63); globulinas (2,76 g/dL ± 0,36); lipase (28,80 UI/L ± 5,11); PST (6,05 g/dL ± 0,56); triglicerídeos (10,71 mg/dL ± 5,29); ureia (53,46 mg/dL ± 18,28). Os resultados da eletroforese permitiram o fracionamento de seis bandas proteicas, classificadas como albumina (2,60 g/dL ± 0,33), alfa-1 (0,33 g/dL ± 0,07), alfa-2 (0,50 g/dL ± 0,17), beta-1 (0,62 g/dL ± 0,17), beta-2 (1,50 g/dL ± 0,25) e gama (1,88 g/dL ± 0,30). Portanto, é possível concluir que existem variações fisiológicas dos parâmetros hematológicos e bioquímicos tamanduás-bandeiras de biomas diferentes. Nestes animais, o padrão eletroforético em gel de agarose são seis bandas proteicas e a porcentagem de albumina é a maior delas. Este fato sugere que fatores extrínsecos como o clima, a nutrição e o manejo possam interferir nessas variáveis. Por isso, propor valores de referências obtidos de espécies análogas é necessário. Os resultados apresentados pelo presente trabalho podem ser utilizados como parâmetros laboratoriais de higidez em tamanduás-bandeiras de cativeiro do cerrado.

The giant anteater (Myrmecophaga tridactyla), wild mammal belonging to the superorder of Xenarthras, who originally inhabited Central and South America. It is currently classified as a vulnerable species, primarily due to deforestation, fires and running over accidents. The absence of reference values for laboratory tests increases the difficulty to assess these animals, especially due to the raising number of attendances in veterinary care institutions. Given the importance of such data, the aim of this study was to establish hematological, biochemical and electrophoretic parameters for captive giant anteaters as well to know which are the effects of storage conditions in hematologic samples with two different concentrations of anticoagulants. Therefore, we used 12 clinically healthy captive animals. The samples were collected from the jugular vein into appropriate blood tubes. The blood analysis was carried out immediately after colletion and in others two different times to check the storage effects (M0 - immediate analysis, M2 - 24 hours after collection, M3 - 48 hours post-collection). Biochemical analysis was performed a week after the harvest. Electrophoresis was carried out in agarose gel support. The mean and standard deviation of haematological variables of samples processed soon after harvest were: RBC (2.07 x 106/uL ± 0.40); packed cell volume (38.08 % ± 5.93); hemoglobin (11.33 g/dL ± 2.15); VCM (186.52 fL ± 21.72); MCHC (29.68 g/dL ± 2.56); MCH (55.08 pcg ± 5.94); total leukocytes (8142/uL ± 2,441); neutrophils (5913/uL ± 2168); lymphocytes (1460/uL ± 740); eosinophils (522/uL ± 385); monocytes (247/uL ± 176); platelets (123.458/uL ± 31.362) and total plasma proteins (6.23 g/dL ± 0.49). The mean and standard deviation corresponding to biochemical analysis was: albumin (3.29 g/dL ± 0.33); ALT (15.49 IU/L ± 7.98); amylase (1037.92 IU/L ± 149.04); AST (21.12 IU/L ± 7.50); total cholesterol (62.79 mg/dL ± 20.08); HDL cholesterol (14.73 mg/dL ± 4.98); LDL cholesterol (26.60 mg/dL ± 11.05); VLDL cholesterol (2.14 mg/dL ± 1.06); CK (111.61 IU/L ± 70.16); creatinine (1.05 mg/dL ± 0.37); iron (194.64 µg/dL ± 81.17); GGT (65.18 IU/L ± 54.57); glucose (103.71 mg/dL ± 29.63); globulins (2.76 g/dl ± 0.36); lipase (28.80 IU/L ± 5.11); PST (6.05 g/dL ± 0.56); triglycerides (10.71 mg/dL ± 5.29); urea (53.46 mg/dL ± 18.28). The results of the electrophoresis led to six protein bands which are classified as albumin (2.60 g/dL ± 0.33), alpha-1 (0.33 g/dL ± 0.07), alpha-2 (0, 50 g/dL ± 0.17), beta 1 (0.62 g/dL ± 0.17), beta 2 (1.50 g/dL ± 0.25) and gamma (1.88 g/dL ± 0.30). Therefore, we conclude that there are physiological changes in hematological and biochemical parameters of giant anteaters from different biomes. In these animals the electrophoretic pattern of the technique in agarose gel are six protein bands and the percentage of albumin is greater than the other fractions. This suggests that extrinsic factors such as climate, nutrition and management can affect these variables. Therefore, the results presented in this work can be used as laboratory parameters of healthiness in cerrado captive giant anteaters.

Cell-free antigens from precocious Paracoccidioides brasiliensis culture induce a typical delayed-type hypersensitivity reaction

Cell-free antigens (CFAg) derived from Paracoccidioides brasiliensis have typically been used in immunodiffusion reactions for serodiagnosis or therapeutic follow-up of paracoccidioidomycosis patients. Thus, we investigated the usefulness of CFAg obtained from cultures at different ages, to evaluate cellular immunity by the footpad test, in experimental murine paracoccidioidomycosis. Male mice infected with P. brasiliensis 265 strain were challenged in the footpad with CFAg obtained from four- (4d CFAg) or 11-day-old cultures (11d CFAg). The increase in footpad swelling provoked by 4d CFAg and 11d CFAg was similar and showed significant difference in relation to control groups. However, the infiltrate pattern was strikingly different: 4d CFAg induced a predominant mononuclear infiltrate whereas 11d CFAg provoked a predominant polymophonuclear infiltrate. These different inflammatory patterns were associated with distinct electrophoretic characteristics. By comparison with 11d CFAg, 4d CFAg showed more numerous and intense bands, including a strong one of 43 kDa (gp43). These results suggest that CFAg derived from Pb 265 isolate can be used as a reagent to evaluate cellular immunity; however, the culture's age is critical because only young cultures are able to induce a typical mononuclear infiltrate. The efficacy of this new paracoccidioidin to assay the cellular immunity in infections caused by other P. brasiliensis isolates is under investigation.(AU)