Microbial diversity in artisanal cheese produced and commercialized in Vale do Taquari in southern Brazil / Diversidade microbiana encontrada em queijos artesanais produzidos e comercializados no Vale do Taquari no sul do Brasil

Researchers have been utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify bacteria and fungi directly from isolates obtained on culture plates; the resulting mass spectrum is then compared with spectra stored in the instrument software. Hence, a fast analytical response is obtained, and the more spectra are known to compare, the safer and more reliable the interpretation of the method will be. Thus, this study sought to identify the diversity of strains found in 10 samples of artisan cheese produced and commercialized in Vale do Taquari (Rio Grande do Sul State, Brazil) using MALDI-TOF MS. From the analyzed cheese, 22 strains were identified at the species level; one sample presented the pathogen Staphylococcus aureus, two showed the presence of lactic acid bacteria (Lactococcus lactis), and the vast majority (68.18%) of strains were composed of species of the Enterobacteriaceae family, with the prevalence of the genera Escherichia, Enterobacter, and Klebsiella. Escherichia coli was present in 50% of the samples analyzed. This demonstrates the need for greater control during all stages of artisanal cheese production and evaluation of the raw material, including safe practices during milking, so that the product meets the identity and quality parameters suitable for human consumption.

MALDI-TOF MS vendo sendo utilizado em laboratórios para identificar bactérias e fungos diretamente de isolados obtidos em placas de cultura. O espectro de massa resultante é então comparado com espectros armazenados no software do equipamento. Assim, obtém-se uma resposta analítica rápida, sendo que, quanto mais espectros forem conhecidos para comparar, mais seguro e confiável será a interpretação do método. Desta maneira, o presente trabalho teve por objetivo, identificar por MALDI-TOF MS, a diversidade de cepas encontrados em 10 amostras de queijos artesanais produzidos e comercializados no Vale do Taquari, Rio Grande do Sul, Brasil. Dos queijos analisados, 22 cepas foram identificadas a nível de espécie, sendo uma (1) amostra apresentou o patógeno Staphylococcus aureus, duas a presença da bactéria ácido láctica (Lactococcus lactis) e a grande maioria (68,18%) das cepas era composta por espécies da família Enterobacteriaceae, com prevalência dos gêneros Escherichia, Enterobacter e Klebsiella. E. coli estava presente em 50% das amostras analisadas. Isso demonstra a necessidade de um maior controle durante todas as etapas de produção do queijo artesanal, bem como a avaliação da matéria-prima, incluindo práticas seguras durante a ordenha para que o produto atenda aos parâmetros de identidade e qualidade, sendo apto ao consumo humano.

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.(AU)

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Abstract Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.

Overview of protein posttranslational modifications in Arthropoda venoms

Accidents with venomous animals are a public health issue worldwide. Among the species involved in these accidents are scorpions, spiders, bees, wasps, and other members of the phylum Arthropoda. The knowledge of the function of proteins present in these venoms is important to guide diagnosis, therapeutics, besides being a source of a large variety of biotechnological active molecules. Although our understanding about the characteristics and function of arthropod venoms has been evolving in the last decades, a major aspect crucial for the function of these proteins remains poorly studied, the posttranslational modifications (PTMs). Comprehension of such modifications can contribute to better understanding the basis of envenomation, leading to improvements in the specificities of potential therapeutic toxins. Therefore, in this review, we bring to light protein/toxin PTMs in arthropod venoms by accessing the information present in the UniProtKB/Swiss-Prot database, including experimental and putative inferences. Then, we concentrate our discussion on the current knowledge on protein phosphorylation and glycosylation, highlighting the potential functionality of these modifications in arthropod venom. We also briefly describe general approaches to study "PTM-functional-venomics", herein referred to the integration of PTM-venomics with a functional investigation of PTM impact on venom biology. Furthermore, we discuss the bottlenecks in toxinology studies covering PTM investigation. In conclusion, through the mining of PTMs in arthropod venoms, we observed a large gap in this field that limits our understanding on the biology of these venoms, affecting the diagnosis and therapeutics development. Hence, we encourage community efforts to draw attention to a better understanding of PTM in arthropod venom toxins.(AU)

Mobilization of seed storage proteins is crucial to high vigor in common bean seeds / Mobilização das proteínas de reserva é crucial para o alto vigor em sementes de feijão

Seed germination is a complex process controlled by many factors, in which physical and biochemical mechanisms are involved and the mobilization of reserves is crucial for this process to occur. Although, seed reserve mobilization is usually thought to be a post-germination process, seed reserve proteins mobilization occurs during germination. This study quantified seed proteins of bean genotypes during different hydration times, in order to understand the process of protein mobilization and whether there is relationship of this biochemical component with seed vigor. This study was conducted using seeds with different levels of vigor, genotypes with highest (13, 42, 55 and 81) and lowest (07, 23, 44, 50, IPR-88-Uirapurú and Iapar 81) physiological quality. High vigor genotypes showed greater efficiency in hydrolysis and mobilization of protein component, because they presented low globulins content in cotyledons at radicle protrusion in relation to low vigor genotypes (07, 23 and 50). The protein alpha-amylase inhibitor, observed in all genotypes, is involved with the longer time needed for radicle protrusion, according to the band intensity difference in genotypes 07, 44 and Iapar 81.

A germinação de sementes é um processo complexo controlado por muitos fatores, nos quais mecanismos físicos e bioquímicos estão envolvidos e a mobilização de reservas é decisiva para que esse processo ocorra. Embora a mobilização de reservas de sementes seja considerada um processo pós-germinativo, a mobilização das proteínas de reserva de sementes ocorre durante a germinação. Este estudo teve como objetivo quantificar as proteínas de sementes de genótipos de feijão durante os diferentes tempos de hidratação, a fim de compreender o processo de mobilização proteica e se há relação desse componente bioquímico com o vigor das sementes. Este estudo foi realizado utilizando sementes com diferentes níveis de vigor, genótipos com maior (13, 42, 55 e 81) e menor (07, 23, 44, 50, IPR-88-Uirapurú e Iapar 81) qualidade fisiológica. Os genótipos de alto vigor apresentaram maior eficiência na hidrólise e mobilização do componente proteico, pois apresentaram baixo teor de globulinas nos cotilédones na protrusão radicular em relação aos genótipos de baixo vigor (07, 23 e 50). A proteína inibidora da alfa-amilase, observada em todos os genótipos, está envolvida com o maior tempo necessário para a protrusão da radícula, de acordo com a diferença de intensidade da banda nos genótipos 07, 44 e Iapar 81.

Acylated anthocyanins from organic purple-fleshed sweet potato (Ipomoea batatas (L. ) Lam) produced in Brazil

Acylated anthocyanins from a purple-fleshed sweet potato (PFSP), obtained by organic cultivation in Brazil, were characterized after separation by a high performance liquid chromatography-diode array detector (HPLC-PDA). These anthocyanins were manually collected at the detector output, concentrated and injected into a high resolution mass spectrometer (ESI-QTOF-MS²). Twenty-two acylated anthocyanins were detected. Among them, sixteen had been reported in the literature and six, derived from peonidin were reported for the first time in sweet potato roots in this study. These compounds showed molecular ions with accurate mass/charge ratios (m/z) of 909.2081, 961.3010, 961.2571, 963.3345, 1123.2932 and 1179.3862. Although anthocyanins in PFSP have already been extensively studied, the variety studied in this work is probably genetically different from all varieties and cultivars already researched, which would explain why these anthocyanins have not been observed in the previously studied varieties.

Acylated anthocyanins from organic purple-fleshed sweet potato (Ipomoea batatas (L. ) Lam) produced in Brazil

Acylated anthocyanins from a purple-fleshed sweet potato (PFSP), obtained by organic cultivation in Brazil, were characterized after separation by a high performance liquid chromatography-diode array detector (HPLC-PDA). These anthocyanins were manually collected at the detector output, concentrated and injected into a high resolution mass spectrometer (ESI-QTOF-MS²). Twenty-two acylated anthocyanins were detected. Among them, sixteen had been reported in the literature and six, derived from peonidin were reported for the first time in sweet potato roots in this study. These compounds showed molecular ions with accurate mass/charge ratios (m/z) of 909.2081, 961.3010, 961.2571, 963.3345, 1123.2932 and 1179.3862. Although anthocyanins in PFSP have already been extensively studied, the variety studied in this work is probably genetically different from all varieties and cultivars already researched, which would explain why these anthocyanins have not been observed in the previously studied varieties.(AU)

Proteomic analysis of Red Sea Conus taeniatus venom reveals potential biological applications

Diverse and unique bioactive neurotoxins known as conopeptides or conotoxins are produced by venomous marine cone snails. Currently, these small and stable molecules are of great importance as research tools and platforms for discovering new drugs and therapeutics. Therefore, the characterization of Conus venom is of great significance, especially for poorly studied species. Methods: In this study, we used bioanalytical techniques to determine the venom profile and emphasize the functional composition of conopeptides in Conus taeniatus, a neglected worm-hunting cone snail. Results: The proteomic analysis revealed that 84.0% of the venom proteins were between 500 and 4,000 Da, and 16.0% were > 4,000 Da. In C. taeniatus venom, 234 peptide fragments were identified and classified as conotoxin precursors or non-conotoxin proteins. In this process, 153 conotoxin precursors were identified and matched to 23 conotoxin precursors and hormone superfamilies. Notably, the four conotoxin superfamilies T (22.87%), O1 (17.65%), M (13.1%) and O2 (9.8%) were the most abundant peptides in C. taeniatus venom, accounting for 63.40% of the total conotoxin diversity. On the other hand, 48 non-conotoxin proteins were identified in the venom of C. taeniatus. Moreover, several possibly biologically active peptide matches were identified, and putative applications of the peptides were assigned. Conclusion: Our study showed that the composition of the C. taeniatus-derived proteome is comparable to that of other Conus species and contains an effective mix of toxins, ionic channel inhibitors and antimicrobials. Additionally, it provides a guidepost for identifying novel conopeptides from the venom of C. taeniatus and discovering conopeptides of potential pharmaceutical importance.(AU)

Proteomic analysis of Red Sea Conus taeniatus venom reveals potential biological applications

Diverse and unique bioactive neurotoxins known as conopeptides or conotoxins are produced by venomous marine cone snails. Currently, these small and stable molecules are of great importance as research tools and platforms for discovering new drugs and therapeutics. Therefore, the characterization of Conus venom is of great significance, especially for poorly studied species. Methods: In this study, we used bioanalytical techniques to determine the venom profile and emphasize the functional composition of conopeptides in Conus taeniatus, a neglected worm-hunting cone snail. Results: The proteomic analysis revealed that 84.0% of the venom proteins were between 500 and 4,000 Da, and 16.0% were > 4,000 Da. In C. taeniatus venom, 234 peptide fragments were identified and classified as conotoxin precursors or non-conotoxin proteins. In this process, 153 conotoxin precursors were identified and matched to 23 conotoxin precursors and hormone superfamilies. Notably, the four conotoxin superfamilies T (22.87%), O1 (17.65%), M (13.1%) and O2 (9.8%) were the most abundant peptides in C. taeniatus venom, accounting for 63.40% of the total conotoxin diversity. On the other hand, 48 non-conotoxin proteins were identified in the venom of C. taeniatus. Moreover, several possibly biologically active peptide matches were identified, and putative applications of the peptides were assigned. Conclusion: Our study showed that the composition of the C. taeniatus-derived proteome is comparable to that of other Conus species and contains an effective mix of toxins, ionic channel inhibitors and antimicrobials. Additionally, it provides a guidepost for identifying novel conopeptides from the venom of C. taeniatus and discovering conopeptides of potential pharmaceutical importance.(AU)

Proteomic analysis of Red Sea Conus taeniatus venom reveals potential biological applications

Abstract Background: Diverse and unique bioactive neurotoxins known as conopeptides or conotoxins are produced by venomous marine cone snails. Currently, these small and stable molecules are of great importance as research tools and platforms for discovering new drugs and therapeutics. Therefore, the characterization of Conus venom is of great significance, especially for poorly studied species. Methods: In this study, we used bioanalytical techniques to determine the venom profile and emphasize the functional composition of conopeptides in Conus taeniatus, a neglected worm-hunting cone snail. Results: The proteomic analysis revealed that 84.0% of the venom proteins were between 500 and 4,000 Da, and 16.0% were > 4,000 Da. In C. taeniatus venom, 234 peptide fragments were identified and classified as conotoxin precursors or non-conotoxin proteins. In this process, 153 conotoxin precursors were identified and matched to 23 conotoxin precursors and hormone superfamilies. Notably, the four conotoxin superfamilies T (22.87%), O1 (17.65%), M (13.1%) and O2 (9.8%) were the most abundant peptides in C. taeniatus venom, accounting for 63.40% of the total conotoxin diversity. On the other hand, 48 non-conotoxin proteins were identified in the venom of C. taeniatus. Moreover, several possibly biologically active peptide matches were identified, and putative applications of the peptides were assigned. Conclusion: Our study showed that the composition of the C. taeniatus-derived proteome is comparable to that of other Conus species and contains an effective mix of toxins, ionic channel inhibitors and antimicrobials. Additionally, it provides a guidepost for identifying novel conopeptides from the venom of C. taeniatus and discovering conopeptides of potential pharmaceutical importance.

Characterization of porcine oocytes stained with Lissamine Green B and their developmental potential in vitro

Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus-oocyte complex, polar body and meiotic spindle. This study is focused on the differences between the morphological assessment of oocyte quality, the assessment based on Lissamine Green B (LB) staining and the analysis of oocytes using a proteomic approach. We evaluated the effectiveness of electrochemical and chemical parthenogenetic activation under our laboratory conditions and evaluated the applicability of Lissamine Green B staining of cumulus-oocyte complexes (COCs) as a non-invasive method for predicting the maturational and developmental competence of porcine oocytes cultured in vitro. We determined that chemical parthenogenetic activation using ionomycin and 6-dimethylaminopurine was slightly more effective than electrochemical activation. After oocyte selection according to LB staining, we found significant differences (P 0.05) between the LB- group and LB+ group and the control group in their maturation, cleavage rate and rate of blastocysts. Proteomic analyses identified a selection of proteins that were differentially expressed in each group of analysed oocytes. Oocytes of the LB- group exhibited an increased variability of proteins involved in transcription regulation, proteosynthesis and the protein folding crucial for oocyte maturation and further embryonic development. These results found a better competence of LB- oocytes in maturation, cleavage and ability to reach the blastocyst stage.

Uterine infusion of conceptus fragments changes the protein profile from cyclic mares

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare's uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares.

Uterine infusion of conceptus fragments changes the protein profile from cyclic mares

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare's uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares.(AU)

Characterization of porcine oocytes stained with Lissamine Green B and their developmental potential in vitro

Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus-oocyte complex, polar body and meiotic spindle. This study is focused on the differences between the morphological assessment of oocyte quality, the assessment based on Lissamine Green B (LB) staining and the analysis of oocytes using a proteomic approach. We evaluated the effectiveness of electrochemical and chemical parthenogenetic activation under our laboratory conditions and evaluated the applicability of Lissamine Green B staining of cumulus-oocyte complexes (COCs) as a non-invasive method for predicting the maturational and developmental competence of porcine oocytes cultured in vitro. We determined that chemical parthenogenetic activation using ionomycin and 6-dimethylaminopurine was slightly more effective than electrochemical activation. After oocyte selection according to LB staining, we found significant differences (P 0.05) between the LB- group and LB+ group and the control group in their maturation, cleavage rate and rate of blastocysts. Proteomic analyses identified a selection of proteins that were differentially expressed in each group of analysed oocytes. Oocytes of the LB- group exhibited an increased variability of proteins involved in transcription regulation, proteosynthesis and the protein folding crucial for oocyte maturation and further embryonic development. These results found a better competence of LB- oocytes in maturation, cleavage and ability to reach the blastocyst stage.(AU)

Streamlined downstream process for efficient and sustainable (Fab)2 antivenom preparation

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)

Streamlined downstream process for efficient and sustainable (Fab)2 antivenom preparation

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(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.(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)

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.

Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk

The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n = 120) were skimmed by centrifugation (10,000 × g/10 min) and pre-incubated at 37 ºC for 12 h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.(AU)

Identification of pathogenic and nonpathogenic Leptospira species of Brazilian isolates by Matrix Assisted Laser Desorption/Ionization and Time Flight mass spectrometry

Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.(AU)