In vitro culture of parasitic stages of Haemonchus contortus / Cultivo in vitro de estádios parasitários de Haemonchus contortus

Haemonchus contortus is a constraint to sheep production. Seeking to reduce the use of hosts and produce parasitic stages in large-scale, a 42-day in vitro culture protocol of H. contortus third-stage larvae was optimized using Dulbecco's modified Eagle's medium (DMEM). In cell-free culture, larvae were maintained at 39.6°C, in acidic media (pH 6.1) for 3 or 6 days with Δ4-dafachronic acid followed by DMEM pH 7.4 supplemented or not with Fildes' reagent. In DMEM pH 7.4 at 37°C, supplementation with Caco-2 cells was compared to Fildes. On Day 14, fourth-stage larvae (L4) development rates in acidic media supplemented (86.8-88.4%) or not (74.4-77.8%) with Fildes and in Caco-2 cell co-culture (92.6%) were similar, and superior to DMEM pH 7.4 with Fildes (0.0%). On Day 21, Caco-2 cell co-culture resulted in higher larvae differentiation (25.0%) and lower degeneration (13.9%) compared to acidic media (1.5-8.1% and 48.6-69.9%, respectively). This is the first report of prolonged in vitro culture of H. contortus larvae using commercial media in co-culture with Caco-2 cells. Although no progression to the adult stage, Caco-2 cell co-culture resulted in morphological differentiation of H. contortus L4 and larval viability for up to 28 days.(AU)

Haemonchus contortus provoca grandes prejuízos à ovinocultura. Visando reduzir a utilização de ovinos hospedeiros e produzir estágios parasitários em larga escala, um protocolo para o cultivo in vitro por 42 dias de larvas de terceiro estádio de H. contortus foi realizado em meio Eagle, modificado por Dulbecco (DMEM). No cultivo sem células, as larvas foram mantidas a 39,6°C e incubadas em DMEM ácido (pH 6,1) por 3 ou 6 dias com 4Δ-ácido dafacrônico seguido por DMEM pH 7,4 suplementado ou não com reagente de Fildes. Em DMEM pH 7,4 a 37°C, a suplementação com células Caco-2 foi comparada à suplementação com Fildes. No Dia 14, as taxas de desenvolvimento até o quarto estádio larvar (L4) foram similares em meio ácido sem células suplementado (86,8-88,4%) ou não (74,4-77,8%) com Fildes e em co-cultura com células Caco-2 (92,6%), e superiores ao desenvolvimento em DMEM pH 7,4 com Fildes (0,0%). No Dia 21, o co-cultivo com células Caco-2 resultou em maior diferenciação (25,0%) e menor degeneração (13,9%) das larvas em comparação ao meio ácido (1,5-8,1% e 48,6-69,9%, respectivamente). Este é o primeiro relato de cultivo in vitro prolongado de H. contortus em meio comercial em co-cultivo com células Caco-2. Apesar da ausência de progressão até o estágio adulto, o co-cultivo com células Caco-2 resultou em diferenciação de L4 e manutenção da viabilidade das larvas de H. contortus por até 28 dias in vitro.(AU)

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37ºC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a [...].

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37ºC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.(AU)

As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a [...].(AU)

Effect of bacterial co-culture and organic amendments on the bioremediation of hydrocarbons in a soil contaminated with spent engine oil

Bioenhancement of hydrocarbonoclastic microorganisms with suitable nutrients has a huge impact in achieving positive bioremediation of polluted environments. This study was conducted to assess the bio-enhancing effect of some organic amendments on Streptococcus pyogenes andEnterococcus faecalis co-culture with a view toremediating spent engine oil (SEO) contaminated soil. Top soil (1.5 kg) was autoclaved and thereafter contaminated with SEO at three levels. The contaminated soil was inoculated with bacterial co-culture (150 mL) and subsequently bioenhanced with compost,processed cocoa pod husk (CPH) and cow dung. The factorial experiment was laid out in completely randomized design. Concentrations of total petroleum hydrocarbon (TPH) and selected polycyclic aromatic hydrocarbons (PAH) were estimated on the first day, 5th week and 10th week of incubation. Results obtained show that bacterial co-culture bioenhanced with compost produced the most significant TPH reductions (1318 and 261 mg kg-1)on 10% SEO contaminated soil at the 5th and 10th week respectively (p<0.05). Again, bacterial co-culture bioenhanced with compost produced the most significant PAH reductions (65.9 and 55.8 mg kg-1) on 10% SEO contaminated soil at the 5th and 10th week respectively (p<0.05). The significant bioremediation capabilities exhibited by the bacterial co-culture bioenhanced with organic amendments in this study has made these bioremediation agents potential candidates in remediating soils impacted with petroleum hydrocarbons.(AU)

Characterization of the antagonism of the mycorrhizal fungus Waitea circinata against Magnaporthe oryzae, Cochliobolus miyabeanus, Monographella albescens and Sarocladium oryzaerice pathogens / Caracterização do antagonismo do fungo micorrízico Waitea circinata contra os patógenos do arroz Magnaporthe oryzae, Cochliobolus miyabeanus, Monographella albescens e Sarocladium oryzae

Rice production is affected by important pathogens such as Magnaporthe oryzae, Cochliobolus miyabeanus, Monographella albescens and Sarocladium oryzae, and orchid mycorrhizal fungi can contribute to the biocontrol of these diseases. The aim of this study was to characterize the antagonism mechanisms of W. circinata against rice pathogens. The indoleacetic acid (IAA) and lytic enzymes of W. circinata were quantified. The effectiveness of the bioagent and its metabolites was assessed in vitro. Antagonism in vivo against rice blast was also investigated. Waitea circinata produced IAA (2.3 µg ml¹), cellulase and polyphenol oxidase in a qualitative test. Glucanase, chitinase and protease were also produced in culture with cell walls and in co-culture with pathogens to explain antibiosis. W. circinata acts on direct antagonism by antibiosis and volatile metabolites. Mycelial suspension reduced M. oryzae conidial germination, appressorium formation, and rice blast by 61, 82 and 84%, respectively. W. circinata, a unique bioagent, controls four rice pathogens from mechanisms of parasitism and antibiosis. The results could contribute to new formulations containing this fungus, enzymes as well as its volatile metabolites.

A produção de arroz é afetada por patógenos importantes como Magnaporthe oryzae, Cochliobolus miyabeanus, Monographella albescens e Sarocladium oryzae, e fungos micorrízicos de orquídeas podem contribuir para o biocontrole dessas doenças. O objetivo deste estudo foi caracterizar os mecanismos de antagonismo de W. circinata contra patógenos do arroz. O ácido indolacético (AIA) e as enzimas líticas de W. circinata foram quantificadas. A eficácia do bioagente e seus metabólitos foi avaliada in vitro. O antagonismo in vivo contra a brusone do arroz também foi investigado. Waitea circinata produziu AIA (2,3 µg ml¹), celulase e polifenol oxidase em teste qualitativo. Glucanase, quitinase e protease também foram produzidas em cultura com paredes celulares e em co-cultura com patógenos para explicar a antibiose. W. circinata atua no antagonismo direto por antibiose e metabólitos voláteis. A suspensão micelial reduziu a germinação de conídios de M. oryzae, a formação de apressórios e a brusone do arroz em 61, 82 e 84%, respectivamente. W. circinata, um bioagente único, controla quatro patógenos do arroz a partir de mecanismos de parasitismo e antibiose. Os resultados podem contribuir para novas formulações contendo este fungo, enzimas, bem como seus metabólitos voláteis.

Atypical mandibular osteomyelitis in an ewe caused by coinfection of Pseudomonas aeruginosa and Lactococcus raffinolactis

Background: Osteomyelitis is defined as a bone inflammation involving the cortical and medullary regions, usually caused by the local invasion of opportunistic microorganisms. The inflammatory reaction of bone may extend to the periosteum and soft tissues, compromising adjacent structures far from the initially infected foci. Different classifications of transmission routes, gravity levels, and tissues involved in animal and human osteomyelitis are available. In humans, the infection can reach bone tissue by exogenous or hematogenous pathways. This paper reports an atypical case of mandibular pyogranulomatous osteomyelitis in an ewe caused by concomitant Pseudomonas aeruginosa and Lactococcus raffinolactis infection. Case: The animal presented a 1-month history of progressive mandibular enlargement refractory to conventional therapy. In a physical examination, an increased volume located in the ventrolateral region of the right ramus of the mandible was observed. Fine-needle aspiration of the lesion enabled isolation in bacteriological culture of Pseudomonas aeruginosa and Lactococcus raffinolactis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS). Besides support care procedures and antimicrobial treatment approaches for the sheep based on in vitro tests, the animal died due to the severity of the clinical signs and the progressive worsening of the general health status. The radiographic image examination of the mandibular region revealed a severe and infiltrative periodontal reaction, with a predominance of a great number of neutrophils and macrophages, necrotic areas, and bone destruction, characterized histologically as a pyogranulomatous rection. At post mortem examination, a large pyogranuloma was observed in the entire horizontal branch of the mandible as well, showing a dark yellowish content of coarse consistency, caseous appearance, and bone fragmentation. Discussion: Ovine mandibular osteomyelitis is a well-established bone inflammation involving the cortical and medullary regions, characterized clinically by local enlargement, asymmetry, pain sensitivity, edema, hyperthermia, infiltrate caseous or suppurative material, and bone rarefaction. In the current report, 1-month history of progressive enlargement of the mandibular region, prostration, and weight loss in an ewe were referred. Where clinical and epidemiological features, bacteriological, cytological, histological, and mass spectrometry diagnostic approaches were assessed to diagnostic. Most reports involving the etiology of ovine mandibular osteomyelitis have been diagnosed based on classical phenotypic tests. Here, the concomitant identification of P. aeruginosa and L. raffinolactis infection was possible using mass spectrometry (MALDI-TOF), highlighting the importance of molecular methods in the diagnosis of animal diseases. In addition, the differentiation between Lactococcus and Enterococcus species is difficult, which could underestimate the diagnosis of Lactococcus species as a primary pathogen from animal diseases. We report, for the first time, a fatal case of mandibular pyogranulomatous osteomyelitis in a sheep caused by Pseudomonas aeruginosa and Lactococcus raffinolactis coinfection.

Pirarucu larviculture in green water provides heavier fish and modulates locomotor activity / Larvicultura de pirarucu em água verde produz peixes mais pesados e modula a atividade locomotora

The green water technique uses microalgae in the water of indoor larviculture, providing a darker environment to favor fish growth, welfare and health. We evaluated growth performance and locomotor activity after light exposure of pirarucu (Arapaima gigas) larvae reared in green or clear water. During one test, pirarucu larvae (3.6 ± 0.3 cm; 0.36 ± 0.1 g) were reared in 50-L circular tanks (n = 3 per treatment, 50 larvae per tank) in a static system containing green water [microalgae (w3algae; Bernaqua® 10 g m-3) added] or clear water (control). Fish weaning was achieved by co-feeding with Artemia nauplii and microdiets for seven days until full microdiet substitution. Larvae were biometrically evaluated on days 10, 17 and 24 to assess growth performance. In a second test, the locomotor activity of the larvae was analyzed before and after light exposure (1400 ± 60 lx) for 48 h according to an ethogram. After 24 days, the larvae reared in the green water were significantly heavier than those from the clear water, and displayed significantly fewer circular swimming movements. Body cortisol increased in both groups after light exposure. The microalgae provided an additional food source for larvae, with positive impact on growth until day 17 of larviculture. Green water can be a strategy to achieve better results in pirarucu larviculture, especially during and up to 10 days after the co-feeding period.(AU)

A técnica de água verde utiliza microalgas na água durante a larvicultura indoor, proporcionando um ambiente mais escuro que favorece o crescimento, bem-estar e saúde dos peixes. Avaliamos o crescimento e a atividade locomotora após exposição à luz de larvas de pirarucu (Arapaima gigas) criadas em água verde ou clara. Em um teste, larvas de pirarucu (3,6 ± 0,3 cm; 0,36 ± 0,1 g) foram criadas em tanques circulares de 50 L (n = 3 por tratamento; 50 larvas por tanque) em sistema estático contendo água verde [microalgas (w3algae; Bernaqua® 10 g m-3) adicionadas] ou água clara (controle). A transição alimentar dos peixes ocorreu por co-alimentação com náuplios de Artemia e microdieta por sete dias até a substituição completa pela microdieta. A biometria das larvas foi avaliada nos dias 10, 17 e 24, para avaliar o crescimento. Um segundo teste avaliou a atividade locomotora das larvas antes e após exposição à luz (1.400 ± 60,47 lx) por 48 horas usando um etograma. Após 24 dias, os peixes criados em água verde pesaram significativamente mais que os da água clara, e apresentaram significativamente menos movimentos circulares de natação. A exposição à luz aumentou o cortisol corporal nos dois grupos depois da exposição à luz. O nível corporal de cortisol aumentou em ambos grupos após exposição à luz. As microalgas forneceram uma fonte adicional de alimento para as larvas, com impacto positivo sobre seu crescimento até o 17º dia de larvicultura. Água verde pode ser uma estratégia para obter melhores resultados na larvicultura de pirarucu, principalmente durante e até 10 dias após o período de co-alimentação.(AU)

Consortium of multifunctional microorganisms in soybean culture / Consórcio de microrganismos multifuncionais na cultura da soja

The use of multifunctional microorganisms (MM) directly benefits the growth and development of plants due to the production of phytohormones and siderophores, supply of nutrients, and assimilation of atmospheric nitrogen (N2), as well as, indirectly, by protecting plants against pathogens. In this way, the search for sustainable agricultural systems is of great importance in searching for cultivation technologiesthat provide productive increments and minimize production costs and negative environmental impacts. The study aimed to determine the effect of multifunctional microorganisms on gas exchange, grain yield, and production components in soybean plants. In the field experiment, 2019/2020 harvest, a randomized block design was used, with 32 replications. Soybean plants were treated with a consortium of microorganisms Serratia marcensens(BRM 32114) + Bacillussp.(BRM63573), and, as a control, soybean plants were treated without intercropping. Soybean plants treated with microorganisms showed an increase in photosynthetic rate (16.65%), stomatal conductance (37.50%), the internal concentration of CO2 (10.36%), a mass of 100 grains (4.04%), and yield of grains (14.83%) about untreated plants. Therefore, using a consortium of multifunctional microorganisms, combining multiple functionalities from different microorganisms, shows the potential to increase the agronomic performance of soybean plants. Co-inoculation technology appears as a strategic component of achieving sustainable agriculture.(AU)

A utilização de microrganismos multifuncionais (MM) beneficia diretamente o crescimento e desenvolvimento de plantas em decorrência da produção de fitormônios e sideróforos, suprimento de nutrientes e assimilação de nitrogênio atmosférico (N2); bem como, indiretamente, por proteger as plantas contra patógenos. Dessa forma, a busca por tecnologias de cultivo que proporcionem incrementos produtivos e minimizem custos de produção e impactos negativos ao ambiente é de grande importância na busca por sistemas agrícolas sustentáveis. O objetivo do estudo foi determinar o efeito de microrganismos multifuncionais, sobre as trocas gasosas, produtividade de grãos e componentes de produção em plantas de soja. No experimento de campo, safra 2019/2020, utilizou-se o delineamento experimental de blocos casualizados, com 32 repetições. As plantas de soja foram tratadas com o consórcio de microrganismos Serratia marcensens(BRM 32114) + Bacillus sp. (BRM63573) e, como controle, plantas de soja tratadas sem o consórcio. Plantas de soja tratadas com microrganismos apresentaram aumento na taxa fotossintética (16,65%), condutância estomática (37,50%), concentração interna de CO2(10,36%), massa de 100 grãos (4,04%) e produtividade de grãos (14,83%) em relação às plantas não tratadas. Portanto, a utilização de consórcio de microrganismos multifuncionais, ou seja, combinação de múltiplas funcionalidades advindas de diferentes microrganismos, mostra potencial em aumentar a performance agronômica de plantas de soja. O uso da tecnologia de co-inoculação figura-se como componente estratégico para alcançar a agricultura sustentável.(AU)

Chemical, mineral composition, in vitro ruminal fermentation and buffering capacity of some rangeland-medicinal plants

A diverse group of rangeland-medicinal plants are being used by ruminant whilst some of them have not been assessed for their nutritional value. This study was aimed to evaluate the chemical and mineral composition, buffering capacity, and in vitro fermentation of some rangeland-medicinal plants including Thymus kotschyanus, Ziziphora persica, Lallemantia royleana, and Scutellaria litwinowii in the family Lamiaceae, and Hypericum scabrum, in the family Hypericaceae. The results indicated that crude protein (CP) content ranged from 8.66% (S. litwinowii) to 12.17% of DM (H. scabrum). It was found that Z. persica had the highest potential gas production, metabolism energy (ME), relative feed value (RFV), and dry matter digestibility (DMD) values of 53.44 (mL 200-1 mg DM), 5.84 (MJ kg-1 DM), 170.66 and 70.88%, respectively. Mineral content differed among plants; Ca ranged from 5.79 to 41.96 g kg-1 DM. The concentrations of Ca, K, Mg, Fe, Zn, and Co were highest for L. royleana. Total volatile fatty acids (TVFA) and propionate concentrations were highest in the culture medium cultured with Z. persica, however, acetate, and butyrate were highest in H. scabrum. Acid-base buffering capacity was lower in T. kotschyanus and H. scabrum compared to other plants, while it was higher in S. litwinowii. Overall, it can be concluded that among plants evaluated in this study, Z. persica had higher nutritional value for sheep feeding.(AU)

Techniques of fragile renal organoids transplantation in mice

Purpose: This study aimed to develop a microsurgical technique to transplant extremely fragile renal organoids in vivo, created by in-vitro reaggregation of metanephros from fetal mice. These organoids in reaggregation and development were examined histologically after transplantation under the renal capsule. Methods: Initially, metanephros from fetal mice were enzymatically treated to form single cells, and spheroids were generated in vitro. Under a microscope, the renal capsule was detached to avoid bleeding, and the outer cylinder of the indwelling needle was inserted to detach the renal parenchyma from the renal capsule using water pressure. The reaggregated spheroid was aspirated from the culture plate using a syringe with an indwelling needle outer cylinder and carefully extruded under the capsule. Pathological analysis was performed to evaluate changes in reaggregated spheroids over time and the effects of co-culture of spinal cord and subcapsular implantation on maturation. Results: In vitro, the formation of luminal structures became clearer on day 5. These fragile organoids were successfully implanted without tissue crapes under the renal capsule and formed glomerular. The effect of spinal cord co-transplant was not obvious histrionically. Conclusions: A simple and easy method to transplant fragile spheroids and renal under the renal capsule without damage was developed.

Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes

Abstract Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.

Analysis on the prokaryotic microbiome in females and embryonic cell cultures of Rhipicephalus sanguineus tropical and temperate lineages from two specific localities in Brazil / Análise do microbioma procarionte de fêmeas e cultura celular embrionária de Rhipicephalus sanguineus linhagens tropical e temperada de duas localidades no Brasil

Abstract Two lineages of Rhipicephalus sanguineus are known in Brazil: the temperate or southern and the tropical or northern populations. The distribution patterns of both lineages of R. sanguineus have epidemiological implications that can affect vectorial competence concerning Ehrlichia canis, the agent of canine monocytic ehrlichiosis. Intending to identify the microbiomes of both lineages and compare microorganisms in R. sanguineus, we used the 16S rRNA (V4-V5 region) gene-based metataxonomic approach, through NGS sequencing on the MiSeq Illumina platform. We selected specimens of females from the environment and samples of primary embryonic cell cultures, from both lineages, and this was the first study to investigate the prokaryotic microbiome in tick cell cultures. The results showed that many bacterial taxa detected in the samples were typical members of the host environment. A significant diversity of microorganisms in R. sanguineus females and in embryonic cell cultures from both lineages was found, with emphasis on the presence of Coxiella in all samples, albeit in different proportions. The Coxiella species present in the two lineages of ticks may be different and may have co-evolved with them, thus driving different patterns of interactions between ticks and the pathogens that they can harbor or transmit to vertebrate hosts.

Resumo Duas linhagens de Rhipicephalus sanguineus são conhecidas no Brasil: populações da linhagem temperada ou do sul, e tropical ou do norte. Os padrões de distribuição de ambas as linhagens de R. sanguineus têm implicações epidemiológicas, podendo afetar a competência vetorial de Ehrlichia canis, o agente etiológico da erliquiose monocítica canina. Com a intenção de identificar os microbiomas de ambas as linhagens e comparar microrganismos de R. sanguineus, foi utilizada a metataxonomia, baseada no gene 16S rRNA (região V4-V5), por meio do sequenciamento de nova geração na plataforma MiSeq Illumina. Foram selecionadas amostras de fêmeas do ambiente e cultivo primário de células embrionárias, considerando-se as duas linhagens conhecidas do Brasil. Este é o primeiro estudo que investiga o microbioma procariótico de células de cultura de carrapato. Os resultados mostram que muitos grupos de bactérias detectadas nas amostras são membros típicos do ambiente do hospedeiro. Uma diversidade significativa de microrganismos em fêmeas e cultura de células embrionárias nas duas linhagens de R. sanguineus foi encontrada, com ênfase na presença de Coxiella em todas as amostras, ainda que em diferentes proporções. Possivelmente, as espécies de Coxiella presentes nas duas linhagens de carrapatos são diferentes e co-evoluíram com essas linhagens, conduzindo a diferentes padrões de interação entre carrapatos e patógenos que podem abrigar ou transmitir aos hospedeiros vertebrados.

Peach palm plantlet growth in different culture media in a temporary immersion system / Crescimento de mudas de pupunheira em diferentes meios de cultura em um sistema de imersão temporária

Peach palm is a domesticated palm commercially important for the production of fruits and hearts of palm. Somatic embryogenesis, an effective technique for mass propagation, was successfully established for this species. Furthermore, a temporary immersion system improved plant regeneration. However, production can be further improved by understanding the peach palms growth dynamic and modifications of culture media. The aims of this study were to evaluate the growth of plantlets cultured in different culture media in a temporary immersion system and to correlate the results with nutrient uptake during the growth period. Somatic embryo-derived young plantlets approximately 1 cm in length were cultivated for 12 weeks in a twin flask system containing MS, Y3 or N6 salts, Morel and Wetmore vitamins and 3% sucrose, with a monthly medium refreshment. Growth was measured and mineral analysis of the plantlets was carried out after 12 weeks of culture. The Y3 and MS salts were the most appropriate for the plant growth. Number of roots was 52.52% higher and the root size was 40.42% between the N6 and MS medium and the root number in Y3 medium was 37.74% greater than in MS medium, which is important for post acclimatization survival. K and Na are important elements for peach palm. N is not required at such a high concentration as in Murashige and Skoog formulation. The Chu (N6) medium did not generate high quality plantlets, possibly due to the absence of some micronutrients, like Mo, Cu and Co.(AU)

A pupunheira é uma palmeira comercialmente importante para a produção de palmito. A embriogênese somática, técnica efetiva para propagação massal, foi estabelecida com sucesso para essa espécie. Além disso, um sistema de imersão temporária aumentou a regeneração de plantas. Entretanto, a produção pode ser melhorada através da compreensão da dinâmica de crescimento e modificações do meio de cultura. O estudo objetivou avaliar o crescimento de plantas em diferentes meios de cultura em um sistema de imersão temporária e correlacionar os resultados com a absorção de nutrientes durante o período de crescimento. Plantas derivadas de embriões somáticos, com aproximadamente 1 cm de comprimento, foram cultivadas por 12 semanas em um sistema tipo frasco gêmeo contendo sais do MS, Y3 ou N6, vitaminas de Morel e Wetmore e 3% de sacarose, com renovação mensal do meio de cultura. O crescimento e os teores de nutrientes nas plantas foram determinados após 12 semanas de cultivo. Os sais do MS e Y3 foram os mais apropriados para o crescimento vegetal. O número e comprimento de raízes foi 52,52% e 40,42% maior no meio MS do que no meio N6, respectivamente, e o número de raízes no meio Y3 foi 37,74% maior que no meio MS, o que é importante para a sobrevivência após a aclimatização. K e Na foram os nutrientes mais importantes para a pupunheira. O N não foi requerido em altas concentrações como verificado na formulação do meio Murashige e Skoog. O meio de Chu (N6) não gerou plantas de boa qualidade, possivelmente devido à ausência dos micronutrientes Mo, Cu e Co.(AU)

Aspergilose sistêmica em potro (Equus caballus) / Systemic aspergillosis in a foals - Equus caballus

O gênero Aspergillus compreende espécies de fungos saprotróficos, globalmente distribuídos, sendo os Aspergillus seção Fumigati conhecidos por causarem doença em humanos e animais imunocomprometidos. Os sinais clínicos dependem do sistema acometido, podendo apresentar febre branda, cólicas intensas, pneumonias, micose de bolsa gutural, e, ocasionalmente, lesões no sistema nervoso central. O presente trabalho objetiva relatar os sinais clínicos, os achados de necropsia e a histologia de um caso de aspergilose sistêmica em um potro de oito meses de idade. O animal apresentava refluxo enterogástrico, peristaltismo aumentado, fezes pastosas, dor abdominal e desidratação intensa. O quadro clínico evoluiu para óbito e o cadáver foi encaminhado para a necropsia. Macroscopicamente, havia sufusões e equimoses na serosa do trato gastrointestinal e ulcerações na mucosa do intestino delgado. No pulmão, havia nódulos multifocais, esbranquiçados e firmes que, ao corte, apresentavam-se císticos, com revestimento interno vinhoso e friável. O coração exibia áreas esbranquiçadas multifocais no miocárdio e os rins continham nódulos avermelhados nas regiões cortical e medular. No encéfalo, havia áreas multifocais amareladas e hemorrágicas com bordos avermelhados. Microscopicamente, observaram-se áreas multifocais de necrose com infiltrado inflamatório granulomatoso no intestino, pulmão, encéfalo, miocárdio e nos rins. Hifas fúngicas intralesionais e intravasculares foram observadas no encéfalo e no pulmão. Estruturas de fenótipo compatível com Aspergillus fumigatus foram observadas na cultura fúngica. Amostras encaminhadas para virologia apresentaram-se positivas para Herpesvírus Equino (HVE) tipo 1 e 4. O diagnóstico de aspergilose sistêmica foi determinado pelo histórico clínico, imunossupressão, achados macroscópicos, histológicos, isolamento do fungo e à infecção concomitante por HVE.

The Aspergillus genus comprises globally distributed species of saprotrophic fungi, with the Aspergillus section Fumigatus known to cause diseases in humans and immunocompromised animals. Clinical signs depend on the affected system, exhibiting mild fever, severe colic, pneumonia, guttural bag mycosis and occasionally lesions in the central nervous system. The present work aims to report the clinical signs, necropsy findings and histology of a case of systemic aspergillosis in an eight-months-old foal. The animal had enterogastric reflux, increased peristalsis, pasty feces, abdominal pain and severe dehydration. The condition evolved to death and the cadaver was sent for necropsy. Macroscopically, there were suffusions and ecchymosis in the serosa of the gastrointestinal tract and ulcerations in the mucosa of the small intestines. In the lung, there were multifocal, whitish and firm nodules that were cystic when cut with a friable winey internal lining. The heart had whitish multifocal areas in the myocardium and the kidneys contained reddish nodules in the cortical and medullary regions. In the brain there were yellowish and hemorrhagic multifocal areas with reddish borders. Microscopically, multifocal areas of necrosis with granulomatous inflammatory infiltrate were observed in the intestine, lung, brain, myocardium and kidneys. Intralesional and intravascular fungal were observed in the brain and lung. Structures with phenotype compatible with Aspergillus fumigatus were observed in the fungal culture. In virology, samples were positive for equine herpesvirus (HVE) types 1 and 4. The diagnosis of systemic aspergillosis was determined due to the clinical history, immunosuppression, macroscopic and histological findings, fungal isolation and concomitant HVE infection.

Meningomielite eosinofílica provavelmente associada à toxoplasmose em um gato / Eosinophilic meningomyelitis probably associated with toxoplasmosis in a cat

Background: The cerebrospinal fluid (CSF) of healthy cats presents up to 5 cells/µL, with predominance of mononuclear cells and the presence of more than 1% eosinophils is rare and should always be considered an abnormal finding. There is no consensus on the term eosinophilic pleocytosis, as it is used to indicate the presence of more than 10 eosinophils/µL or more than 10% of the total leukocytes. The increase in eosinophils in the CSF may result from infectious, inflammatory, neoplastic and idiopathic diseases. The objective of this paper is to report a case of marked pleocytosis in CSF, with 84% eosinophils, probably due to toxoplasmosis, in a cat with paraparesis and diffuse spinal pain. Case: A mixed breed female cat, neutered, adult and domiciled in a rural area was presented due to gait abnormalities in the pelvic limbs that started one day before presentation. The general physical examination was unremarkable. On neurological examination it was observed asymmetric deficit of postural reactions in pelvic limbs, patellar reflex normal to increased and pain elicited on palpation of the thoracic and lumbar spine. Based on these findings, the neurological syndrome was classified as thoracolumbar, but with diffuse pain, and the main differential diagnoses were inflammatory/ infectious and neoplastic diseases. The leukogram showed eosinophilia and the serum biochemistry showed no significant changes. Serological assays for feline immunodeficiency virus and feline leukemia virus were negative. Analysis of cerebrospinal fluid (CSF) identified marked pleocytosis with 84% eosinophils and increase in protein concentration. Myelography showed no compressive or expansive changes. Fungal culture for CSF cryptococcosis was negative. Serum immunofluorescence antibody titer for Toxoplasma gondii (IgG) was 1:256. There was a marked improvement after treatment with sulfametoxazole/trimethoprim and pyrimethamine and after 3 weeks of treatment, there was almost complete recovery of neurological signs and after 9 months the cat was neurologically normal. Discussion: The most common causes of acute-onset thoracolumbar spinal cord syndrome in cats, with diffuse pain on spinal palpation, are meningomyelitis of inflammatory/infectious origin, such as feline infectious peritonitis (FIP) and neoplasms such as lymphoma. Other meningomyelitis of inflammatory origin, such as infectious and immune-mediated meningomyelitis of unknown origin are considered uncommon in cats. Although the clinical, systemic and neurological signs of FIP and toxoplasmosis may have similarities, in the present case FIP was not considered responsible for the observed signs, as the evolution of the case and the analysis of the CSF tend to be different. The peripheral eosinophilia, the cytological analysis of the CSF, characterized by marked eosinophilic pleocytosis, associated with a positive titer for toxoplasmosis, good response to treatment and improvement in the neurological condition, with survival for more than 9 months after treatment, rules out the possibility of FIP. Neurological signs observed in the absence of systemic signs are more common in cases of protozoan reactivation, which probably occurred in the present case. The possibility of toxoplasmosis in the patient in this report was reinforced by the fact that the animal came from a rural area. Eosinophilia of CSF is most commonly associated with parasitic infections, although it can be caused by a variety of infectious agents, but in the cat of the present report, the marked eosinophilic pleocytosis was likely due to toxoplasmosis, which is a rare occurrence in this specie. In conclusion, toxoplasmosis should be considered in the differential diagnosis of focal spinal cord lesions in cats. The identification of laboratory findings as well as the appropriate therapy favored the good evolution of the condition.

Aspergilose sistêmica em potro (Equus caballus) / Systemic aspergillosis in a foals - Equus caballus

O gênero Aspergillus compreende espécies de fungos saprotróficos, globalmente distribuídos, sendo os Aspergillus seção Fumigati conhecidos por causarem doença em humanos e animais imunocomprometidos. Os sinais clínicos dependem do sistema acometido, podendo apresentar febre branda, cólicas intensas, pneumonias, micose de bolsa gutural, e, ocasionalmente, lesões no sistema nervoso central. O presente trabalho objetiva relatar os sinais clínicos, os achados de necropsia e a histologia de um caso de aspergilose sistêmica em um potro de oito meses de idade. O animal apresentava refluxo enterogástrico, peristaltismo aumentado, fezes pastosas, dor abdominal e desidratação intensa. O quadro clínico evoluiu para óbito e o cadáver foi encaminhado para a necropsia. Macroscopicamente, havia sufusões e equimoses na serosa do trato gastrointestinal e ulcerações na mucosa do intestino delgado. No pulmão, havia nódulos multifocais, esbranquiçados e firmes que, ao corte, apresentavam-se císticos, com revestimento interno vinhoso e friável. O coração exibia áreas esbranquiçadas multifocais no miocárdio e os rins continham nódulos avermelhados nas regiões cortical e medular. No encéfalo, havia áreas multifocais amareladas e hemorrágicas com bordos avermelhados. Microscopicamente, observaram-se áreas multifocais de necrose com infiltrado inflamatório granulomatoso no intestino, pulmão, encéfalo, miocárdio e nos rins. Hifas fúngicas intralesionais e intravasculares foram observadas no encéfalo e no pulmão. Estruturas de fenótipo compatível com Aspergillus fumigatus foram observadas na cultura fúngica. Amostras encaminhadas para virologia apresentaram-se positivas para Herpesvírus Equino (HVE) tipo 1 e 4. O diagnóstico de aspergilose sistêmica foi determinado pelo histórico clínico, imunossupressão, achados macroscópicos, histológicos, isolamento do fungo e à infecção concomitante por HVE.(AU)

The Aspergillus genus comprises globally distributed species of saprotrophic fungi, with the Aspergillus section Fumigatus known to cause diseases in humans and immunocompromised animals. Clinical signs depend on the affected system, exhibiting mild fever, severe colic, pneumonia, guttural bag mycosis and occasionally lesions in the central nervous system. The present work aims to report the clinical signs, necropsy findings and histology of a case of systemic aspergillosis in an eight-months-old foal. The animal had enterogastric reflux, increased peristalsis, pasty feces, abdominal pain and severe dehydration. The condition evolved to death and the cadaver was sent for necropsy. Macroscopically, there were suffusions and ecchymosis in the serosa of the gastrointestinal tract and ulcerations in the mucosa of the small intestines. In the lung, there were multifocal, whitish and firm nodules that were cystic when cut with a friable winey internal lining. The heart had whitish multifocal areas in the myocardium and the kidneys contained reddish nodules in the cortical and medullary regions. In the brain there were yellowish and hemorrhagic multifocal areas with reddish borders. Microscopically, multifocal areas of necrosis with granulomatous inflammatory infiltrate were observed in the intestine, lung, brain, myocardium and kidneys. Intralesional and intravascular fungal were observed in the brain and lung. Structures with phenotype compatible with Aspergillus fumigatus were observed in the fungal culture. In virology, samples were positive for equine herpesvirus (HVE) types 1 and 4. The diagnosis of systemic aspergillosis was determined due to the clinical history, immunosuppression, macroscopic and histological findings, fungal isolation and concomitant HVE infection.(AU)

Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes

Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.(AU)

Lúpus eritematoso sistêmico associado a manifestações neurológicas em cadela da raça Border Collie / Systemic lupus erythematosus associated with neurological manifestations in a Border Collie bitch

Background: Systemic lupus erythematosus (SLE) is an immune-mediated and multisystemic disorder which etiology is believed to be multifactorial. Its clinical signs vary accordingly to affected organs, cutaneous lesions being the most frequently observed. There are few reports of SLE in dogs with neurological manifestations. Therefore, the aim of this report is to describe a case of SLE in a dog with indicative signs of nervous system involvement. Case: A 6-year-old Border Collie bitch was referred to the Veterinary Hospital (HVU) of the University of Uberaba (UNIUBE) with a history of cluster seizures, inappetence and urinary incontinence. Erythema and flaking of nasal plan were noted on physical examination, and splenomegaly on abdominal palpation. Thrombocytopenia and slightly increased ALT were found on blood tests. Ehrlichiosis was suspected and doxycycline was prescribed along with phenobarbital for the control of seizures. In the follow-up visit, the dog was still presenting urinary incontinence, thrombocytopenia and splenomegaly. Also, an ulcer on the nasal mucocutaneous junction was observed. The patient went through a neurological examination which indicated thalamocortical lesion. Cerebrospinal fluid samples were obtained for cytology, culture and canine distemper test, and serology tests for leishmaniasis, toxoplasmosis and neosporosis were done. No alterations were found in these exams. The histopathology of the nasal lesion was proceeded and showed results consistent with lupus erythematosus. It was prescribed a 15-day course of prednisolone at immunosuppressive dose. The patient showed clinical improvement with this treatment. Azathioprine was started along with gradual removal of prednisolone. After twenty days of discontinuation of this drug, the dog presented epileptic seizures, urinary incontinence, thrombocytopenia, increased ALT and worsened nasal lesion. Prednisolone at immunosuppressive dose was reintroduced and the dose of phenobarbital, increased. One week past this, the patient showed inappetence and an extensive hematoma in the thoracic region. Lab exams confirmed drug-induced acute pancreatitis. All medications were interrupted, the patient was hospitalized, and treatment for pancreatitis was initiated, but the dog passed away. Discussion: For involving multiple body systems and for presenting varied clinical signs, diagnosing SLE can be challenging in clinical routine. The dog from this report was a Border Collie; this breed is considered to be predisposed to this disease. The animal had a history of being exposed to solar radiation for a large part of the day, had dyspigmentation of nasal plan and had no application of sunscreen, predisposing the occurrence of SLE. Neurological signs are uncommon in SLE, but the seizures and the urinary incontinence were the main reasons for the dog's guardian to look for medical assistance. The suspicion for SLE was raised due to cutaneous manifestations and persistent thrombocytopenia along with splenomegaly. Histopathological findings are essential for diagnosing SLE, as well as antinuclear antibody tests. Nonetheless, due to financial limitations, this last test was not performed. Azathioprine is an immunomodulating drug largely used along with glucocorticoids when treating SLE; however, this medication is prone to induce side effects as the ones presented by the dog from this report. Therefore, it is concluded that SLE should be considered as a differential diagnosis in patients showing cutaneous, hematological, systemic and neurological manifestations, considering the variety of signs caused by this disorder.(AU)