Impact of Spray-Drying on Biological Properties of Chitosan Matrices Supplemented with Antioxidant Fungal Extracts for Wine Applications.

Black aspergilli produce many bioactive compounds: enzymes, organic acids, and secondary metabolites. One such fungus, Aspergillus tubingensis G131, isolated from French Mediterranean vineyards, produces secondary metabolites with antioxidant properties that can be extracted with ethanol. In this study, crude antioxidant extracts obtained from A. tubingensis G131 cultures were encapsulated with two types of chitosan matrix. Spray-drying was used to obtain dried particles from a dispersion of fungal crude extracts in a solution of the coating agent chitosan. This process appeared to be an efficient method for obtaining a dry extract with antioxidant activity. Three types of fungal extracts, with different antioxidant capacities, were produced: two different concentrations of crude extract and a semi-purified extract. In this study, the chitosan matrices for encapsulation were chosen on the basis of their antimicrobial activities for wine applications. Classical low molecular weight chitosan was compared with NoBrett Inside® which is already used to prevent the development of Brettanomyces spp. in wine. The objective of this study was to confirm that both antioxidant (fungal extract) and antimicrobial (chitosan) properties were preserved after spray-drying. The combination of these two properties and the powder formulation of this entirely natural product would make it a good alternative to chemicals, such as sulfites, in the food and wine industries.

Whole-genome sequencing of Aspergillus tubingensis G131 and overview of its secondary metabolism potential.

BACKGROUND: Black Aspergilli represent one of the most important fungal resources of primary and secondary metabolites for biotechnological industry. Having several black Aspergilli sequenced genomes should allow targeting the production of certain metabolites with bioactive properties. RESULTS: In this study, we report the draft genome of a black Aspergilli, A. tubingensis G131, isolated from a French Mediterranean vineyard. This 35 Mb genome includes 10,994 predicted genes. A genomic-based discovery identifies 80 secondary metabolites biosynthetic gene clusters. Genomic sequences of these clusters were blasted on 3 chosen black Aspergilli genomes: A. tubingensis CBS 134.48, A. niger CBS 513.88 and A. kawachii IFO 4308. This comparison highlights different levels of clusters conservation between the four strains. It also allows identifying seven unique clusters in A. tubingensis G131. Moreover, the putative secondary metabolites clusters for asperazine and naphtho-gamma-pyrones production were proposed based on this genomic analysis. Key biosynthetic genes required for the production of 2 mycotoxins, ochratoxin A and fumonisin, are absent from this draft genome. Even if intergenic sequences of these mycotoxins biosynthetic pathways are present, this could not lead to the production of those mycotoxins by A. tubingensis G131. CONCLUSIONS: Functional and bioinformatics analyses of A. tubingensis G131 genome highlight its potential for metabolites production in particular for TAN-1612, asperazine and naphtho-gamma-pyrones presenting antioxidant, anticancer or antibiotic properties.

Fungal naphtho-γ-pyrones--secondary metabolites of industrial interest.

Naphtho-γ-pyrones (NGPs) are secondary metabolites mainly produced by filamentous fungi (Fusarium sp., Aspergillus sp.) that should be considered by industrials. Indeed, these natural biomolecules show various biological activities: anti-oxidant, anti-microbial, anti-cancer, anti-HIV, anti-hyperuricuric, anti-tubercular, or mammalian triacylglycerol synthesis inhibition which could be useful for pharmaceutical, cosmetic, and/or food industries. In this review, we draw an overview on the interest in studying fungal NGPs by presenting their biological activities and their potential values for industrials, their biochemical properties, and what is currently known on their biosynthetic pathway. Finally, we will present what remains to be discovered about NGPs.

Association between haptoglobin and IgM levels and the clinical progression of caseous lymphadenitis in sheep.

BACKGROUND: Sheep caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis (Cp), is associated with direct economic losses and presents significant zoonotic potential. Despite the importance of the disease, a satisfactory vaccine model has not been developed. Thus, this study aimed to investigate the association between haptoglobin (Hp) and IgM levels and the clinical progression of CLA in primarily infected sheep and in sheep immunized with Cp- secreted antigens adjuvanted with Quillaja saponaria saponins. These animals were kept with CLA-positive sheep to simulate natural exposure that occurs in field conditions. During the experiment, the Hp and IgM levels were monitored for 21 days, and the development of internal CLA lesions was investigated through necropsies on day182 post-immunization. RESULTS: Primarily infected sheep in Group 2 (inoculated with 2x105 Cp virulent strain) had higher Hp values between the first and ninth days post inoculation (PI) than sheep in Group 1 (control; P < 0.05). Immunized animals in Group 3 had significantly higher Hp values between the third and seventh days PI, compared with the control group (P < 0.01). Binary logistic regression (BLR) analysis of primarily infected sheep indicated an association between Hp concentration and CLA clinical progression: animals with high Hp values had 99.9% less risk of having CLA abscesses than animals with low Hp levels (Odds ratio = 0.001, P < 0.05). Both experimental groups had significantly higher IgM titers than the control group around the ninth and eleventh days PI (P < 0.05). The BLR analysis for immunized sheep indicated an association between IgM levels and clinical progression: sheep with high IgM titers had 100.0% less risk of having CLA abscesses than animals with low IgM levels (Odds ratio = 0.000, P < 0.05). CONCLUSIONS: Resistance to C. pseudotuberculosis infection is supported by the early acute phase response, in which up-regulation of Hp and IgM were predictive of a lower risk of CLA lesion development. Because the immunogen used in this study induced a high production of both Hp and IgM, Q. saponaria saponin should be considered a promising candidate in vaccine formulations against sheep CLA.

Chemokine production induced by Corynebacterium pseudotuberculosis in a murine model.

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis. The main clinical sign of this disease is the development of granulomas, especially in small ruminants; however, the pathways that are involved in the formation and maintenance of these granulomas are unknown. Cytokines and chemokines are responsible for the migration of immune cells to specific sites and tissues; therefore, it is possible that chemokines participate in abscess formation. This study aimed to evaluate the induction of chemokine production by two C. pseudotuberculosis strains in a murine model. A highly pathogenic (VD57) and an attenuated (T1) strain of C. pseudotuberculosis, as well as somatic and secreted antigens derived from these strains, was used to stimulate murine splenocytes. Then, the concentrations of the chemokines CCL-2, CCL-3, CCL-4, and CCL-5 and the cytokines IL-1 and TNF were measured in the culture supernatants. The VD57 strain had a higher ability to stimulate the production of chemokines when compared to T1 strain, especially in the early stages of stimulation, which can have an impact on granuloma formation. The T1 lysate antigen was able to stimulate most of the chemokines studied herein when compared to the other antigenic fractions of both strains. These results indicate that C. pseudotuberculosis is a chemokine production inducer, and the bacterial strains differ in their induction pattern, a situation that can be related to the specific behavior of each strain.

Cell wall glycolipids from Corynebacterium pseudotuberculosis strains with different virulences differ in terms of composition and immune recognition.

Caseous lymphadenitis (CLA) is an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants and is characterized by the development of granulomas in the lymph nodes, spleen, liver, and lungs. Although little is known about the host-pathogen relationship of this bacterium, it was previously reported that the pathogen's lipids are important for its taxonomic classification and survival inside macrophages. However, there are no studies regarding the composition of these molecules. In this study, cell wall glycolipids from two C. pseudotuberculosis strains presenting different virulence profiles were purified and its composition was characterized. A difference was observed between the electrophoretic and chromatogram profiles for cell wall components from the two strains, mainly among molecules with low molecular weights. IgM from sheep with acute CLA recognized antigens with an estimated molecular weight of 11 kDa of the low-pathogenicity strain, while low-molecular weight antigens from the high-pathogenicity strain presented a lower recognition by these antibodies. Mass spectrometry analysis showed that the cell wall of the high-pathogenicity strain contained glycolipids with high amounts of unsaturated fatty acids and glycerophosphoinositols, which may contribute to the capacity of this strain to cause severe disease. In conclusion, it is indicated that cell wall non-protein antigens can play a key role in C. pseudotuberculosis virulence.

Influence of the culture conditions on the production of NGPs by Aspergillus tubingensis.

The filamentous fungus Aspergillus tubingensis that belongs to the black Aspergillus section has the capacity to produce high-value metabolites, for instance, Naphtho-Gamma-Pyrones (NGPs). For these fungal secondary metabolites, numerous biological properties of industrial interest have been demonstrated, such as antimicrobial, antioxidant and anti-cancer capacities. It has been observed that these secondary metabolites production is linked with the fungal sporulation. The aim of this research was to apply environmental stresses to trigger the production of NGPs in liquid cultures with CYB (Czapek Dox Broth): osmotic and oxidative stresses. In addition, numerous parameters were tested during the experiments, such as pH value, incubation time, container geometry, and static and agitation conditions. Results demonstrate that the produced amount of NGPs can be enhanced by decreasing the water activity (aw) or by adding an oxidative stress factor. In conclusion, this study can contribute to our knowledge regarding A. tubingensis to present an effective method to increase NGPs's production, which may support the development of current industrial processes.

Identification of membrane-associated proteins with pathogenic potential expressed by Corynebacterium pseudotuberculosis grown in animal serum.

OBJECTIVE: Previous works defining antigens that might be used as vaccine targets against Corynebacterium pseudotuberculosis, which is the causative agent of sheep and goat caseous lymphadenitis, have focused on secreted proteins produced in a chemically defined culture media. Considering that such antigens might not reflect the repertoire of proteins expressed during infection conditions, this experiment aimed to investigate the membrane-associated proteins with pathogenic potential expressed by C. pseudotuberculosis grown directly in animal serum. RESULTS: Its membrane-associated proteins have been extracted using an organic solvent enrichment methodology, followed by LC-MS/MS and bioinformatics analysis for protein identification and classification. The results revealed 22 membrane-associated proteins characterized as potentially pathogenic. An interaction network analysis indicated that the four potentially pathogenic proteins ciuA, fagA, OppA4 and OppCD were biologically connected within two distinct network pathways, which were both associated with the ABC Transporters KEGG pathway. These results suggest that C. pseudotuberculosis pathogenesis might be associated with the transport and uptake of nutrients; other seven identified potentially pathogenic membrane proteins also suggest that pathogenesis might involve events of bacterial resistance and adhesion. The proteins herein reported potentially reflect part of the protein repertoire expressed during real infection conditions and might be tested as vaccine antigens.

In vivo and in vitro expression of five genes involved in Corynebacterium pseudotuberculosis virulence.

Caseous lymphadenitis (LC) is a chronic contagious disease caused by Corynebacterium pseudotuberculosis, which mainly affects goats and sheep. Vaccination is an effective but not yet well-established method, partly due to a lack of knowledge surrounding the most effective immunoprotective components. The present study aimed to quantify and compare the in vivo expression of genes pld (phospholipase D), cpp (CP40), nanH (neuraminidase H), sodC (superoxide dismutase C) and spaC (adhesin) using qRT-PCR, with the respective expression in vitro. Caseous material of abscesses removed from five animals was cultured, with colonies suggestive of C. pseudotuberculosis identified. RNA extraction was performed on these samples, as well as on the respective pellets derived from liquid cultures brain heart infusion. After evaluating RNA integrity, complementary DNA was synthesized, followed by the relative quantification each of the genes of interest. Mean mRNA expression of the five genes found in abscesses and in cultures differed significantly, with respective values of: nanH 811.50 ± 198.27 and 359.35 ± 75.45 (p = 0.009); cpp 856.31 ± 385.11 and 154.54 ± 94.34 (p = 0.0039); plD 922.70 ± 450.73 and 212.41 ± 153.10 (p = 0.016); sodC 1,293.53 ± 564.75 and 223.63 ± 145.58 (p = 0.016); spaC 1,157.10 ± 525.13 and 214.26 ± 125.70 (p = 0,016). Expression was observed to be 6-8 times higher in abscesses than in cultures, Indicative that is a genetic expression of the in vitro bacterium exists, yet in vivo has a greater magnitude corroborating to one of these virulence factors in the pathogenesis of LC.

Acaricides efficiency on Rhipicephalus (Boophilus) microplus from Bahia state North-Central region.

The cattle tick Rhipicephalus (Boophilus) microplus is responsible for major losses in the Brazilian livestock, mainly due to reduction in cattle productive performance. Resistance development to major classes of acaricide widely used nowadays has been extensively reported, as well as the occurrence of residues from these compounds in animal products and the environment. This study aimed to evaluate the efficiency of acaricides on R. (B.) microplus collected from rural properties in the North-Central region of Bahia State. Ticks were collected in several cattle farms in the cited region, and an in vitro acaricide efficiency assay was performed based on the immersion of ticks in acaricide solutions made according to manufacturers' recommendations. The results obtained in the experiments indicated varying degrees of efficiency of the several bases studied, with the products benzofenilurea, macrocyclic lactone and fipronil showing the highest levels of efficiency, 100%, 100% and 97.34%, respectively. It was possible to conclude that, for the region studied, there are different degrees of commercial acaricides efficiency, and many present less than 95% effectiveness, value determined as acceptable by the Brazilian legislation.

Determinação de valores de referência séricos para os eletrólitos magnésio, cloretos, cálcio e fósforo em ovinos das raças Dorper e Santa Inês / Determination of magnesium, chloride, calcium and phosphorus serum reference values for Dorper and Santa Inês sheep breeds

A ovinocultura no Brasil é uma atividade em grande expansão e, com o aumento da demanda mundial por carne ovina, aumentou-se o interesse no monitoramento da sanidade do rebanho, utilizando diversas ferramentas como auxiliares no diagnóstico clínico, tais como os intervalos de referência séricos. Os elementos minerais constituem 2 a 5,5% do corpo dos vertebrados, exercendo diversas funções no organismo. O objetivo deste trabalho foi obter intervalos de referência para os eletrólitos magnésio, fósforo, cloreto e cálcio para ovinos das raças Dorper e Santa Inês. Foram coletados soros de 487 animais clinicamente sadios, sendo 146 da raça Dorper e 341 da raça Santa Inês. Os eletrólitos foram mensurados utilizando-se kits comerciais. Os dados foram analisados quanto à raça, sexo e idade, e os intervalos de referência determinados. Os resultados revelaram diferenças significativas nos intervalos de referência obtidos para os eletrólitos cálcio e magnésio na variável raça, e para o eletrólito fósforo na variável faixa etária e, quando confrontados com valores de referência já publicados, comprovou-se a existência de diferença estatística significativa entre os mesmos em todos os analitos estudados.

The sheep industry in Brazil is an important economic activity, and with the increasing global demand for sheep meat there is a great interest in the monitoring of the herd health, and serum reference ranges are basic tools for veterinary clinical pathology assays. Mineral elements correspond to 2-5.5% of the body of vertebrates, holding different functions in their physiology. The objective of this study was to obtain reference intervals of the electrolytes magnesium, phosphorus, chloride and calcium for the Dorper and Saint Ines sheep breeds. Sera samples were collected from 487 clinically healthy sheep, 146 from Dorper and 341 from Santa Ines breed. Electrolytes were measured using commercial kits. Data were analyzed taking the race, sex and age variables in account, and reference ranges were established. The results revealed significant statistical differences in reference ranges obtained for the electrolytes calcium and magnesium concerning the variable race, and for the electrolyte phosphorus in the variable age and, when compared with reference values already published, proved the existence of significant differences.

Acaricides efficiency on Rhipicephalus (Boophilus) microplus from Bahia state north-central region / Eficiência de acaricidas sobre Rhipicephalus (Boophilus) microplus na região centro-norte, Bahia

The cattle tick Rhipicephalus (Boophilus) microplus is responsible for major losses in the Brazilian livestock, mainly due to reduction in cattle productive performance. Resistance development to major classes of acaricide widely used nowadays has been extensively reported, as well as the occurrence of residues from these compounds in animal products and the environment. This study aimed to evaluate the efficiency of acaricides on R. (B.) microplus collected from rural properties in the North-Central region of Bahia State. Ticks were collected in several cattle farms in the cited region, and an in vitro acaricide efficiency assay was performed based on the immersion of ticks in acaricide solutions made according to manufacturers' recommendations. The results obtained in the experiments indicated varying degrees of efficiency of the several bases studied, with the products benzofenilurea, macrocyclic lactone and fipronil showing the highest levels of efficiency, 100%, 100% and 97.34%, respectively. It was possible to conclude that, for the region studied, there are different degrees of commercial acaricides efficiency, and many present less than 95% effectiveness, value determined as acceptable by the Brazilian legislation.

O carrapato Rhipicephalus (Boophilus) microplus é responsável por grandes perdas na pecuária brasileira, principalmente devido à redução no desempenho produtivo dos animais. A resistência do parasita a diferentes classes de acaricidas tem sido amplamente relatada, bem como os problemas de ocorrência de resíduos desses compostos em produtos de origem animal e no ambiente. Esse estudo teve como objetivo avaliar a eficiência de diversos acaricidas sobre populações de R. (B.) microplus provenientes de propriedades rurais da Região Centro-Norte do Estado da Bahia, Brasil. Teleóginas ingurgitadas foram colhidas em diferentes propriedades da região e submetidas a ensaio in vitro de eficiência de acaricidas, baseado na imersão dos carrapatos em soluções de acaricidas feitas seguindo as recomendações dos fabricantes. Os resultados obtidos indicam níveis variados de eficiência das bases estudadas, com os acaricidas benzofenilureia, lactona macrocíclica e fipronil apresentando os índices mais altos de eficiência, 100%, 100% e 97.34%, respectivamente. Pode-se concluir que, para a região estudada, os acaricidas apresentam níveis diversos de eficiência, e muitos deles possuem eficácia de menos de 95%, como recomendado pela legislação brasileira.