Fist report on fumagillin production by Aspergillus fumigatus sensu stricto gliotoxigenic strains recovered from raw cow milk and clinical samples in Argentina / Primer reporte en Argentina sobre la producción de fumagilina por cepas deAspergillus fumigatus sensu stricto productoras de gliotoxina, aisladas de lechecruda y de casos clínicos

Abstract In Argentina there are no reports on Aspergillus fumigatus fumagillin-producingstrains. In this study we describe the isolation and mycotoxin production capacity of ten A.fumigatus strains isolated from farm and clinical samples. Farm strains were isolated frommilk samples taken from dairy cows in Córdoba province, some of which were associated withsubclinical mastitis. A culture medium was defined to optimize fumagillin production and adetection method was developed by HPLC chromatography. It is known that in addition to thehost immune status, strain virulence is a fundamental characteristic that will determine itspathogenicity and, in this sense, fumagillin is considered to be among the virulence factors. Inthe present work, all the strains tested for the production of fumagillin were able to synthesizeit, highlighting that the strain A. fumigatus RC2243, from a milk sample from a cow with clinicalmastitis, was the most productive. The existence of fumagillin-producing strains represents apotential risk of mycotoxins being transferred to raw milk, constituting a public health risk.

Resumen En Argentina no existen reportes sobre cepas de Aspergillus fumigatus productoras de fumagilina. En este trabajo se describe el aislamiento y la producción de dicha micotoxina clínicaspor 10 cepas, provenientes del medioambiente rural y aisladas de muestras clínicas. Las cepasde origen rural fueron aisladas de vacas lecheras en tambos de la provincia de Córdoba, yalgunas de esas cepas se asociaron a casos de mastitis subclínica. Se definió la composición deun medio de cultivo para optimizar la producción de fumagilina y se desarrolló un método decromatografía HPLC para su determinación. Es conocido que, además del estado inmunitario delhuésped, la virulencia de la cepa es una de las características fundamentales que determinansu potencial patogénico y, en este sentido, la fumagilina es considerada un factor de virulencia. En el presente trabajo todas las cepas estudiadas fueron capaces de sintetizarla y la cepa A.fumigatus RC2243, proveniente de leche de una vaca con mastitis subclínica, se destacó comola cepa más productora. La existencia de cepas productoras de fumagillina representa un riesgopotencial por el pasaje de dicha micotoxina a la leche, lo cual constituye un problema para lasalud pública.

Isolation, identification and technological properties of lactic acid bacteria from raw cow milk / Isolamento, identificação e propriedades tecnológicas de bactérias ácido lácticas a partir de leite de vaca cru

Lactic acid bacteria are used as starter culture for the production of fermented dairy products, and that occur naturally as indigenous microbiota of the raw milk. In this study, lactic acid bacteria were isolated from raw cow milk samples. The serial dilutions of raw milk were made and plated onto LM17 agar and MRS agar adjusted to pH 5.4. The isolates were firstly identified based on cell morphology, reaction to gram stain, catalase production, growth in MRS broth containing 2%, 4%, and 6.5% NaCl, growth ability of different temperatures and formation of gas in MRS broth. Acid formation in 10% reconstituted skim milk and antimicrobial activity against foodborne pathogens Listeria monocytogenes ATCC 7644, Stapylococcus aureus ATCC 25923, and Clostridium perfringens ATCC 13124 were examined using agar well diffusion method. The total of 90 LAB isolates were classified as Lactobacillus (37.78%), Lactococcus (36.67%), Enterococcus (20.00%), Streptococcus (4.44%), and Leuconostoc (1.11%). Based on technological properties, 56 of 90 isolates (42 cocci, 14 rods) were selected, and further identified at the species level using API 20 Strep and API 50 CH identification system, respectively. The Lactobacillus isolates were identified as Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, Lactobacillus delbrueckii ssp. lactis, and Lactobacillus rhamnosus. The enzymatic profiles of the 17 selected isolates were studied with API ZYM system. The Lactobacillus spp. strains tested displayed high leucine arylamidase activity. Two Lactococcus lactis ssp. lactis AKS320.1 and AKS320.2 strains and one Enterococcus faecalis AKS424 strain were able to produce bacteriocin. In conclusion, some of these isolates could be considered as potential starter culture candidates for fermented milk products.

Bactérias de ácido lático são utilizadas como cultura inicial para a produção de produtos lácteos fermentados e que ocorrem naturalmente como microbiota indígena do leite cru. Neste estudo, as bactérias ácido lácticas foram isoladas a partir de amostras de leite cru de vaca. As diluições em série do leite cru foram feitos e plaqueadas em ágar LM17 e agar MRS ajustado para pH 5,4. Os isolados foram primeiramente identificados com base na morfologia das células, coloração pelo Método de Gram, produção de catalase, crescimento em caldo MRS contendo 2%, 4%, e 6,5% de NaCl, a capacidade de crescimento de diferentes temperaturas e formação de gás em caldo MRS. Foram avaliadas a formação de ácido em 10% de leite desnatado reconstituído e propriedades antagonistas contra os agentes patogênicos de origem alimentar Listeria monocytogenes ATCC 7644, Stapylococcus aureus ATCC 25923, e Clostridium perfringens ATCC 13124, usando o método de difusão em agar. O total de 90 isolados de laboratório foram classificados como Lactobacillus (37,78%), de Lactococcus (36,67%), Enterococcus (20,00%), Streptococcus (4,44%), e Leuconostoc (1,11%). Com base em propriedades tecnológicas, foram selecionados 56 dos 90 isolados (42 cocos, 14 hastes) e identificados ao nível das espécies usando o sistema de identificação API 20 Strep e API 50 CH, respectivamente. Os isolados de Lactobacillus foram identificados como Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, Lactobacillus delbrueckii ssp. lactis e Lactobacillus rhamnosus. Os perfis enzimáticos dos 17 isolados selecionados foram estudados por meio do sistema API ZYM. As cepas de Lactobacillus possuem alta atividade de arilamidase leucina. Duas cepas de Lactococcus lactis ssp. lactis AKS320,1 e AKS320,2 cepas e um Enterococcus faecalis AKS424 estirpe foram capazes de produzir bacteriocina. Em conclusão, alguns destes isolados poderiam ser considerados como potenciais candidatos de cultura iniciadora para produtos à base de leite fermentados.

Effect of Raw Cow Milk and Gliocladium virens in Pearl Millet against Downy Mildew Disease Caused by Sclerospora graminicola.

Pearl millet [Pennisetum glaucum (L.) R. Br.] downy mildew (DM) is caused by the fungus Sclerospora graminicola (SACC.) SCHRŐT. is the most widespread and destructive disease of pearl millet affecting yield and quality in all the millet cultivating tracts of India. Since pearl millet is a crop of low economic value grown by resource-poor farmers, conventional technological interventions are not cost feasible. Integration of indigenous knowledge with biocontrol agents appeared as a logical strategy in the present case. Studies were, therefore, undertaken to manage DM in rainfed crop of pearl millet using raw cow milk together with Gliocladium virens. Seed and soil treatments resulted in the lowest disease incidence. Biochemical constituents (metabolites and oxidative enzymes) were analysed to determine possible mode of action of Raw Cow Milk (RCM) and Gliocladium virens. A considerable increase in sugars, phenols and ortho-dihydroxy phenols (OD) in healthy and DM infected leaves of treated pearl millet plants was recorded when compared to untreated controls. A marked increase in all the photosynthetic pigments in both healthy and diseased treated plants was observed. The induction of resistance was accompanied by increased activities of defense related enzymes. It is assumed that the combination of RCM and G. virens is capable of stimulating different systemic responses in host plant.