Advances in the design of a multi-strain homologous probiotic formula for cattle

Microorganisms colonizing different mucosas can exert diverse type of beneficial effects if they are included in probiotic formula. Then, the objective of this work was to complete the set of probiotic-related characteristics of different lactic acid bacteria strains previously isolated from different bovine mucosas to further include them in a probiotic product. The studies include some complementary assays related with their surface-adhesive characteristics (autoaggregation, biofilm formation, hydrophobicity, production of exopolisaccharides), production of inhibitory substances against pathogenic bacteria (hydrogen peroxide, bacteriocins). Also, the strains compatibility to go further in the design of a multi?strain probiotic formula to be used either to pregnant cows through the vaginal tract (for metritis prevention), later to the newborn calves (for diarrhea prevention), and to the mammary gland of the cows (for mastitis prevention). The formula will be integrated by strains from the three bovine tracts, being host? specific and able to promote the mucosal colonization and the beneficial effect in different ages and physiological states of bovines.Fil: Miranda, Maria Hortencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Nader, Maria Elena Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

Effects of plant extracts on the growth of beneficial indigenous lactic acid bacteria (BLAB) for their potential use in preventing bovine reproductive tract infections

There is a renewed interest in products based on phytocompounds, prebiotics and probiotics in recent years, applied to different hosts to exert a wide variety of effects. The microbiome of the bovine reproductive tract can become unbalanced for many reasons, favoring the entry and proliferation of pathogenic microorganisms, currently treated with antibiotics that exert adverse effects and generate antimicrobial resistance. To deal with this situation, "phytobiotic" formulas are proposed that combine phytocompounds and probiotics. This study aims to determine the compatibility of beneficial autochthonous lactic acid bacteria (BALB) with plant extracts, prebiotics and vitamins, to incorporate them into intravaginal formulas with therapeutic activity. Nine beneficial strains isolated from different bovine ecosystems were evaluated against nine phytocompounds, two prebiotics and five vitamins. Compatibility was assayed using the diffusion technique on agar plates, and the effect of the phytocompounds on the growth of lactic acid bacteria by microplates. The growth of all the strains was affected by some plant extracts, showing a stimulating or inhibitory effect. By the qualitative method, only vitamin A affected the viability of Lactobacillus johnsonii CRL1702 at concentrations higher than 7.5 mg/ml, however, when studying the growth kinetics of the strains with the phytocompounds, the results show that the effect was different in each of one the associated strains + plant extracts, indicating a strain specific effect of plant extacts on each BALB strain. Lapacho and Malva have a stimulating effect on most of the microorganisms, while Garlic and Belladonna inhibited the growth of all of them. Plant extracts at different concentrations did not inhibit the growth of most of the pathogens responsible for endometritis. On the other hand, the highest concentrations of phenolic compounds were detected in Echinacea, Lapacho and Llantén; and the best percentages of antioxidant activity were evidenced in Garlic, Blueberry and Chamomile (<60%). The results obtained in this stage are original, since the combination of natural extracts with lactobacillus strains for veterinary application was not studied previously

Diseño y formulación de medio de cultivo que simula fluido vaginal bovino (MSFVB): adaptación, crecimiento y propiedades de bacterias lácticas autóctonas benéficas (BLAB)

La aplicación de una fórmula veterinaria probiótica en vagina establece contacto con el fluido del tracto, ambiente en que las bacterias se adaptan, permanecen y sobreviven para reestablecer el equilibrio ecológico de la mucosa. El diseño de productos probióticos requiere que las BLAB resistan condiciones in vitro comparables a las del tracto en que se aplicarán, y continúen expresando sus propiedades benéficas (PB). Para ello se formuló un medio de cultivo similar al fluido vaginal bovino, diseñado con ingredientes de secreciones vaginales, para evaluar resistencia, viabilidad y propiedades benéficas de BLAB durante las fases foliculares (FF) y luteales (FL) del ciclo reproductivo. El medio contiene (mg/100ml): fructosa, 0.5; glucosa, 7; albúmina, 130; mucina, 6; caseína, 30 (FF) y 20 (FL); NaCl, 500; KCl, 150; KHPO4, 1,5; CaCl2, 10; Na2HPO4, 170 y Tween 80 0,1; pH 7.6 (FF) y 6.5 (FL). 11 BLAB de mucosas bovinas con PB se inocularon al 4% (MRS caldo como control), y se incubaron 32 h a 37°C, determinando número de UFC/ml, pH y propiedades probióticas. En MSFVB FL crecieron todas las BLAB, con mayor crecimiento a las 6 h, e incrementos de 1.00x106 a 1.00x108 ufc/ml hasta las 32 h. CRL1702 incrementó de 3.00x106 a 6.00x108 ufc/ml y se mantuvo hasta el final del ensayo, mientras CRL1696 fue el que menos creció. En FF, todas las BLAB crecieron de manera diferente. CRL1702 y CRL1695 de 1.50x106 a 1.00x1010 a las 24 h, mientras las restantes solo incrementaron 2 U.log. CRL1461 y CRL1696 crecieron a menores niveles. Todas las BLAB disminuyeron el pH en los medios ensayados, y las cepas con mayor descenso de pH fueron CRL1831, CRL1833 y CRL 1421 (pH=3,26; 3,35 y 3,46 y 3,40; 3,56 y 3,66 a las 24 h, respectivamente) tanto en MSFVB FL y FF. Todas las BLAB cultivadas en MSFVB mantuvieron las PB por las que se seleccionaron. CRL1412, CRL1421, CRL1655 y CRL1831 mantuvieron la producción de H2O2. La auto-agregación de CRL1693 en MSFVB FF y FL fue 60% y 85%, respectivamente, mientras CRL 1461 la incrementó (63%) en MSFVB FF, al comparar con la inicial (51%). CRL1693 continuó inhibiendo a S. aureus. Las tres cepas EPS+ (CRL1412, CRL1831 y CRL1833) produjeron exopolisacáridos y 1833 incrementó su hidrofobicidad (91%) en MSFVB.Los resultados obtenidos muestran que las cepas ensayadas se conservan viables y mantienen las PB en un medio que simula el tracto en que se aplicarán, y pueden incluirse en el diseño de fórmulas probióticas veterinarias de aplicación vaginal.Fil: Miranda, Maria Hortencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Nader, Maria Elena Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad del Norte Santo Tomás de Aquino; ArgentinaLII Reunión Anual y Primer Congreso Virtual de la Asociación Argentina de Farmacología ExperimentalCiudad Autónoma de Buenos AiresArgentinaAsociación Argentina de Farmacología Experimenta

Safety, environmental and technological characterization of beneficial autochthonous lactic bacteria, and their vaginal administration to pregnant cows for the design of homologous multi-strain probiotic formulas

In recent years, veterinary probiotic formulations constitute an interesting alternative to the use of antibiotics in animals for human consumption, but beneficial microorganisms must meet certain requirements to be included in these products. The objective of this work was to evaluate the safety and innocuity of beneficial autochthonous lactic bacteria (BALB) as well as to determine their beneficial, environmental, and technological characterization. Antibiotic resistance was assayed using phenotypic and genotypic methodology. A bovine vaginal fluid simulated medium (MSBVF) was designed where growth, pH changes, and expression of beneficial characteristics of lactic bacteria were evaluated; additionally, the optimal culture conditions in commercial media were determined in order to obtain the highest biomass production of the strains. Finally, the best strains were lyophilized and administered intravaginally to pregnant cows and their permanence in the vagina and adverse effects were evaluated. The results show that most of the strains were resistant to vancomycin, tetracycline, and streptomycin, with a high sensitivity to ampicillin, gentamicin, and clindamycin. The strains evaluated did not show gelatinase or hyaluronidase activity; however, 11 strains produced α-type hemolysis. The optimal growth of the microorganism was obtained in MRS broth, under slight agitation and without pH control. The strains grown in the MSBVF grew well and maintained the probiotic properties. Animals treated with probiotics bacteria did not show systemic or local inflammation. These strains can be included in a probiotic veterinary product to be applied to different bovine mucosa.Fil: Miranda, Maria Hortencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Aristimuño Ficoseco, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Nader, Maria Elena Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

Safety and growth optimization of lactic acid bacteria isolated from feedlot cattle for probiotic formula design

In order to eliminate the widespread use of antibiotics in livestock production, the research for alternatives has increased lately. This study examined the safety of 40 lactic acid bacteria (LAB) isolated from bovine feedlot environment and previously selected as potential probiotics. A high sensitivity prevalence to ampicillin (AMP, 100%), gentamicin (GEN, 96.3%), kanamycin (KAN, 96.3%), clindamycin (CLI, 85.2%), chloramphenicol (CHL, 92.6%) and streptomycin (STR, 88.9%) while moderate and high resistance against erythromycin (ERY, 48%) and tetracycline (TET, 79%) respectively, were determined. Feedlot enterococci and pediococci displayed high resistance to CLI, ERY, GEN and TET (73, 100, 54.5, and 73%, respectively). Among fifteen resistance genes investigated, seven were identified in lactobacilli; their presence not always was correlated with phenotypic resistance. STR resistance genes, aadA and ant(6) were observed in 7.4 and 3.7% of isolates, respectively; genes responsible for aminoglycosides resistance, such as bla (7.4%), and aph(3?)-III (3.7%) were also recognized. In addition, resistance cat and tetS genes (3.7 and 7.4%, respectively) were harbored by feedlot lactobacilli strains. The presence of ermB gene in 22.3% of isolates, including two of the six strains phenotypically resistant to ERY, exhibited the highest prevalence among the assessed antibiotics. None of the feedlot lactobacilli harbored virulence factors genes, while positive PCR amplification for ace, agg, fsrA, and atpA genes was found for enterococci. With the objective of producing large cell biomass for probiotic delivery, growth media without peptone but containing glucose and skim milk powder (Mgl and Mlac) were selected as optimal. Lactobacillus acidophilus CRL2074, L. amylovorus CRL2115, L. mucosae CRL2069, and L. rhamnosus CRL2084 were strains selected as free of antibiotic resistance and virulence determinants, able to reach high cell numbers in non-expensive culture media and being compatible among them.Fil: Aristimuño Ficoseco, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Mansilla, Flavia Ivana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Maldonado, Natalia Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Miranda, Maria Hortencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Nader, Maria Elena Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Vignolo, Graciela Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

Effect of probiotic lactobacilli supplementation on growth parameters, blood profile, productive performance, and fecal microbiology in feedlot cattle

Lactic acid bacteria (LAB) selected on the basis of probiotic characteristics were administered to beef feedlot catlle and the effect on body condition/growth and nutritional-metabolic status as well as on E. coli O157:H7 fecal shedding, were investigated. A feeding trials involving 126 steers were used to evaluate the effects of Lactobacillus acidophilus CRL2074, Limosilactobacillus fermentum CRL2085 and Limosilactobacillus mucosae CRL2069 and their combinations (5 different probiotic groups and control) when 107-108 CFU/animal of each probiotic group were in-feed supplemented. Cattle were fed a high energy corn-based diet (16 to 88%) and samples from each animal were taken at 0, 40, 104 and 163 days. In general, animals body condition and sensorium state showed optimal muscle-skeletal development and behavioral adaption to confinement; no nasal/eye discharges and diarrheic feces were observed. The nutritional performance of the steers revealed a steady increase of biometric parameters and weight. Animals supplied with L. mucosae CRL2069 for 104 days reached the maximum mean live weight (343.2 kg), whereas the greatest weight daily gain (1.27 ± 0.16 Kg/day) was obtained when CRL2069 and its combination with L. fermentum CRL2085 (1.26 ± 0.11 kg/day) were administered during the complete fattening cycle. With several exceptions, bovine cattle blood and serum parameters showed values within referential ranges. As a preharvest strategy to reduce Escherichia coli O157:H7 in cattle feces, CRL2085 administered during 40 days decreased pathogen shedding with a reduction of 43% during the feeding period. L. fermentum CRL2085 and L. mucosae CRL2069 show promise for feedlot cattle feeding supplementation to improve metabolic-nutritional status, overall productive performance and to reduce E. coli O157:H7 shedding, thus decreasing contamination chances of meat food products.Fil: Mansilla, Flavia Ivana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Miranda, Maria Hortencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Uezen, Jose David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Maldonado, Natalia Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: D'urso Villar, Marcela Adriana. Universidad Nacional de Tucumán; ArgentinaFil: Merino, Luis Antonio. Universidad Nacional del Nordeste; ArgentinaFil: Vignolo, Graciela Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Nader Macias, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

Brazilian Flora 2020: Leveraging the power of a collaborative scientific network

International audienceThe shortage of reliable primary taxonomic data limits the description of biological taxa and the understanding of biodiversity patterns and processes, complicating biogeographical, ecological, and evolutionary studies. This deficit creates a significant taxonomic impediment to biodiversity research and conservation planning. The taxonomic impediment and the biodiversity crisis are widely recognized, highlighting the urgent need for reliable taxonomic data. Over the past decade, numerous countries worldwide have devoted considerable effort to Target 1 of the Global Strategy for Plant Conservation (GSPC), which called for the preparation of a working list of all known plant species by 2010 and an online world Flora by 2020. Brazil is a megadiverse country, home to more of the world's known plant species than any other country. Despite that, Flora Brasiliensis, concluded in 1906, was the last comprehensive treatment of the Brazilian flora. The lack of accurate estimates of the number of species of algae, fungi, and plants occurring in Brazil contributes to the prevailing taxonomic impediment and delays progress towards the GSPC targets. Over the past 12 years, a legion of taxonomists motivated to meet Target 1 of the GSPC, worked together to gather and integrate knowledge on the algal, plant, and fungal diversity of Brazil. Overall, a team of about 980 taxonomists joined efforts in a highly collaborative project that used cybertaxonomy to prepare an updated Flora of Brazil, showing the power of scientific collaboration to reach ambitious goals. This paper presents an overview of the Brazilian Flora 2020 and provides taxonomic and spatial updates on the algae, fungi, and plants found in one of the world's most biodiverse countries. We further identify collection gaps and summarize future goals that extend beyond 2020. Our results show that Brazil is home to 46,975 native species of algae, fungi, and plants, of which 19,669 are endemic to the country. The data compiled to date suggests that the Atlantic Rainforest might be the most diverse Brazilian domain for all plant groups except gymnosperms, which are most diverse in the Amazon. However, scientific knowledge of Brazilian diversity is still unequally distributed, with the Atlantic Rainforest and the Cerrado being the most intensively sampled and studied biomes in the country. In times of “scientific reductionism”, with botanical and mycological sciences suffering pervasive depreciation in recent decades, the first online Flora of Brazil 2020 significantly enhanced the quality and quantity of taxonomic data available for algae, fungi, and plants from Brazil. This project also made all the information freely available online, providing a firm foundation for future research and for the management, conservation, and sustainable use of the Brazilian funga and flora