Morphometric and gut microbial evaluation of Tilapia (Oreochromis niloticus) fed on different levels of Moringa oleifera.

In current study, different feeding levels of Moringa oleifera formulated diet was compared to analyze the growth performance, feed conversion ratio, feed conversion efficiency and gut microbiology of Oreochromis niloticus. The study was comprised of four treatment groups including 4%, 8% and 12% Moringa oleifera and one control group which was devoid of Moringa leaves. The experimental trial was conducted at the Zoology laboratory of Pakistan Institute of Applied and Social Sciences, (PIASS) Kasur. The physicochemical parameters of water such as temperature, dissolve oxygen, pH, total dissolved solids and salinity in all aquaria were found non-significantly different from each other. In control condition T1, the average weight gain was 14.89±16.90a grams, while average length gain was 11.52±7.444a cm. However, the total viable count on Eosin methylene blue was 7.4×107, 5.8×107 on Tryptic soy agar and 5.8×107on Nutrient agar. In T2, the average weight gain was 16.22±16.09b grams and average length gain was 12.97±7.79b cm. The total viable count on Eosin methylene blue was 7×107, 5.5×107 on Tryptic soy agar and 5.8×107on Nutrient agar. In T3, the average weight gain was 37.88±27.43c grams, while the average length gain was recorded as 16.48±12.56c cm. However, the total viable count for treatment 3 was 6.4×10 on Eosin methylene blue, 4.8×107 on Tryptic soy agar and 5.2×107on Nutrient agar. In T4, the average weight gain was 44.22±31.67d grams, while the average length gain was 15.25±10.49d cm. The total viable count was 4.3×107on Eosin methylene blue, 3.1×107 on Tryptic soy agar and 3.8×107 on Nutrient agar. The effect of Moringa oleifera on the growth of Oreochromis niloticus was found to be significant and 12% Moringa extract showed maximum length and weight gain and minimum feed conversion ratio with the least microbial count in fish intestine.

Fatores de virulência e susceptibilidade a antifúngicos de Cryptococcus Spp / Virulence factors and susceptibility to Cryptococcus Spp. antifungals

Criptococose é uma doença grave que afeta tanto imunocomprometidos quanto imunocompetentes, com isso analisar a virulência é fundamental para novas terapêuticas. Objetivo: Analisar a capacidade de virulência e susceptibilidade aos antifúngicos de Cryptococcus spp. isolados de líquor de pacientes de hospital do norte do Paraná. Métodos: A partir de dois isolados clínicos C. neoformans e C. gattii, realizou-se a confirmação da identificação. Para a virulência, avaliou-se o tamanho da cápsula, capacidade de sobrevivência após exposição a neutrófilos, produção de melanina e urease. No antifungigrama por difusão em disco utilizou-se: anfotericina B, cetoconazol, voriconazol, itraconazol e miconazol. Resultados: C. gattii destaca-se por maior desenvolvimento da cápsula além da melhor capacidade de sobreviver a fagocitose em relação ao C. neoformans. No antifungigrama, ambos os isolados se apresentam sensíveis às drogas estudadas. Conclusão: Esses achados contribuem para a compreensão das diferentes patogêneses entre C. gattii e C. neoformans.

Cryptococcosis is a serious disease that can affect both immunocompromised and immunocompetent individuals, thus the virulence analysis is fundamental for the development of new treatments. Objective: To analyze the virulence and susceptibility of Cryptococcus spp. isolated from cerebrospinal fluid of patients from a hospital in the north of Paraná. Methods: From two clinical isolates, C. neoformans and C. gattii were confirmed and identified. For virulence, capsule size, survival capacity after exposure to neutrophils, melanin production and urease were evaluated. In the disc-diffusion method, the following antifungals were used: amphotericin B, ketoconazole, voriconazole, itraconazole and miconazole Results: It was observed that C. gattii presents greater results for development of the capsule beside presenting the best ability to survive phagocytosis in relation to C. neoformans. In the disc-diffusion method, both isolates presented sensitivity to the studied drugs. Conclusion: These findings contribute to the understanding of the different pathogens between C. gattii and C. neoformans.

Importance of the agar-media in the evaluation of bacteriocin activity against the same test-microorganisms

Abstract Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria, which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. Successful application of techniques for quantitative or qualitative bacteriocin determination relies not only on the sensitivity of the test-microorganisms, but also on the agar-medium employed. Cell free supernatants are routinely used to preliminary screen for antimicrobial activity of bacteria by means of the agar well diffusion method, but the supernatant may also include other molecules (such as medium components and/or intracellular compounds) accidentally released during cell free supernatant preparation, which may interfere with the assay. Reproducibility of bacteriocin activity against the same test-microorganisms is an important factor to be considered. Unfortunately, no specific information about bioassays standardization to determine bacteriocin activity is available in the literature. In this work, growth inhibition by means of the agar well diffusion assays were carried out on different agar-media showing a strong dependence on the agar-medium used, indicating that the inhibitory effects could also depend on the diffusion of exudates that are included in the cell-free supernatant. The results presented in this communication show that selection of the agar-medium is crucial for the bioassay response.

Avaliação das técnicas, imunodifusão em gel de agar (IDGA), ensaio imunoenzimático (ELISAi) e reação em cadeia da polimerase (PCR), para o diagnostico da brucelose ovina / Evaluation of technical, immunodiffusion agar gel (AGID), enzyme-linked immunosorbent assay (ELISAi) and polymerase chain reaction (PCR) for the diagnosis of ovine brucellosis

Brucellosis in sheep has received a major focus, since it is a disease that affects the reproductive systemof animals, causing serious impairment in the productive sector. Thus, three methods for the diagnosis of ovinebrucellosis were evaluated as goal, the indirect ELISA test, the AGID technique and the PCR. Therefore, weused 211 sheep blood samples arising from properties of nine municipalities of the homogeneous micro-regionof Teresina, Piaui. The 211 blood samples were subjected to the serologic testing and PCR to detect anti-B. ovisantibodies, and Brucella ovis DNA, respectively. Positive results in serological tests were obtained, 36 (17.06%)positive in the AGID test and seven (3.31%) positive to the ELISA test, however, there were no positive results inthe PCR technique. The use of the techniques for the diagnosis of B. ovis infection is satisfactory, but the choiceof blood as the biological sample for performing the direct diagnosis of ovine brucellosis is not recommended.

Avaliação das técnicas, imunodifusão em gel de agar (IDGA), ensaio imunoenzimático (ELISAi) e reação em cadeia da polimerase (PCR), para o diagnostico da brucelose ovina / Evaluation of technical, immunodiffusion agar gel (AGID), enzyme-linked immunosorbent assay (ELISAi) and polymerase chain reaction (PCR) for the diagnosis of ovine brucellosis

Brucellosis in sheep has received a major focus, since it is a disease that affects the reproductive systemof animals, causing serious impairment in the productive sector. Thus, three methods for the diagnosis of ovinebrucellosis were evaluated as goal, the indirect ELISA test, the AGID technique and the PCR. Therefore, weused 211 sheep blood samples arising from properties of nine municipalities of the homogeneous micro-regionof Teresina, Piaui. The 211 blood samples were subjected to the serologic testing and PCR to detect anti-B. ovisantibodies, and Brucella ovis DNA, respectively. Positive results in serological tests were obtained, 36 (17.06%)positive in the AGID test and seven (3.31%) positive to the ELISA test, however, there were no positive results inthe PCR technique. The use of the techniques for the diagnosis of B. ovis infection is satisfactory, but the choiceof blood as the biological sample for performing the direct diagnosis of ovine brucellosis is not recommended.(AU)

Isolation of Campylobacter from Brazilian broiler flocks using different culturing procedures.

Conventional culturing methods enable the detection of Campylobacter in broiler flocks. However, laboratory culture of Campylobacter is laborious because of its fastidious behavior and the presence of competing nontarget bacteria. This study evaluated different protocols to isolate Campylobacter from broiler litter, feces, and cloacal and drag swabs. Samples taken from commercial Brazilian broiler flocks were directly streaked onto Preston agar (PA), Campy-Line agar (CLA), and modified charcoal cefoperazone deoxycholate agar (mCCDA) and also enriched in blood-free Bolton broth (bfBB) for 24 and 48 h followed by plating onto the different selective media. Higher numbers of Campylobacter-positive cloacal and drag swab samples were observed using either direct plating or enrichment for 24 h before plating onto PA, compared with enrichment for 48 h (P < 0.05). Furthermore, direct plating was a more sensitive method to detect Campylobacter in broiler litter and feces samples. Analysis of directly plated samples revealed that higher Campylobacter levels were detected in feces streaked onto PA (88.8%), cloacal swabs plated onto mCCDA (72.2%), drag swabs streaked onto CLA or mCCDA (69.4%), and litter samples inoculated onto PA (63.8%). Preston agar was the best agar to isolate Campylobacter from directly plated litter samples (P < 0.05), but there was no difference in the efficacies of PA, mCCDA, and CLA in detecting Campylobacter in other samples. The isolated Campylobacter strains were phenotypically identified as Campylobacter jejuni or Campylobacter coli. The predominant contaminant observed in the Campylobacter cultures was Proteus mirabilis, which was resistant to the majority of antimicrobial agents in selective media. Together, these data showed that direct plating onto PA and onto either CLA or mCCDA as the second selective agar enabled the reliable isolation of thermophilic Campylobacter species from broiler samples. Finally, Campylobacter was detected in all broiler flocks sampled.

Comparison of selective agars recommended by method ISO 11290-1 and chromogenic agars for the isolation of Listeria sp. in refrigerated sausages

The aim of this study was to determine the prevalence of Listeria sp. in refrigerated sausages, and to compare the performance of the selective plating media employed in the ISO 11290-1 method (PALCAM and Oxford agars) with chromogenic agars (Chromogenic Listeria agars CM 1080 (OCLA) and CM 1084). The prevalence of Listeria sp. detected was 52.9%, comprising 13.7% L. monocytogenes strains. The efficacy of the four agars for the isolation of L. monocytogenes proved to be satisfactory. Despite differences in composition of the chromogenic media assessed, these disparities did not affect concordance among results. However, PALCAM agar was shown to suppress other microorganisms more effectively, being more applicable for detecting Listeria strains present in lower quantities. Based on these results, the use of PALCAM agar, in combination with a chromogenic media, is recommended for enhanced isolation of atypical Listeria sp. strains in meat products.

Este estudo teve como objetivo a análise da prevalência de Listeria sp. em linguiças resfriadas e a comparação dos meios seletivos utilizados no plaqueamento do método ISO 11290-1 (Ágar PALCAM e Ágar Oxford), e ágares cromogênicos (Ágares Listeria Cromogênico CM 1080 (OCLA) e CM 1084 (ISO)). A frequência de Listeria sp. foi de 52,9%, sendo que destas, 13,7% corresponderam à L. monocytogenes. A eficácia dos quatro ágares para o isolamento de L. monocytogenes demonstrou-se satisfatória. Apesar de haver algumas diferenças nas composições dos meios cromogênicos analisados, estas não pareceram influenciar nas concordâncias entre os resultados expressos. Contudo, o ágar PALCAM mostrou-se mais eficaz na supressão de outros micro-organismos, aumentando, assim, a possibilidade de detecção de espécies de Listeria presentes em número reduzido. Através deste trabalho sugere-se a utilização do ágar PALCAM associado a um meio cromogênico para aumentar a chance de isolamento de cepas atípicas de Listeria sp. em produtos cárneos.

Physicochemical properties of biodegradable polyvinyl alcohol-agar films from the red algae Hydropuntia cornea.

Agar obtained from the red alga Hydropuntia cornea was blended with polyvinyl alcohol (PVOH) in order to produce biodegradable films. In this study, we compare the properties of biopolymeric films formulated with agars extracted from H. cornea collected at different seasons (rainy and dry) in the Gulf of Mexico coast and PVOH as synthetic matrix. The films were prepared at different agar contents (0%, 25%, 50%, 75%, and 100%) and their optical, mechanical, thermal, and morphological properties analyzed. The tensile strength of PVOH-agar films increased when agar content was augmented. The formulation with 50% agar from rainy season (RS) had a significant higher tensile strength when compared to those from dry season (DS; p < 0.05). Tensile modulus also displayed an increasing trend and likewise, for 50% and 75% agar blends from RS showed higher values than those from DS (p < 0.05). In contrast, elongation at break decreased as the agar content increased, independently of the season. Environmental scanning electron microscopy images of PVOH-agar 75% biofilms from RS showed a homogeneous structure with good interfacial adhesion between the two components. The changes evidenced in the FTIR spectrum of this blend suggest that hydrogen bonding is taking place between the agar ether linkages (C-O-C) and the hydroxyl groups (OH) of the PVOH. Based on the above mentioned results, blends of PVOH and 75% agar from H. cornea collected in rainy season showed good properties for applications in the biodegradable packaging industry.

DRBC agar: a new tool for Candida dubliniensis identification.

Candida dubliniensis pathogenic species, which shares many phenotypic features with C. albicans, may be misidentified in the microbiology laboratory. The growth on DRBC agar at 25 degrees C was shown to be a new tool for differentiation between C. dubliniensis and C. albicans. All 27 isolates of C. dubliniensis showed in this medium rough colonies (peripheral hyphal fringes) and abundant chlamydospore production, while all 103 isolates of C. albicans showed smooth colonies without fringes or chlamydospores. DRBC agar allowed the differentiation of C. albicans from C. dubliniensis with 100 % sensitivity and specificity.

Influence of probiotics on Candida presence and IgA anti-Candida in the oral cavity

Probiotics are defined as microorganisms that promote benefits to host health, mainly by regulating resident microbiota. Disequilibrium in microbiota can favor the growth of opportunist microorganisms and the development of pathologies, like candidosis caused by yeasts of the Candida genus. This work evaluated whether probiotics consumption was able to influence a specific immunological response to Candida and the presence of these yeasts in the oral cavity. Saliva samples were collected from healthy individuals and plated in Dextrose Saboraud Agar with chloramphenicol. Individuals presenting Candida in the oral cavity used the probiotic Yakult LBâ for 20 days, after which new collections and identifications were performed. Anti-Candida IgA analysis was conducted using the ELISA technique. Analysis of the results showed a significant reduction in Candida prevalence (46 percent) and mean Candida CFU/mL counts (65 percent). The Candida species identified were C. albicans (98 percent) and C.tropicalis (2 percent), before and after probiotics consumption. Immunological analysis demonstrated a significant reduction in anti-Candida IgA levels after probiotics use, probably due to less antigenic stimulation. In conclusion, in the individuals studied, probiotics use significantly reduced the amount of Candida in the oral cavity, possibly due to competition between the yeasts rather than by specific secretory immune response stimulation.

Detection of salmonella sp in chicken cuts using immunomagnetic separation

The immunomagnetic separation (IMS) is a technique that has been used to increase sensitivity and specificity and to decrease the time required for detection of Salmonella in foods through different methodologies. In this work we report on the development of a method for detection of Salmonella in chicken cuts using in house antibody-sensitized microspheres associated to conventional plating in selective agar (IMS-plating). First, protein A-coated microspheres were sensitized with polyclonal antibodies against lipopolysacharide and flagella from salmonellae and used to standardize a procedure for capturing Salmonella Enteritidis from pure cultures and detection in selective agar. Subsequently, samples of chicken meat experimentally contaminated with S. Enteritidis were analyzed immediately after contamination and after 24h of refrigeration using three enrichment protocols. The detection limit of the IMS-plating procedure after standardization with pure culture was about 2x10 CFU/mL. The protocol using non-selective enrichment for 6-8h, selective enrichment for 16-18h and a post-enrichment for 4h gave the best results of S. Enteritidis detection by IMS-plating in experimentally contaminated meat. IMS-plating using this protocol was compared to the standard culture method for salmonellae detection in naturally contaminated chicken cuts and yielded 100 percent sensitivity and 94 percent specificity. The method developed using in house prepared magnetic microespheres for IMS and plating in selective agar was able to diminish by at least one day the time required for detection of Salmonella in chicken products by the conventional culture method.

A separação imunomagnética (IMS) é uma técnica que tem sido associada a diferentes métodos de detecção de Salmonella em alimentos para aumentar a sensibilidade e a especificidade e diminuir o tempo de análise. Neste trabalho é comunicada a obtenção de microesferas magnéticas sensibilizadas com anticorpos anti-Salmonella e seu uso em associação com a metodologia de cultivo convencional para desenvolver um método de detecção de salmonelas em cortes de frango (IMS-plaqueamento). Inicialmente, microesferas cobertas com proteína A foram sensibilizadas com anticorpos policlonais contra lipopolissacarídeo e flagelo de salmonelas e usadas na padronização de um procedimento que captura Salmonella Enteritidis em cultivo puro e faz posterior detecção em ágar seletivo. A seguir, amostras de carne de frango experimentalmente contaminadas com S. Enteritidis foram analisadas imediatamente após a contaminação e após 24h de refrigeração utilizando três protocolos de enriquecimento. O limite de detecção foi cerca de 2x10 UFC/mL. O protocolo que incluiu enriquecimento não-seletivo por 6-8h, enriquecimento seletivo por 16-18h e pós-enriquecimento por 4h foi o que proporcionou melhor resultado na detecção de S. Enteritidis em carne de frango experimentalmente contaminada. Este protocolo foi comparado à metodologia convencional em estudo de detecção de salmonelas em cortes de frango naturalmente contaminados e obteve 100 por cento de sensibilidade e 94 por cento de especificidade. O método desenvolvido foi capaz de diminuir em pelo menos um dia o tempo de detecção de salmonelas em cortes de frango pela metodologia convencional.

Comparison of microbiological and UV-spectrophotometric assays for determination of voriconazole in tablets.

Two methods have been developed for the determination of voriconazole, a new antifungal drug, in tablets. A UV method, with detection at 255 nm, was compared with a diffusion agar bioassay, which used Sacharomyces cerevisiae ATCC 2601 as the assay organism. The developed methods were linear in the range of 3.0-12.0 and 12.0-24.0 microg/mL, for the microbiological and UV methods, respectively, both exhibiting a coefficient correlation of 0.9999. The UV method demonstrated an improved precision compared to the bioassay method (1.0 versus 2.4%). The average recovery, 99.8 and 100.9%, was suitable in both methods. The results obtained by these 2 methods were compared with those of a high-performance liquid chromatography (HPLC) method published previously, and no evidence of significant difference was observed. The proposed methods are appropriate for the determination of voriconazole in tablets and can be used in routine quality control.

Seasonal variation in the biomass and agar yield from Gracilaria cervicornis and Hydropuntia cornea from Brazil.

Seasonality of biomass and agar yield from two agarophytes (G. cervicornis and H. cornea) was determined. The biomass from G. cervicornis was higher (390 g m-2) during the dry season and lower during the rainy season (129 g m-2). The data analysis for G. cervicornis revealed a significant seasonal variation (P < 0.05). H. cornea did not show a clear seasonal variation and was present only from March to August. The peak in biomass for this species was recorded in April (383 g m-2) and was significantly different from the other months (P < 0.05). The agar yield for G. cervicornis varied from 11% to 20%, with generally higher values recorded during the dry season. The agar yield showed a highly significant variation (P < 0.001). Agar yield from H. cornea ranged from 29% to 41%, with a peak recorded in June. The results above indicate that H. cornea can be considered a good candidate for commercial use.

Determinación del crecimiento de saccharornyces cerevisiae en agares formulados con extractos de desechos agroindustriales I / Determination of growth of saccharomyces cerevisiae in agars formulated with agroindustrial waste extracts I

Se utilizó saccharomyces cerevisiae para determinar cuantitativamente su crecimiento en agares formulados en base a extractos obtenidos de desechos de lactuca sativa (lechuga) y brassica oleracea (repollo), aserrín de pinus radiata, papel de diario de desecho y melaza como sustratos alternativos para el cultivo de esta levadura, con miras a la producción de proteínas unicelulares (SCP). Se prepararon los siguientes medios de cultivos: agar extracto vegetal (AFV), agar extracto aserrín (AFA), agar extracto papel (AEP) y agar extracto melaza (AEME), solos y suplementados con glucosa y NH4NO3. Estos se sembraron con diluciones de la levadura e incubaron por 48 h a 25ºC, tras lo cual se hizo el recuento de las colonias. Como control se utilizó agar extracto de malta al 2 porciento (AEM). El mayor recuento poblacional se registró en AEV (7.2x 105 ufc/ml). A los 10 días de incubación, se obtuvieron colonias de 5mm de diám. en AEV y solo puntiformes en AEP

Detection of extracellular proteases from microorganisms on agar plates

We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin) incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity od proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced proyease liberation. Our results that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

Inhibición del crecimiento de pseudomonas aeroginosa en la determinación de salmonellae / Growth inhibit of pseudomonas aeroginosa to salmonellae determination

Los caldos selectivos con cisteina HCL 0.05 por ciento, cubiertos con parafina resularon ser mas efectivas para la inhibición de pseudomonas aeroginosa que los caldos cisteinadas sin cubiertas de parafina. El caldo selenita fue más efectivo para dicha inhibición que el calcio tetrationate. El agar desoxicolato citrato (incubado aeróbica o anaeróbicamente) resultó el más efectivo para la inhibición, especialmente cuando se aisló a partir de caldos cisteinados parafinadas. El agar verde brillante rojo de fenal-lactosa incubada aeróricamente tambien resultó util para dicha inhibición, cuando se aisló a partir de caldo solenito cisteinado y parafinado. Siendo no recomendable la incubación anaeróbica debido a las variaciones de forma y color de las colonias. El agar bismuto sulfito es muy buen medio de cultivo respecto a la diferenciación de las colonias de ambos microorganismos, pero no se logró inhibir totalmente el crecimiento de pseudomonas, aunque hubo una notable disminución del crecimiento, cuando se incubó el agar bajo condiciones anaeróbicas.