Effects of probiotic Lactobacillus gasseri strains of breast-fed infant in a murine model

The ingestion of probiotic lactic acid bacteria has been evaluated and noted that it has an effect on the balance of desirable microbiota in the gastrointestinal tract. Lactobacillus gasseri demonstrates good survival in the gastrointestinal tract, and it has been associated with a variety of probiotic activities and roles, including the reduction of fecal mutagenic enzymes, the production of bacteriocins and the stimulation of macrophages immunomodulation. The aim of the study was to evaluate the effects of a pool of L. gasseri strains isolated from the feces of breastfed infants added in the human milk of healthy women. The milk was both pasteurized and unpasteurized, to verify the cell cytotoxicity of macrophages and to quantify the production of immunologic mediators such as IL-4, IL-6, IFN-g, TNF-a, NO and oxygen intermediary compounds (H2O2). The administration of raw human milk and pasteurized human milk to infants is a regular, encouraged practice in units of intensive therapy (UITs) and our present investigation verified the beneficial effect of addition of a pool of L. gasseri to pasteurized human milk (PHML). Our results show that probiotic supplementation helped to maintain cell viability, reduced IL-6 and IFN-γ production and stimulated TNF-α, NO, H2O2, IL-4 production. Nevertheless, the results indicate that the addition of lactobacillus to human milk was not a determinant in the production of TNF-α. L. gasseri added to breast milk did not present a cytotoxic risk, and the addition of L. gasseri to pasteurized milk of human milk bank would benefit newborns that depend on milk banks for the colonization of more desirable microbiota.

Unraveling Trichoderma species in attine ant environment: description of three new taxa

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Taxonomia e potencial antimicrobiano de fungos depositados no acervo de pesquisa da Central de Recursos Microbianos da UNESP

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo de genes de Candida albicans com função desconhecida quanto à formação de biofilme, características biológicas e interação patógeno- hospedeiro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação in vitro do crescimento micelial de cinco linhagens de Agaricus blazei em duas temperaturas

The objective of the work was to evaluate the in vitro mycelial growth of five A. blazei strains (ABL-05/53, ABL-04/49, ABL-03/44, ABL-99/30 and ABL-02/51) when submitted to the temperatures of 20 and 25 ºC. In a laminar flow chamber, discs of the strains were inoculated in the middle of Petri’s plates containing CA (compost-agar) medium and incubated in BOD. After 48 hours, measurements of the mycelial growth began, with the help of a ruler with scale in millimeters, by means of four equidistant measurements, until the moment when the fungal colony reached near the edges of the Petri’s plate in one of the treatments. The experimental design was totally randomized, in 5 x 2 factorial design. Each treatment consisted of seven repetitions, corresponding to one Petri’s plate, totalizing seventy experimental units. We verified that A. blazei growth is influenced by incubation temperature, being that the temperature of 25 ºC was more favorable for the mycelial growth of all A. blazei strains tested, with attention for ABL-04/49 and ABL-03/44 strains, which obtained the highest averages for mycelial growth under this temperature condition at the end of the cultivation cycle.

Strains of Coniothyrium minitans reduce the emission of apothecia of Sclerotinia sclerotiorum

Sclerotia of Sclerotinia sclerotiorum (Ss) can survive for long time in soil and are the main inoculum source of the white mold disease. An alternative for reducing this inoculum is the use of parasites, such as Coniothyrium minitans (Cm). We evaluated the potential of Cm isolates for the biological control of Ss in beans. The effect of the temperature on the growth of 15 isolated of Cm was evaluated in vitro. The hyperparasitism ability of Cm was evaluated in soil infested with sclerotia and conditioned in pots. The infested soil was treated with conidia suspension of the antagonists, fluazinan or sterile distilled water. After seven days at 20°C, the sclerotia were removed from soil and placed inside Petri dishes over bean leaves previously disinfested. The germination and parasitism of sclerotia were evaluated after 7 to 10 days. To evaluate the apothecia emission, soil infested with sclerotia of Ss and treated as described was maintained at 18°C and the number of emerged apothecia was counted up to 84 days after inoculation. The emergence of bean plants in soil infested with sclerotia and mycelium of the pathogen and treated as described was evaluated in greenhouse. The ideal temperature for growth of Cm isolates varied from 18 to 19°C and at 30-35°C they were complete inhibited. The isolates of Cm promoted less than 10% of reduction in viability of the sclerotia, but they significantly reduced the emission of apothecia. Two isolates increased the emergence of plants in relation to the inoculated check, but was significantly lower than the non-inoculated check. Field tests will be conduct to confirm the potential of the selected isolates to reduce the inoculum source of the pathogen.

Yield of different white button strains in sugar cane by product-based composts

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular characterization of Pleurotus ostreatus commercial strains by random amplified polymorphic DNA (RAPD)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Monitoring of Transmission of Tuberculosis between Wild Boars and Cattle: Genotypical Analysis of Strains by Molecular Epidemiology Techniques

An epidemiological survey for the monitoring of bovine tuberculosis transmission was carried out in western Liguria, a region in northern Italy. Fifteen Mycobacterium bovis strains were isolated from 63 wild boar samples (62 from mandibular lymph nodes and 1 from a liver specimen). Sixteen mediastinal lymph nodes of 16 head of cattle were collected, and 15 Mycobacterium bovis strains were subsequently cultured. All M. bovisstrains isolated from cattle and wild boars were genotyped by spoligotyping and by restriction fragment length polymorphism (RFLP) analysis with the IS6110 and IS1081 probes. All M. bovis strains showed the typical spoligotype characterized by the absence of the 39 to 43 spacers in comparison with the number in M. tuberculosis. A total of nine different clusters were identified by spoligotyping. The largest cluster included 9 strains isolated from wild boars and 11 strains isolated from cattle, thus confirming the possibility of transmission between the two animal species. Fingerprinting by RFLP analysis with the IS6110 probe showed an identical single-band pattern for 29 of 30 strains analyzed, and only 1 strain presented a five-band pattern. The use of IS1081 as a second probe was useful for differentiation of M. bovis from M. bovis BCG but not for differentiation among M. bovis strains, which presented the same undifferentiated genomic profile. In relation to the epidemiological investigation, we hypothesized that the feeding in pastures contaminated by cattle discharges could represent the most probable route of transmission of M. bovis between the two animal species. In conclusion, our results confirmed the higher discriminatory power of spoligotyping in relation to that of RFLP analysis for the differentiation of M. bovis genomic profiles. Our data showed the presence of a common M. bovis genotype in both cattle and wild boars, confirming the possible interspecies transmission of M. bovis.

Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White-tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock and the cause for many faltering bovine tuberculosis eradication programs. One approach in dealing with wildlife reservoirs of disease is to interrupt inter-species and intraspecies transmission through vaccination of deer or cattle. To evaluate the efficacy of BCG vaccination in white-tailed deer, 35 deer were assigned to one of three groups; one s.c. dose of 107 CFU of M. bovis BCG Pasteur (n = 12); 1 s.c. dose of 107 CFU of M. bovis BCG Danish (n = 11); or unvaccinated deer (n = 12). After vaccination, deer were inoculated intratonsilarly with virulent M. bovis. Lesion severity scores of the medial retropharyngeal lymph node, as well as all lymph nodes combined, were reduced in vaccinated deer compared to unvaccinated deer. BCG Danish vaccinated deer had no late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid-fast bacilli compared to BCG Pasteur vaccinated or unvaccinated deer where such lesions were present. Both BCG strains were isolated as late as 250 days after vaccination from deer that were vaccinated but not challenged. In white-tailed deer, BCG provides protection against challenge with virulent M. bovis. Issues related to vaccine persistence, safety and shedding remain to be further investigated.

Physiological Studies on Candida albicans

Candida albicans is a common opportunistic, dimorphic human fungal pathogen. One of its virulence factors is the morphological switch between yeasts and hyphal or pseudohyphal forms, which can invade tissues and cause damage. Our studies focus on factors regulating pseudohyphae and epigenetic modifications of C. albicans. Regulating factors of pseudohyphae are aromatic alcohols and high phosphate. At low concentrations, exogenous aromatic alcohols induced pseudohyphae, as did high phosphate. For addressing the pathways involved in inducing pseudohyphae by aromatic alcohols or high phosphate, we used mutants defective in cAMP dependent PKA pathway (efg1/efg1), MAP kinase pathway (cph1/cph1), or both (cph1/cph1/efg1/efg1). These mutants failed to produce either hyphae or pseudohyphae in the presence of aromatic alcohols; but high phosphate still stimulated pseudohyphae. Gcn4, a transcription activator of more than 500 amino acid related genes, is turned-on in response to amino acid starvation. The accumulation of aromatic alcohols sends nitrogen starvation signals, which inhibit eIF2B, which in turn derepresses Gcn4p. High phosphate also induces pseudohyphae by derepressing Gcn4p, although the pathways involved are still unknown. In sum, aromatic alcohols and high phosphate induce pseudohyphae by derepressing Gcn4. In this study we found a novel posttranslational histone modification in C. albicans, which is biotinylation. Western blot and Mass spectrometry techniques were used to find that Histones H2B and H4 were biotinylated at every condition tested such as yeast vs. hyphae, aerobic growth vs. anaerobic growth, rich medium vs. defined medium. In C. albicans lysines K8, K11 in histone H4 and lysines K17, K18, K31 in histone H2B are biotin attachment sites as found using mass spectrometry. Biotin was also found to enhance the germ tube formation of C. albicans. Germ tube formation assays with biotin-starved cells as inoculum showed low percent of germ tubes (1-5%). Addition of biotin to the media showed 100% germ tubes. Biotinylation of histones were not detected from biotin-starved cells. Appendix-A details work related to Farnesol quantification assays in several strains of C.albicans and Ceratocystis ulmi, and growth studies of class E VPS strains of Saccharomyces Cerevisiae. Adviser: Kenneth W. Nickerson

Experimental Infection of Richardson’s Ground Squirrels (Spermophilus Richardsonii ) With Attenuated and Virulent Strains pf Brucella abortus

A previous investigation of the safety of Brucella abortus strain RB51 (sRB51) in various nontarget species suggested that Richardson’s ground squirrels (Spermophilus richardsonii) may develop persistent infections when orally inoculated with the vaccine. In the present study, sRB51, B. abortus strain 19 (s19), and virulent B. abortus strain 9941 (s9941) were administered orally to Richardson’s ground squirrels to further characterize B. abortus infection in this species. Six groups of nongravid ground squirrels were orally inoculated with 6x108 colony forming units (cfu) sRB51 (n=10), 2.5x104 cfu s19 (n=10), 2.5x107 cfu s19 (n=6), 1.3x106 cfu s9941 (n=5), 2.1x108 cfu s9941 (n=5), or vaccine diluent (control; n=4). One of five animals in the lower-dose s19 group and two of three animals in the higher-dose s19 group showed persistence of bacteria in various tissues at 14 wk post-inoculation (PI). At 18 wk PI, one of five animals in the sRB51 group and one of five animals in the high-dose s9941 group were culture positive. Although we did detect some persistence of B. abortus strains at 18 wk, we found no evidence of pathology caused by B. abortus strains in nonpregnant Richardson’s ground squirrels based on clinical signs, gross lesions, and microscopic lesions.

Host-Specificity of Myxoma Virus: Pathogenesis of South American and North American Strains of Myxoma Virus in Two North American Lagomorph Species

The pathogenesis of South American and North American myxoma viruses was examined in two species of North American lagomorphs, Sylvilagus nuttallii (mountain cottontail) and Sylvilagus audubonii (desert cottontail) both of which have been shown to have the potential to transmit the South American type of myxoma virus. Following infection with the South American strain (Lausanne, Lu), S. nuttallii developed both a local lesion and secondary lesions on the skin. They did not develop the classical myxomatosis seen in European rabbits (Oryctolagus cuniculus). The infection at the inoculation site did not resolve during the 20-day time course of the trial and contained transmissible virus titres at all times. In contrast, S. audubonii infected with Lu had very few signs of disseminated infection and partially controlled virus replication at the inoculation site. The prototype Californian strain of myxoma virus (MSW) was able to replicate at the inoculation site of both species but did not induce clinical signs of a disseminated infection. In S. audubonii, there was a rapid response to MSW characterized by a massive T lymphocyte infiltration of the inoculation site by day 5. MSW did not reach transmissible titres at the inoculation site in either species. This might explain why the Californian myxoma virus has not expanded its host-range in North America.

SIGNIFICANCE AND CONTROL OF FUNGAL DISEASES RELATED TO BIRD ROOSTS

Certain fungi have been found frequently as saprophytes in areas containing large amounts of bird excreta. These fungi have the ability to survive, multiply, and cause disease once they have entered a host. Two of these are Crypto-coccus neoformans and Histoplasma capsulatum. Both may easily become airborne and be disseminated throughout an area by the prevailing winds. C. neo-formans is commonly isolated from the excreta of pigeon habitats, and in turn has been associated with clinical cases of cryptococcosis, while blackbird roosts, harboring H. capsulatum, have been responsible for several outbreaks of histoplasmosis. When either of these fungi have become established in nature, the sites may become foci for infection and epidemics may occur if the sites are disturbed. This has led to investigation of these organisms with respect to: 1) the frequency of isolation of H. capsulatum from the soil beneath blackbird roosts in a histoplasmosis endemic area; 2) the infectivity of undisturbed roosts positive for H. capsulatum; and 3) the effectiveness of chemical decontamination of areas containing C. neoformans or H. capsulatum.

Diversity of hydrocarbon-degrading Klebsiella strains isolated from hydrocarbon-contaminated estuaries

To investigate the diversity and the catabolic capacity of oil-degrading Klebsiella strains isolated from hydrocarbon-contaminated sediments in Santos-Sao Vicente estuary systems in Brazil. Klebsiella strains obtained from the estuary were characterized using 16S rRNA gene sequencing and BOX-PCR patterns, testing their catabolic capacity to degrade toluene, xylene, naphthalene and nonane, and identifying the catabolic genes present in the oil-degrading strains. Results show that Klebsiella strains were widespread in the estuary. Twenty-one isolates from the Klebsiella genus were obtained; 14 had unique BOX patterns and were further investigated. Among four distinct catabolic genes tested (todC1, ndoB, xylE and alkB1), only the todC1 gene could be amplified in two Klebsiella strains. The biodegradation assay showed that most of the strains had the ability to degrade all of the tested hydrocarbons; however, the strains displayed different efficiencies. The oil-degrading Klebsiella isolates obtained from the estuary were closely related to Klebsiella pneumoniae and Klebsiella ornithinolytica. The isolates demonstrated a substantial degree of catabolic plasticity for hydrocarbon degradation. The results of this study show that several strains from the Klebsiella genus are able to degrade diverse hydrocarbon compounds. These findings indicate that Klebsiella spp. can be an important part of the oil-degrading microbial community in estuarine areas exposed to sewage.