Escherichia coli strains isolated from the uteri horn, mouth, and rectum of bitches suffering from pyometra: virulence factors, antimicrobial susceptibilities, and clonal relationships among strains

Pyometra is recognized as one of the main causes of disease and death in the bitch, and Escherichia coli is the major pathogen associated with this disease. In this study, 70 E. coli isolates from the uteri horn, mouth, and rectum of bitches suffering from the disease and 43 E. coli isolates from the rectum of clinically healthy bitches were examined for the presence of uropathogenic virulence genes and susceptibility to antimicrobial drugs. DNA profiles of isolates from uteri horn and mouth in bitches with pyometra were compared by REP, ERIC, and BOX-PCR. Virulence gene frequencies detected in isolates from canine pyometra were as follows: 95.7% fim, 27.1% iss, 25.7% hly, 18.5% iuc, and 17.1% usp. Predominant resistance was determined for cephalothin, ampicillin, and nalidixic acid among the isolates from all sites examined. Multidrug resistance was found on ∼ 50% pyometra isolates. Using the genotypic methods some isolates from uteri, pus, and saliva of the same bitch proved to have identical DNA profiles which is a reason for concern due to the close relationship between household pets and humans.

Fluorescent antibody test, quantitative polymerase chain reaction pattern and clinical aspects of rabies virus strains isolated from main reservoirs in Brazil

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

Effect of essential oils of Syzygium aromaticum and Cinnamomum zeylanicum and their major components on biofilm production in Staphylococcus aureus strains isolated from milk of cows with mastitis

Bovine mastitis is an inflammation of the mammary glands of cows and causes significant economic losses in dairy cattle. Staphylococcus aureus is one of the microorganisms most commonly isolated. Novel agents are required in agricultural industries to prevent the development of mastitis. The production of biofilm by Staph. aureus facilitates the adhesion of bacteria to solid surfaces and contributes to the transmission and maintenance of these bacteria. The effect of the essential oils of Syzygium aromaticum (clove; EOSA) and Cinnamomum zeylanicum (cinnamon; EOCZ) and their major components, eugenol and cinnamaldehyde, on Staph. aureus biofilm formation on different surfaces was investigated. The results showed a significant inhibition of biofilm production by EOSA on polystyrene and stainless steel surfaces (69.4 and 63.6%, respectively). However, its major component, eugenol, was less effective on polystyrene and stainless steel (52.8 and 19.6%, respectively). Both EOCZ and its major component, cinnamaldehyde, significantly reduced biofilm formation on polystyrene (74.7 and 69.6%, respectively) and on stainless steel surfaces (45.3 and 44.9%, respectively). These findings suggest that EOSA, EOCZ, and cinnamaldehyde may be considered for applications such as sanitization in the food industry.

Induction, expression and characterisation of laccase genes from the marine-derived fungal strains Nigrospora sp. CBMAI 1328 and Arthopyrenia sp. CBMAI 1330

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

Photodynamic inactivation of virulence factors of Candida strains isolated from patients with denture stomatitis

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

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.

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.

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.

Xenia Effects on Quality of Maize Female Inbred in Production of Hybrid Seeds

In Maize (Zea maize L.), cost of hybrid seed production is directly related to the yield and quality of seed obtained per hectare of female parent. It is also important to consider the effects that a male parent can exert on the development of hybrid seed in the female parent. This effect is known as xenia. The objectives of this study were to evaluate xenia effects on 1) yield as 80K units, 2) germination of the hybrid seed and 3) susceptibility of the hybrid seed to mechanical damage. One female inbred and four male inbred lines were selected from a parent list of hybrids. The experiment was designed to allow individual cross pollination between each male inbred and the female inbred line. For use as a control, the female inbred was allowed to self pollinate. Experiments were conducted in Illinois and Iowa during 2008 and 2009 and in Nebraska during 2009. A significant inbred effect was detected on yield as 80k (α=0.001). The selfed female and pollination with male inbred B resulted in lower yields of hybrid seed. For germination, a significant inbred effect was detected (α=0.001), but was due to lower germination percentage of seed produced on the selfed female. All hybrid combinations resulted in higher germination percentages with no significant differences among hybrids. The inbred x mechanical damage interaction was significant (P=0.04) for effects on cold saturated soil germination tests. Use of inbred B resulted in a two-percentage-point reduction in cold germination when treated with the impact simulator. In a maize seed company, the production research group provides yield estimates for production of new hybrid combinations. Results from this study indicate that using only the female inbred yield may provide inaccurate estimates. Therefore to improve yield estimation, experiments should be designed to include male inbreds. Male inbreds can also impart a negative effect to the hybrid seed on tolerance to mechanical damage, thus lowering quality and increasing seed discard. When testing for hybrid seed germination, there is no need to consider distinct hybrid combinations. Female inbreds can be grown in open-pollinated fields to avoid loss of vigor observed with selfing. Advisor: George Graef

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.