Synergistic action of both Aspergillus niger and Burkholderia cepacea in co-culture increases phosphate solubilization in growth medium

Co-inoculation of the fungus Aspergillus niger and the bacterium Burkholderia cepacia was undertaken to understand the interaction between different species of phosphate-solubilizing microorganisms (PSM). PSM were inoculated in a single or mixed (A. nigerB.similar to cepacia) culture. During 9 similar to days of incubation, microbial biomass was enhanced, accompanied with increases in the levels of soluble phosphate and titratable acidity, as well as increased acid phosphatase activity. Production of acids and levels of phosphate solubilization were greater in the co-culture of A.similar to nigerB.similar to cepacia than in the single culture. The quantity of phosphate solubilized by the co-culture ranged from 40.51 +/- 0.60 to 1103.64 +/- 1.21 similar to mu g similar to PO4 3-similar to mL-1 and was 922% higher than single cultures. pH of the medium dropped from 7.0 to 3.0 in the A.similar to niger culture, 3.1 in the co-culture, and 4.2 in the B.similar to cepacia culture. on the third day of postinoculation, acid production by the co-culture (mean 5.40 +/- 0.31 similar to mg NaOH mL-1) was 1990% greater than single cultures. Glucose concentration decreased almost completely (9799% of the starting concentration) by the ninth day of the incubation. These results show remarkable synergism by the co-culture in comparison with single cultures in the solubility of CaHPO4 under in vitro conditions. This synergy between microorganisms can be used in poor available phosphate soils to enhance phosphate solubilization.

Relationship between incubation temperature and egg size with heart hypoplasy in broiler chicks at hatching

It was analyzed if the effects of continuous incubation temperature deviations during the second half on the development of body, organs and hematological respiratory and energetic parameters differ between male and female from 30- and 60-week-old breeder eggs. From day 13, Cobb eggs were exposed to 36°C, 37.5°C, or 39°C. At 3, 6, 12, 24, 48 and 72 h after this change in the temperature and at hatch, red cells count, hematocrit, hemoglobin, mean corpuscular volume, plasma glucose level and body, liver and heart weights were evaluated. Independent of incubation temperature, sexes and breeder ages, mean corpuscular volume decreased and the other variables increased during late incubation. In 30-week-old breeder eggs, body weights and erythrocytic parameters were not influenced by temperature but liver and heart weights decreased increasing incubation temperature and glucose level increased at 36 and 39°C. In 60-week-old breeder eggs, males were heavier at hatching with incubation at 36°C and females had smaller body weights with incubation at 39°C. In both sexes, liver weight decreased and glucose concentration was higher at 36 and 39°C and heart weights and erythrocytes parameters were not influenced by temperature. Independent of breeder age, hatchability was lower at 39°C. The data show that high temperature from day 13 of incubation reduced more intensively the hatching success and caused cardiac hypoplasia in chicks from 30-week-old breeder eggs only, revealing for the first time that the susceptibility for ascites syndrome, by reduced heart development at hatching, is associated to a relationship between incubation temperature and egg size. © Asian Network for Scientific Information, 2012.

Inoculation of salivary gland extracts obtained from female of Rhipicephalus sanguineus (Latreille, 1806) (Acari, Ixodidae) with 2, 4, and 6 days of feeding in rabbit: I - Histopathology of the feeding lesion

This study analyzed the histopathology of rabbit skin, previously immunized with SGE2, SGE4, and SGE6 gland extracts prepared from salivary glands of Rhipicephalus sanguineus female with 2, 4, and 6 days of feeding, at the region of the R. sanguineus female feeding lesion 2, 4, and 6 days after tick attachment. In this work, infestation-naïve New Zealand White rabbits were inoculated either with the extracts (test group (TG)) or with phosphate buffer and complete Freund's adjuvant mixture (control group 2 (CG2)). Each extract-inoculated- (TG and CG2) and non-inoculated (CG1) rabbit was subsequently infested with R. sanguineus. Skin biopsies were collected from the rabbit at the tick feeding lesion at 2, 4, and 6 days of feeding. Results revealed that rabbit immunization with gland extracts induced acquisition of resistance against this species. It should be stated that the SGE4 extract was the most effective in developing an immune-inflammatory response against ectoparasites, being this process characterized by the presence of an early and intense inflammatory cell infiltrate. On the other hand, SGE6 extract caused a later appearance of resistance with less infiltrate occurrence and intense edema at the feeding lesion site. As to the inflammatory process deriving from SGE2 extract inoculation, it was the less intense. It was concluded that immunization with different extracts from R. sanguineus female salivary glands did not change microscope features of the inflammatory process, although an earlier or more intense and later response, which was also dependent on the inoculate extract, was noticed. © 2012 Springer-Verlag Berlin Heidelberg.

Raising of horn fly haematobia irritans (L.) (diptera: muscidae) in laboratory by means of egg and larva inoculation

Several studies have required Haematobia irritans (L.) raising in laboratory. The present study assessed two methods of inoculating immature forms of H. irritans to obtain adults. In 2007, 15 Nellore steers (Bos indicus) (L.) were used for the collection of feces free of anthelmintic treatment and flies to produce for eggs and larva. For method I, 30 eggs were incubated in square filter paper (5 × 5 cm) and deposited on bovine feces (500 g) where they were kept until hatching and spontaneous penetration of larvae (L1) into the fecal mass. After 24 h, eggs were analyzed under a stereoscope microscope (40×) for the number of larvae that instinctively penetrated the feces. In method II, larvae were obtained only by natural egg hatching. At birth, 30 larvae were collected and individually inoculated, directly onto the fecal plate by employing a moistened brush. The tests were carried out at controlled temperature (28˚C ± 2˚C) and saturated humidity (80%) until the emergence of flies with both methods. The number of emerged flies was considered in the result. Using method I, 276 (76.7%) flies emerged from 360 inoculated eggs, while using method II, 283 (78.6%) flies emerged from 360 inoculated larvae. There was no significant difference (P = 0.7821) between methods for the number of flies; however, the proportion between males and females by means of larva inoculation was different from 1:1 (P = 0.0146). Results indicated that both methods led to a satisfactory production of flies and egg inoculation provided an easier establishment.

Experimental Inoculation of Coyotes with Mycobacterium bovis Susceptibility and Shedding

Several wildlife species have tested positive for bovine tuberculosis in Michigan and may potentially transmit the disease to other animals. Coyotes have the highest known prevalence in the endemic area and thus, our objective was to investigate the shedding of Mycobacterium bovis by coyotes. Four coyotes were orally inoculated with 1 ml of 1 x 105 CFU/ml of M. bovis. Oral and nasal swabs, and feces were collected regularly and tested by culture. Fecal samples were also tested by exposing guinea pigs to the coyotes' feces. All animals were necropsied to determine if infection occurred. All swabs, feces and tissues were negative on culture. The dosage of M. bovis given to these coyotes was considered biologically relevant, but was insufficient for causing infection. Due to the lack of infection, we still do not know the risk coyotes pose for shedding M. bovis.

Dynamics of free and H-3-labelled glutamine concentrations during zygotic and somatic embryogenesis of Feijoa [Acca sellowiana (O. Berg.) Burret]

Amino acids play fundamental roles in plant morphogenesis. Among sources of organic nitrogen (N), glutamine has frequently been used during the establishment and maintenance of cell and tissue cultures. The aim of this study was analyse endogenous levels of glutamine during somatic and zygotic embryogenesis of Acca sellowiana (Feijoa or pineapple guava). The in vitro absorption of H-3-labelled glutamine was investigated. Zygotic embryos and embryogenic cultures (EC) were evaluated at 30 d and 70 d after explant inoculation onto the medium. Endogenous levels of glutamine were similar during zygotic and somatic embryogenesis, and showed a gradual decline until day-24 in culture. The highest rates of H-3-labelled glutamine uptake were observed during the first 2 h of incubation, resulting in values of 6.29 mu mol g(-1) fresh weight (FW) for zygotic embryos, 14.43 mu mol g(-1) FW for EC after 30 d, and 13.85 mu mol g(-1) FW for EC after 70 d. These results showed that the decreased levels of glutamine observed during the initial phase of development may be related to de novo protein synthesis and mobilisation during embryo maturation. The absorption of glutamine in the first 2 h of incubation also emphasises its involvement as an important source of N during morphogenesis of somatic and zygotic embryos.

Comparison of indigestible markers from in situ and in vivo incubation to predict apparent digestibility in hay- and corn-fed horses

Four castrated crossbred horses were used in a randomized block design to study the use of indigestible internal markers iNDF and iADF obtained in situ (from bovines) or in vivo (from equines). Treatments consisted of determining digestibility by the direct method comprising total feces collection (TC) and by the indirect method comprising internal markers iNDF and iADF obtained by in situ incubation in bovine rumen or in vivo by the mobile nylon bag (MNB) technique with horses. iNDF-IV and iADF-IV resulted in better marker recovery rate (RR) (91.50%), similar to TC. The in situ technique resulted in lower RR values for the two indigestible markers, averaging 86.50% (p < 0.05). Estimates of the nutrient coefficient of digestibility (CD) were adequately predicted by iADF-IV, for horses fed on hay exclusively, with rates 46.41, 48.16, 47.92 and 45.51% for dry matter (DM), organic matter (OM), FDN and gross energy, respectively. Results show that MNB may be used to obtain iADF in horses fed on coast-cross hay exclusively, whereas NDFi and ADFi were selected for horses fed on mixed diets to predict the coefficient of nutrient digestibility.

Bottle effects, toxicity and cell viability during incubation experiments to asses community respiration

Máster en Oceanografía

Emerging contaminants in agricultural ecosystems: impact of selected pharmaceutical on water and soil ecology and pratical implications

Pharmaceuticals are useful tools to prevent and treat human and animal diseases. Following administration, a significant fraction of pharmaceuticals is excreted unaltered into faeces and urine and may enter the aquatic ecosystem and agricultural soil through irrigation with recycled water, constituting a significant source of emerging contaminants into the environment. Understanding major factors influencing their environmental fate is consequently needed to value the risk, reduce contamination, and set up bioremediation technologies. The antiviral drug Tamiflu (oseltamivir carboxylate, OC) has received recent attention due to the potential use as a first line defence against H5N1 and H1N1 influenza viruses. Research has shown that OC is not removed during conventional wastewater treatments, thus having the potential to enter surface water bodies. A series of laboratory experiments investigated the fate and the removal of OC in surface water systems in Italy and Japan and in a municipal wastewater treatment plant. A preliminary laboratory study investigated the persistence of the active antiviral drug in water samples from an irrigation canal in northern Italy (Canale Emiliano Romagnolo). After an initial rapid decrease, OC concentration slowly decreased during the remaining incubation period. Approximately 65% of the initial OC amount remained in water at the end of the 36-day incubation period. A negligible amount of OC was lost both from sterilized water and from sterilized water/sediment samples, suggesting a significant role of microbial degradation. Stimulating microbial processes by the addition of sediments resulted in reduced OC persistence. Presence of OC (1.5 μg mL-1) did not significantly affect the metabolic potential of the water microbial population, that was estimated by glyphosate and metolachlor mineralization. In contrast, OC caused an initial transient decrease in the size of the indigenous microbial population of water samples. A second laboratory study focused on basic processes governing the environmental fate of OC in surface water from two contrasting aquatic ecosystems of northern Italy, the River Po and the Venice Lagoon. Results of this study confirmed the potential of OC to persist in surface water. However, the addition of 5% of sediments resulted in rapid OC degradation. The estimated half-life of OC in water/sediment of the River Po was 15 days. After three weeks of incubation at 20 °C, more than 8% of 14C-OC evolved as 14CO2 from water/sediment samples of the River Po and Venice Lagoon. OC was moderately retained onto coarse sediments from the two sites. In water/sediment samples of the River Po and Venice Lagoon treated with 14C-OC, more than 30% of the 14C-residues remained water-extractable after three weeks of incubation. The low affinity of OC to sediments suggests that the presence of sediments would not reduce its bioavailability to microbial degradation. Another series of laboratory experiments investigated the fate and the removal of OC in two surface water ecosystems of Japan and in the municipal wastewater treatment plant of the city of Bologna, in Northern Italy. The persistence of OC in surface water ranged from non-detectable degradation to a half-life of 53 days. After 40 days, less than 3% of radiolabeled OC evolved as 14CO2. The presence of sediments (5%) led to a significant increase of OC degradation and of mineralization rates. A more intense mineralization was observed in samples of the wastewater treatment plant when applying a long incubation period (40 days). More precisely, 76% and 37% of the initial radioactivity applied as 14C-OC was recovered as 14CO2 from samples of the biological tank and effluent water, respectively. Two bacterial strains growing on OC as sole carbon source were isolated and used for its removal from synthetic medium and environmental samples, including surface water and wastewater. Inoculation of water and wastewater samples with the two OC-degrading strains showed that mineralization of OC was significantly higher in both inoculated water and wastewater, than in uninoculated controls. Denaturing gradient gel electrophoresis and quantitative PCR analysis showed that OC would not affect the microbial population of surface water and wastewater. The capacity of the ligninolytic fungus Phanerochaete chrysosporium to degrade a wide variety of environmentally persistent xenobiotics has been largely reported in literature. In a series of laboratory experiments, the efficiency of a formulation using P. chrysosporium was evaluated for the removal of selected pharmaceuticals from wastewater samples. Addition of the fungus to samples of the wastewater treatment plant of Bologna significantly increased (P < 0.05) the removal of OC and three antibiotics, erythromycin, sulfamethoxazole, and ciprofloxacin. Similar effects were also observed in effluent water. OC was the most persistent of the four pharmaceuticals. After 30 days of incubation, approximately two times more OC was removed in bioremediated samples than in controls. The highest removal efficiency of the formulation was observed with the antibiotic ciprofloxacin. The studies included environmental aspects of soil contamination with two emerging veterinary contaminants, such as doramectin and oxibendazole, wich are common parasitic treatments in cattle farms.

Uncertainty About the Incubation Period of AIDS and its Impact on Backcalculation

We analyze three sets of doubly-censored cohort data on incubation times, estimating incubation distributions using semi-parametric methods and assessing the comparability of the estimates. Weibull models appear to be inappropriate for at least one of the cohorts, and the estimates for the different cohorts are substantially different. We use these estimates as inputs for backcalculation, using a nonparametric method based on maximum penalized likelihood. The different incubations all produce fits to the reported AIDS counts that are as good as the fit from a nonstationary incubation distribution that models treatment effects, but the estimated infection curves are very different. We also develop a method for estimating nonstationarity as part of the backcalculation procedure and find that such estimates also depend very heavily on the assumed incubation distribution. We conclude that incubation distributions are so uncertain that meaningful error bounds are difficult to place on backcalculated estimates and that backcalculation may be too unreliable to be used without being supplemented by other sources of information in HIV prevalence and incidence.

Influence of temperature of incubation and type of growth medium on pigmentation in Serratia marcescens.

Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize prodigiosin when suspended in saline at 27 C when casein hydrolysate was added. These suspensions produced less prodigiosin at a slower rate than did cultures growing in casein hydrolysate at 27 C without prior incubation at 38 C. Optimal concentration of casein hydrolysate for pigment formation by suspensions was 0.4%; optimal temperature was 27 C. Anaerobic incubation, shift back to 38 C, killing cells by heating, or chloramphenicol (25 mug/ml) inhibited pigmentation. Suspensions of washed cells forming pigment reached pH 8.0 to 8.3 rapidly and maintained this pH throughout incubation for 7 days. Measurements of viable count and of protein, plus other data, indicated that cellular multiplication did not occur in suspensions of washed cells during pigment formation. By this procedure utilizing a shift down in temperature, biosynthesis of prodigiosin by washed cells could be separated from multiplication of bacteria.

Microbial formation and degradation of oxygen-containing polycyclic aromatic hydrocarbons (OPAHs) in soil during short-term incubation

We tested whether OPAHs were formed during 19-wk incubation of a fertile soil at optimum moisture in the dark. The soil had initial mean (±s.e., n = 3) concentrations of 22 ± 1.7 (Σ28PAHs) and 4.2 ± 0.34 μg g−1 (Σ14OPAHs). After 19 wk, individual PAH and OPAH concentrations had decreased by up to 14 and 37%, respectively. Decreases in % of initial concentrations were positively correlated with their KOW values for PAHs (r = 0.48, p = 0.022) and 9 OPAHs (r = 0.78, p = 0.013) but negatively, albeit not significantly, for 5 OPAHs (r = −0.75, p = 0.145) suggesting net formation of some OPAHs. The latter was supported by significantly increasing 1-indanone/fluorene ratios while the other OPAH to parent-PAH ratios remained constant or tended to increase. We conclude that OPAHs are formed in soils during microbial turnover of PAHs in a short time.