Fresh Inoculum Research Articles

Timing of seroconversion and acquisition of positive polymerase chain reaction assay results in calves experimentally infected with bovine leukemia virus

To determine the interval to provirus and serum antibody detection (via PCR assay and ELISA, respectively) in calves after experimental inoculation with bovine leukemia virus (BLV). 8 colostrum-deprived, BLV-negative Holstein bull calves (> or = 6 weeks old). Via IM injection, each calf received a fresh whole-blood inoculum (day 0) calculated to contain 2 x 10(6) lymphocytes. Blood samples for the ELISA and PCR assay were collected from calves immediately prior to inoculation and weekly thereafter for 7 weeks. Mean and median number of weeks to PCR-detected conversion of BLV status and seroconversion were calculated. Point sensitivity and cumulative sensitivity of the 2 assays were calculated at each sample collection. At each sampling time, the proportion of calves identified as infected by the cumulative weekly ELISA and PCR assay results was compared by use of a Fisher exact test. In 5 calves, conversion of BLV status was detected via PCR assay before seroconversion was identified. However, seroconversion preceded PCR-detected conversion in 2 calves. In 1 calf, both assays yielded positive results at the same test date. These differences were not significant. In experimentally inoculated BLV-negative calves, conversion of BLV status was detected via PCR assay more quickly than via ELISA; this difference was not significant and probably not clinically important. The PCR assay may be useful as a confirmatory test in animals of exceptional value; tests based on viral identification may become critically important if vaccines against BLV infection are developed and marketed.

Effect of natural dolomites on the in vitro fermentation and rumen protozoan population using rumen fluid and fresh faeces inoculum from sheep

The objective of the study was to determine the effect of dolomites from five different sources upon the end products of in vitro fermentation (total gas, methane, total and individual fatty acids, hydrogen recovery) and protozoan population. Dolomites as natural products in the dose of 0.1 g were added to the fermentation bottles containing inoculum from sheep and substrates. Both rumen fluid (RF) and fresh faeces (FF) from sheep as the sources of inocula for in vitro fermentation were used. Meadow hay (MH) and barley grain (BG) were used as fermentation substrates and incubated with the buffered rumen fluid using an in vitro gas measuring technique in separate incubation during 72 h. Both inocula (RF and FF) and dolomites impact in vitro fermentation characteristics. The gas volume was significantly increased with dolomites with RF or FF, respectively, by 20% or 20–40% (MH) and by 10% or 10–30% (BG). The methane production was significantly decreased with dolomite additives with RF inocula by 15–32% (MH) and by 50–70% (BG). A significant effect of the dolomite additives on the rumen protozoan population was observed during fermentation of MH; the total protozoan concentration and the number of Entodinium spp. was decreased ( P < 0.05). Populations of Isotrichids and large Entodiniomorphids were not influenced by experimental incubations. More studies are needed to optimize the combination of different diets with dolomite additives for practical feeding conditions.

THE EFFECT OF INTESTINAL MICROFLORA ON ANTIOXIDANT ACTIVITY OF APPLE JUICE

ABSTRACT The influence of intestinal microflora on the antioxidant activity of apple juice was assessed using the scavenging of DPPH radical. The culture medium contained 20% of laboratory‐made apple juice of Idaret cultivar and 0.2% of sodium citrate neutralizing solution. Thirty‐hour anaerobic cultures were characterized by comparable acidity and bacterial profiles of the genera determined, regardless of pure strain coculture or fresh fecal inoculum used. Apple juice showed the highest DPPH scavenging capacity of 59.9%, which was significantly reduced upon fermentation of fecal bacteria to 37.4%, or pure strain coculture to 33.4%. In the 24‐h anaerobic monocultures, the degree of DPPH scavenging was differentiated: Lactobacillus 21.4–30.2%, Bifidobacterium 28.4–51.4%, Escherichia 50.9% and Enterococcus 35.9–37.9%. The degree of DPPH scavenging was highly positively correlated (0.824) with pH, and highly/moderately negatively correlated (−0.660) with bacterial count. The results indicate reduction of antioxidant activity of apple juice as influenced by intensive bacterial growth, irrespective of species or strain used.

Optimization of storage conditions for adequate shelf-life of ‘pesta’ formulation of Fusarium oxysporum ‘foxy 2’, a potential mycoherbicide for Striga: Effects of temperature, granule size and water activity

An adequate shelf-life of mycoherbicidial products is an essential requirement for their acceptance and commercialization. Therefore, attempts were made to study the effects of temperature, granule size, and water activity (R.H./100) on the viability of the encapsulated propagules of Fusarium oxysporum ‘Foxy 2’ in ‘Pesta’ granules during storage. ‘Pesta’ granules were made with different inocula of Foxy 2, including: microconidia; mixture of mycelia and microconidia; fresh and dried chlamydospore-rich biomass. Two sizes of each granular preparation (0.5–2 and 0.25–0.5 mm) were stored in the refrigerator at 4°C as well as at room temperature (21±3°C) for 1 year. Additional samples were also stored at water activities (aw) of 0.12 and 0.41 at 25°C. Regardless of the type of formulated propagules and the granule size, all samples stored at 4°C maintained a significantly higher viability compared to those kept under room temperature. At 4°C, the ‘Pesta’ preparations with the larger granule size (0.5–2 mm) maintained more viable propagules than those with the smaller one (0.25–0.5 mm) in case of microconidia, mycelia plus microconidia and fresh chlamydospore inoculum after 1 year of storage. Granule size did not affect the viability of the dried chlamydospores. At 25°C, shelf-life of all ‘Pesta’ granules was significantly prolonged when stored at a low water activity of 0.12 compared to the storage at 0.41 aw. The results of the combined effect of water activity and temperature also revealed clearly that all formulated propagules in ‘Pesta’ granules retained a significantly higher viability when stored at 0.62 aw and 4°C than at 0.12 aw and 25°C, indicating the most pronounced effect of storage temperature.

Fructooligosaccharide production using fructosyl transferase obtained from recycling culture of Aspergillus oryzae CFR 202

Fructooligosaccharide (FOS) production was carried out using fructosyl transferase (FTase) produced by Aspergillus oryzae CFR 202 under submerged fermentation conditions. The pellets of A. oryzae CFR 202 obtained after 48 h of fermentation were supplemented with fresh media after every 24 h and fermentation was carried out to produce FTase. FTase so obtained was used to produce FOS using 60% sucrose as substrate at 55 °C at pH 5.15. FTase activity was maintained in the range of 15 ± 2 U/ml/min up to six recycles. FOS yields were maintained at 53% up to 6th cycle. Recycling of pellets could not be carried out after 6th cycle due to disintegration. The system is advantageous and economical in that it does not require supplementation of any additional nutrients nor it requires the development of fresh inoculum.

Fibrobacter succinogenes S85 ferments ball-milled cellulose as fast as cellobiose until cellulose surface area is limiting.

Fibrobacter succinogenes S85 grew rapidly on cellobiose (0.31 h(-1) and the absolute rate of increase in fermentation acids was 0.68 h(-1). Cultures that were provided with ball-milled cellulose initially produced fermentation acids and microbial protein as fast as those provided with cellobiose, but the absolute cellulose digestion rate eventually declined. If the inoculum size was increased, the kinetics decayed from first to zero order (with respect to cells) even sooner, but in each case the absolute rate declined after only 20 to 30% of the cellulose had been fermented. Congo red binding indicated that the cellulose surface area of individual cellulose particles was not decreasing, and the transition of ball-milled cellulose digestion corresponded with the appearance of unbound cells in the culture supernatant. When bound cells from partially digested cellulose were removed and the cellulose was re-incubated with a fresh inoculum, the initial absolute fermentation rate was as high as the one observed for undigested cellulose and cellobiose. Based on these results, cellulose digestion by F. succinogenes S85 appears to be constrained by cellulose surface area rather than cellulase activity per se.

Antimicrobial Substantivity of Chlorhexidine-Treated Bovine Root Dentin

Previous studies have demonstrated antimicrobial substantivity in root canal dentin up to 7 days after treatment with chlorhexidine. This in vitro study assessed the antimicrobial substantivity of chlorhexidine-treated bovine root dentin over a period of 21 days. Sixty standardized bovine root sections were randomly divided into three equal groups, and their canals immersed in one of the following solutions: (i) sterile saline; (ii) 2.5% NaOCl; or (iii) 0.2% chlorhexidine (CHX). Half the specimens in each group were treated with the solution for 5 min and the other half for 7 days. After solutions were removed, the specimens were incubated at 37 degrees C in Brain Heart Infusion broth containing Enterococcus faecalis (ATCC 29212). A fresh inoculum was added to the broth every other day over a 21-day period. The canals were then enlarged with sterile burs, and the dentin shavings collected and cultured for the presence of cultivable bacteria in the dentinal tubules. Specimens treated with CHX for 7 days demonstrated significantly less dentin colonization by E. faecalis than the other specimens. CHX has potential as an intracanal medicament, if it can be applied for a period of at least 7 days.

Plasmodium coatneyi in the rhesus monkey (Macaca mulatta) as a model of malaria in pregnancy.

Pregnant women with Plasmodium falciparum infection are at increased risk for complications such as anemia and cerebral malaria. In addition, the infants of these women suffer intrauterine growth retardation (IUGR), low birth weight (LBW), congenital infection, and high infant mortality. Although much has been learned from studies of malaria during human pregnancy, progress has been limited by the lack of a suitable animal model. Nonhuman primates are of particular interest because, other than the armadillo, they are the only animals with a discoidal, villous, hemochorial placenta like that of humans. We have established a model of malaria during human pregnancy by inoculating pregnant rhesus monkeys (Macaca mulatta) with Plasmodium coatneyi (a sequestering parasite) during the first trimester. In our initial experiment, four monkeys were inoculated with a fresh inoculum containing 10(8) viable parasites from an infected donor monkey. All four monkeys became parasitemic seven days postinoculation (PI) and three monkeys aborted 7-10 days PI coincident with high peak parasitemias (41,088-374,325 parasites/mm3). Although abortion is one of the outcomes observed in Plasmodium-infected women, the intent of this study was to examine the effects of Plasmodium infection throughout gestation. Since the rapid onset of high parasitemia may have been responsible for the abortions, a decision was made to reduce the size of the effective inoculum. Six additional pregnant monkeys were inoculated with a frozen isolate taken from the same donor containing 10(6) parasites. These six animals became parasitemic by 14 days PI and, along with monkey E412, carried their infants to term. These seven infants weighed significantly less at term than the infants of uninfected mothers (P = 0.0355). Symmetrical IUGR was detected by ultrasound in one fetus with an LBW of 334 g. Another LBW infant (300 g) had asymmetrical growth retardation, which has been associated with uteroplacental insufficiency and was consistent with the lower placental weights found in infected dams compared with controls (P = 0.0455). The infant with symmetric IUGR died at five days of age, while the other is alive but congenitally infected. The IUGR, LBW, congenital infection, postnatal infant mortality, and early abortions observed in these animals suggest that P. coatneyi in pregnant rhesus monkeys is a valid model of malaria in human pregnancy. This model should provide the opportunity to study questions about malaria in pregnancy that have been difficult to study in humans.

The adverse effect of nitrogen limitation and excess-cellobiose on Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 cultures that were cellobiose-limited converted cellobiose to succinate and acetate, produced little glucose or cellotriose, maintained an intracellular ATP concentration of 4.1 mM and a membrane potential of 140 mV for 24 h, did not lyse at a rapid rate once they had reached sta- tionary phase, and had a most probable number of viable cells that was greater than 10 6 /ml. When the cel- lobiose concentration was increased 6-fold (5 mM to 30 mM), ammonia was depleted and the cultures left 10 mM cellobiose. Cultures provided with excess cello- biose produced succinate and acetate while they were growing, but there was little increase in fermentation acids after the ammonia was depleted and growth ceased. The stationary-phase, cellobiose-excess cultures had a lysis rate that was 7-fold faster than that of the cellobiose-limited cultures, and the most probable number was only 3.3 · 10 3 cells/ml. The stationary- phase, cellobiose-excess cultures had 2.5 times as much cellular polysaccharide as the cellobiose-limited cultures, but the intracellular ATP and membrane potential were very low (0.1 mM and 40 mV respectively). Met- hylglyoxal, a potentially toxic end-product of carbohy- drate fermentation, could not be detected, and fresh inocula grew rapidly in spent medium that was supple- mented with additional ammonia. Stationary-phase, cellobiose-excess cultures converted cellobiose to glucose and cellotriose, but the apparent Km of cellotriose for- mation was 15-fold lower than the Km of glucose pro- duction (0.7 mM compared to 10 mM).

A retrospective analysis of microbial contaminants in outdated random-donor platelets from multiple sites.

Platelet components contaminated with bacteria are an important source of transfusion-associated bacterial sepsis. Estimates of contamination rates vary widely (0-10%) and are highly controversial. The present study, designed with stringent testing regimens, retrospectively determined the prevalence of microbial contaminants in platelets from four collection regions. During a 9-month period, outdated platelet units were assayed by spreading aliquots from the unit, and from thioglycollate broth medium inoculated with part of the unit, onto 5-percent sheep blood agar media. Cultures were examined after 72-hour incubation at 37 degrees C, and, if bacterial growth was present, the assay processes were repeated with fresh inocula. Units were considered contaminated only if repeatedly positive. Four (0.08%) of 4995 units sampled were contaminated, two with Corynebacterium sp. and one each with Propionibacterium acnes and Aspergillus terreus. Contaminants were present at low, subclinical levels and were detected only after amplification in thioglycollate. The contaminated units were cultured 1, 2, 3, and 7 days after expiration. Contamination rates were low and did not vary by region. The identification of A. terreus suggests the role that transfusion may play in transmitting fungal infections should be reassessed. The persistent detection of contaminated platelet units supports the need for a test to detect clinically relevant levels of microbial contaminants in blood components.

Immunity to reinfection and immunization of male guinea pigs against urethral infection with the agent of guinea pig inclusion conjunctivitis.

There is little information available on immunity of males to chlamydial infection after recovering from a primary urethral infection or after immunization with chlamydial antigen. The guinea pig model of genital infection using the chlamydial agent of guinea pig inclusion conjunctivitis was utilized to evaluate the protective immune response in these circumstances. To determine whether immunity to reinfection develops after a primary urethral infection and whether immunity develops as a result of immunization with inactivated chlamydiae. Groups of five male guinea pigs each in two separate experiments were infected in the urethra with chlamydiae and challenged with a fresh inoculum at either 30, 75, or 150 days after infection. The course of the challenge infection was then determined. Similarly, guinea pigs were immunized subcutaneously with ultraviolet-inactivated chlamydial elementary bodies and the course of urethral infection was determined when inoculated 2 weeks after immunization. Male guinea pigs were highly resistant to reinfection after a primary urethral infection. Animals that were immunized with inactivated chlamydiae generally became infected upon challenge, but the intensity of the infection was markedly reduced. Male guinea pigs possess protective mechanisms that make them more resistant to repeat chlamydial genital infection for a longer period of time than is seen in female guinea pigs. In addition, immunization of males with inactivated chlamydial antigen by a parenteral route is able to elicit a protective response to urethral infection with chlamydiae.

Increased incidence of oviduct pathology in the guinea pig after repeat vaginal inoculation with the chlamydial agent of guinea pig inclusion conjunctivitis.

Although it has been hypothesized that repeated infections with Chlamydia trachomatis result in an increased potential for the development of infertility, it is not know whether repeated chlamydial infection by the vaginal route will result in an increased incidence of upper tract pathology or enhanced pathology. To determine whether guinea pigs given two infections with the chlamydial agent of guinea pig inclusion conjunctivitis would experience an increased incidence of pathologic changes compared with animals having only a single infection. Guinea pigs previously infected with guinea pig inclusion conjunctivitis were challenged with a fresh intravaginal inoculum 73-77 days after the primary infection. Oviducts were examined either nine or 30 to 37 days after the challenge infection for pathologic changes and compared with control unchallenged animals 75 to 85 days after a primary infection. A significant increase in the number of animals with oviducts demonstrating marked tubal dilatation was observed in the challenged animals 30 to 37 days after the challenge infection. There was no association of increased antibody titer and chlamydial Hsp60 with the presence of tubal dilatation. These data strongly indicate that repeated infection via the natural vaginal route does increase the risk of tubal damage.

Studies on dissipation and degradation of 14C-ddt and 14C-dde in Pakistani soils under field conditions

Abstract Dissipation and degradation of 14C‐p,p'‐DDT (Faisalabad and Peshawar locations) and 14C‐p,p'‐DDE (Faisalabad location) were studied for one year in soils under field conditions. Both DDT and DDE dissipated more rapidly under the Pakistani subtropical climate than reported for temperate regions. More binding to soil of 14C‐DDT was observed at Peshawar than that at Faisalabad. Overall halflives were 144 and 313 days in Faisalabad and Peshawar respectively. The main degradation products of p,p'‐DDT extracted from soils at the two locations were p,p'‐DDE and p,p'‐DDD. 14C‐DDE showed an overall halflife of 191 days under Faisalabad field conditions. Since the degradation of both DDT and DDE was not substantial, it is maintained that dissipation of both chemicals was largely due to volatilization/codistillation. About 68–92% of bound 14C‐residues from soils previously treated with 14C‐DDT were released by sulphuric acid treatment. Fresh soil inoculum also released up to 77% after three months. The resi...

Laboratory studies on volatilization and mineralization of 14c-p,p'-DDT in soil, release of bound residues and dissipation from solid surfaces

Abstract In support of field data, laboratory studies were conducted on volatilization, mineralization and binding of 14C‐p,p'‐DDT in soils at Sao Paulo. Incubation of soil for 6 weeks did not result in volatilized organics or mineralization; with >95% extractable radiocarbon in the form of p,p'‐DDT. Small amounts of bound residues (1.8%) were detected in soil. These data confirm the very slow dissipation of DDT in the field which presumably relates to the acidic pH of soil (4.5–4.8). Bound 14C‐residues in soils treated with 14C‐p,p'‐DDT at Praia Grande and Sao Paulo could be released (5–21%) by sulphuric acid treatment. The released residue had the composition: 69–90% DDT, 7–32% DDD and 0–3% DDE. Incubation of soil bound 14C‐residues with fresh inoculum for 3 months did not result in release of 14C. Dissipation from wooden surfaces was fairly slow. After 20 weeks, 74% of the applied radioactivity could be recovered; 44% hexane‐non‐extractable.

Preservation of unicellular gree algae by a liquid-drying method

Successful drying of fresh water unicellular green algae ( Chlorococcales) for long-term maintenance and preservation is described. The cultures of Chlorella pyrenoidosa, Chlorella vulgaris, Selenastrum capricormutum, Scenedesmus subspicatus and Euglena gracilis which are generally used for ecotoxicity testing were freeze-dried and liquid-dried. The liquid-dried cells proved viable and stable after reactivation. There was a slight drop in viability during one year storage at −30°C. However, poor viabilities were demostrated after freeze-drying. The method is first of its kind which is applicable for the drying of eukaryotic algae. The dried cells can be stored at low cost with out specialized equipment for a long time and within few days of reactivation a fresh inoculum (pre-culture with normal cells) can be obtained easily for ecotoxicity testing and other purposes.

Controls for rhizosphere microorganisms to study effects of vesicular-arbuscular mycorrhizae on Artemisia tridentata

Seven treatments were set up to test the effects of vesicular-arbuscular (VA) mycorrhizal fungi and other rhizosphere microorganisms on the growth of Artemisia tridentata ssp. tridentata. Soil sievings had no significant effect on root or shoot mass. Spores and surface-sterile spores were a poor inoculum source, but roots and fresh soil caused 45–75% mycorrhizal infection. Whereas root-inoculated plants still had low growth responses by the end of the experiment, fresh soil inoculum caused the greatest response, and partial fresh inoculum caused a lesser response. These results suggest that fresh soil is an appropriate inoculum for this plant-fungal-soil system, and that the major effect on plant growth of the fresh soil inoculum is from the mycorrhizal fungi and not from the other microorganisms, because the sievings had no effect on plant growth. In addition, soil dilution plating of saprophytic fungi showed 85% species similarity between sterile and fresh soil inoculum by the end of the experiment. Since the effects of non-VA microorganisms are complex and varied, we suggest that researchers work out the type of mycorrhizal controls that best suit their system.

Rice performance and natural infection with blast (Pyricularia oryzae Cav.) under different algalization techniques and rates of fertilizer nitrogen

Japonica rice, Giza 171, was inoculated with either a dry or fresh soil-based inoculum of cyanobacteria containingAnabaena cylindrica, Anabaena oryzae, Nostoc muscorum andTolypothrix tenuis together with fertilization with urea at 0, 36, 72, or 108 kg N/ha. Fresh inoculum enhanced plant growth, yield and N content in comparison with the dry one. The efficiency of nitrogen utilization from the urea at all N concentrations was improved by using the fresh inoculum. Natural infection with leaf and neck blast caused byPyricularia oryzae Cav. increased with increasing N fertilization. Algalization with the fresh inoculum decreased leaf blast while neck blast was slightly higher in the algalized sub-plots but without considerable yield damage.

Characteristics of Inhibition of Suppressive Soil Created hy Monoculture with Radish in the Presence of Rhizoctonia solani

AbstractSoils collected from five districts of Hawaii county were infested with Rhtzoctonia solani in small inoculum particles and successfully planted with radish to induce suppression, Suppressiveness was induced in some, but not all, replicates of all. soils. When fresh inoculum was added, suppressiveness was demonstrated in some, but not all, replicates of two soils, but not in the other three soils. Acidity of soil was not important in successful induction of suppression. Characteristics of induced suppression in soil from one site (S. Kohala) were further investigated.Reduction of microbial population by heat treatment of suppressive soil completely nullified its inhibitory effect. The populations of actinomycetes, fungi in general and Trichoderma spp. in suppressive and conducive soil were not significantly different. However, the population of bacteria in suppressive soil was almost four times higher than that in conducive soil. The survival time of R. solani in suppressive soil was shorter than that in conducive soil. Hyphae of R. solani also lysed faster in suppressive soil than in conducive soil. It is suggested that suppressiveness of the South Kohala soil created by monoculture is due to enhanced competitive pressure generated by an increased bacterial population, which in turn causes the rapid autolysis of R. solani hyphae.

Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L.

A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 x 10(-5) molar abscisic acid (ABA) for 7 days at 25 degrees C survived slow cooling to -60 degrees C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N(2) after slow cooling to -40 or -60 degrees C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of -28 degrees C after 5 days of ABA treatment. Ethanol (2 x 10(-2) molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25 degrees C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10(-4) molar) treated alfalfa cells (Medicago sativa L.) grown at 25 degrees C hardened from an initial LT(50) of -5 degrees C to an LT(50) of -23 degrees C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days.

Vesicular–arbuscular endomycorrhizal inoculum production. I. Exploratory experiments with beans (Phaseolus vulgaris) in nutrient flow culture

A system is described in which typical vesicular–arbuscular (VA) mycorrhizal infections were produced in bean plants (Phaseolus vulgaris) grown in trays in which the roots were bathed in a shallow layer of recirculating nutrient solution (nutrient film technique, NFT). Infections were compared in solutions containing 1, 3, and 8 mg∙L−1 P, bonemeal, and rock phosphate. The infectivity of the NFT-grown mycorrhizal roots was tested using 1.2, 0.24, and 0.05 g of fresh root inoculum on maize and bean seedlings. The inoculum had good infectivity and even 0.05 g produced 5–10% infection in test seedlings after 6 weeks.