THE NUMBERS AND GROWTH RATES OF THE BACTERIA IN HEVEA LATEX, AMMONIATED FIELD LATEX AND AMMONIATED LATEX CONCENTRATE

SUMMARY: The rates of growth of bacteria on Hevea latex systems in the presence and absence of ammonia have been derived from colony count data obtained on a modified Kligler's iron agar medium. For fresh latex, ammoniated field latex, and ammoniated latex concentrate, variations in the bacterial populations encountered are given for a period of about one year's testing, with some data on commercial samples of latex concentrate. Field latex at routine collection contained about 8 × 10⁶ bacteria/ml. This could be reduced by 62% by the use of sterile tapping cups. In the routine latex, the mixed population grew logarithmically after a lag phase of 2·1/2–3 hr. In clean latex the lag phase was extended to 4 hr. On ammoniation to 0.3% (w/w), the population was reduced over about 2 hr, but subsequently logarithmic growth recurred, without a lag phase. On concentration and further ammoniation to 0·7% (w/w), the count dropped still further, levels of 10/ml or less being achieved in clean conditions, but very slow logarithmic growth again occurred with a lag phase preceding it.     

A MEDIUM FOR THE CULTIVATION OF BACTERIA FROM HEVEA LATEX

SUMMARY: A medium has been devised for the culture of bacteria from fresh Hevea latex, ammoniated field latex and ammoniated latex concentrate. It is a modification of Kligler's iron agar, and can be used as a counting medium.     

The effect of ammoniation on the intake and nutritive value of straw

Studies were conducted to compare the increases in dry matter digestibility (DMD) in vitro and in vivo and to determine the metabolisable energy (ME) value of straw ammoniated at ambient temperature. Two stacks of straw sealed with polyethylene were allowed to react with 3% (w/w) anhydrous NH₃ for 30 and 56 days, respectively. Both DMD in vitro and nitrogen tests were carried out over an eight-week period subsequent to opening the stacks. Digestibility in vivo was measured with 12 wether lambs. The non-treated and ammoniated straws were given ad libitum, with a supplement of either ground barley or a lamb concentrate which contained 16% crude protein (CP).There was a mean increase of 15 percentage units in DMD in vitro for the ammoniated straw irrespective of whether it was treated for 30 or 56 days. The corresponding increase in mean DMD in vivo was 14.2 units. The CP content of the straw was increased from 3.1 to 7.6%. The increase in DMD in vitro and total N content was maintained throughout the sampling period. Approximately 58% of the anhydrous NH₃ added to the straw appeared to have been irreversibly “bound” to the straw. The ME values for the ammoniated straw were 6.78 and 7.49 MJ/kg when the straw was supplemented with either barley or the lamb concentrate, respectively. Straw ammoniation had a marked effect on intake. The overall increase in intake was 70% for the treated compared with the non-treated material.     

Studies on blood biochemical constituents and rumen fermentation in growing buffalo calves fed ammoniated straw-based rations supplemented with different protein sources

Studies were done on blood biochemical constituents and rumen fermentation pattern of growing buffalo calves fed ammoniated wheat straw (UAS)-based rations supplemented with three different protein supplements. Eighteen buffalo calves (140 kg) were divided into three groups of six calves each and were fed on UAS supplemented with a concentrate mixture (roughage:concentrate ratio 58:42) containing 8% (or 3.5% of total ration) untreated groundnut cake (GNC) (group I), 8% formaldehyde treated GNC (group II) and 8% fish meal (group III), respectively, for 19 weeks. During this growth study, blood biochemistry was studied. Rumen fermentation study was done on three fistulated growing buffalo calves using a 3×3 Latin square switch over design. Blood glucose and ammonia-N did not differ significantly among the treatments. However, blood urea-N of calves in group III was higher (P<0.05) than in groups I and II, but the latter two did not differ from each other in this respect. Total protein in group I was comparable to group II and group III which were significantly different from each other. The rumen pH, total volatile fatty acids, ammonia-N and total protozoal count were similar, however, total N and TCA insoluble N in group II and group III were higher (P<0.01) than in group I. Fish meal containing diet favoured holotrichs, whereas, protected GNC containing diet favoured spirotrichs as indicated by significantly higher count. It was concluded that the beneficial effects of supplementing formaldehyde protected GNC at 8% in the concentrate mixture to the UAS-based diets were greater than supplementation of unprotected GNC or low quality fish meal in ration of buffalo calves growing at 400 g because of improved rumen fermentation, absorption and metabolism of nutrients as observed in blood biochemistry.     

THE ENUMERATION OF WATERBORNE BACTERIA BY A NEW DIRECT COUNT METHOD

SUMMARY: A direct method has been devised to enumerate bacteria in lake water, using a concentration technique. The method involves the staining of 10⁻² ml samples of water with gentian violet dissolved in a glycerol-water base, and concentrating in vacuo . The residue remaining after concentration consists of glycerol and stained bacteria. Glass capillaries are filled from the concentrate and the volume of the contents determined. The organisms in this are counted microscopically. Results by the direct count were from six times to eleven thousand times greater than by the colony count.     

Effect of long term feeding of ammoniated wheat straw treated with or without HCl on blood biochemical parameters in growing male buffalo (Bubalus bubalis) calves

Twenty-four growing male buffalo calves (one year of age; 88.54 +/- 3.81 kg average body weight) were divided into three comparable groups (I, II and III) on the basis of their body weight (BW) in a completely randomised design to study the effect of long term feeding of ammoniated wheat straw (AWS) and hydrochloric acid treated ammoniated wheat straw (HCl-AWS) on blood biochemical changes. The animals were offered a concentrate mixture (CM) along with wheat straw (WS), ammoniated wheat straw (AWS) (4% urea at a 50% moisture level) and hydrochloric acid treated ammoniated wheat straw (HCI-AWS) (4% urea at a 50% moisture level and HCI added to trap 30% of NH3 evolved) in groups I, II and III, respectively for an average daily gain (ADG) of 500 g. All the diets were made iso-nitrogenous by preparing three types of concentrate mixtures of different CP levels. The blood was collected from the jugular vein randomly from three animals of each group initially after 8 months post feeding and subsequently after two months interval up to 14 months of experimental feeding. Due to urea ammoniation, the CP content of WS increased from 3.66 to 8.51 and was further increased to 11.35 due to the addition of HCl during urea-ammoniation of wheat straw. The cumulative period mean plasma glucose values (mg %), in group II (53.13) were significantly (P < 0.001) higher than those in groups I (48.44) and III (50.60). The cumulative period mean values of serum albumin and globulin (g %) were not significantly different and were comparable among the groups I (3.33 and 3.06), II (3.53 and 2.97) and III (3.49 and 2.94). The cumulative period mean values of serum albumin: globulin ratio and total protein values were not significantly different among the different groups. Serum urea and creatinine values were significantly (P < 0.001) higher in group III (58.66 and 2.24) as compared to groups I and II. The cumulative period mean values of serum alkaline phosphatase (ALP) (KA units) did not differ significantly, but serum glutamate pyruvate transaminase (SGPT) and glutamate oxaloacetate transaminase (SGOT) values (units x mL(-1) were significantly (P < 0.001) higher in groups II and III than in group I. The cumulative period mean values of T3 (ng x mL(-1)) did not differ significantly among the groups, but T4 values were significantly (P < 0.001) higher in group III (22.74) than in groups 1 (21.41) and II (20.89), respectively. Since the mean values of all the blood parameters were within the normal range, it may be concluded that feeding of ammoniated wheat straw treated with and without HCl to growing male buffalo calves for fourteen months has no adverse effect on the blood biochemical parameters.     

Ammoniated Forage Poisoning: Concentrations of Alkylimidazoles in Ammoniated Forage and in Milk,Plasma and Urine in Sheep and Cow

4-methylimidazolc (4-MeI) has until now been the only identified toxic compound in ammoniated forage considered to be of possible etiological significance in ammoniated forage poisoning. However, several authors have concluded that 4-MeI alone cannot explain the toxicity observed (Morgan & Edwards 1986, Nielsen et al 1986, Motoi et at. 1997). On this background, we have examined samples of ammoniated forage and of milk, plasma, and urine collected from ewes and lambs during a previous experimental poisoning study (Sivertsen et al. 1993). We have also studied similar samples from a Norwegian Red Cattle dairy cow fed ammoniated hay for the first 5 days after calving. We have, in addition to the previously known ingredients 4-MeI and 2-methylimida-zole (2-MeI), identified 5 new compounds in these samples, all of which were found to be di-alkylsubstituted imidazoles: 1,2-dimcthylimi-dazole (1,2-diMeI), 1,4-dimethyl imidazole (1,4-diMeI), 1,5-dimethyl imidazole (1,5-diMeI), 2,4-dimethylimidazole (2,4-diMeI) and 2-ethyl, 4-methylimidazole (2Et-4MeI) (Müller et al. 1998). In this paper we present quantitative measurements of these di- and monoalky 1 imidazoles in a selection of the collected samples.     

Subchronic toxicological investigations of Fusarium moniliforme-contaminated corn,culture material,and ammoniated culture material

The fungus Fusarium moniliforme is ubiquitous on corn throughout the world and is a likely co-contaminant on corn infested with aflatoxin-producing Aspergillus flavus. Ammoniation has been used to detoxify aflatoxin-contaminated commodities. To determine the effect of ammoniation on the toxic potential of Fusarium moniliforme, male Sprague-Dawley rats were fed either diets containing 10% sound corn, ammoniated corn, corn culture material of hepatotoxic F. moniliforme strain MRC 826 (CM), or ammoniated CM for four weeks. They were observed for signs of toxicity and hematological, serum chemical and histopathological evaluations were made. Groups of male Balb/c mice were fed diets fortified with 10% sound corn or CM for four weeks and evaluated by serum chemical and histopathological means to determine the suitability of mice as a model species for investigation of F. moniliforme-induced hepatotoxicity. Ammoniation was ineffective for detoxification of the CM. Hepatotoxicity and renal toxicity of CM and ammoniated CM were qualitatively similar, although renal tubular lesions appeared more advanced in rats fed ammoniated CM. Adrenal cortical cellular vacuolation was also found in CM and ammoniated CM-fed rats, while focal seminiferous tubular degeneration and aspermia were found only in the testes of ammoniated CM-fed rats. Fumonisin B₁ concentrations of the CM and ammoniated CM diets averaged 99 and 75 ppm, respectively. CM containing 99 ppm fumonisin B₁ also produced hepatotoxicity in mice similar to that found in CM-fed rats. Thus, mice may be useful for investigations of F. moniliforme-induced hepatotoxicity.Mention of a trademark, proprietary name or vendor does not imply its approval by the US Department of Agriculture to the exclusion of others that may also be suitable.     

The Bielschowsky Staining Technic a Study of the Factors Influencing Its Specificity for Nerve FIBERS

By comparing results obtained with adult mammalian tissue from introducing variables into each separate step in block-staining by the Bielschowsky silver method, the following conclusions were reached:

1. No specific means for inhibiting the staining of connective tissue and still permitting complete staining of nerve fibers was found, but the avoidance of overstaining was very helpful toward such differentiation.
2. Overstaining could be corrected by reducing the concentration of the silver nitrate bath or by adding an excess of ammonia to the ammoniated silver bath.
3. Staining of fine fibers was favored by adding acetic acid to the formaldehyde used for fixation or by adding pyridin to the silver nitrate bath.
4. Addition of protein-precipitating organic acids (trichloracetic or sulfosalicylic) to the fixative was disadvantageous.
5. Prolonged fixation favored an increase in intensity of the stain. Four days' time was sufficient.
6. Extraction of lipids with ammoniated alcohol gave results similar to those obtained after extraction with pyridin, but the stain was lighter.
7. Ammoniated silver carbonate without excess ammonia had an action similar to ammoniated silver hydroxide with excess ammonia.
8. An excess of ammonia in the ammoniated silver solution (Ag 0.1 N) was tolerated, without apparent impairment of nerve-fiber staining, up to 6 M NH₃, altho the use of more than 3 M excess (2 cc. concentrated ammonia water added to 100 cc. of balanced ammoniated silver hydroxide solution) seemed unnecessary.
9. Impregnation with 1.7% (0.1 N) silver nitrate solution was quite satisfactory and variations in the concentrations of this bath suggested that the practical limits of concentrations that would be generally satisfactory lay between 0.3 and 3.0%.
10. The writers' experiences agreed with Agduhr's relative to the advantage of washing in 2.5% acetic acid between the ammoniated silver bath and formaldehyde reduction.

     

A method for protein extraction from different subcellular fractions of laticifer latex in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> compatible with 2-DE and MS

Background  

Proteomic analysis of laticifer latex in Hevea brasiliensis has been received more significant attentions. However, the sticky and viscous characteristic of rubber latex as cytoplasm of laticifer cells and the complication of laticifer latex membrane systems has made it challenge to isolate high-quality proteins for 2-DE and MS.     

Plant latex and other exudates as plant defense systems: roles of various defense chemicals and proteins contained therein

Plant latex and other exudates are saps that are exuded from the points of plant damage caused either mechanically or by insect herbivory. Although many (ca. 10%) of plant species exude latex or exudates, and although the defensive roles of plant latex against herbivorous insects have long been suggested by several studies, the detailed roles and functions of various latex ingredients, proteins and chemicals, in anti-herbivore plant defenses have not been well documented despite the wide occurrence of latex in the plant kingdom. Recently, however, substantial progress has been made. Several latex proteins, including cysteine proteases and chitin-related proteins, have been shown to play important defensive roles against insect herbivory. In the mulberry (Morus spp.)-silkworm (Bombyx mori) interaction, an old and well-known model system of plant-insect interaction, plant latex and its ingredients - sugar-mimic alkaloids and defense protein MLX56 - are found to play key roles. Complicated molecular interactions between Apocynaceae species and its specialist herbivores, in which cardenolides and defense proteins in latex play key roles, are becoming more and more evident. Emerging observations suggested that plant latex, analogous to animal venom, is a treasury of useful defense proteins and chemicals that has evolved through interspecific interactions. On the other hand, specialist herbivores developed sophisticated adaptations, either molecular, physiological, or behavioral, against latex-borne defenses. The existence of various adaptations in specialist herbivores itself is evidence that latex and its ingredients function as defenses at least against generalists. Here, we review molecular and structural mechanisms, ecological roles, and evolutionary aspects of plant latex as a general defense against insect herbivory and we discuss, from recent studies, the unique characteristics of latex-borne defense systems as transport systems of defense substances are discussed based on recent studies.     

Applications of dielectrophoretic/electrohydrodynamic "zipper" electrodes for detection of biological nanoparticles

A major problem for surface-based detection techniques such as surface plasmon resonance and quartz crystal microbalances is that at low concentrations, diffusion is an insufficient driving force to bring colloidal submicron-scale particles to the detection surface. In order to overcome this, it has previously been demonstrated that a combination of dielectrophoresis and AC-electro-hydrodynamic flow can be used to focus cell-sized particles from suspension onto a large metal surface, in order to improve the detection capabilities of such systems. In this paper we describe how the combination of these two phenomena, using the so-called "zipper" electrode array, can be used to concentrate a wide range of nanoparticles of biological interest, such as influenza virus, dissolved albumin, and DNA molecules as well as latex beads of various sizes. We also demonstrate that the speed at which particles are transported towards the centre of the electrode pads by dielectrophoresis and electro-hydrodynamic flow is not related to the particle size for colloidal particles.     

Visualization and identification of hepatitis C viral particles by atomic force microscopy combined with MS/MS analysis

A possibility of detection and identification of hepatitis C viral (HCV) particles by atomic force microscopy (AFM) in combination with mass spectrometry (MS) has been investigated. The AFM/MS approach is based on two technologies: 1. AFM-biospecific fishing that allows to detect, concentrate from solution and to count protein complexes on the surface of a AFM-nanochip; 2. mass spectrometric identification of these complexes. AFM-biospecific fishing of HCVcoreAg from solution was carried onto the surface of AFM-nanochips with immobilized anti-HCVcoreAg. It was shown that HCVcoreAg/anti-HCVcore_im complexes were formed on AFM-nanochips in quantity sufficient for their subsequent mass spectrometric identification. Thus, the AFM/MS approach allows to identify fragments of hepatitis C virus fished on the surface of AFM-nanochip from serum.     

In-depth exploration of Hevea brasiliensis latex proteome and “hidden allergens” via combinatorial peptide ligand libraries

The proteome of Hevea brasiliensis latex has been explored in depth via combinatorial peptide ligand libraries. A total of 300 unique gene products have been identified in this latex, whose proteome has been largely unknown up to the present. In search for unknown allergens, control latex and eluates from the ligand libraries have been fractionated by two-dimensional mapping, blotted and confronted with sera of 18 patients. In addition to the already known and named Hevea major allergens, we have unambiguously detected several others like, for instance: heat shock protein (81 kDa), proteasome subunit (30 kDa), protease inhibitor (8 kDa), hevamine A (43 kDa) and glyceraldehyde-3-phosphate dehydrogenase (37 kDa). Gene Ontology analysis of analyzed fractions has shown that major functions are substantially unchanged after sample treatment, while novel biological functions appeared that were undetectable in the crude sample.     

The feeding value of ammoniated flax straw,wheat straw and wheat chaff for beef cattle

Two experiments were conducted to assess the feeding value of ammoniated and untreated flax straw, wheat straw and wheat chaff in comparison to a mixed bromegrass/alfalfa hay. Anhydrous ammonia was applied to the crop residues at the rate of 35 kg t⁻¹ dry matter. In the first experiment, the effect of ammoniation on crude protein, acid detergent fibre (ADF), neutral detergent fibre (NDF) and acid detergent lignin (ADL), digestible organic matter in vitro and in vivo (DOM%), ADF and NDF digestibility of the crop residues was determined. In the second experiment, ammoniated flax straw, ammoniated wheat straw, ammoniated and untreated wheat chaff, each supplemented with barley, were compared to bromegrass/alfalfa hay as feed sources for wintering beef cows.Ammoniation increased the crude protein content of the crop residues ∼2-fold. Wheat straw DOM in vitro and in vivo was not increased by ammoniation. Ammoniation increased the DOM in vitro of wheat chaff from 36.3 to 46% and flax straw from 35.2 to 46.3%. The DOM in vivo increased from 53.3 to 63.4% (P < 0.05) for wheat chaff and from 33.9 to 58.4% (P < 0.05) for flax straw following ammoniation. Digestibility of ADF increased from 9.9 to 43.9% (P < 0.05) and of NDF from −0.6 to 37.9% (P < 0.05) in flax straw with ammoniation. Non-significant increases in ADF and NDF digestibility were observed for all other crop residues. Lignin content was not changed in the crop residues by ammoniation.In the winter feeding trial, young cows gained more weight than older cows (P < 0.05). Average daily gains of cows were greatest for hay followed by ammoniated flax straw, ammoniated chaff, untreated chaff and ammoniated wheat straw rations (P < 0.05). Increases in backfat in the younger cows was greatest with hay and ammoniated flax straw, followed by ammoniated chaff and ammoniated wheat straw (P < 0.05). Untreated chaff caused no increase in backfat thickness.Ammoniated flax straw (3.2 kg day⁻¹) given with barley (5.6 kg day⁻¹), is similar in feeding value to medium quality bromegrass/alfalfa hay. Furthermore, wheat chaff and ammoniated wheat chaff show good potential as alternatives to hay in winter feeding.     

High-performance liquid chromatography with fluorescence detection for quantitation of tryptophan and tyrosine in a shrimp waste protein concentrate

A new, simple, and reproducible isocratic high-performance liquid chromatography (HPLC) method has been developed for the determination of free and total tyrosine and tryptophan in a protein concentrate. To determine total amino acids, the method involves alkaline hydrolysis of the proteins with sodium hydroxide at 120 degrees C for 4h in the absence of air. Best results were achieved with a SS Exil ODS column 5microm (25cmx0.46cm i.d.), with an eluent of methanol: 40mM sodium acetate buffer (adjusted to pH 4.5 with acetic acid; 20:80, v/v), a flow rate of 0.80mL/min at 26 degrees C, and with programmable fluorescence detection. Under optimum conditions excellent linearity was obtained, and the overall recovery was 90.5, and 95.9% for total tryptophan and tyrosine, respectively. The precision results showed that the relative standard deviation of the repeatability and reproducibility were < or =4.78 and < or =4.65, respectively. This method was used to quantify the cited analytes in the protein concentrate obtained during the lactic acid fermentation of shrimp waste.     

Adenosine Triphosphate and Deoxyribonucleic Acid in the Alimentary Tract of Cattle Fed Different Nitrogen Sources

Total microbial cell counts and the content of deoxyribonucleic acid (DNA) and adenosine triphosphate (ATP) were determined in ruminal and abomasal digesta and in faeces from heifers fed a basal diet of barley and straw supplemented with urea, casein, soybean-protein concentrate or feather meal. The concentration of ATP in the rumen content varied independently of the DNA content and total cell count, but dependently upon the nitrogen sources, the highest ATP/DNA ratio being obtained with casein. The ATP/DNA ratio in faeces was only one-tenth of that found in the digesta of the rumen and abomasum, indicating either an extremely low level of activity of the microbial cells or more probably a very large number of dead organisms. It is suggested that DNA and ATP might be useful indices of the microbial status in terms of biomass and metabolic activity. The total cell count should still be included in routine studies to determine the proportion of protozoa to bacteria.     

A SIMPLE APPARATUS FOR THE COLLECTION OF HEVEA LATEX UNDER ASEPTIC CONDITIONS

SUMMARY: A simple apparatus, designed to eliminate the bacterial infection of Hevea latex which occurs on tapping, is described. It produces two materials, ultraviolet irradiated latex and non-irradiated latex. An attempt has been made to maintain normal tapping conditions on a half-spiral cut. Under these conditions the latex flow time and the dry rubber content were both increased.     

Measurement of latex proteins and assessment of latex protein exposure

Reduction of protein levels in manufactured natural rubber latex products is important for preventing sensitization and adverse allergic reactions to latex. Because of the complex nature of latex extracts, accurate protein measurement is a challenge. Standard total protein assays were effective in reducing protein levels from what were once extremely high levels, but these assays are plagued with false-positive reactions and limited sensitivity. An ELISA for antigenic protein has been standardized and promises to provide more consistent measurement of the proteins with potential to cause adverse reactions. Antigenic proteins represent the total protein fraction with potential to be allergenic. Measuring antigenic protein in a consistent manner should help to further reduce the level of sensitizing protein and further reduce allergic reactions to latex-medical products.     

Use of platelet concentrate in eastern Ontario.

To better understand the reasons for the increasing use of platelet concentrate in Canada, we undertook a 4-month study of platelet concentrate transfusion in six eastern Ontario hospitals in 1985. A total of 4801 units of platelet concentrate were transfused on 687 occasions to 303 patients; the average number of transfusions per patient was 2.3, the average number of units per transfusion 7.0 and the average number of units per patient 15.8. The cardiovascular service used the largest proportion of units (28%), aortocoronary bypass grafting being the most common procedure. The mean pretransfusion platelet count for the medical and oncology services was about 30.0 X 10(9)/L, compared with 155.5 X 10(9)/L for the cardiovascular service. An increment in platelet count 1 hour after transfusion was noted with 238 (75%) of the transfusions for which the data were available; the average increment was 3.4 X 10(9)/L per unit of platelet concentrate transfused. When the data for patients who did not respond were excluded, the average increment was 6.9 X 10(9)/L. Single-donor platelet concentrate was requested for only half of the transfusions to which no response was detected. The current medical literature supports the appropriate use of platelet concentrate in patients with thrombocytopenia due to chemotherapy, but prophylactic platelet transfusion for patients undergoing cardiovascular bypass procedures is being questioned. We advise continued surveillance of the use of these products and re-evaluation of the aims of platelet transfusion therapy.