A Comparison of Methods for the Enrichment of Salmonellae from Sheep Faeces

S ummary : A 10-tube MPN technique was used to test the efficiency of nutrient, tetrathionate, mannitol-selenite and mannitol-selenite-cystine broths as enrichment media for detecting salmonellae. Small numbers of broth grown salmonellae could be detected in all 4 media in the presence of 5% of sheep faeces. In naturally infected sheep faeces small numbers of salmonellae were not detected with either nutrient or tetrathionate broths. With mannitol-selenite the sensitivity of salmonella detection increased with both incubation temperature (37–43°) and the addition of faeces. The most sensitive and reliable medium for detecting salmonellae in naturally infected sheep faeces was mannitol-selenite-cystine broth. Neither incubation temperature (37–43°) nor the addition of faeces had a statistically significant effect on its sensitivity.     

Effects of an Abrupt Change in Ration from All Roughage to High Concentrate upon Rumen Microbial Numbers in Sheep

When three sheep were abruptly changed from a ration of 100% orchardgrass hay to 60% cracked corn-40% orchardgrass hay, fed at equal dry-matter intakes, significant increases in concentration were observed in the rumen microbial population. Bacterial numbers (colony counts) per gram of rumen contents did not appear to have stabilized within 21 days after the ration change; however, protozoan numbers per milliliter plateaued after 5 days. The concentration of cellulose-digesting bacteria varied considerably between animals and decreased in all animals with the change. Changes were observed in total and molar percentages of volatile fatty acids, which were typical for the two types of rations. Although the concentration of protozoa increased after the ration change, only minor differences were observed in their percent generic distribution. A significant decrease in rumen volume was measured in two of the three sheep with the change in ration; however, fluid turnover rates were not significantly affected. Rates of rumen dry-matter turnover were slower with the concentrate ration, although rumen dry-matter digestion was increased. Calculation of total bacterial numbers based on total rumen volume completely negated the effect of ration change in one animal, whereas total numbers in the other two animals were still significantly different between rations and very similar between animals. Adjustment of total protozoa numbers did not alter the trends seen previously with concentration values.     

Examination of Feces from Food Handlers for Salmonellae, Shigellae, Enteropathogenic Escherichia coli, and Clostridium perfringens

Duplicate fecal specimens from food handlers were collected in Louisiana. One set of specimens was examined immediately for salmonellae and shigellae by the Central Laboratory of the Louisiana State Board of Health in New Orleans; the other set was shipped to the Food Microbiology Unit at the Robert A. Taft Sanitary Engineering Center in Cincinnati, Ohio, where it was examined for enteropathogenic Escherichia coli (EEC) and Clostridium perfringens. A total of 219 specimens were examined by both laboratories. None yielded salmonellae or shigellae; 171 (78.1%) yielded C. perfringens; 175 (79.9%) yielded E. coli; and 14 (6.4%) yielded EEC. The 14 isolates of EEC were distributed among eight serotypes; one specimen yielded two serotypes. Multiple isolations of C. perfringens strains (two to four) were made from 64 (37.4%) of the specimens, and a total of 244 strains were isolated and studied for identifying characteristics. Of the total, only 87 (35.5%) could be identified serologically by a battery of 67 antisera; only 4 (1.6%) possessed the characteristics of the English “food-poisoning type.” The hemolytic activity on agar containing horse, ox, or sheep blood showed that 140 (57.1%) were “hemolytic,” 81 (33.1%) were “nonhemolytic,” and 23 (9.8%) gave varied results. Only 12 (4.9%) of the strains produced spores that resisted boiling for 30 min or more.     

Rumen contents as a reservoir of enterohemorrhagic Escherichia coli

Abstract We investigatedthe role of the rumen fermentation as a barries to the foodborne pathogen, Escherichia coli O157:H7. Strains of E. coli , including several isolates of O157:H7, grew poorly in media which simulated the ruminal environment of a well-fed animal. Strains of E. coli O157:H7 did not display a superior tolerance to ruminal conditions which may facilitate their colonization of the bovine digestive tract. Unrestricted growth of E. coli was observed in rumen fluid collected from fasted cattle. Growth was inhibited by rumen fluid collected from well-fed animals. Well-fed animals appear less likely to become reservoirs for pathogenic E. coli . These results have implications for cattle slaughter practices and epidemiological studies of E. coli O157:H7.     

Rumen Anaerobic Fungi of Cattle and Sheep

A system for the large-scale production and purification of mouse mammary tumor virus in the absence of detectable endogenous murine leukemia virus is described. By utilizing the Mm5mt/c1 cell line established from an adenocarcinoma of a C3H mouse, the continuous production of over 25,000 liters of mouse mammary tumor virus-containing tissue culture fluids has been achieved. By the strict adherence to well-defined tissue culture conditions, mammary tumor virus production was accomplished without the expression of murine leukemia virus. Various biochemical and immunological systems were established for the rapid and precise detection of the endogenous leukemia virus, the expression of which could be enhanced under conditions of culture stress.     

The Effects of NPY and Insulin on Food Intake Regulation in Fish

Recent abundant studies report that in rodents starvation inducesincreased neuropeptide Y (NPY) mRNA expression and peptide secretionin the hypothalamus which reduces autonomic nervous activityand promotes food intake, and intracerebroventricular (ICV)injection of NPY has potent orexigenic effects. Conversely,the effect of insulin in the central nervous system is to inhibitfood intake and NPY biosynthesis and secretion. In mammals bodyfatness is regulated and insulin acts as one intake inhibitorysignal related to fatness. In salmon (Oncorhynchus sp.) we havedemonstrated a rise in NPY-like mRNA expression and a coincidentdecrease in plasma insulin levels during 2 to 3 weeks of starvation.Additionally, experimentally manipulating body fatness withhigh and low fat diets has demonstrated that body fatness affectsfood intake in teleost fishes, raising the possibility thatNPY and insulin act to regulate their food intake. Therefore,we hypothesized that as in rodents, ICV treatment with NPY wouldstimulate food intake while ICV insulin would reduce food intake.Preliminary results suggest that ICV NPY administration doesstimulate food intake in channel catfish (Ictalurus punctatus),but central injection of insulin has no effect. Results of treatmentswith the sulfated octapeptide of cholecystokinin and the recombinantfragment of rat leptin 22–56 are also discussed.     

Basis for the Exclusion of Escherichia coli from the Rumen Ecosystem

The possible causes for the exclusion of Escherichia coli from the rumen ecosystem were investigated. Evidence presented indicates that oxidation-reduction potential, temperature, pH, CO₂, volatile fatty acids, traces of heavy metals, and bacteriophage are not environmental factors which select against E. coli in the rumen ecosystem. The feeding of nutrients utilizable by E. coli to an artificially inoculated continuous-culture rumen ecosystem did not prevent the washout of the E. coli from the continuous cultures. The presence of an inhibitor of E. coli growth was demonstrated. High concentrations of rumen fluid (50 to 100%) were necessary to inhibit growth in Antibiotic Medium 3. The inhibitor may operate to control the growth of E. coli in the rumen ecosystem. Because of the presence of an inhibitor, the possibility that rumen fluid is deficient in nutrients to support the growth of E. coli cannot be eliminated.     

Effects of moenomycin on Escherichia coli

The antibiotic moenomycin is a valuable biochemical tool for studying the metabolism of peptidoglycan and the autolytic system in Escherichia coli, since as a specific inhibitor of peptidoglycan polymerases it can efficiently promote cell lysis. In liquid media the bacteriolytic effect on E. coli K12 was dependent on the concentration of moenomycin, on growth phase and on growth rate. Before lysis cells underwent major morphological alterations. In sucrose-containing medium complete transformation to osmotically sensitive spheroplasts was easily achieved by addition of moenomycin. The minimum inhibitory concentration of the antibiotic varied with the strain of E. coli and was highly dependent on the growth medium. A tritiated derivative of moenomycin, [3H]decahydromoenomycin A, was prepared and found to have the same inhibiting efficiency. Its binding to E. coli membranes and membrane proteins was investigated. The absence of irreversible binding suggested that moenomycin might be a competitive inhibitor of the peptidoglycan polymerases. Spontaneous moenomycin resistant variants were isolated at a frequency of about 10(-9).     

Effects of toluene on Escherichia coli

Jackson, Robert W. (University of California, San Diego, La Jolla), and J. A. DeMoss. Effects of toluene on Escherichia coli. J. Bacteriol. 90:1420-1425. 1965.-When toluene is added at appropriate levels to exponentially growing cultures of Escherichia coli, a time-dependent loss of turbidity is observed which is concurrent with a loss of material to the medium and with unmasking of beta-galactosidase. In addition, the galactoside permease system is totally destroyed. Electron micrographs confirm the indications that the cells are not being lysed by toluene, although the cytoplasm collapses to the interior of the cell. Included in the material lost from the cell after toluene treatment is 85% of the total ribonucleic acid (RNA), the principal source of which appears to be the ribosomes. The loss of RNA is temperature-dependent. Protein is also lost to the medium as a function of both temperature and available toluene. Up to 25% of the total protein is found in the medium, the precise amount depending on the level of toluene employed. Zone centrifugation studies of extracts from treated cells indicate that toluene elicits a rapid disaggregation of ribosomes that is terminated, at any stage, by disruption of the cells. The disaggregation is temperature-dependent and does not occur at 4 C. It appears to be distinct from the actual degradation of ribosomal RNA and is accompanied by an accumulation of small particles during the initial phases of treatment at 21 C. Toluene added to crude extracts of normal E. coli cells is unable to cause detectable ribosome destruction.     

Volatile Fatty Acids and the Inhibition of Escherichia coli Growth by Rumen Fluid

Concentrations of volatile fatty acids (VFA) normally found in bovine rumen fluid inhibited growth of Escherichia coli in Antibiotic Medium 3. Acetic, propionic, and butyric acids each produced growth inhibition which was markedly pH-dependent. Little inhibition was observed at pH 7.0, and inhibition increased with decreasing pH. A combination of 60 mumoles of acetate, 20 mumoles of propionate, and 15 mumoles of butyrate per ml gave 96, 69, and 2% inhibition at pH 6.0, 6.5, and 7.0, respectively. Rumen fluid (50%) gave 89 and 48% inhibition at pH 6.0 and 6.5, respectively, and growth stimulation (22%) at pH 7.0. Rumen fluid inhibitory activity was heat-stable, was not precipitated by 63% ethyl alcohol, and was lost by dialysis and by treatment with anion-exchange resins but not with cation-exchange resins. These results are consistent with the idea that VFA are the inhibitory substances in rumen fluid. Previous results which indicated that rumen fluid VFA did not inhibit E. coli growth were due to lack of careful control of the final pH of the growth medium. The E. coli strain used does not grow in rumen fluid alone at pH 7.0.     

Concentrations of Free Sugars in Sheep Rumen Fluids

A convenient method was proposed for the estimation of glucosamine content in koji. In this method, the time of acid hydrolysis of the mold sample was markedly shortened modifying the previously reported method as follows; the mold sample was immersed in 60% of sulfuric acid for 24 hr at 25°C, and then the mixture was diluted with water to make the concentration of sulforis acid 1 n and autoclaved under the pressure at 1 kg/cm² for 1 hr. The liberated glucosamine was assayed by Blix’s method. The glucosamine contents of mycelia obtained from the submerged and surface cultures were increased about 2 times of initial stages in prolonged incubation. The significant differences of the glucosamine contents in various kojis were seen depending on the raw materials used; the values were 18~26mg/g in wheat bran koji and 6~16 mg/g in rice and defatted soybean meal koji.     

Plasmid Effects on Escherichia coli Metabolism

ABSTRACT:?

The idea that plasmids replicate within hosts at the expense of cell metabolic energy and preformed cellular blocks depicts plasmids as a kind of molecular parasites that, even when they may eventually provide plasmid-carrying strains with growth advantages over plasmid-free strains, doom hosts to bear an unavoidable metabolic burden. Due to the consistency with experimental data, this idea was rapidly adopted and used as a basis of different hypotheses to explain plasmid-host interactions. In this article we critically discuss current ideas about plasmid effects on host metabolism, and present evidence suggesting that the complex interaction between plasmids and hosts is related to the alteration of the cellular regulatory status.     

Specificity of Rumen Ciliate Protozoa in Cattle and Sheep*

SYNOPSIS. Six protozoa-free sheep, 3 fed alfalfa hay and 3 fed a concentrate diet, were inoculated with rumen contents from a steer fed the same alfalfa hay. All 24 species of protozoa in the inoculum became established in the sheep fed alfalfa hay, while only 9 species established in the sheep fed concentrate. Percentage species composition in the alfalfa-fed sheep was fairly similar to that of the inoculum. Rumen volumes of the alfalfa hay-fed sheep were significantly higher than those of the concentrate-fed sheep; however, fluid turnover rates were similar. Total protozoan numbers per ml of rumen contents were significantly higher in the concentrate-fed sheep, but after adjustment for rumen volume, there was no significant difference in the total number of protozoa in the rumen.     

Inactivation of Enterohemorrhagic Escherichia coli in Rumen Content- or Feces-Contaminated Drinking Water for Cattle

Cattle drinking water is a source of on-farm Escherichia coli O157:H7 transmission. The antimicrobial activities of disinfectants to control E. coli O157:H7 in on-farm drinking water are frequently neutralized by the presence of rumen content and manure that generally contaminate the drinking water. Different chemical treatments, including lactic acid, acidic calcium sulfate, chlorine, chlorine dioxide, hydrogen peroxide, caprylic acid, ozone, butyric acid, sodium benzoate, and competing E. coli, were tested individually or in combination for inactivation of E. coli O157:H7 in the presence of rumen content. Chlorine (5 ppm), ozone (22 to 24 ppm at 5°C), and competing E. coli treatment of water had minimal effects (<1 log CFU/ml reduction) on killing E. coli O157:H7 in the presence of rumen content at water-to-rumen content ratios of 50:1 (vol/wt) and lower. Four chemical-treatment combinations, including (i) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.05% caprylic acid (treatment A); (ii) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.1% sodium benzoate (treatment B); (iii) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.5% butyric acid (treatment C); and (iv) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 100 ppm chlorine dioxide (treatment D); were highly effective (>3 log CFU/ml reduction) at 21°C in killing E. coli O157:H7, O26:H11, and O111:NM in water heavily contaminated with rumen content (10:1 water/rumen content ratio [vol/wt]) or feces (20:1 water/feces ratio [vol/wt]). Among them, treatments A, B, and C killed >5 log CFU E. coli O157:H7, O26:H11, and O111:NM/ml within 30 min in water containing rumen content or feces, whereas treatment D inactivated approximately 3 to 4 log CFU/ml under the same conditions. Cattle given water containing treatment A or C or untreated water (control) ad libitum for two 7-day periods drank 15.2, 13.8, and 30.3 liters/day, respectively, and cattle given water containing 0.1% lactic acid plus 0.9% acidic calcium sulfate (pH 2.1) drank 18.6 liters/day. The amounts of water consumed for all water treatments were significantly different from that for the control, but there were no significant differences among the water treatments. Such treatments may best be applied periodically to drinking water troughs and then flushed, rather than being added continuously, to avoid reduced water consumption by cattle.     

Effects of p-Fluorophenylalanine and Chloramphenicol on Chemotaxis in Escherichia coli

The effects of chloramphenicol and p-fluorophenylalanine (p-FPA) on growth, proportion of motile cells, average rate of motility, and the chemotactic response of a methionine auxotroph of Escherichia coli K-12 were studied. Kinetic studies revealed that the inhibition of chemotaxis by p-FPA can be explained by the effect on growth, proportion of motile cells, and average rate of motility rather than a selective inhibition of chemotaxis per se. The effect of chloramphenicol on chemotaxis could not be explained in terms of these characteristics. It is concluded that low concentrations of chloramphenicol, unlike p-FPA, selectively inhibit chemotaxis.     

Breed Difference in Water Content of Faeces From Five Breeds of Sheep

Water content of faeces from five breeds was examined (14 weekly observations — eight sheep of each breed). A higher water content was found of faeces from New Zealand Romney Marsh than from their pasture mates of breeds originating from drier areas — Somali, Merino, Karakul; but Nandi originating from a medium to wet area had a similar dry matter content of faeces as the dry area breeds mentioned above. The growth rate in New Zealand Romney Marsh is greater than in the other breeds.     

Common Antigenic Determinant in a Rumen Organism and in Salmonellae Containing the Antigen O4

Nineteen of 28 strains of rumen organisms isolated from a cow on a high roughage diet and identified morphologically as butyrivibrios, reacted to a low agglutinin titer with salmonella antisera, forming five groups. However only one strain reacted with polyvalent O salmonella antiserum. This strain reacted with O4 factor serum and with antisera to Salmonella strains containing the antigen O4, and agglutinin absorption tests showed the presence of an antigen identical to O4. When 16 further strains of butyrivibrio-like rumen organisms isolated from three cows and one steer were examined, one possessed an antigen similar to but not identical with the antigen O9, and two strains reacted with specific O6,7 factor serum but were not examined further. These four strains were presumptively identified by physiological tests as butyrivibrios. The possible site of antigenic stimulation by such organisms is discussed.