IgG anti-D MAbs in tests by flow cytometry

The faunistic results regarding intestinal parasitism by protozoa and helminths in 21 primate species (three Cebidae, thirteen Cercopithecidae, one Hylobatidae, one Lemuridae, three Pongidae) are reported. The primate species were housed in four separate galleries. Six faecal samples of each host species were subjected to coprological analysis. Fifteen parasite species were detected: 11 protozoa (Entamoeba coli, E. chattoni, E. hartmanni, Iodamoeba bütschlii, Endolimax nana, Giardia intestinalis, Chilomastix mesnilii, Enteromonas hominis, Trichomonas intestinalis, Balantidium coli, and Blastocystis hominis) and 4 helminths (Ancylostoma sp., Strongyloides fuelleborni, Strongyloides sp., and Trichuris trichiura). The results reveal certain parasitic similarities between host species housed in the same gallery; however, these primate species do not always carry identical parasite species.     

Over-expression of acetylcholinesterase stimulates the expression of agrin in NG108-15 cells

A 16-month old female child living on an Ontario dairy farm was taken to hospital suffering from bloody diarrhoea. Escherichia coli O157:H7 was isolated from her stool. Initial tests of well water samples were negative for E. coli by standard methods but culture of selected coliform colonies on sorbitol-MacConkey agar led to isolation of E. coli O157:H7. E. coli O157:H7 was also isolated from 63% of cattle on the farm. The E. coli O157:H7 isolates from the child, the water and the cattle were phage type 14, produced verotoxins 1 and 2, and were highly related on analysis by pulsed field gel electrophoresis. The child did not have known direct contact with the cattle and did not consume unpasteurized milk. Hydrogeological investigation revealed the design and location of the well would allow manure-contaminated surface water to flow into the well. This investigation demonstrates that cattle farm well water is a potential source of E. coli O157:H7 which may not be identified by standard screening for E. coli in water.     

Magnetic resonance tomography in neurocutaneous melanosis

Beef lean, fat, and connective tissues were inoculated with Escherichia coli O157:H7 before and after a prewashing procedure to compare the efficacy of prewashing and no prewashing on bacterial adherence and, consequently, on the removal of bacteria from the inoculated surfaces. Prewashing consisted of spraying tissues with tap water before inoculation. Final washing with disinfectant solutions compared the efficacy of several chemicals for the removal or destruction of E. coli O157:H7. The results showed that prewashing was very effective in reducing the numbers of bacterial cells on beef tissues, mainly lean tissue, in the control samples which received final washing with water. An opposite effect of prewashing was observed when disinfectant solutions were used for final washing; this may be due to dilution by water carried on the tissues after prewashing. The efficacy of chemicals was dependent on the type of exposed tissue. Hydrogen peroxide (3%) was more efficient in the removal of E. coli O157:H7 from connective tissues, with reductions greater than 4 log CFU/cm2, compared to a normally washed control (P < 0.01). Chlorhexidine (0.1%) was very efficient on fat and lean tissues, causing reductions over 5 log CFU/cm2 on not prewashed fat and lean tissues, compared to the control (P < 0.01). Acetic acid (5%) was the least effective, decreasing the number of CFU by under 1 log/cm2 as compared to the control; and no statistically significant difference was found among tissues, even though the removal of bacteria seemed less in lean tissue compared to fat or connective tissues.     

A case control study of potential enteric pathogens for calves raised in cow-calf herds

A matched case control study was performed to describe the epidemiological features of potential enteric pathogens for calves reared in 53 cow-calf herds located in western Switzerland. A total of 106 diarrhoeic calves and 126 healthy control calves were collected, all calves were less than 4 months old. Faecal samples were analysed for presence of infectious agents related to calf diarrhoea including enterotoxigenic E. coli, Verotoxin producing E. coli (VTEC), Campylobacter sp., Yersinia sp., Salmonella sp., rotavirus, coronavirus, helminths and coccidian protozoa. Multivariate logistic models were used to analyse the relationship between presence of infection and onset of diarrhoea. The study provided evidence of significant associations between diarrhoea and infection with rotavirus, Campylobacter coli and the presence of Verotoxin in faecal samples. With the exception of Cryptosporidium parvum intestinal parasites including Strongylidae and Eimeria sp. were found to be less prevalent in cases than in controls. Control calves were significantly more frequently infected with Strongyloides papillosus than case animals.     

Experimental investigation of the distribution of residual strains in the artery wall

Diarrheal diseases often result from ingestion of contaminated water or food. The population of La Paz, Bolivia is directly or indirectly exposed to the sewage-contaminated La Paz River. We conducted a bacteriologic survey of the La Paz River to quantify the level of bacterial contamination, with particular reference to enteropathogens. A total bacterial count exceeding 10(6) colony-forming units (CFU)/ml, including lactose fermenting and nonfermenting, gram-negative bacilli of approximately 10(5) CFU/ml, respectively, were detected in river water samples collected near two densely populated areas. A total bacterial count of 10(5) CFU/ml was also detected at the most downstream area of the river near a sparsely populated area. At four sampling locations, several enteropathogens were detected, including five enterotoxigenic Escherichia coli (ETEC) (serotype O6, O15, and O159), two enteropathogenic E. coli (EPEC) (serotype O44), two enteroinvasive E. coli (EIEC) (serotype O29), and three Salmonella O4 group isolates. The heat-labile enterotoxin gene and the invasive toxin gene were detected in all ETEC and EIEC isolates by polymerase chain reaction analysis. Nine isolates of E. coli were found by the agar dilution method to be susceptible to ampicillin, kanamycin, nalidixic acid, tetracycline, and chloramphenicol, and ampicillin resistance was found in only two isolates of EIEC 7-4 (serotype O29) and EPEC 7-5 (serotype O44). Ampicillin resistance was coded on plasmids and transferred conjugatively to E. coli chi1037 at a frequency of 10(-5) CFU/donor by the broth mating method. Strains of Aeromonas caviae, which can cause diarrheal disease in infants, were detected in vegetables grown in fields irrigated by water from the La Paz River. The survival of nine isolates of E. coli in filtered river water was compared with that of laboratory strains (E. coli chi1037, W3110, and ATCC29577). The survival time of seven isolates, excluding two ampicillin-resistant isolates, was markedly longer than that of the laboratory strains. Our results show a high bacterial contamination of the La Paz river and suggest that such levels may contribute to the high incidence of diarrheal disease in the city of La Paz.     

Survival and recovery of Escherichia coli O157:H7 in inoculated bottled water

A methodology used to isolate Escherichia coli O157:H7 from water and survival of this pathogen in inoculated water is described. The methodology used in the isolation of E. coli O157:H7 included the use of selective plating on Sorbitol MacConkey agar (supplemented with potassium tellurite [2.5 mg/liter], cefixime [0.05 mg/liter], and cefsulodin [10 mg/liter], and modified hemorrhagic colitis agar (also supplemented with potassium tellurite [2.5 mg/liter]) and cefsulodin [10 mg/liter]). There were no significant differences (P < 0.05) between the recoveries of E. coli O157:H7 on these two selective media. Direct plating on these selective agars was used to determine the length of time that E. coli O157:H7 was able to grow, remain viable, and be resistant to the selective agents. E. coli O157:H7 survived in inoculated water for up to > 300 days, depending on the type of water. Observation by scanning electron microscopy indicated that E. coli O157:H7 cells attached to, and multiplied on, the container walls.     

Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E. coli (STEC) strains, including enterohaemorrhagic E. coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII. Both ETEC and STEC are pathogenic to humans, pigs and cattle. As contamination of environmental water by any of these pathogenic E. coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E. coli was developed. The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories. The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E. coli cells as well as non-E. coli bacteria. Its detection limit was 10(2)-10(3) cfu each of the target cells per assay. When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10(0) cells 100 ml-1 of the water sample could be detected. Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples.     

Effect of compressed carbon dioxide on microbial cell viability

In order to study the influence of compressed carbon dioxide, over a range of pressures (1.5 to 5.5 MPa) and exposure times (up to 7 h), on the survival of Escherichia coli, Saccharomyces cerevisiae, and Enterococcus faecalis, a new pressurizable reactor system was conceived. Microbial cells were inoculated onto a solid hydrophilic medium and treated at room temperature; their sensitivities to inactivation varied greatly. The CO2 treatment had an enhanced efficiency in cell destruction when the pressure and the duration of exposure were increased. The effects of these parameters on the loss of viability was also studied by response-surface methodology. This study showed that a linear correlation exists between microbial inactivation and CO2 pressure and exposure time, and in it models were proposed which were adequate to predict the experimental values. The end point acidity was measured for all the samples in order to understand the mechanism of microbial inactivation. The pHs of the treated samples did not vary, regardless of the experimental conditions. Other parameters, such as water content and pressure release time, were also investigated.     

Comparative evaluation of modified m-FC and m-TEC media for membrane filter enumeration of Escherichia coli in water

Two media used to detect fecal coliforms in water by membrane filtration, m-FC and m-TEC, were modified and supplemented with the chromogenic substrate 5-bromo-6-chloro-3-indoyl-beta-D-glucuronide (BCIG) and were compared for quantitative recovery of Escherichia coli. Student's t test of data from 181 water samples of sewage, rivers, lakes, and wells did not demonstrate any statistically significant differences (P = 0.05) in the enumeration of E. coli with these media. Target colonies were confirmed to be E. coli at rates of 98.6 and 97.3% by using FC-BCIG and TEC-BCIG media, respectively. Glucuronidase-negative isolates of E. coli were encountered at the same frequency (6.0%) on both media. This collaborative study demonstrated that either modified basal medium could be used successfully for detection of E. coli in various nontreated waters within 24 h.     

Comparison of survival of diarrhoeagenic agents in two local weaning foods (ogi and koko)

The pH values of both cooked and uncooked ogi and koko samples were determined and the survival rate of four diarrhoeagenic agents, enteroinvasive Escherichia coli, Salmonella typhi, Shigella flexneri, and Vibrio cholerae were studied after they were seeded into cooked ogi and koko. Analysis of the pH of the cooked inoculated samples showed that there was a slight increase in pH (decrease in acidity) during storage for 48 h and 37 degrees C (from 3.5 to 3.7 for ogi and from 3.7 to 4.1 for koko). The study also showed that ogi had a slightly lower pH value than koko both before and after cooking. In both cases, the cooked samples had a slightly lower pH value than the uncooked samples. The pH value of ogi ranged from 3.0 to 3.6 and that of koko from 3.5 to 3.9. The survival experiment showed that the inoculated enteric pathogens were inhibited in cooked ogi and koko during storage for 24-48 h. The antibacterial effect of cooked koko was more pronounced, on the four enteric pathogens studied, than that of cooked ogi. Except for Shigella flexneri and E. coli in ogi, non of the other bacteria studied was recovered after 24 h.     

Effectiveness of salt, pH, and diacetyl as inhibitors for Escherichia coli O157:H7 in dairy foods stored at refrigeration temperatures

The behavior of Escherichia coli O157:H7 inoculated in 10% rehydrated nonfat dry milk adjusted to pH levels between 3.8 and 5.4 with lactic acid, salt levels of 0 to 6%, and diacetyl levels of 0, 5, and 10 micrograms/g was determined at 4 and 12 degrees C. Cell populations were determined by surface plating on tryptic soy agar after 7 and 35 days of incubation. Survival was also determined using retail cultured diary products. E. coli O157:H7 did not survive in skim milk at pH 3.8 and was reduced by 3 log cycles at pH 4.1, regardless of salt, diacetyl, and temperature levels. At pH levels above 4.4, survival was observed at lower salt concentrations for up to 35 days at both 12 and 4 degrees C. The organism grew (up to a 2.2-log increase) at pH 5.0 at 2% salt levels after 35 days of storage at 12 degrees C. Diacetyl at a concentration of 10 ppm had no effect on survival and growth. In all but one case, E. coli O157:H7 was inactivated in yogurt, sour cream, and buttermilk at a rate similar to or greater than what was consistent with the acidified skim milk data. Also consistent with the skim milk data, growth occurred in two of the three cottage cheese samples at 12 degrees C after 7 days but not after 35 days or at 4 degrees C, when a 1- to 2-log decline was observed.     

Role of leuX in Escherichia coli colonization of the streptomycin-treated mouse large intestine

Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18 Col-, a derivative of E. coli F-18 that no longer makes the E. coli F-18 colicin, colonizes the mouse large intestine as well as E. coli F-18 when fed alone, but is eliminated when fed together with E. coli F-18. Recently, a random bank of E. coli F-18 DNA was transformed into E. coli F-18 Col-, the resultant population was fed to streptomycin-treated mice, and the intestine was used to select the best colonizer. In this fashion, a 6.5 kb E. coli F-18 DNA fragment was isolated. This fragment was shown to enhance E. coli F-18 Col- mouse large intestinal colonizing ability and survival during stationary phase in intestinal mucus in vitro, as well as stimulate the synthesis of type-1 fimbriae. Here, we present evidence that the gene responsible for the enhanced E. coli F-18 Col- colonizing ability and survival during stationary phase in vitro is leuX. This gene encodes a rare leucine tRNA specific for the UUG codon. In addition, we show that the presence of a functional leuX gene is necessary for E. coli K-12 intestinal colonization and for survival in stationary phase.     

城市再生水中大肠杆菌在浮选过程中迁移规律

通过再生水模拟浮选试验和吸附、解吸附试验分析研究了城市再生水中大肠杆菌在浮选过程中的迁移规律.研究结果表明：再生水中大肠杆菌能够快速被矿物颗粒吸附,尾矿废水继续回用于浮选流程是安全的,但精矿、中矿、尾矿中吸附了大量的大肠杆菌,在特定暴露水平下会对从业人员构成健康风险;矿物颗粒对大肠杆菌的吸附是影响再生水中大肠杆菌在浮选过程迁移的主要因素;随着大肠杆菌浓度的升高,大肠杆菌衰减率、吸附速率随之下降;矿物颗粒对大肠杆菌的吸附对pH变化敏感,随着pH升高,矿物颗粒对大肠杆菌的吸附呈下降趋势,捕收剂煤油、Pj053和调整剂CaO的加入会明显提高矿物颗粒对大肠杆菌的吸附.     

Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin

A survey was conducted between March and October of 1994 to determine the prevalence and identify the sources of serotype O157:H7 isolates of Escherichia coli in Wisconsin dairy herds. A stratified sample of 400 farms was identified, and 70 farms with weaned calves less than 4 months old were included in the study. During the prevalence study, 5 of the 70 farms (herd prevalence, 7.1 +/- 4.5%) and fecal samples from 10 of 560 calves (animal prevalence, 1.8%) tested positive for serotype O157:H7. In a follow-up study, the five O157:H7-positive farms and seven of the O157:H7-negative farms identified in the prevalence study were visited again. An additional 517 fecal samples from cattle of various ages were tested, and a total of 15 animals from four of the five herds that were previously positive and 4 animals from two of seven herds that were previously negative tested positive for E. coli O157:H7. Observations made during the follow-up study suggested that horizontal transmission was an important means of E. coli O157:H7 dissemination on the farms. A total of 302 environmental samples, were examined, and 2 animal drinking water samples from one previously negative farm and 1 animal drinking water sample from a previously positive farm contained E. coli O157:H7. Analyses by the pulsed-field gel electrophoresis technique of contour-clamped homogeneous electric field electrophoresis revealed that isolates from the same farm displayed identical or very similar XbaI restriction endonuclease digestion profiles (REDP), whereas isolates from different farms typically displayed different REDP. However, more than one REDP was usually observed for a given herd over the 8-month sampling period. Analyses of multiple isolates from an animal revealed that some animals harbored O157:H7 strains that had different REDP, although the REDP of isolates obtained from the same fecal sample were very similar. Collectively, 160 bovine isolates obtained from 29 different animals and three water isolates displayed 20 distinct XbaI REDP. Our data revealed that there are several clonal types of serotype O157:H7 isolates in Wisconsin and indicated that there is probably more than one source of this pathogen on the dairy farms studied. However, animal drinking water was identified as one source of E. coli O157:H7 on one farm.     

Protozoa and the decline of Rhizobium populations added to soil

A fall in Rhizobium abundance occurred in nonsterile soil inoculated with large numbers of the root-nodule bacteria, but many of the rhizobia still survived. No such decline was evident in sterile soil. Protozoa feeding on these bacteria were isolated from soil and other environments. As the abundance of Rhizobium meliloti and a cowpea Rhizobium strain in soil decreased, the protozoan density increased. The inability of the predators to eliminate their prey from soil was not the result of the presence of organisms feeding on the protozoa because many rhizobia survived in sterile soil inoculated with the prey and cultures of individual protozoa, nor was it the result of the rapid multiplication of the bacteria to replace those consumed because survivors were still numerous in essentially organic matter free soil in which the bacteria did not grow appreciably. The lack of elimination also was not associated with a protective effect of soil particles because survivors were still abundant in solutions inoculated with protozoa and bacteria. It is suggested that the size of the prey population diminishes until a density is attained at which the energy used by the predator in hunting for the survivors equals that obtained from the feeding.     

Effect of urokinase on disseminated intravascular coagulation

Our study evaluated the possible therapeutic effect of urokinase in treating the microthrombiotic effects of disseminated intravascular coagulation by assisting the activation of endogenous plasminogen. Twenty-six pigs were anesthetized, intubated, mechanically ventilated, and surgically catheterized. Septic shock was induced in all 26 pigs by an intravenous infusion of heat-killed Escherichia coli. The pigs were divided into two sets of experiments: in experiment 2 (n = 14), one-half received an intravenous dose of urokinase 1 h after heat-killed E. coli infusion and in experiment 3 (n = 12) one-half received an intravenous bolus dose and a continuous drip of urokinase 2 h after heat-killed E. coli infusion. The untreated pigs served as controls. Hemodynamic parameters, blood chemistries, and blood gases were analyzed. Urokinase given 1 h after bacterial toxin infusion significantly restored blood flow, resulting in an increase in cardiovascular and pulmonary function and improved survival rate (43% control vs. 100% treated, 24-h experimental period). Treatment given after 2 h showed some significant effect on pulmonary function; however, within 10 h of E. coli infusion, mortality rates in control and treated groups were 100 and 83%, respectively. Early administration of urokinase after onset of disseminated intravascular coagulation restored blood flow and helped resolve organ damage.     

Bacterial cross-contamination of meat during liquid nitrogen immersion freezing

Prerigor beef carcass surface tissue (BCT) was used to simulate lamb carcasses on a processing line with a 15-min liquid nitrogen (LN) immersion freezing step, and the potential for the dissemination of bacteria during freezing was examined. Streptomycin-resistant strains of Listeria innocua and Escherichia coli O157:H7 spiked into a fecal slurry were inoculated onto BCT pieces that were introduced into the freezing process to represent contaminated carcasses. Following this introduction, subsequently frozen uninoculated BCT, LN, and LN containers were examined for the inoculated organisms. In the first study, BCT samples were inoculated with ca. 7 log CFU/cm2 of both L. innocua and E. coli O157:H7, spray washed with water and frozen, distributed among uninoculated BCT, in LN for 15 min. In two separate trials, L. innocua was recovered by enrichment from all uninoculated BCT and LN samples. E. coli O157:H7 was also recovered from uninoculated BCT and LN, but this cross-contamination was more sporadic. Both species were recovered from the LN container following freezing. Attempts to enumerate cross-contaminating bacteria in the second trial indicated that contaminating levels were low (< 1.0 CFU/cm2 BCT). In a second study, a 2.0% lactic acid spray wash was used to reduce further the numbers of L. innocua introduced into the freezing system and resulted in fewer positive samples, although this organism was still recovered from many uninoculated BCT samples. When either bacterium was inoculated at lower initial levels (1.35 to 1.77 log CFU/cm2) and BCT was water or 2.0% lactic acid spray washed prior to freezing, neither L. innocua nor E. coli O157:H7 was recoverable by enrichment from uninoculated BCT, LN, or from the freezing container. Results demonstrate that bacterial cross-contamination of meat during LN immersion freezing can occur but indicate that the use of good sanitation practices and product with low microbial numbers can limit this occurrence.     

Rapid, automated separation of specific bacteria from lake water and sewage by flow cytometry and cell sorting

The use of fluorescence-activated flow cytometric cell sorting to obtain highly enriched populations of viable target bacteria was investigated. Preliminary studies employed mixtures of Staphylococcus aureus and Escherichia coli. Cells of S. aureus, when mixed in different proportions with E. coli, could be selectively recovered at a purity in excess of 90%. This was possible even when S. aureus composed only approximately 0.4% of the total cells. Cell sorting was also tested for the ability to recover E. coli from natural lake water populations and sewage. The environmental samples were challenged with fluorescently labelled antibodies specific for E. coli prior to cell sorting. Final sample purities of greater than 70% were routinely achieved, as determined by CFU. Populations of E. coli released into environmental samples were recovered at greater than 90% purity. The use of flow cytometry and cell sorting to detect and recover viable target bacteria present at levels of less than 1% within an indigenous microflora was also demonstrated.     

Severe community-acquired pneumonia. Assessment of severity criteria

Sixty-three of 209 (30.1%) samples of cattle feed that were collected from multiple commercial sources and from farms were found to contain Escherichia coli. However, none of the feed samples examined were culture-positive for E. coli O157. Replication of fecal E. coli, including E. coli O157, was demonstrated in a variety of feeds at temperatures that were similar to those found on farms in summer months. Fresh mixed rations containing corn silage were sampled from 16 dairies. Rations from 12 of these dairies were found to contain E. coli, and the rations from 5 dairies had concentrations of E. coli that were greater than 1000 cfu/g. The ability of experimental mixed rations to support the replication of E. coli was correlated with the concentration of organic acids in the corn silage that was used in the ration. Widespread contamination of cattle feeds with E. coli and the ability of E. coli to replicate in feeds suggest that feeds are a potentially important factor in the ecology of organisms that can be transmitted from feces to mouth, such as E. coli O157.     

rexB of bacteriophage lambda is an anti-cell death gene

In Escherichia coli, programmed cell death is mediated through "addiction modules" consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of lambdarexB, one of the few genes expressed in the lysogenic state of bacteriophage lambda, prevents cell death directed by each of two addiction modules, phd-doc of plasmid prophage P1 and the rel mazEF of E. coli, which is induced by the signal molecule guanosine 3',5'-bispyrophosphate (ppGpp) and thus by amino acid starvation. lambdaRexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of lambdaRexB through its action on the ClpP proteolytic subunit. We also propose that the lambdarex operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the rex operon may be considered as the "survival operon" of phage lambda.