Impact of Infection Dose and Previous Serum Antibodies against the Locus of Enterocyte Effacement Proteins on Escherichia coli O157:H7 Shedding in Calves following Experimental Infection.

Escherichia coli O157:H7 is the main causative agent of haemolytic uremic syndrome. Cattle are the main reservoir of these bacteria, and have been shown to develop immune response to colonization. Our aim was to investigate the faecal shedding pattern of E. coli O157:H7 in calves challenged intragastrically with either 10(8) or 10(10) CFU, as well as the ability of specific preexisting antibodies to reduce shedding of the pathogen. Shedding was analysed by direct counting as well as enrichment of rectoanal mucosal swabs. Statistical analysis was performed using a linear model for repeated measures with and without the inclusion of preexisting antibodies against the carboxy-terminal fraction of intimin-γ (γ-intimin C280) as a covariable. Results suggest that there is a statistical difference in the area under the shedding curves between both doses for 14 as well as 28 days after challenge (p = 0.0069 and 0.0209, resp.). This difference is increased when the prechallenge antibodies are taken into account (p = 0.0056 and 0.0185). We concluded that the bacterial dose influences shedding on calves experimentally challenged and that preexisting antibodies against E. coli O157:H7 γ-intimin C280 could partially reduce faecal excretion.

Effect of Saccharomyces cerevisiae fermentation product on ruminal fermentation and nutrient utilization in dairy cows.

The goal of this experiment was to investigate the effect of yeast culture (Saccharomyces cerevisiae) on rumen fermentation, nutrient utilization, and ammonia and methane emission from manure in dairy cows. Eight ruminally cannulated Holstein cows were allocated to 2 dietary treatments in a crossover design. Treatments were control (no yeast culture) and XP (yeast culture, fed at 56 g/head per day; XP, Diamond V Mills Inc., Cedar Rapids, IA). Dry matter intake, milk yield, milk composition, and body weight were similar between treatments. Milk urea nitrogen concentration was also not affected by treatment. Rumen pH was similar between the control and XP treatments, but rumen ammonia concentration tended to be lower with XP than with the control. Treatment had no effect on concentrations of total or individual volatile fatty acids, protozoal counts, polysaccharide-degrading activities (except amylase activity that tended to be increased by XP), or methane production in the rumen. Urinary N losses did not differ significantly between treatments, but allantoin and total purine derivative excretions and the estimated microbial N outflow from the rumen tended to be increased by XP compared with the control treatment. Total-tract apparent digestibility of dietary nutrients was not affected by XP. Milk fatty acid composition was also not altered by XP supplementation. Cumulative (253 h) ammonia and methane emissions from manure, measured in a steady-state gas emission system, were slightly decreased by XP. Overall, the yeast culture tested had little effect on ruminal fermentation, digestibility, or N losses, but tended to reduce rumen ammonia concentration and increase microbial protein synthesis in the rumen, and decreased ammonia and methane emissions from manure.

Comparison of a waterless hand-hygiene preparation and soap-and-water hand washing to reduce coliforms on hands in animal exhibit settings.

Outbreaks of enteric disease associated with exposure to live animals on exhibit have occurred with increasing frequency in recent years. Possibly the most important pathogen causing such outbreaks is Escherichia coli O157:H7, because of the serious illness it can cause. Hand hygiene is consistently protective against disease among persons exposed to animals implicated in these outbreaks. Livestock barns have limited hand-washing facilities, therefore a waterless hand-sanitizing gel would be a potentially preventive measure readily available to visitors and animal exhibitors. This study compared the reduction of bacterial counts on hands of animal exhibitors when soap and water was used or when an ethanol-based hand gel was used after animal handling. Participants were youth and adults involved with showing livestock. The sanitation methods were similar in reducing the total bacteria and coliform counts on the hands of the participants (Wilcoxon rank sum test P values 0.12 and 0.69 respectively).

Correlation between geographic distance and genetic similarity in an international collection of bovine faecal Escherichia coli O157:H7 isolates.

Evidence from epidemiological and molecular studies of bovine Escherichia coli O157:H7 suggests that strains are frequently transmitted across wide geographic distances. To test this hypothesis, we compared the geographic and genetic distance of a set of international bovine Escherichia coli O157:H7 isolates using the Mantel correlation. For a measure of genetic relatedness, pulsed-field gel electrophoresis of six different restriction enzyme digests was used to generate an average Dice similarity coefficient for each isolate pair. Geographic distance was calculated using latitude and longitude data for isolate source locations. The Mantel correlation between genetic similarity and the logarithm of geographic distance in kilometers was -0.21 (P<0.001). The low magnitude of the Mantel correlation indicates that transmission over long distances is common. The occurrence of isolates from different continents on the same cluster of the dendrogram also supports the idea that Escherichia coli O157:H7 strains can be transferred with considerable frequency over global distances.

Acid resistance of Escherichia coli O157:H7 from the gastrointestinal tract of cattle fed hay or grain.

There has been strong debate as to whether feeding cattle hay prior to slaughter will reduce the number and/or virulence of Escherichia coli O157:H7 in the bovine gastrointestinal tract (GIT). This study addressed this issue by comparing numbers, persistence, and acid resistance of generic coliforms and E. coli O157:H7 from various gastrointestinal tract sites of cattle fed grain or hay. Mature Angus steers, doubly cannulated into the rumen and duodenum were inoculated with E. coli O157:H7. Aliquots of digesta from the rumen, duodenum, and rectum were cultured directly or acid shocked (pH 2.0) and then cultured to determine acid resistance. The culture technique used was as sensitive as standard immunomagnetic bead separation protocols. E. coli O157:H7 from hay-fed or grain-fed cattle were similarly acid resistant in all GIT locations. In contrast, generic coliforms from the rumen and rectum of hay-fed animals were more sensitive to an acid shock than coliforms from those GIT locations in grain-fed animals. E. coli O157:H7 colonized the most distal region of the GIT and was not consistently cultured from the rumen or the duodenum. Numbers in the upper GIT did not predict numbers or persistence of E. coli O157:H7 in rectal samples. Grain-feeding or hay-feeding did not affect survival of E. coli O157:H7 in the rumen, nor its passage through the abomasum (pH 2.0) to the duodenum. These data show that generic coliforms behave differently in the bovine host than E. coli O157:H7 and that E. coli O157:H7 acid resistance was independent of animal diet.

Antiviral activity of shiga toxin 1: suppression of bovine leukemia virus-related spontaneous lymphocyte proliferation.

Human infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemorrhagic colitis. The Stxs belong to a large family of ribosome-inactivating proteins (RIPs) that are found in a variety of higher plants and some bacteria. Many RIPs have potent antiviral activity for the plants that synthesize them. STEC strains, both virulent and nonvirulent to humans, are frequently isolated from healthy cattle. Interestingly, despite intensive investigations, it is not known why cattle carry STEC. We tested the hypothesis that Stx has antiviral properties for bovine viruses by assessing the impact of Stx type 1 (Stx1) on bovine peripheral blood mononuclear cells (PBMC) from cows infected with bovine leukemia virus (BLV). PBMC from BLV-positive animals invariably displayed spontaneous lymphocyte proliferation (SLP) in vitro. Stx1 or the toxin A subunit (Stx1A) strongly inhibited SLP. Toxin only weakly reduced the pokeweed mitogen- or interleukin-2-induced proliferation of PBMC from normal (BLV-negative) cows and had no effect on concanavalin A-induced proliferation. The toxin activity in PBMC from BLV-positive cattle was selective for viral SLP and did not abrogate cell response to pokeweed mitogen- or interleukin-2-induced proliferation. Antibody to virus or Stx1A was most effective at inhibiting SLP if administered at the start of cell culture, indicating that both reagents likely interfere with BLV-dependent initiation of SLP. Stx1A inhibited expression of BLV p24 protein by PBMC. A well-defined mutant Stx1A (E167D) that has decreased catalytic activity was not effective at inhibiting SLP, suggesting the inhibition of protein synthesis is likely the mechanism of toxin antiviral activity. Our data suggest that Stx has potent antiviral activity and may serve an important role in BLV-infected cattle by inhibiting BLV replication and thus slowing the progression of infection to its malignant end stage.

Ruminant gastrointestinal cell proliferation and clearance of Escherichia coli O157:H7.

Human infections with Escherichia coli O157:H7 cause hemorrhagic colitis that can progress to a life-threatening sequelae. The most common mode of disease transmission is ingestion of contaminated bovine food products, and it is well established that E. coli O157:H7 is a transient member of the bovine microbiota. However, the conditions that induce acquisition and subsequent clearance of this bacterium from the ruminant gastrointestinal tract (GIT) are not understood. Evidence that the rates of epithelial cell proliferation in the lower GIT of cattle are associated with the duration animals remained E. coli O157:H7 culture positive is presented. Cattle with slower rates of intestinal cell proliferation in the cecum and the distal colon were culture positive significantly longer than cohort cattle with faster cell proliferation rates. Cell death rates (apoptotic indices) between the short- and long-term culture-positive animals were not different. Typical grain-based finishing diets and forage-based growing diets did not effect GIT cell proliferation or the duration animals remained E. coli O157:H7 culture positive. To identify a dietary intervention that would effect GIT cell proliferation, we used sheep as a model ruminant. A fasting-refeeding regime that increased the rate of GIT cell proliferation was developed. The fasting-refeeding protocol was used in cattle to test the hypothesis that feeding interventions that increase the rate of GIT cell proliferation induce the clearance of E. coli O157:H7 from the bovine GIT.

Biocontrol of Escherichia coli O157 with O157-specific bacteriophages.

Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coli O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4 degrees C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. coli cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4 degrees C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.

Effect of cattle diet on Escherichia coli O157:H7 acid resistance.

The duration of shedding of Escherichia coli O157 isolates by hay-fed and grain-fed steers experimentally inoculated with E. coli O157:H7 was compared, as well as the acid resistance of the bacteria. The hay-fed animals shed E. coli O157 longer than the grain-fed animals, and irrespective of diet, these bacteria were equally acid resistant. Feeding cattle hay may increase human infections with E. coli O157:H7.

Disruption of an internal membrane-spanning region in Shiga toxin 1 reduces cytotoxicity.

Shiga toxin type 1 (Stx1) belongs to the Shiga family of bipartite AB toxins that inactivate eukaryotic 60S ribosomes. The A subunit of Stxs are N-glycosidases that share structural and functional features in their catalytic center and in an internal hydrophobic region that shows strong transmembrane propensity. Both features are conserved in ricin and other ribosomal inactivating proteins. During eukaryotic cell intoxication, holotoxin likely moves retrograde from the Golgi apparatus to the endoplasmic reticulum. The hydrophobic region, spanning residues I224 through N241 in the Stx1 A subunit (Stx1A), was hypothesized to participate in toxin translocation across internal target cell membranes. The TMpred computer program was used to design a series of site-specific mutations in this hydrophobic region that disrupt transmembrane propensity to various degrees. Mutations were synthesized by PCR overlap extension and confirmed by DNA sequencing. Mutants StxAF226Y, A231D, G234E, and A231D-G234E and wild-type Stx1A were expressed in Escherichia coli SY327 and purified by dye-ligand affinity chromatography. All of the mutant toxins were similar to wild-type Stx1A in enzymatic activity, as determined by inhibition of cell-free protein synthesis, and in susceptibility to trypsin digestion. Purified mutant or wild-type Stx1A combined with Stx1B subunits in vitro to form a holotoxin, as determined by native polyacrylamide gel electrophoresis immunoblotting. StxA mutant A231D-G234E, predicted to abolish transmembrane propensity, was 225-fold less cytotoxic to cultured Vero cells than were the wild-type toxin and the other mutant toxins which retained some transmembrane potential. Furthermore, compared to wild-type Stx1A, A231D-G234E Stx1A was less able to interact with synthetic lipid vesicles, as determined by analysis of tryptophan fluorescence for each toxin in the presence of increasing concentrations of lipid membrane vesicles. These results provide evidence that this conserved internal hydrophobic motif contributes to Stx1 translocation in eukaryotic cells.

Analysis of Escherichia coli O157:H7 survival in ovine or bovine manure and manure slurry.

Farm animal manure or manure slurry may disseminate, transmit, or propagate Escherichia coli O157:H7. In this study, the survival and growth of E. coli O157:H7 in ovine or bovine feces under various experimental and environmental conditions were determined. A manure pile collected from experimentally inoculated sheep was incubated outside under fluctuating environmental conditions. E. coli O157:H7 survived in the manure for 21 months, and the concentrations of bacteria recovered ranged from <10(2) to 10(6) CFU/g at different times over the course of the experiment. The DNA fingerprints of E. coli O157:H7 isolated at month 1 and month 12 were identical or very similar. A second E. coli O157:H7-positive ovine manure pile, which was periodically aerated by mixing, remained culture positive for 4 months. An E. coli O157:H7-positive bovine manure pile was culture positive for 47 days. In the laboratory, E. coli O157:H7 was inoculated into feces, untreated slurry, or treated slurry and incubated at -20, 4, 23, 37, 45, and 70 degreesC. E. coli O157:H7 survived best in manure incubated without aeration at temperatures below 23 degreesC, but it usually survived for shorter periods of time than it survived in manure held in the environment. The bacterium survived at least 100 days in bovine manure frozen at -20 degreesC or in ovine manure incubated at 4 or 10 degreesC for 100 days, but under all other conditions the length of time that it survived ranged from 24 h to 40 days. In addition, we found that the Shiga toxin type 1 and 2 genes in E. coli O157:H7 had little or no influence on bacterial survival in manure or manure slurry. The long-term survival of E. coli O157:H7 in manure emphasizes the need for appropriate farm waste management to curtail environmental spread of this bacterium. This study also highlights the difficulties in extrapolating laboratory data to on-farm conditions.

Shiga toxin attacks bacterial ribosomes as effectively as eucaryotic ribosomes.

Several pathogenic bacteria, including Shigelladysenteriae and certain strains of Escherichia coli, produce potent class 2 ribosome inhibiting proteins (RIPs) termed Shiga toxins (Stx). The toxins are bipartite molecules composed of a single A chain (StxA) noncovalently associated with a pentamer of receptor-binding B subunits (StxB). StxA and Stx1A from E. coli are protoxins. Proteolysis generates an A1 enzyme (28 kDa) and an A2 fragment (3 kDa), which remain bound, inactivating the enzyme, until a disulfide bond linking them is reduced. Efforts to express active recombinant Stx1A1 in the cytoplasm of E. coli were very difficult and led to the hypothesis that Stx1A1 is toxic to E. coli. We created the gene for a His-tagged Stx1A1 (cStx1A1) and expressed it in E. coli from a tightly controlled expression vector. About 1-2 mg of protein can be purified in a one-step isolation from 1 L of culture. cStx1A1, RTA, and PAP exhibited similar high toxicity against the Artemia ribosomes with IC50 values near 1 nM. Surprisingly, Stx1A1 had an IC50 of 0.8 nM against E. coli ribosomes, about the same as it had for Artemia ribosomes. This is about 250 times more active than PAP against bacterial targets, making Stx1A1 the most powerful RIP toxin presently known against E. coli ribosomes.

Evaluation of dietary influences on Escherichia coli O157:H7 shedding by sheep.

The effect of diet, an abrupt diet change, and fasting on the shedding of Escherichia coli O157:H7 was investigated with experimentally inoculated sheep as a ruminant model. Sheep were fed a grass hay diet (G), which was low in protein and digestible energy and high in fiber, or a mixture of corn and pelleted alfalfa (C), which was high in protein and digestible energy and low in fiber. After a single oral inoculation of E. coli O157:H7, all the animals shed fecal E. coli O157:H7. However, sheep that were fed G shed the bacterium almost twice as long as, and in larger numbers than, did sheep that were fed C. The number of culture-positive animals increased after the diet was abruptly changed from C to G and decreased with the opposite change (G to C). A 24-h fast did not influence E. coli O157:H7 shedding. Horizontal transmission of infection between animals occurred. Recent shedding of E. coli O157:H7 did not affect recolonization with E. coli O157:H7. The findings presented in this study indicate that preharvest control of diet may reduce the risk of E. coli O157:H7-positive animals entering the food chain.

Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep.

The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) strains from sheep are described. One flock was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7-positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis. Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E. coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E. coli O157:H7 strains, and that the strains shed by individuals changed over time. E. coli O157:H7 was isolated only by selective enrichment culture off 10 g of ovine feces. In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment. The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM. Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx1, stx2, and eae), suggesting their potential for human pathogenicity. The most common toxin-eae genotype was positive for stx1, stx2, and eae. A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene. The report demonstrates that sheep transiently shed a variety of STEC strains, including E. coli O157:H7, that have potential as human pathogens.

Purification of Staphylococcus aureus beta-toxin: comparison of three isoelectric focusing methods.

beta-Toxin (beta-hemolysin) is one of several extracellular proteins produced by Staphylococcus aureus. It is a sphingomyelinase which disrupts the membranes of erythrocytes and other mammalian cells. Despite its characterized mechanism of action, the role of beta-toxin in human and animal disease remains unclear. In this report, we compare three gentle, rapid methods to purify enzymatically active beta-toxin. Extracellular proteins in S. aureus strain RN4220 cell supernatants, containing a high concentration of the toxin, were precipitated by ethanol, dialyzed, and separated by preparative isoelectric focusing (IEF). We compared the efficiency of three preparative IEF methods: a Sephadex flat-bed IEF using pH 3.5-10.0 Ampholine, a liquid IEF using pH 7.8-8.9 Rotolyte buffers, and a liquid IEF with two consecutive steps using pH 3.0-10.0 Bio-Lytes in the first separation followed by a second step using pH 6.0-8.0 Bio-Lytes. All three IEF methods purified milligram amounts of enzymatically and biologically active beta-toxin. Typically, 2-5 mg of purified beta-toxin was obtained from 1.2 liters of culture medium. The total enzymatic activity recovered and overall yield were similar for all three methods. However, the single-step liquid IEF separation using Rotolyte buffers was the most preferred method because it purified beta-toxin to >95% purity, did not require dialysis to remove ampholytes, and was the most rapid of the three methods.

A minimal medium for growth of Pasteurella multocida.

We developed a minimal medium supporting the growth of both toxigenic and nontoxigenic strains of Pasteurella multocida to optical densities of > 0.5 (600 nm). P. multocida P1059 (ATCC 15742), one of a number of strains which can cause fowl cholera, was used as the model strain in this study. The medium was composed of 17 ingredients including cysteine, glutamic acid, leucine, methionine, inorganic salts, nicotinamide, pantothenate, thiamine, and an energy source. Leucine was not required for growth but was stimulatory, and thiamine could be replaced by adenine. An additional 46 strains of P. multocida were tested, and 40 out of 46 (87%) strains grew as well as strain P1059 through a minimum of 10 serial transfers. P. multocida toxin (PMT) was produced when cells of a known toxigenic strain (P4261) were cultivated in the minimal medium. No growth of Pasteurella Haemolytica or Pasteurella trehalosi strains was observed in this minimal medium.

Escherichia coli O157:H7 in microbial flora of sheep.

We found naturally occurring, potentially virulent Escherichia coli O157:H7 strains in sheep. The incidence of E. coli O157:H7 was transient and ranged from 31% of sheep in June to none in November. The use of a sensitive culture technique and the choice of the proper sampling season were both essential for detecting this bacterium in sheep. DNA hybridizations showed that 80% of the E. coli O157:H7 isolates had at least two of the Shiga-like toxin types I or II or the attaching-effacing lesion genes.

Purification of recombinant shiga-like toxin type I B subunit.

Shiga-like toxin type I (SLT-I) is a cytotoxin produced by certain strains of Escherichia coli. SLT-I is a bipartite molecule comprised of A (SLT-IA) and B (SLT-IB) subunits. In holotoxin, five B subunits are arranged in a pentameric ring and bind to globotriaosylceramide receptors on the surface of susceptible eukaryotic cells. The SLT-IB pentamer is noncovalently associated with one A subunit that has N-glycosidase activity and ultimately causes the death of targeted cells. Using a previously described overexpression vector, plasmid SBC32, we developed a two-step procedure for the purification of biologically active recombinant SLT-IB. Periplasmic proteins were extracted from E. coli JM105(pSBC32), fractionated by ammonium sulfate precipitation, and separated by isoelectric focusing in a pH 3-10 gradient. SLT-IB was present in fractions with pH values between 5.0 and 6.0, consistent with its reported pI of 5.8. SLT-IB was purified to homogeneity in a second step by native polyacrylamide gel electrophoresis. Purified SLT-IB was characterized for biological and biochemical activity. When analyzed by native polyacrylamide gel electrophoresis, the majority of SLT-IB had an apparent molecular weight of 38,900, consistent with a pentameric subunit association. Chemical cross-linking of SLT-IB with disuccinimidyl suberate resulted in species with molecular weights corresponding to dimeric, trimeric, tetrameric, and pentameric forms of B subunit. SLT-IB was not cytotoxic to Vero cells at concentrations as high as 10 micrograms/ml and protected Vero cells from native SLT-I. Purified SLT-IB maintained its ability to associate with SLT-IA to form holotoxin that exhibited toxicity similar to native toxin.

Effect of diet on the shedding of Escherichia coli O157:H7 in a sheep model.

The purpose of this study was to develop a sheep model to investigate reproduction, transmission, and shedding of Escherichia coli O157:H7 in ruminants. In addition, we investigated the effect of diet change on these parameters. Six groups of twin lambs given oral inoculations of 10(5) or 10(9) CFU of E. coli O157:H7 and their nondosed mothers were monitored for colonization by culture of fecal samples. A modified selective-enrichment protocol that detected E. coli O157:H7 at levels as low as 0.06 CFU per g of ovine feces was developed. Horizontal transmission of infection occurred between the lambs and most of the nondosed mothers. When animals were kept in confinement and given alfalfa pellet feed, lambs receiving the higher dose shed the bacteria sooner and longer than all other animals. However, when the animals were released onto a sagebrush-bunchgrass range, every animal, regardless of its previous status (dosed at one of the inoculum levels tested or nondosed) shed E. coli O157:H7 uniformly. Shedding persisted for 15 days, after which all animals tested negative. E. coli O157:H7 reproduction and transmission and the combined effect of diet change and feed withholding were also investigated in a pilot study with experimentally inoculated rams. Withholding feed induced animals to shed the bacteria either by triggering growth of E. coli O157:H7 present in the intestines or by increasing susceptibility to infection. Introduction of a dietary change with brief starvation caused uniform shedding and clearance of E. coli O157:H7, and all animals then tested negative for the bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)