Escherichia coli O157:H7 lacking the qseBC-encoded quorum-sensing system outcompetes the parental strain in colonization of cattle intestines.

The qseBC-encoded quorum-sensing system regulates the motility of Escherichia coli O157:H7 in response to bacterial autoinducer 3 (AI-3) and the mammalian stress hormones epinephrine (E) and norepinephrine (NE). The qseC gene encodes a sensory kinase that autophosphorylates in response to AI-3, E, or NE and subsequently phosphorylates its cognate response regulator QseB. In the absence of QseC, QseB downregulates bacterial motility and virulence in animal models. In this study, we found that 8- to 10-month-old calves orally inoculated with a mixture of E. coli O157:H7 and its isogenic qseBC mutant showed significantly higher fecal shedding of the qseBC mutant. In vitro analysis revealed similar growth profiles and motilities of the qseBC mutant and the parental strain in the presence or absence of NE. The magnitudes of the response to NE and expression of flagellar genes flhD and fliC were also similar for the qseBC mutant and the parental strain. The expression of ler (a positive regulator of the locus of enterocyte effacement [LEE]), the ler-regulated espA gene, and the csgA gene (encoding curli fimbriae) was increased in the qseBC mutant compared to the parental strain. On the other hand, growth, motility, and transcription of flhD, fliC, ler, espA, and csgA were significantly reduced in the qseBC mutant complemented with a plasmid-cloned copy of the qseBC genes. Thus, in vitro motility and gene expression data indicate that the near-parental level of motility, ability to respond to NE, and enhanced expression of LEE and curli genes might in part be responsible for increased colonization and fecal shedding of the qseBC mutant in calves.

Fermentation products as feed additives mitigate some ill-effects of heat stress in pigs.

Heat stress (HS) may result in economic losses to pig producers across the USA and worldwide. Despite significant advancements in management practices, HS continues to be a challenge. In this study, an in-feed antibiotic (carbadox, CBX) and antibiotic alternatives ( [XPC], and [SGX] fermentation products) were evaluated in a standard pig starter diet as mitigations against the negative effects of HS in pigs. A total of 100 gilts were obtained at weaning (6.87 ± 0.82 kg BW, 19.36 ± 0.72 d of age) and randomly assigned to dietary treatments (2 rooms/treatment, 2 pens/room, 6 to 7 pigs/pen). After 4 wk of dietary acclimation, half of the pigs in each dietary group (1 room/dietary treatment) were exposed to repeated heat stress conditions (RHS; daily cycles of 19 h at 25°C and 5 h at 40°C, repeated for 9 d), and the remaining pigs were housed at constant thermal neutral temperature (25°C, [NHS]). Pigs subjected to RHS had elevated skin surface temperature ( < 0.05; average 41.7°C) and respiration rate ( < 0.05; 199 breaths per minute (bpm) during HS, and overall reduced ( < 0.05) BW, ADG, ADFI, and G:F regardless of dietary treatment. Independent of diet, RHS pigs had significantly shorter ( < 0.05) jejunum villi on d 3 and d 9 compared to NHS pigs. Heat stress resulted in decreased villus height to crypt depth ratio (V:C) in pigs fed with control diet with no added feed additive (NON) and CBX diets at d 3, whereas the pigs fed diets containing XPC or SGX showed no decrease. Transcriptional expression of genes involved in cellular stress (, , , ), tight junction integrity (, , ), and immune response (, , and ) were measured in the ileum mucosa. Pigs in all dietary treatments subjected to RHS had significantly higher ( < 0.05) transcript levels of and , and an upward trend ( < 0.07) of mRNA expression. RHS pigs had higher ( < 0.05) transcript levels of and in NON diet, in XPC and CBX diets, and in SGX diet compared to the respective diet-matched pigs in the NHS conditions. Neither RHS nor diet affected peripheral natural killer () cell numbers or NK cell lytic activity. In conclusion, pigs subjected to RHS had decreased performance, and supplementation with fermentation products in the feed (XPC and SGX) protected pigs from injury to the jejunum mucosa.

Characterization of Serpulina (Treponema) hyodysenteriae and related intestinal spirochetes by ribosomal RNA gene restriction patterns.

Serpulina (Treponema) hyodysenteriae strain A-1 partially purified rRNA, labelled with photobiotin, was used as a non-radioactive probe to identify the rRNA gene restriction patterns of S. hyodysenteriae strains and other spirochetes. Sau3A restriction enzyme digests resulted in similar rRNA gene restriction patterns in S. hyodysenteriae strains from five different countries. Some S. hyodysenteriae strains could be differentiated by variations in their rRNA gene restriction patterns after cleavage of DNA by restriction enzymes SspI or BglII. S. innocens and Treponema succinifaciens, non-pathogenic pig intestinal spirochetes, had rRNA gene restriction patterns that differed markedly from the S. hyodysenteriae patterns, and from each other.

Rumen contents as a reservoir of enterohemorrhagic Escherichia coli.

We investigated the role of the rumen fermentation as a barrier 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.

A clear view trephine and lamellar dissector for corneal grafting.

We designed a corneal trephine that permits a full view of the cornea while cutting. The drive mechanism of the trephine enables the surgeon to keep the central cutting axis of the instrument stable while the trephine is rotated. The instrument accepts variable sizes of disposable trephine blades, making it versatile and economical. We also devised a lamellar dissector with two blades to negate the splitting force that occurs during a closed lamellar dissection. It enables the surgeon to perform a closed lamellar dissection with minimal force applied to the globe and minimal risk of perforation.

Penetrating keratoplasty for keratoconus: complications and long-term success.

A series of 100 penetrating keratoplasties for keratoconus performed between 1968 abd 1986 were reviewed for long-term results. The mean follow-up was 6.1 years with a range of 4-16 years. The systemic associations and the postoperative complications were analysed. Grafting in cases associated with Down's syndrome had a higher incidence of complications. 93% of grafts remained clear and 81% achieved a final corrected visual acuity of 6/12 or better. 21% of eyes developed a homograft reaction, with 50% of rejection episodes occurring in the first year after operation. Factors associated with higher incidence of rejection included loose sutures, traumatic wound dehiscence, and grafts larger than 8.5 mm. Only three grafts with rejection episodes lost graft clarity, while rejection in the rest was successfully reversed with topical steroid therapy. No relationship was found between donor age and long-term graft clarity.

Posterior polymorphous keratopathy.

Seven cases with posterior polymorphous changes of the cornea are reported. After clinical and pathological examination of the above cases, as well as a short review of the literature, the following points are made: (1) Some cases are congenital, being either familial or sporadic, but others are acquired. (2) The term "posterior polymorphous keratopathy" covers all the variants of the condition and is preferred to the traditional "posterior polymorphous dystrophy". (3) The congenital type is a mild variant of the mesodermal dysplasia, whereas the acquired type follows local disease. (4) The condition can be static, but over 50% of cases are slowly progressive, calling for penetrating keratoplasty.

Influence of topical human epidermal growth factor on postkeratoplasty re-epithelialisation.

AIM: To test the efficacy and safety of recombinant human epidermal growth factor (hEGF) on corneal re-epithelialisation following penetrating keratoplasty. METHODS: A prospective, randomised, placebo controlled study was carried out in which patients were matched for diagnosis and received either hEGF ophthalmic solution (30 micrograms/ml or 100 micrograms/ml) or placebo in a double masked fashion. Matched pairs of patients received donor corneas from the same donor and were operated by the same surgeon on the same day. At the end of surgery all donor epithelium was removed mechanically. Patients were examined twice daily and fluorescein stained photographs were taken until the epithelium had closed. The area of the defect was measured by planimetry of the fluorescein stained defect on the photographs. RESULTS: There were no significant differences in re-epithelialisation of the donor cornea between the placebo group and the group treated with 30 micrograms/ml hEGF. Time until complete closure was slightly longer with 100 micrograms/ml hEGF compared with 30 micrograms/ml hEGF and with placebo. Mean healing rate of the epithelial defect with 100 micrograms/ml hEGF was significantly slower than in the other groups. CONCLUSION: No significant acceleration of corneal re-epithelialisation was demonstrated with the use of recombinant hEGF after penetrating keratoplasty in humans.

A monoclonal antibody identifies 2134P fimbriae as adhesins on enterotoxigenic Escherichia coli isolated from postweaning pigs.

Fimbriae (pili) of enterotoxigenic Escherichia coli (ETEC), including K88, K99, 987P, and F41, are adhesins that facilitate intestinal colonization in neonatal pigs. K88 is also associated with some ETEC isolated from weaned pigs. Many ETEC isolates from weaned pigs do not express known adhesins and are termed 4P-. A novel bacterial adhesin, 2134P, was recently identified on two 4P- ETEC isolates from weaned pigs. In this study, we identified a 2134P-specific monoclonal antibody, mAb 6C7/C1, that blocked the binding of 2134P+ bacteria to intestinal epithelial cells. Indirect immunofluorescent antibody and immunoperoxidase assays using mAb 6C7/C1 confirmed that the 2134P adhesin is expressed in vivo by adherent bacteria in pigs challenge-exposed with 2134P+ ETEC. 2134P was detected on 31% of 189 postweaning diarrhea 4P- ETEC isolates from the National Animal Disease Center's culture collection by dot blot immunoperoxidase assays using mAb 6C7/C1. We conclude that 2134P is a bacterial adhesin and is an important virulence attribute of some ETEC that cause diarrhea in weaned pigs.

Reducing the risk of corneal graft rejection. A comparison of different methods.

Corneal graft rejection represents the leading cause of failure in corneal transplantation. Two of the major risk factors for graft rejection are previous sensitization, usually in the form of a previous rejected corneal graft and corneal vascularization. The major histocompatibility MHC antigens (HLA, A, B and DR) are the target of the corneal graft rejection process. Because HLA, A, B, and DR antigens have been found in the corneal epithelium, the corneal stroma, and the corneal endothelium, matching patients and donors would seem to reduce the incidence of rejection. The results of studies on HLA, A, B, and DR matching are discussed. Cyclosporin, a fungal by-product, prevents the proliferation of sensitized cytotoxic T cells. Its use topically in corneal transplant patients in a controlled series has also reduced the incidence of rejection. Its use systemically has also been tried in an effort to prevent corneal graft rejection.

Penetrating keratoplasty for bilateral acute corneal calcification.

A 76-year-old man with bilateral practolol-induced dry eyes developed atypical acute bilateral corneal calcification. Serum calcium, phosphate, and urea levels were within normal limits. The calcium deposition progressed rapidly to involve 90% of the right cornea. Right penetrating keratoplasty was performed with subsequent visual rehabilitation of the patient. Left tectonic penetrating keratoplasty was performed 8 weeks later after corneal perforation. The corneal specimens were examined by light and electron microscopy, which showed an atypical calcareous degeneration involving Bowman's layer as well as the full thickness of the stroma. Transmission electron microscopy showed the granular calcification to consist of extracellular, radially orientated aggregates of fine, needle-shaped crystals. Both transplants remained clear with no evidence of postoperative recurrence. To our knowledge this is the first report of bilateral penetrating keratoplasty for acute calcareous degeneration.

Steady-state and time-resolved spectroscopy of F420 extracted from methanogen cells and its utility as a marker for fecal contamination.

Methanogenic bacteria, which are common inhabitants of the animal digestive tract, contain the fluorescent compound F420 (coenzyme 420), a 7,8-didemethyl-8-hydroxy-5-deazariboflavin chromophore. F420 was characterized as an initial step in determining if this compound would be useful as a fluorescent marker for the detection of fecal and ingesta contamination. Using a single anion exchange chromatographic process, F420 was separated from other cell components of a Methanobrevibacter sp. cell culture. The extent of separation was determined spectroscopically. To aid in the development of possible techniques for the detection of fecal contamination using F420 as a marker, further spectroscopic investigation of F420 was conducted using steady-state and time-resolved fluorescence methods. The fluorescence lifetime of F420 in an elution buffer of pH 7.5 was found to be 4.2 ns. At higher pH values, the fluorescence decay, F(t), was best described by a sum of two exponentials: at pH 13, F(t) = 0.31 exp(-t/4.20 ns) + 0.69 exp(-t/1.79 ns). Further investigation using front-faced fluorescence techniques has shown that emission from F420 can be collected efficiently from samples of methanogen cell cultures as well as from fecal material.

Pathogenicity of porcine enterotoxigenic Escherichia coli that do not express K88, K99, F41, or 987P adhesins.

Three-week-old weaned and colostrum-deprived neonatal (less than 1 day old) pigs were inoculated to determine the pathogenicity of 2 enterotoxigenic Escherichia coli isolates that do not express K88, K99, F41, or 987P adhesins (strains 2134 and 2171). Strains 2134 and 2171 were isolated from pigs that had diarrhea after weaning attributable to enterotoxigenic E coli infection. We found that both strains of E coli adhered in the ileum and caused diarrhea in pigs of both age groups. In control experiments, adherent bacteria were not seen in the ileum of pigs less than 1 day old or 3 weeks old that were noninoculated or inoculated with a nonpathogenic strain of E coli. These control pigs did not develop diarrhea. Antisera raised against strains 2134 and 2171 and absorbed with the autologous strain, grown at 18 C, were used for bacterial-agglutination and colony-immunoblot assays. Both absorbed antisera reacted with strains 2134 and 2171, but not with strains that express K99, F41, or 987P adhesins. A cross-reaction was observed with 2 wild-type K88 strains, but not with a K12 strain that expresses K88 pili. Indirect immunofluorescence with these absorbed antisera revealed adherent bacteria in frozen sections of ileum from pigs infected with either strain. We concluded that these strains are pathogenic and express a common surface antigen that may be a novel adhesin in E coli strains that cause diarrhea in weaned pigs.

Postweaning diarrhea in swine: experimental model of enterotoxigenic Escherichia coli infection.

A reproducible model of postweaning colibacillosis was obtained by controlling management and environmental variables to simulate conditions often seen at weaning. Suckling pigs were exposed briefly to starter diet at 1 week of age, weaned at 3 weeks of age, held at an ambient temperature of 20 +/- 2 C, and again given the starter diet. One day after weaning, each pig was given 10(10) colony-forming units of enterotoxigenic Escherichia coli strain M1823B (O157:K88ac:H43-LT+ STb+) in broth containing 1.2% sodium bicarbonate via stomach tube. In vitro adhesion by strain M1823B to isolated intestinal branch borders was used to test pigs for susceptibility to K88. In this model, 3 syndromes were induced in susceptible pigs: (1) peracute fatal diarrhea; (2) moderate diarrhea, weight loss, and fecal shedding of the inoculum strain; and (3) no diarrhea, weight loss, and fecal shedding of the inoculum strain. Rotavirus particles were not found in fecal specimens of pigs with diarrhea. The K88-susceptible, noninoculated control pigs remained clinically normal. It was concluded that susceptibility to adhesion by K88+ enterotoxigenic Escherichia coli was a requirement for the production of disease in this model; inoculation with rotavirus was not necessary.

Fecal shedding of coliform bacteria during the periparturient period in dairy cows.

OBJECTIVE: To determine whether numbers of coliform bacteria in feces of dairy cattle changed during the periparturient period and whether fluctuations were associated with changes in dry-matter intake. ANIMALS: 12 healthy Holstein cows. PROCEDURE: Fecal samples were collected on a semi-regular basis (i.e., 3 to 7 times/wk) beginning 4 to 6 weeks before the anticipated parturition date and continuing through the third day (5 cows) or second week (7 cows) after parturition, and total numbers of fecal coliform bacteria were determined. Daily feed intake of 7 cows was monitored. RESULTS: For 11 cows, fecal coliform bacterial counts between 34 and 25 days prior to parturition were low and relatively constant (< 102 change in number of bacteria). Coliform bacteria were not detected in 4 to 8% of fecal samples from 10 cows. All cows had a 10(4) to 10(7) increase in number of colony forming units/g of feces near the time of parturition. Number of fecal coliform bacteria peaked within 7 days of parturition in 9 cows and within 12 days of parturition in 3. Number of fecal coliform bacteria was not correlated with feed intake. CONCLUSIONS AND CLINICAL RELEVANCE: Cows may have large increases in fecal coliform bacteria count during the periparturient period; however, periparturient cows do not continually shed high numbers of coliform bacteria, and coliform bacteria may not always be detectable by conventional culture methods. Changes in fecal coliform bacteria count did not correlate with changes in dry-matter intake.

Shiga-like toxin production and attaching effacing activity of Escherichia coli associated with calf diarrhea.

Four hundred twenty-nine isolates of Escherichia coli from calves were tested for the production of HeLa cell cytotoxin(s). Isolates that produced enough cytotoxin to be detected in culture supernatants of iron-depleted broth were considered to produce increased amounts of cytotoxins. Isolates also were tested for homology with a DNA probe for a gene that encodes localized adherence of human enteropathogenic E coli. Four isolates produced increased amounts of cytotoxin that was neutralized by Shiga antitoxin (toxin designated as Shiga-like toxin-I [SLT-I]). A 5th isolate produced increased amounts of cytotoxin (SLT+) that was not neutralized by the Shiga antitoxin, but was neutralized by antitoxin against a variant of SLT (toxin designated as SLT-II). None of the isolates hybridized with the probe for the localized adherence gene. Three of the SLT+ isolates belonged to human enteropathogenic E coli serogroups O26 and O111. All 5 of the SLT+ isolates were from calves with diarrhea, but none of the 5 SLT+ isolates contained genes for classic heat-labile or heat-stable enterotoxins, for K99 fimbriae, or for invasiveness; neither did any of them adhere to HeLa cells in culture. Three of the 5 SLT+ isolates had attaching and effacing activities when inoculated into ligated intestinal loops of rabbits. One of the isolates with attaching and effacing activity in rabbits was originally isolated from a calf with lesions characteristic of those produced by attaching effacing E coli (AEEC). Calves inoculated with this SLT+ AEEC isolate developed focal colonic lesions characteristic of those produced by AEEC, but did not develop diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS)

Hybridization of bovine Escherichia coli isolates with gene probes for four enterotoxins (STaP, STaH, STb, LT) and one adhesion factor (K99).

Colony hybridizations with 4 enterotoxins (STaP, STaH, STb, and LT) and 1 adherence factor (K99) gene probes were done on Escherichia coli isolated from calves. Agreement between the K99 probing and a serologic assay to detect the K99 antigen was 99% for the identification of K99+ and K99- isolates. Ninety-five of the isolates (22%) hybridized with at least 1 enterotoxin gene probe (Ent+ isolates), and 82 (19%), with the K99 gene probe. The majority of Ent+ isolates (85%) reacted with probes for STaP and K99 genes. The STaP gene was present by itself in 4 of the Ent+ isolates (4%) and with the STb gene in 6 of the Ent+ isolates (6%). Five of the Ent+ isolates (5%) carried the STb and LT genes, and none (0%) of the isolates carried the STaH gene. All but 2 of the isolates with the K99 gene also had the STaP gene. Twenty-eight isolates shown to produce STa enterotoxin in previous studies failed to hybridize with any of the enterotoxin gene probes. These 28 isolates were also phenotypically negative when the tests for enterotoxin production were repeated. These isolates probably lost their genes for enterotoxin production during storage in the laboratory.

Prevalence of mitral valve prolapse in keratoconus patients.

Fifty patients with advanced degrees of keratoconus, requiring corneal transplantation, were screened for mitral valve prolapse by two dimensional echocardiography. The overall prevalence of 58% was found to be statistically higher than the prevalence of 7% found in a group of age and sex-matched controls. It was also found to be higher than the previously reported prevalence of 38% in a group of keratoconus patients with similar age and sex match to our series. The findings of our study in conjunction with the histopathological and biochemical similarities between the two conditions strongly suggest that they may be different manifestations of similar defects in collagen metabolism.