Helicobacter pylori physiology predicted from genomic comparison of two strains.

Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outcome of H. pylori-related disease.

Binding of nonmucoid Pseudomonas aeruginosa to normal human intestinal mucin and respiratory mucin from patients with cystic fibrosis.

Lung infections due to Pseudomonas aeruginosa and Pseudomonas cepacia are common in patients with cystic fibrosis. Initial colonization is due to nonmucoid P. aeruginosa, while later mucoid variants emerge and are associated with chronic infection. P. cepacia colonization tends to be more prevalent in older patients. The present study was conducted to discover whether highly purified mucins (from cystic fibrosis sputum and control intestinal secretions) exhibited specific binding of nonmucoid P. aeruginosa. In vitro solid phase microtiter binding assays (with or without a blocking agent) as well as solution phase assays were conducted. Bacteria bound to both mucins via bacterial pili, but no differences in binding capacity were noted between the mucins. Unlike P. cepacia (described in the accompanying manuscript) there was also no preferential binding of P. aeruginosa to mucins versus bovine serum albumin, casein, gelatin, or a host of structurally unrelated proteins and glycoproteins. Carbohydrate hapten inhibition studies did not suggest the existence of specific mucin carbohydrate receptors for P. aeruginosa. In solid phase assays a low concentration (0.05 M) of tetramethylurea abolished P. aeruginosa bacterial binding to both mucins as well as to BSA, whereas in solution phase assays mucin binding to bacteria was not completely disrupted by tetramethylurea. Specific monoclonal antipilus antibodies did not inhibit binding to a greater extent than did Fab fragments of normal mouse IgG. Binding of strains PAO1 and PAK (and isolated PAK pili) to buccal epithelial cells was not influenced by the presence of mucin in binding assay mixtures. Our findings do not support the widely held notion that specific mucin receptors are responsible for the attachment of P. aeruginosa pili, nor do they support the idea that there is a competitive interference by mucins of bacterial binding to respiratory cells. In patients with cystic fibrosis, it would seem unlikely therefore that initial colonization of the lungs by P. aeruginosa is due to a 'selective tropism' of these bacteria for respiratory mucin.

Treatment of log yard runoff using a r-circulating sand filtration process.

A re-circulating filtration process using oxide-coated sand successfully removed COD and turbidity from log yard runoff. After passing only one pore volume of the runoff through the sand column, 72% COD was removed. The 2.4% Fe and Al oxide coating on the sand contributed to better COD removal than was obtained when the sand was stripped of oxide coating (86% versus 52%, respectively), at least initially before saturation of adsorption sites on the oxide coating occurred. The best COD removal performance came from conditioned sand. This sand, from the same original source and identical to the oxide-coated sand used in all experiments, came from an existing experimental sand column that had been treating log yard runoff for 1 year. The "conditioning" resulted in the sand having a higher TOC content (0.26% wt) and smaller particle sizes. This sand was able to consistently remove 80% COD from repeated batches of log yard runoff with strengths up to 3690 mg l(-1).

Distribution of surface-exposed and non-accessible amino acid sequences among the two major structural domains of the S-layer protein of Aeromonas salmonicida.

The tetragonally arranged crystalline surface protein array (A-layer) of the fish pathogenic bacterium Aeromonas salmonicida is a virulence factor. Circular dichroism studies in the presence or absence of 0.1% sodium dodecyl sulfate showed that the secondary structure of A-protein, and its 39,439 molecular weight amino-terminal trypsin-resistant peptide, were altered. In both cases alpha-helix was increased significantly at the expense of beta-structure when SDS was added. Western and dot immunoblotting, immuno-microscopy and enzyme-linked immunosorbent assay with monospecific polyclonal antiserum and eight monoclonal antibodies specific for epitopes exposed on the surface of native A-layer showed that the 481 residue A-protein subunit and the surface of A-layer were conserved antigenically. Mimeotope analysis of nonapeptides representing the sequence of A-protein allowed identification of 146 residues in presumed linear epitopes accessible on the surface of A-layer. Inaccessible or non-epitopic residues accounted for 70% of the protein. The majority of inaccessible residues were in the N-terminal 301 residues of A-protein. Dispersed among these were 65 surface-accessible residues in five linear epitope clusters illustrating the complex folding of this major structural domain of A-protein. The C-terminal 180 residues carried fewer linear epitopes but contained the major region of A-layers surface-accessible sequence, including four linear epitopes in predominantly hydrophobic sequence. Four A-layer surface-binding monoclonal antibodies also bound to this minor structural domain, although the epitopes of only two were identified by mimeotope analysis. The epitopes of six A-layer surface-binding monoclonals could not be identified, suggesting that A-layer may also contain conformation dependent surface epitopes.

Molecular Characterization of H. pylori Surface Antigens.

Despite its clinical significance, relatively little is known about the components of Helicobacter pylori that allow it to colonize, persist, and elicit an inflammatory response within the host. Bacterial surface components frequently influence colonization and persistence of a pathogen, as well as the disease process (1). In the case of H. pylori, one macromolecular assembled protein component that is unequivocally located on the surface of the bacterium is the sheathed flagellum. Two other proteins that can be isolated in abundance from suspensions of H. pylori cells, but that are normally regarded as intracellular in other bacterial species, are urease and a GroEL analog, Hp60K (2-6). Whether these large macromolecular assemblies are surface proteins on H. pylori or are released as a result of cellular lysis is uncertain. However, numerous other intracellular proteins can be readily isolated from culture supernatants after mild shearing and extraction procedures, and these released proteins have initially been incorrectly interpreted as representing surface proteins (7-9).

Bovine abortion and neonatal death associated with Ureaplasma diversum.

Ureaplasma diversum was isolated from the lungs and/or stomach fluid and placentas of five aborted bovine fetuses and four newborn calves. All isolates were serotype D48. Placentitis was observed in all instances in which the placenta was examined. Gross lesions consisted of focal or diffuse reddening of the chorioallantois and amnion and thickening of the amnion. Microscopically there were fibrosis, edema and inflammation of the amnion. Microscopic lesions in the lung consisted of diffuse pneumonitis with thickening of the alveolar walls and in some cases peribronchiolar lymphoid accumulations. Macrophages and granulocytes were present in the alveoli. Inoculation of the vulva of a virgin heifer with one of the isolates from a fetal lung produced hyperemia and profuse purulent discharge with slight granularity.

The effects of Ureplasma diversum inoculated into the amniotic cavity in cows.

Ureaplasma diversum was inoculated into the amniotic cavity in four cows. Two calves were aborted and two were born alive. One of the latter died shortly after birth and the other was killed. The cows remained clinically normal except that three retained their placenta. On microscopic examination there was a severe placentitis and an alveolitis was present in the lungs of all calves. Ureaplasma was recovered from four placentas and three lungs. Cows remained infected for a maximum of 132 days following inoculation and the organism was recovered in urine and vulvar swabs for a maximum of 17 and 60 days respectively following expulsion of the calf. Ureaplasma diversum has been isolated from natural cases of abortion with similar lesions. This experiment strongly supports a causal relationship between abortion, birth of calves with pneumonia and U. diversum infection.

The effect of intrauterine inoculation with Ureaplasma diversum on bovine fertility.

To determine the influence of Ureaplasma diversum on bovine fertility 11 uninfected virgin heifers with normal ovarian cyclic activity were randomly allocated to test or control groups. At a synchronized estrus, five test heifers were given an intrauterine broth inoculum containing 1.09 x 10(8) to 1.4 x 10(9) colony forming units of U. diversum and six control animals were infused with sterile ureaplasma broth medium. All animals were artificially inseminated within one hour of infusion. Pregnancy was diagnosed in one of five test heifers and all of six controls by serum progesterone concentrations measured to 25 days postinsemination. The difference in pregnancy rates between the two groups was statistically significant (p = 0.0152). It was concluded that under the conditions of this experiment U. diversum is capable of causing infertility in cattle.

Identification of a mutant bovine herpesvirus-1 (BHV-1) in post-arrival outbreaks of IBR in feedlot calves and protection with conventional vaccination.

Outbreaks of infectious bovine rhinotracheitis (IBR) have recently been observed in vaccinated feedlot calves in Alberta a few months post-arrival. To investigate the cause of these outbreaks, lung and tracheal tissues were collected from calves that died of IBR during a post-arrival outbreak of disease. Bovine herpesvirus-1 (BHV-1), the causative agent of IBR, was isolated from 6 out of 15 tissues. Of these 6 isolates, 5 failed to react with a monoclonal antibody specific for one of the epitopes on glycoprotein D, one of the most important antigens of BHV-1. The ability of one of these mutant BHV-1 isolates to cause disease in calves vaccinated with a modified-live IBR vaccine was assessed in an experimental challenge study. After one vaccination, the majority of the calves developed humoral and cellular immune responses. Secondary vaccination resulted in a substantially enhanced level of immunity in all animals. Three months after the second vaccination, calves were either challenged with one of the mutant isolates or with a conventional challenge strain of BHV-1. Regardless of the type of virus used for challenge, vaccinated calves experienced significantly (P < 0.05) less weight loss and temperature rises, had lower nasal scores, and shed less virus than non-vaccinated animals. The only statistically significant (P < 0.05) difference between the 2 challenge viruses was the amount of virus shed, which was higher in non-vaccinated calves challenged with the mutant virus than in those challenged with the conventional virus. These data show that calves vaccinated with a modified-live IBR vaccine are protected from challenge with either the mutant or the conventional virus.

Prevalence and distribution of Haemophilus somnus in the male bovine reproductive tract.

"Haemophilus somnus' was isolated from 77% of 31 reproductive tracts of bulls from an Ontario slaughterhouse. Identification of H somnus was based on morphologic and cultural characteristics and on fluorescent antibody and immunodiffusion tests, using antisera prepared against a known pathogenic encephalitic isolate of H somnus. The infection rate and distribution of H somnus within the tract were as follows: preputial orifice--71% preputial cavity--71%; urinary bladder--26%; accessory sex glands--19% and ampulla of ductus deferens--10%. Isolates were not obtained from the testes or epididymides. On 2 occasions, H somnus was isolated in pure culture from the preputial cavity. A higher prevalence of infection was found in young bulls. There were no differences found in infection rates between breeds. Differences in hemolytic activity and minor antigenic variation between isolates indicated that a series of biotypes within the species H somnus may exist. The study indicates that organisms presently identified as H somnus may normally form part of the flora of the bovine prepuce and that dissemination from the male bovine reproductive tract is one possible means of infection in H somnus-associated diseases. The pathogenic significance of genital isolates of H somnus awaits further study.

Bovine genital mycoplasmosis.

Infection, lesions and clinical significance of Acheloplasmas, Mycoplasma bovis and Mycoplasma bovigenitalium in genital disease of cattle are described. A more detailed account is given of ureaplasma infections. Acute and chronic forms of granular vulvitis in both field and experimental disease are described as well as the role of the organism in abortion. Recovery rates of ureaplasma and mycoplasma from semen and preputial washings in bulls are outlined and their significance in disease is discussed. There are problems in differentiating pathogenic from nonpathogenic isolates. Methods are being developed to treat semen for these organisms. This paper provides a concise summary of clinical and microbiological aspects of bovine genital mycoplasmosis.

Pregnancy and peripheral plasma progesterone levels in cows inseminated after synchronization of estrus with prostaglandin F2 alpha.

Fifteen Holstein cows were treated with two doses of 25 mg of a prostaglandin F(2)alpha (PGF(2)alpha as dinoprost tromethamine) administered intramuscularly 11 days apart. The cows were then divided into three groups and inseminated either at 72, 80 or 72 and 96 hours after the second dose of PGF(2)alpha. Thirteen cows ovulated after the second prostaglandin treatment. Eight cows were diagnosed pregnant by rectal palpation 42 days after insemination but only five calved. PGF(2)alpha induced luteolysis in cows with active corpora lutea as evidenced by the dramatic decreases in the plasma progesterone concentrations after treatment. In contrast, following PGF(2)alpha administration to cows in follicular or late luteal phase the concentrations of plasma progesterone either increased gradually or remained low for several days before increasing to maximal levels. The ovulatory rate after the two doses of PGF(2)alpha11 days apart was high. However, the pregnancy rate after this treatment was influenced by other factors including abnormal ovarian function.

The use of endometrial biopsy in the infertile mare.

The results of a study on endometrial biopsies obtained from 700 infertile mares are reported. Infiltrative endometritis was present in 51% consisting of a combination of an acute and chronic cellular response in 6%, mild chronic infiltrations in 35% and moderate to severe chronic infiltrations in 10%. Demonstrable endometrial fibrosis was found in 88% of the mares with the majority having mild (51%) or moderate (35%) changes. The age of the mares and the average number of years barren gradually increased with the severity of endometrial fibrosis, as did the combined incidence of fetal loss (early embryonic death and abortion) during the three year period preceding the biopsy.A significant decrease in foaling percentage for the year following the biopsy was observed with increasing severity of endometrial fibrosis. Chisquare analysis projected a decrease in foaling rate of 22.8% for each category increase in severity. The number of years barren and the farm management system used also had a significant effect on foaling probability. The foaling rates for mares that were bred artificially (82%) under strict veterinary supervision using an extender incorporating gentamicin were significantly (P < 0.05) higher than for mares bred in a conventional manner under average management conditions (48%). It was concluded that endometrial biopsy is a valuable diagnostic and prognostic technique. When the degree of fibrosis is used to predict foaling probability, the number of years barren and the breeding method to be used should also be considered to arrive at a more accurate prediction.

A study of drug residues in milk following intrauterine infusion of antibacterial drugs in lactating cows.

Intrauterine infusion of nine antibacterial compounds caused detectable drug residues in 17 out of 165 cows or in 25 out of 1110 posttreatment milkings. Four cows treated with pyrolidinomethyl tetracycline suspension had drug residues at the first milking. One cow had residues after oxytetracycline treatment, two after procaine penicillin G, three after acriflavin and after chloramphenicol-dapsone and four after hibitane. Nitrofurazone, nitrofurathiazide and Hibitane Compound(R) did not cause detectable inhibitory residues in any milk sample.

Clinical efficacy and tolerance of meloxicam in dogs with chronic osteoarthritis.

A clinical trial was conducted to evaluate the safety and efficacy of the nonsteroidal anti-inflammatory drug meloxicam in dogs with chronic osteoarthritis. Forty clinical cases were enrolled in the 2-phase study. Phase 1 compared therapeutic efficacy and tolerance of meloxicam or placebo for 1 week. Phase 2 involved a 4-week evaluation of the drug's clinical efficacy and tolerance. Clinical efficacy was evaluated by using a scoring system that assessed specific lameness, general stiffness, painful rise, exercise intolerance, and behavior. Evaluations demonstrated significant reductions (P < 0.05) in clinical signs of osteoarthritis following 4 weeks of drug therapy. Side effects were minimal in extent and duration. The drug was accepted without problems in the majority of cases. The findings of this investigation suggest that the efficacy, tolerance, and formulation of meloxicam oral suspension make it well suited for the treatment of chronic osteoarthritis in the dog.

Bovine granular vulvitis associated with ureaplasma infection.

A granular vulvitis syndrome associated with ureaplasma infection was first recognized in Ontario dairy herds in 1972. The acute form of the disease was characterized by a purulent vulvar discharge, an inflamed hyperemic vulvar mucosa and varying degrees of granularity. In the chronic form, there was an absence of a purulent discharge and a gradual decline in the severity of the hyperemia and granularity. Epithelial inclusion cysts were observed in the vulvar epithelium of approximately 10% of affected cows.A seasonal variation in the incidence of the disease was observed. Herd morbidities during the summer months reached a low of 37% and increased to 75% during the winter months with constant housing.When widespread in herds, the acute form of the disease had a significant effect on fertility. In four herds examined, first service conceptions dropped on average by 27%. The chronic form of the disease had a less detrimental effect on fertility with first service conceptions being reduced on average by 13%. Intrauterine infusions of a tetracycline 24 hours postbreeding were found to be of value in improving conception rates in acutely affected herds.