Correlation between in vitro secretion of virulence-associated proteins of Campylobacter jejuni and colonization of chickens.

The mechanisms used by Campylobacter jejuni to colonize the (chicken) intestinal tract have not been defined. In this study, we obtained evidence that in the presence of chicken serum and mucus, C. jejuni secreted proteins that may play a role in the colonization of chicken gut (Campylobacter invasion antigen = Cia). C. jejuni strains NCTC11168V1 and 81-176, as well as an NCTC11168V1 flaA mutant, were found to colonize intestinal tract and secrete proteins in the presence of chicken mucus, chicken serum, or fetal bovine serum in cell culture-conditioned medium. C. jejuni strain NCTC11168V26, which was observed to be a poor colonizer compared with the other C. jejuni isolates, did not secrete Cia proteins. Secreted proteins were also recognized by Western immunoblot using sera from birds that had been colonized by C. jejuni. These data suggest that C. jejuni secretes Cia proteins during colonization of chicken gut and that these Cia proteins play an important role in colonization.

An experimental and theoretical study of the reactions OIO+NO and OIO+OH.

The kinetics of the reaction OIO+NO were studied by pulsed laser photolysis/time-resolved cavity ring-down spectroscopy, yielding k(235-320 K)=7.6(+4.0)(-3.1) x 10(-13) exp[(607+/-128)/T] cm3 molecule-1 s-1. Quantum calculations on the OIO+NO potential-energy surface show that the reactants form a weakly bound OIONO intermediate, which then dissociates to the products IO+NO2. Rice-Ramsberger-Kassel-Markus (RRKM) calculations on this surface are in good accord with the experimental result. The most stable potential product, IONO2, cannot form because of the significant rearrangement of OIONO that would be required. The reaction OIO+OH was then investigated by quantum calculations of the relevant stationary points on its potential-energy surface. The very stable HOIO2 molecule can form by direct recombination, but the bimolecular reaction channels to HO2+IO and HOI+O2 are closed because of significant energy barriers. RRKM calculations of the HOIO2 recombination rate coefficient yield krec,0=1.5x10(-27) (T/300 K)(-3.93) cm6 molecule-2 s-1, krec,infinity=5.5x10(-10) exp(46/T) cm3 molecule-1 s-1, and Fc=0.30. The rate coefficients of both reactions are fast enough around 290 K and 1 atm pressure for these reactions to play a potentially important role in the gas phase and aerosol chemistry in the marine boundary layer of the atmosphere.

Attenuation of an avian pathogenic Escherichia coli strain due to a mutation in the rpsL gene.

The diseases caused by pathogenic Escherichia coli constitute a major economic loss to the poultry industry. The development of a live oral E. coli vaccine to prevent or reduce diseases in poultry had been the objective of our work. Four spontaneous streptomycin-dependent (str-dependent) mutants were generated from a virulent avian strain that contains a mutation in the fur region of the chromosome. Genetic analysis of the mutants indicated that the str-dependent phenotype was due to a base change of C --> T at base 272 in the rpsL gene. The mutants were tested for attenuation using the day-old chick model. Day-old birds, in groups of 20, were either challenged with 10(6) colony-forming units (CFU) of the str-dependent mutant, the parent strain (containing the fur mutation), or the wild-type strain without the fur mutation. The parent strain and the wild-type strain were highly virulent, and 80% or more of the birds died. None of the birds challenged with the str-dependent mutants died, indicating attenuation of the mutants. The protective effect of the mutant as a live vaccine against the challenge with 10(6) CFU of the wild-type strain EC317 was investigated. Vaccination by both aerosol (day 1) and oral (days 14 and 28) routes using 10(8) CFU of the str-dependent mutant (EC1598) had no effect on the occurrence of cellulitis in the birds. Two vaccinations given as aerosol on day 1 and given orally on day 14 also had no significant effect on the occurrence of systemic lesions. Three immunizations on days 1, 14, and 28 resulted in a significant reduction in the number of birds with systemic lesions. Antibody titers prior to challenge were not predictive of outcome of challenge.

Phenotypic and genotypic characterization of virulence factors of Escherichia coli isolated from broiler chickens with simultaneous occurrence of cellulitis and other colibacillosis lesions.

The objective of this study was to characterize virulence factors of Escherichia coli isolates from broilers with simultaneous occurrence of cellulitis and other colibacillosis lesions. Thirty flocks were sampled and 237 birds with cellulitis were examined. Eighty-two (34.6%) of 237 birds condemned for cellulitis had gross lesions in the heart, air sacs, joints, or liver. In 58 chickens, E. coli was isolated from both the cellulitis and other lesions of colibacillosis, and 18.9% of the E. coli isolates from the 2 types of lesions belonged to the same O group. Escherichia coli of serogroups O78, O1, and O2 predominated. Isolates of the same serogroup that were derived from different lesions in the same birds had similar patterns of biotype, aerobactin production, serum sensitivity profile, antibiotic sensitivity, and K1 capsule production. Escherichia coli derived from cellulitis lesions produced virulence factors similar to those found in E. coli isolated from other colibacillosis lesions in poultry.

Studies on cellulitis and other disease syndromes caused by Escherichia coli in broilers in Sri Lanka.

Cellulitis caused by Escherichia coli in broilers results in substantial losses to the broiler industry in North America and Europe due to condemnations at slaughter. The objective of this study was to identify cellulitis in broilers in Sri Lanka and to characterize the E. coli from cellulitis and other colibacillosis lesions. Twenty-four farms from the low- and mid-country were selected and bacterial isolations were obtained from 241 birds. Two hundred and ninety-one gross lesions were observed in these 241 birds and 162 E. coli isolates were obtained. Cellulitis was observed in 21% of the birds. Twenty-one per cent of the birds had multiple lesions due to E. coli. The frequency of detection of other disease syndromes was 162 (67%) birds with pericarditis, 26 (11%) airsacculitis, 24 (10%) hepatitis, 12 (5%) perihepatitis, and 16 (7%) polyserositis (a combination of pericarditis, perihepatitis and airsacculitis). Serogroups O78, O2, O85 and O88 were distributed among the 32% of typable E. coli and 81% of isolates were assigned to three biotypes. Forty-four per cent of the E. coli isolates produced aerobactin and 88% demonstrated resistance to the bactericidal effect of normal chicken serum. The majority of the E. coli isolates were resistant to the antibiotics commonly used in poultry. All the E. coli isolates were non-haemolytic and 25% of the isolates produced K1 capsule. This study demonstrated the presence of cellulitis in Sri Lanka and this report describes some of the phenotypic characteristics of the E. coli isolates.

Cellulitis in broiler chickens: epidemiological trends, meat hygiene, and possible human health implications.

The present work evaluates trends in the incidence of cellulitis during the last decade using Canadian National Poultry Condemnation Records. In 1986, only 0.048% of the total slaughter broilers were condemned as a result of cellulitis lesions. Over the next 10 yr, steady increments in cellulitis condemnations were observed, and between 1986 and 1996, the percentage of cellulitis condemnation increased 11.8-fold. In 1996, more than 2.6 million broilers (0.568% of total slaughter) were condemned due to cellulitis; this constituted 30.1% of total condemnations, making it the number one condemnation category in 1996. In the context of dynamic increase in cellulitis, the problems concerning meat hygiene and possible health risk to the consumer are deliberated.

Isolation of Escherichia coli from cellulitis and other lesions of the same bird in broilers at slaughter.

Cellulitis results in substantial losses to the broiler industry due to condemnations at slaughter. This study was conducted to clarify the association between Escherichia coli isolated from cellulitis and other lesions caused by E. coli in individual birds. Fourteen flocks were sampled and 118 birds with cellulitis were examined. Escherichia coli was isolated from all but 2 of the cellulitis lesions, and serogroups O78, O1, and O2 predominated. Thirty-six birds had at least 1 other lesion in addition to the cellulitis lesion. Isolation of E. coli from cellulitis and other lesions occurred in 7 of the 14 flocks. Escherichia coli of the same serogroup were isolated from cellulitis and other lesions in some birds, suggesting that a single E. coli may sometimes be responsible for both types of lesions.

Experimental reproduction of Escherichia coli cellulitis and septicemia in broiler chickens.

Experimental reproduction of avian cellulitis was conducted by subcutaneous inoculation of 25-day-old broiler chickens with a field isolate of serogroup O78 Escherichia coli. Development of the cellulitis lesion occurred as early as 24 h post-infection. Reproduction of cellulitis occurred in 98% of inoculated birds, and E. coli was isolated from > 75% of cellulitis lesions. In addition to cellulitis, other lesions, including pericarditis, airsacculitis, osteomyelitis, arthritis, and perihepatitis, occurred in > 80% of birds inoculated with E. coli. Bacteremia occurred as early as 6 h post-infection and dramatically declined by 5 days post-infection. Seventeen of 59 (29%) birds inoculated with E. coli developed a fatal infection between 1 and 6 days post-infection, and bacteria were isolated from lesions in 98% birds. In contrast, E. coli was not isolated from lesions in birds that survived until days 7-14 post-infection. Birds that survived with cellulitis and other lesions until day 14 post-infection had a significantly lower body weight compared with the control group. This avian model of cellulitis and other lesions will be useful for studying the development of vaccination strategies for E. coli in broilers.

Effect of infection with hemorrhagic enteritis virus on susceptibility of turkeys to Escherichia coli.

The present study was designed to investigate hemorrhagic enteritis virus (HEV) as a predisposing factor influencing the susceptibility of young turkeys to Escherichia coli infections. In addition, the pathologic changes caused by administration of E. coli by various routes were compared. Following oral infection with HEV, groups of turkeys were inoculated with various doses of pathogenic E. coli by intravenous (IV), intra-air sac (IA), or intratracheal (IT) routes. A synergistic effect was observed in birds that were exposed to a combined HEV-E. coli challenge, resulting in higher mortality than that caused by either pathogen alone. This synergy was more evident when the bacteria were administered by the IT route than when it was administered by the two other routes. Turkeys infected with HEV and then inoculated IT with E. coli O78 had higher mortality (61%) and higher occurrence of gross body lesions (74%) than birds given E. coli alone (0% mortality and 16% gross lesions). After E. coli inoculation by the IA and IT routes, lesions observed were mainly pericarditis, perihepatitis, lung and air-sac lesions, splenic enlargement, and occasional arthritis. The incidence of lesions was affected by HEV exposure. In contrast, IV inoculation with E. coli O78 usually resulted in arthritis, and its incidence was independent of previous HEV exposure. The synergistic effect between HEV and E. coli administered IT can be used as a challenge model for testing E. coli vaccines.

A carAB mutant of avian pathogenic Escherichia coli serogroup O2 is attenuated and effective as a live oral vaccine against colibacillosis in turkeys.

Colibacillosis is a serious and economically important disease of the respiratory tract of chickens and turkeys. The serogroups of Escherichia coli commonly associated with colibacillosis in poultry are O1, O2, and O78. Although previous attempts to develop a vaccine have not been very successful, vaccination is still considered the most effective way of controlling the disease. Therefore, our laboratory has been involved in the development of an attenuated live vaccine that will be effective in the prevention of colibacillosis. The carAB operon coding for carbamoyl-phosphate synthetase, an essential enzyme in arginine and pyrimidine metabolism, was selected for study. Generalized transduction was used to transfer a Tn10-generated mutation from a laboratory strain to virulent avian field isolates of E. coli. Molecular techniques were used to determine the point of Tn10 insertion within the carAB operon. The insertion mutants were then cured of the tetracycline resistance gene of the transposon to select for antibiotic-sensitive and stable carAB mutants. The degree of attenuation obtained by the mutation was determined in day-old chickens. Typically, when 100-fold the 50% lethal dose (for the wild type) was given, no more than 50% mortality in the day-old chickens was observed. The deletion mutant of serotype O2 was also found to be avirulent in turkeys rendered susceptible to infection with hemorrhagic enteritis virus A. Turkey poults vaccinated orally at 4 weeks old with either the wild-type E. coli EC317 strain or its carAB mutant EC751 were completely protected from infection following challenge with the homologous wild-type strain. Our data indicate that carAB mutants of virulent avian strains of E. coli will be effective and safe as live oral vaccines for prevention of colibacillosis in poultry.

Cloning of the early promoters of Pseudomonas aeruginosa bacteriophage D3: sequence of the immunity region of D3.

The early promoters of bacteriophage D3 of Pseudomonas aeruginosa were cloned and physically mapped to the right 25% of the phage genome. The promoters were cloned into promoter selection vector pQF26, and their relative strengths, the direction of transcription, and whether they were directly regulated by repressor were determined. A 3.3-kb fragment of the genome containing the immunity region was sequenced and analyzed (GenBank accession number: L22692). The promoter activity associated with this region was determined to be bidirectional and repressible, indicating that this region contains operator-promoter complexes. Sequence and functional analyses suggest that this region is analogous to the immunity region of coliphage lambda. Two strong promoters, one of which was repressible, were found to be located adjacent to the immunity region. Clear-plaque mutant phage D3c contains insertion element IS222, which causes it to behave as a repressor-negative (c1) variant. The site of insertion of IS222 was sequenced and determined to lie within the c1 gene open reading frame. This phage shows remarkable similarity in genomic organization to coliphage lambda and its relatives.

Characterization of Escherichia coli isolated from cases of avian colibacillosis.

Forty-four western Canadian isolates of Escherichia coli associated with colibacillosis of turkeys and chickens were examined for serotype, antibiotic resistance, and production of aerobactin. The isolates belonged to fourteen O serogroups, with 39% of the strains being non-typeable. A high frequency of resistance to tetracycline, kanamycin, neomycin, cephalothin, streptomycin and erythromycin was observed. Most isolates produced aerobactin. Ten E. coli belonging to serogroups O1, O2 and O78 were also examined for pili production, hemagglutination, serum sensitivity, production of iron-regulated outer membrane proteins (IROMPS), and virulence. All isolates examined produced pili, exhibited mannose-sensitive hemagglutination of avian red blood cells and produced IROMPS under iron-restricted growth conditions. The five isolates of serogroup O1 and O2 were resistant to killing by turkey serum and were highly virulent. Only two of the five isolates of serogroup O78 were serum resistant. No correlation between serum resistance and virulence was observed in serogroup O78.

Cyclic nucleotide responses in control and cystic fibrosis labial glands.

Adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) levels were measured in labial gland slices from controls and patients with cystic fibrosis (CF). Incubation in vitro with 10 microM epinephrine, 50 microM isoproterenol, or 10 microM carbachol increased cAMP levels by 2.3-fold, 3.1-fold, and 1.8-fold, respectively, in control glands and by similar amounts in CF glands. The only statistically significant CF-related difference was a decreased response to isoproterenol. Addition of MIX (3-isobutyl-1-methylxanthine) increased cAMP levels in control and CF glands by an order of magnitude under all conditions but did not eliminate the CF-related decrease in cAMP level obtained with isoproterenol. cGMP levels were measured only in the presence of MIX. Incubation with carbachol nearly doubled cGMP levels in control and CF glands but only the control gland response approached statistical significance (P = 0.06). cGMP levels in CF glands were nearly threefold greater than those in control glands, and disease-related differences obtained in the presence of carbachol and isoproterenol were statistically significant.

Characterization of the genome of Pseudomonas aeruginosa bacteriophage phi PLS27 with particular reference to the ends of the DNA.

The DNA of Pseudomonas aeruginosa rough-specific bacteriophage phi PLS27 was studied. The genome size as determined by summing the sizes of restriction fragments was 42.7 kilobase pairs. Of particular interest was the fact that the DNA was insensitive to certain common restriction endonucleases including EcoRI, BamHI, and HindIII. The ends of the phage DNA were cloned and sequenced, revealing direct repeats of 318 nucleotides. The left end of the genome when cloned into the promoter selection vector pKK232-8 exhibited promoter activity in Escherichia coli. Two promoters bearing greater than 70% sequence homology to the plasmid pNM74 TOL operon and PAK pilin promoters were identified.

Potassium release in labial glands from controls and patients with cystic fibrosis.

Labial glands from patients with cystic fibrosis (CF) were tested for a disease-related decrease in cholinergically-induced K release. Labial gland slices from normal controls and patients with cystic fibrosis were incubated in vitro in the presence or absence of cholinergic and adrenergic agonists and with or without a phosphodiesterase inhibitor. Both control and CF glands released K in response to cholinergic stimulation only; no K release response was detected to alpha- or beta- adrenergic stimulation. In contrast to previous results reported for parotid glands, no CF-related decrease in cholinergically-induced K release was detected. Both normal and CF glands released significantly less K with carbachol stimulation in the presence of the phosphodiesterase inhibitor. Overall, the results suggest considerable interglandular differences in disease sensitivity and functional regulation of K release.

Human pancreatic secretory protein profiles in pancreas cancer and chronic pancreatitis.

Pancreatic secretory protein profiles differed significantly between patients with chronic pancreatitis (CP) and pancreatic carcinoma (CA). Specific regions of the patterns were altered when CP versus CA and when CP versus normals were compared. Bands isoelectric in the region of pH 9-11 were elevated in CP. The possible identification of this band as lactoferrin is discussed.

Characterization of tetracycline resistance plasmids from Campylobacter jejuni and Campylobacter coli.

Tetracycline resistance in strains of Campylobacter jejuni and Campylobacter coli was mediated by plasmids. Intra- and interspecies transfer was demonstrated within the genus Campylobacter. Buoyant densities of plasmid DNAs ranged from 1.691 to 1.694 g/cm3 (31 to 33% guanine plus cytosine). Restriction enzymes AccI, BclI, BglII, and PstI were found to be most useful for comparing the plasmids. The molecular weight of C. jejuni plasmid pMAK175 was 44.7 kilobases (29 X 10(6), and the other plasmids had similar sizes. Two plasmids from Belgian isolates of C. coli of human origin had very similar restriction enzyme profiles and are probably identical. Plasmids from human isolates of C. jejuni originating in Canada and the animal isolate of C. coli showed greater diversity. DNA homology among the campylobacter plasmids was assessed by probing the digests with a nick-translated campylobacter plasmid, pMAK175. All restriction fragments showed significant homology with pMAK175 probe DNA. No homology was noted between campylobacter plasmid DNA and plasmids specifying the four classes of tetracycline resistance determinants found in Enterobacteriaceae.

Human pancreatic secretory protein profiles in the search for tumor markers.

Pancreatic cancer secretory protein profiles were shown to be different from normals by a microcomputer-assisted analysis of isoelectric focusing patterns. Two acidic protein band regions (pI 3.0 and 4.5) of the pancreatic carcinoma profiles were significantly increased, and eight protein bands (pI 3.7, 5.0, 5.5, 6.5, 6.9, 7.3, 8.0, greater than 10.0) were significantly decreased. Highly significant decreases occurred at pH 7.3 (chymotrypsinogen) and at pH 5.0 (procarboxypeptidase Al, DNase I) in nonactivated specimens. The ratio between the absorbance points at 2.08 and 2.63 cm from the anode in each protein pattern differentiated the pancreas cancer specimens from the control group. Profiles found for the control group gave pI values similar to those found in the literature. The potential value of these findings in the search for tumor markers warrants further investigation as to whether these specimens can be differentiated from those from chronic pancreatitis patients.