Influence of mutation in cj0183 and cj0588 genes for colonization abilities of Campylobacter jejuni in Caco-2 cells using confocal laser scanning microscope.

The cj0183 and cj0588 genes identified in the Campylobacter jejuni NCTC 11168 genome encode proteins with homology to virulence factors found in other bacteria. Previous studies showed that single mutation in the cj0183 gene does not affect adhesion of C. jejuni to the Caco-2 cell line whereas protein encoded by cj0588 is involved in adherence to the Caco-2 cells. In the presented study differences in invasion index were observed between mutants in both genes and single mutation of cj0588 in 81116 and 81-176 C. jejuni strains This fact indicates that Cj0183 protein might play some role in invasion of bacteria into host cells.

The viability of the genetically diverse C. jejuni and C. coli strains in the macrophage J774 cell line.

The intracellular survival of Campylobacter has been described within epithelial cells as well as in macrophages in vitro. The goal of this study was to estimate the viability of the genetically diverse C. jejuni and C. coli strains in the macrophage J774 cell line. Strains selected for analysis differed with regard to the occurrence of genes encoding specific virulence factors. The present work indicates that was no correlation between the source of isolates and relative intracellular survival.

Construction of a reporter system for Lactobacillus sp. using the gfpuv gene.

Lactobacilli are bacteria commonly associated with the gastrointestinal tract of animals and humans. They are able to produce antimicrobial substances such as bacteriocins, lactic acid and hydrogen peroxide, the factors which have been shown to be beneficial for controlling overgrowth of potentially pathogenic bacteria. For this reason lactobacilli are often applied in probiotics. The aim of present study was to construct a reporter strain based on the gfpuv expression system which can bee used as a strain with the ability to colonize the intestinal tract in future experiments.

Genotyping and PCR detection of potential virulence genes in Campylobacter jejuni and Campylobacter coli isolates from different sources in Poland.

The prevalence of potential virulence markers was determined among the population of Polish Campylobacter jejuni and Campylobacter coli isolates from children, chickens, pigs and dogs. The presence of the flaA, flaB, cdtA, cdtB, cdtC, cdtABC, virB11, and cj0588 genes among 74 C. jejuni and 15 C. coli isolates was detected by PCR. High prevalence of five different putative virulence and toxin genes (flaA, cdtA, cdtB, cdtC, and cj0588) was found among isolates obtained from children, chickens and dogs. The occurrence of these genes among isolates obtained from pigs was significantly different than for strains isolated from other sources. Two methods for genotyping Campylobacter spp. strains were applied - flaA-typing, and ADSRRS-fingerprinting method, which was used for the first time for Campylobacter spp. strains. Similarity of the genetic profiles was demonstrated in strains isolated from chickens and dogs, and in isolates from chickens and children. Strains isolated from pigs, both C. jejuni as well as C. coli, did not group with isolates from other sources.

Strategies of constructing recombinant vaccines.

Vaccines are the most effective prophylactic tool in veterinary medicine. Despite the great success of many vaccines used currently, there is still a constant need for their improvement. An ideal vaccine should contain a variety of immunogens, be safe and efficacious and induce broad humoral and cell-mediated immunity with one or, at most, two administrations given orally rather than by injection, and should be inexpensive. Traditional approaches include attenuated live vaccines, inactivated vaccines and subunit vaccines. Recently, scientific advances in molecular biology, immunology and bioinformatics, as well as the growing number of sequenced genomes of pathogens, have led to significant progress in respect to understanding virulence mechanisms at the molecular level. Genetic engineering has been applied to obtain recombinant bacterial and viral genomes in order to produce a modified and safe product useful in vaccine development. This article presents the progress and novel strategies used in creating new generation vaccines. It focuses on methods of searching for vaccine candidates to construction of vaccines based on recombinant DNA or proteins.

Resistance to antimicrobial agents of Campylobacter spp. strains isolated from animals in Poland.

A total of 69 Campylobacter jejuni and 16 Campylobacter coli strains isolated from chicken, dog and pig stool samples were characterized based on their resistance to five antimicrobial agents and on plasmid pTet profiles. Antimicrobials used in this study were: amoxicillin/clavulanic acid, ciprofloxacin, erythromycin, tetracycline and trimethoprim/sulfamethoxazole. Among the isolates studied, 91.7% were resistant to one or more antimicrobial agent. The highest level of resistance for the whole test group was to trimethoprim/sulfamethoxazole (57.6%), followed by ciprofloxacin (44.2%) and tetracycline (20%). All isolates were susceptible to amoxicillin/clavulanic acid. Strains isolated from chickens were susceptible to erythromycin. Few erythromycin-resistant strains were isolated from dogs and pigs (5.8%). C. coli strains exhibited a higher antibiotic resistance than C. jejuni strains, excluding resistance to trimethoprim/sulfamethoxazole. The pTet plasmid harboring the tet(O) gene was detected in 14 Campylobacter spp. strains. Our studies demonstrate that the majority (71.4%) of tetracycline-resistant isolates carry a plasmid-borne tet(O) gene, particularly strains for which the minimum inhibitory concentration (MIC) are > or = 256 microg/ml. In conclusion, we have found high-level trimethoprim/sulfamethoxazole, ciprofloxacin and tetracycline resistance in Polish strains isolated from different sources. This study has demonstrated that resistance of Campylobacter species differs depending on both the bacterial species and animal origins. All strains that displayed resistance to four antimicrobial agents were isolated from pigs. Localization of the tet(O) gene on either plasmid or chromosome was not found to be correlated with tetracycline resistance.

The cytotoxic properties of Serpulina hyodysenteriae.

Examination of colonic enterocytes inoculate with pure culture of S. hyodysenteriae by phase-contrast microscopy revealed that only few spirochaetes adhere to epithelial cells. S. hyodysenteriae was observed to be highly motile, showed corkscrew-like movement which might suggest that bacteria were trying to penetrate and damaged the host cells. The pattern of motility provide evidence of a chemotaxis. Supernatant of S.hyodysenteriae lysate were found to cause CTE in CHO, Vero and PK-15 culture. This support the hypothesis that damage is consistent with the presence of toxin. Inhibition activity of serpulinas hemolysin preparation with streptolysin S inhibitors confirms the suggestion that the mechanism by which S. hyodysenteriae toxin effects the cells seems to be similar to the action of streptococcal toxin S.

Transfer of plasmid Hly in vivo in pigs intestine.

The results of our study suggest the in vivo transfer of Hly plasmid from native pathogenic and enterotoxigenic Escherichia coli strain to autochtonous Escherichia coli, using ileal loop test. To confirm this hypothesis pHly::Tn5 and PHly::Tn3 were obtained using an in vitro recombination method, and introduced to Escherichia coli laboratory strain. For experiments the laboratory strain, carrying pHly::Tn5 and pHly::Tn3 and pHly::Tn5 strain which acquired K88(F4) by means of conjugation, were used. In the study in which the donor Escherichia coli pHly::Tn5 strain, carrying antigen K88(F4), was injected into the ileum, pHly conjugants were isolated from faeces after 48 h in 2 out of 5 pigs, which was a low frequency. After the oral introduction of 10(9) cells of pHly::Tn5 and pHly::Tn3 Escherichia coli strains without the colonizing factor K88(F4), conjugants were not isolated from faeces of experimental animals. However when the pigs received donor CSH55 pHly::Tn5 Escherichia coli strain orally, which were also carrying plasmid K88(F4), transconjugants were obtained in a low frequency of 3 out of 9 pigs. Our experiments confirmed the suggestion of Smith that in vivo transfer of plasmid in the intestine of animals is only possible when the donor transfers the plasmid with high frequency and readily colonizes the intestine. The pHly::Tn5 plasmid acquired by in vitro recombination does not spread with time throughout the autochtonic population of Escherichia coli present in the intestine of swine. The results of our study showed the in vivo transfer in pigs intestine of Hly pathogenicity marker from both native pathogenic strains carrying antigen K88(F4) and constructed donor laboratory strain of Escherichia coli pHly::Tn5 also carrying antigen K88(F4) to autochtonous intestinal strains.

Characterization of alcohol dehydrogenase in young soybean seedlings.

Molecular properties of alcohol dehydrogenase (ADH) were examined in young soybean seedlings. Soybean radicle tissue is ADH-rich. Enzyme specific activity decreases slowly with the development of roots and becomes almost undetectable when the first true leaves appear. Soybean ADH was not found to be inducible by flooding. 2,4-Dichlorophenoxyacetic acid (2,4-D) treatment increased ADH specific activity as much as 14-fold. Only one ADH isozyme was detected by isoelectric focusing. By DNA-DNA hydridization, soybean ADH genomic sequences were shown to be partly homologous to maize ADH1 cDNA. The presence of more than one Adh gene in soybean is discussed.

Production of enterotoxins by strains of Escherichia coli isolated from pigs.

The production of enterotoxins by 237 hemolytic strains of Escherichia coli isolated from pigs was determined with the use of CTE in CHO. Vero and Hela cells and ILT. More frequent (p less than 0.01) production of enterotoxins, determined by ILT, was found for the serotypes being pathogenic for the animals (63.8% of the strains). No correlation between intensity of ILT and particular serotype was observed. Both the serotypes pathogenic for pigs and other serotypes produced LT enterotoxins and ST toxin. The frequency of LT enterotoxin production was statistically insignificant compared to the frequency of ST enterotoxin production by strains with serotypes pathogenic for the pigs. Strains of E. coli producing only enterotoxin ST belonged both to the pathogenic serotypes as well as to other hemolytic serotypes. The cytotoxic activity of supernatants of E. coli strains with different serotypes isolated from pigs in Vero and Hela cells and simultaneous CTE in CHO cells was observed. This suggests the production by the strains of enterotoxin LT and cytotoxin VT. Seven out of the 96 isolates showing CTE in CHO cells gave no reaction in the ILT in pigs. This suggests the production by these isolates of a toxin (toxins) differing from the E. coli enterotoxins.

Evaluation of different cell cultures for the detection of heat-labile enterotoxins of Escherichia coli strains isolated from pigs.

The sensitivity of various cell cultures to heat-labile enterotoxins (LT) and Verocytotoxin (VT) of fifteen E. coli strains isolated from cases of pig colibacillosis in Poland was estimated and compared with the effect of enterotoxins of four standard E. coli strains. Of ten tested cell cultures, only the following were susceptible: CHO, Vero, GMK, and HeLa. Eight strains showed CTE in He La and CHO cells and five of these reacted in Vero cells. The results appear to suggest that some of the tested E. coli strains isolated from pigs produced VT enterotoxin. Morphological changes caused by the above mentioned E. coli toxins in Vero and GMK cells took the form of cell rounding, followed by cell dissolution.

Effect of ts mutation in gene 43 of bacteriophage T4 on recombination of the bacteriophage.

Certain ts mutations in gene 43 of bacteriophage T4 are known to exhibit mutator or antimutator activities with respect to different rII mutations of this phage. The effect of these mutations on recombination frequencies of double mutant strains of phage T4 with genotype rII ts--homoallelic with respect to the ts trait--was examined. The results implicate the essential role of the genetic background in the investigated process.