Biomedical subjects

L K Nolan

Publications and source records attributed to L K Nolan.

29 records · Page 2Linked to original sources

PubMedDate not supplied

Comparison of a complement resistance test, a chicken embryo lethality test, and the chicken lethality test for determining virulence of avian Escherichia coli.

Results with four pathogenic avian Escherichia coli isolates and one avirulent isolate in a complement resistance test, a chicken lethality test, and a chicken embryo lethality test were compared. Results of the complement resistance test with these isolates were highly correlated to results of the chicken lethality test of virulence. The chicken embryo test yielded results that were of a medium positive correlation with the chicken lethality results. The results of the complement resistance and chicken embryo lethality tests were highly correlated.

Animals↗

PubMedDate not supplied

Comparison of a quantitative microtiter method, a quantitative automated method, and the plate-count method for determining microbial complement resistance.

A quantitative microtiter method for determining the degree of complement resistance or sensitivity of microorganisms is described. The microtiter method is compared with a quantitative automated system and the standard plate-count technique. Data were accumulated from 30 avian Escherichia coli isolates incubated at 35 C with either chicken plasma or heat-inactivated chicken plasma. Analysis of data generated by the automated system and plate-count techniques resulted in a classification of the microorganisms into three groups: those sensitive to the action of complement; those of intermediate sensitivity to the action of complement; and those resistant to the action of complement. Although the three methods studied did not agree absolutely, there were statistically significant correlations among them.

Animals↗

PubMedDate not supplied

Comparison of chicken plasma and guinea pig serum in a quantitative microtiter method of determining microbial complement resistance.

A quantitative microtiter method using chicken plasma is described for determining the degree of complement resistance or sensitivity of avian Escherichia coli isolates. Results obtained with the microtiter method using chicken plasma were compred with results obtained using commercially available standardized guinea pig serum as the source of complement. The test organisms consisted of five isolates of E. coli isolated from chickens. Three isolates were from flocks with colisepticemia; one was from a flock with omphalitis; and one isolate was a non-pathogenic control. Data were accumulated from the five avian E. coli isolates incubated at 35 C with either chicken plasma or guinea pig serum and with heat-inactivated chicken plasma or guinea pig serum. The microtiter results of the chicken plasma and guinea pig serum had a statistically positive correlation. The use of commercially available guinea pig serum in the test system will allow for standardization of this method.

Animals↗

PubMedDate not supplied

Phenotypic expression of recombinant plasmids pKT107 and pHK11 in an avirulent avian Escherichia coli.

An avirulent wild-type avian Escherichia coli strain (Av) was electrotransformed with plasmids coding for complement resistance (pKT107) and Colicin V (ColV) production (pHK11) in order to study the effects of complement resistance and ColV production on virulence. Transformants were also compared with the wild type for embryo lethality, uptake by macrophages, motility, growth rate, plasmid content, and hemolysis. Growth rates and complement resistance patterns of strain Av and transformant Av+pHK11 were similar, but Av+pHK11 caused a significantly greater number of deaths in embryos and acquired motility. Transformant Av+pKT107 had a lower rate of phagocytosis, a slower growth rate, and a greater sensitivity to complement, and it changed from being non-hemolytic to expressing alpha-hemolytic action. The 35-kb plasmid present in the wild type was not present in the transformants. Although some of the results demonstrate the difficulties encountered in using wild-type organisms as recipients in virulence studies, the results with Av+pHK11 indicate that ColV production plus the acquisition of motility contributes to the virulence of avian E. coli.

Animals↗

PubMedDate not supplied

Effect of normal intestinal flora of chickens on colonization by virulent colicin V-producing, avirulent, and mutant colicin V-producing avian Escherichia coli.

Colonization of the intestinal tracts of newly hatched chicks with Escherichia coli was attempted by swabbing test organisms onto the air-shell of 19-day-old embryos. Test organisms consisted of two virulent E. coli isolates, one avirulent isolate, and one laboratory-derived mutant of the avirulent isolate carrying a recombinant plasmid coding for Colicin V production. Chicks were cultured weekly for 3 weeks for total E. coli and for the test organisms using selective media. Control chicks were sampled on weeks 1 and 5, and the normal E. coli intestinal microflora were examined for the production of colicins. The two virulent E. coli isolates maintained colonization of the chicks for the 3-week test period, with titers decreasing from 10' to 10'- colony-forming units (CFU)/g of intestine. The avirulent isolate and laboratory mutant did not consistently colonize the intestinal tracts. The majority of intestinal samples taken from the control chicks at 1 and 5 weeks had colicin-producing E. coli that were inhibitory to the test organisms.

Analysis of Variance↗

PubMedDate not supplied

Characterization of an avirulent mutant of a virulent avian Escherichia coli isolate.

A virulent, complement-resistant avian Escherichia coli isolate and its avirulent, complement-sensitive, transposon-insertion mutant were compared for the purpose of revealing structures associated with complement resistance. Both had a smooth lipopolysaccharide layer, contained traT, and lacked a capsule, but the mutant possessed a 16.2-kilodalton outer-membrane protein (OMP) not present in the wild-type. This protein may be the product of a coding region interrupted by transposon insertion. Such results suggest that an OMP greater than 16.2 kilodaltons in size may be responsible for the complement resistance and virulence of this wild-type E. coli.

Animals↗

PubMedDate not supplied

Association of K-1 capsule, smooth lipopolysaccharides, traT gene, and Colicin V production with complement resistance and virulence of avian Escherichia coli.

A group of complement-resistant, virulent avian Escherichia coli isolates were compared with a group of complement-sensitive, avirulent avian isolates for the presence of K-1 capsule, smooth lipopolysaccharides (LPS), the traT gene, and Colicin V (ColV) production. These parameters were selected because of their reported association with complement resistance and virulence in E. coli. Lethality in chicken embryos has also been shown to be correlated with virulence of avian E. coli for chickens. The complement-resistant, virulent E. coli isolates did not possess a K-1 capsule. Production of ColV and the presence of smooth LPS were significantly correlated with embryo lethality. There was no correlation between the presence of traT and embryo lethality. These results suggest that complement resistance and virulence in avian E. coli are associated with ColV production and smooth LPS but not with K-1 antigen or traT.

Animals↗

PubMedDate not supplied

Invasion of Caco-2 cells by Salmonella typhimurium (Copenhagen) isolates from healthy and sick chickens.

In a previous study, Salmonella isolates of sick birds were distinguished from those of apparently healthy birds by their high degree of invasion of tissue culture cells. In this study, a single pair of Salmonella isolates was examined to determine the source of this observed difference in invasion. When isolates were allowed to invade Caco-2 cells for 8 hours, the isolate from the sick bird (S) appeared to invade in greater numbers than did the isolate from the healthy bird (H). However, when invasion was distinguished from intracellular growth/survival, it was found that H invaded in greater numbers than S, but once inside the cell, H declined in number, and S increased. Inhibition of RNA, protein, and DNA syntheses lessened the degree to which both invaded. The presence of mannose inhibited invasion by S but did not appear to inhibit invasion by H. Trypsin treatment of monolayers affected invasion of S and H, whereas neuraminidase treatment did not. There was no significant difference noted between S and H in ability to adhere to fixed monolayers. Therefore, the two isolates tested differ in their mechanisms of entry into Caco-2 cells, the efficiency with which they invade, and their ability to survive within Caco-2 cells.

Animals↗

PubMedDate not supplied

Analysis of plasmids cloned from a virulent avian Escherichia coli and transformed into Escherichia coli DH5 alpha.

Three of four plasmids from a virulent wild-type avian Escherichia coli were cloned or transformed into an avirulent laboratory recipient E. coli DH5 alpha and tested for the ability to confer a virulence phenotype. The three plasmids transformed into E. coli DH5 alpha were 5, 6, and 56 kb. A fourth plasmid of 64 kb was not successfully transformed. Parameters used to measure virulence included presence of type 1 pili and a smooth lipopolysaccharide (LPS) layer, motility, production of Colicin V, resistance to host complement, and embryo lethality. The 5-kb plasmid encoded for ampicillin resistance, whereas the 6-kb plasmid encoded for tetracycline resistance. The 56-kb plasmid encoded for streptomycin, sulfisoxazole, and tetracycline resistance. Twelve-day embryos inoculated with 467 colony-forming units of E. coli DH5 alpha containing the 56-kb plasmid had increased death rates (45%) in the embryo lethality assay and a decreased weight of surviving embryos with cranial hemorrhages as compared with embryos inoculated with similar amounts of E. coli DH5 alpha (0%) and phosphate-buffered saline (0%). Embryos inoculated with the wild-type virulent E. coli had 90% deaths. The 56-kb plasmid also had homology with a probe for Colicin V production (cvaC). No differences in LPS layer, complement resistance, motility, Colicin V activity, type 1 pili, cell-free supernatant proteins, or outer membrane proteins were observed in the transformants when compared with nontransformed E. coli DH5 alpha.

Animals↗

PubMedDate not supplied

Large plasmids of avian Escherichia coli isolates.

The plasmid DNA of 30 Escherichia coli isolates from chickens was extracted and examined using techniques designed to isolate large plasmids. This plasmid DNA was examined for the presence of certain known virulence-related genes including cvaC, traT, and some aerobactin-related sequences. Seventeen of the 30 isolates contained from one to four plasmids greater than 50 kb in size. Eleven of these 17 strains possessed plasmids greater than 100 kb in size. Therefore, E. coli isolates of chickens frequently contain large plasmids, and many of these plasmids are likely to contain virulence-related sequences.

Animals↗

PubMedDate not supplied

Further characterization of a complement-sensitive mutant of a virulent avian Escherichia coli isolate.

An attempt was made to characterize the mechanism of complement resistance operating in a virulent avian Escherichia coli isolate. Using flow cytometry to detect antibody to C3, we found that there was significantly more antibody bound to a complement-sensitive mutant of this wild type than to the parent organism, suggesting that more C3 subunits were bound to the wild type. Neither the wild type nor the mutant degraded C3. Further, the mutant was phagocytosed to a significantly greater degree than the wild type by cultured phagocytes in the presence of C5-deficient serum. These data suggest that the wild type is resistant to complement, at least in part, because of its ability to restrict C3 deposition on its surface. Therefore, the decrease in virulence seen in the mutant may be related to its increased sensitivity to complement-mediated bacteriolysis or its enhanced susceptibility to complement-opsonized phagocytosis or both.

Animals↗