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Biomedical subjects

P Timms

Publications and source records attributed to P Timms.

At least 55 records · Page 3Linked to original sources
PubMed1994

Genetic modification of a vaginal strain of Lactobacillus fermentum and its maintenance within the reproductive tract after intravaginal administration.

Many micro-organisms cause important diseases of the female genital tract. Because systematic vaccination does not usually provide a good immune response at mucosal sites, commensal lactobacilli from the female genital tract were developed as vehicles to deliver continued doses of foreign antigen directly to the genital mucosal surface with the aim of stimulating strong local mucosal immune responses. Lactobacilli were shown to be common inhabitants of the genital tract of the animal model studied, the guinea-pig. One species, Lactobacillus fermentum, was found in all guinea-pigs studied and was chosen for genetic manipulation. Improved methods of electroporation were developed to enable the routine transformation of L. fermentum BR11 strain with the broad host range plasmid pNZ17. This recombinantly modified Lactobacillus strain was shown to possess good segregational stability over 120 generations in the absence of antibiotic selection. When this recombinant L. fermentum strain was administered to the vaginal tract of three guinea-pigs it persisted for only 5 days. Despite the relatively short period of persistence in these initial experiments, this novel vaccine approach could provide an effective means of stimulating mucosal immunity in the female genital tract.

Animals↗
PubMed1994

Comparison of Chlamydia pneumoniae isolates by western blot (immunoblot) analysis and DNA sequencing of the omp 2 gene.

The 60-kDa cysteine-rich outer membrane protein gene (omp 2) from nine Chlamydia pneumoniae isolates (TW-183, CM-1, CWL-050, CWL-011, IOL-207, FIL, Kajaani-6, Helsinki-12, and Parjaanonen) was amplified by PCR and sequenced from positions 1 to 580. In contrast to the sequence differences previously observed in this gene in other chlamydial species, all nine C. pneumoniae isolates were 100% identical. However, when sera from C. pneumoniae microimmunofluorescence-positive patients (with clinical signs of persistent cough or asthma) were immunoblotted against five C. pneumoniae isolates, distinct antigenic differences were observed. TW-183 was characterized by major bands at 35 and 43 kDa. In contrast, the other four isolates tested produced similar, though not identical, immunoblot profiles, characterized by strong bands at 18, 25, 29, 40, 46, and 53 kDa. These data support the fact that significant differences do exist between C. pneumoniae isolates, but unlike the case with other chlamydial species, these differences do not reside in either of the commonly studied outer membrane protein genes, omp 1 or omp 2.

Antigens, Bacterial↗
PubMed1993

Detection of Chlamydia trachomatis in urine using enzyme immunoassay and DNA amplification.

The suitability of urine specimens for the detection of Chlamydia trachomatis infections in men was assessed. Urethral swabs from 301 patients were cultured for C. trachomatis, and the results were compared with results obtained from Chlamydiazyme. The results of 298 specimens were also compared with results obtained from PCR analysis of first-void urine specimens. The sensitivity of confirmed Chlamydiazyme analysis was 93% and the specificity was greater than 99% compared with culture. The sensitivity of the PCR method was 100% compared with culture. Chlamydia trachomatis was detected by PCR in an additional three specimens from which C. trachomatis could not be cultured. Urine appears to be an appropriate specimen for the detection of C. trachomatis antigens and nucleic acids.

Adolescent↗
PubMed1993

DNA fingerprint analysis of a free-range koala population.

Thirty-six koalas in a free-range Queensland population were fingerprinted using an M13 probe in combination with MspI digestion. The technique was found to be highly repeatable, with estimates of 0.1-1.6% within-gel error and 0.1-2.5% between-gel error. Of the 43 different-size fingerprint bands produced in the population, only 2 bands were common to all 36 koalas. Ten bands were quite rare, occurring at a frequency of 0.2 or less. All 36 koalas had unique DNA fingerprints (probability of 1.88 x 10(-7)), which enabled them each to be uniquely identified. Despite this, there was still a high level of band sharing in the population (mean number of shared bands = 0.749). This level is much higher than that reported for humans, birds, cats, dogs, and cattle but not as high as that reported previously for Victorian koalas. This lack of genetic variation may influence the ability of the population to respond to stress situations, such as lack of food, habitat destruction, and disease.

Animals↗
PubMed1992

Proof of differentiative mode of action of all-trans retinoic acid in acute promyelocytic leukemia using X-linked clonal analysis.

Using X-linked clonal analysis, mechanism of action of all-trans retinoic acid (ATRA) was sought in a 16-year-old female with relapsed clonally evolved acute promyelocytic leukemia (APL), who achieved complete remission. On ATRA, metamorphosis of peripheral blood leukemic promyelocytes to mature neutrophils was observed, despite the persistence of t(15;17) in 100% of bone marrow metaphases. DNA was extracted from fractionated serial blood specimens, collected at diagnosis, in first complete remission (CR), relapse, and during ATRA treatment. Using a phosphoglycerokinase (PGK) probe, the patient was heterozygous for both Bgl I and Bst XI PGK polymorphisms. Methylation analysis showed monoclonal leukemic promyelocytes with a polyclonal first CR achieved by standard chemotherapy. Subsequent examination, in relapse, of granulocytes appearing during ATRA treatment showed these to be monoclonal, proving these were derived from the neoplastic clone. The X-linked clonal analysis methodology has provided in vivo evidence of cellular differentiation as the mechanism of action of ATRA. Parallel studies of cytogenetic and clonal analysis showed a regression of the t(15;17) cytogenetic abnormality and return of a polyclonal PGK methylation pattern in 5 weeks, indicating a repopulation of marrow by normal stem cells. As standard cytogenetic techniques are inappropriate for nondividing cells, X-linked clonal analysis provides a marker system to allow insight into mechanism of drug action in malignant hematologic disease.

Adolescent↗
PubMed1992

PCR detection and differentiation of Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia trachomatis.

A PCR-based system was developed for the detection and differentiation of Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae. A conserved 145 bp fragment of the chlamydial omp1 gene was amplified from all three species. The three species were then differentiated from each other by digestion of this PCR product with restriction enzymes Eco RI and either Hind III or Pst I. The system was shown to work for two strains of C. pneumoniae, 11 strains of C. psittaci and 10 serovars of C. trachomatis, and had a sensitivity of less than 10 chlamydial elementary bodies. This method was also applicable to the detection of C. trachomatis in conjunctival and nasopharyngeal swabs.

Adult↗
PubMed1992

Comparison of nine antigen detection kits for diagnosis of urogenital infections due to Chlamydia psittaci in koalas.

Chlamydia psittaci is the major cause of infectious disease in the koala (Phascolarctos cinereus). It causes four disease syndromes in the koala, namely, conjunctivitis, rhinitis, cystitis, and infertility (females only). Diagnosis of chlamydial infections in koalas relies primarily on isolation of the organism in cell culture. Serology has generally not been useful, and little use has previously been made of the commercially available antigen detection kits. We examined the sensitivity, specificity, and usefulness of three direct fluorescent-antibody kits (Vet-IF [Cell Labs], IMAGEN [Celltech], Chlamydia-Direct IF [Bio Merieux]) and six antigen detection enzyme-linked immunosorbent assay (ELISA) kits (Clearview [Unipath], Surecell [Kodak], Pathfinder [Kallestad], Chlamydia-EIA [Pharmacia], Chlamydiazyme [Abbott], IDEIA [Celltech]) for the detection of urogenital infections in koalas. Laboratory studies showed that the direct fluorescent-antibody kits were the least sensitive in this case and did not detect fewer than 10(4) elementary bodies per ml, while most ELISA kits detected between 130 and 600 elementary bodies per ml. Field study results showed that the Clearview kit was the most sensitive (91%) compared with the IDEIA (88%) and the Surecell (73%) kits. All three kits were more sensitive than cell culture (36%), highlighting viability loss problems that occur during transport. This study showed that the Clearview kit is sensitive, specific, and easy to use for the detection of type II (urogenital) C. psittaci from koalas in the field and warrants further evaluation.

Animals↗
PubMed1991

Detection of Chlamydia psittaci using DNA probes and the polymerase chain reaction.

Fewer than 10(5) elementary bodies of Chlamydia psittaci could be detected by using DNA hybridisation with a plasmid probe specific for avian chlamydial strains. PCR amplification of chlamydial DNA using primers specific for conserved regions of the major outer membrane protein gene enabled the detection of fewer than 10 elementary bodies. DNA could be amplified from 22 of the 24 chlamydial strains tested including avian, feline, ovine, caprine, koala and lymphogranuloma venereum strains.

Animals↗
PubMed1991

Serological assessment of chlamydial infection in the koala by a slide EIA technique.

A rapid and simplified slide enzyme immunosorbent assay (EIA) was developed for the diagnosis of chlamydial infection in the koala. HeLa 229 cells infected with koala strain Chlamydia psittaci were fixed on the surface of multiwell slides and used as the antigen. The assay consisted of first reacting koala antiserum with the fixed C psittaci antigen, followed by reaction with biotinylated rabbit anti-koala IgG, ABC reagent and substrate. The chlamydial EIA antibody titres obtained were compared with those of a complement fixation (CF) test using koala strain C psittaci as antigen. Of 35 koala sera tested, 16 CF positive sera (greater than or equal to 1:8) also had a positive titre (greater than or equal to 1:200) in the slide EIA test (sensitivity 93.8%, 15/16). Nineteen CF negative sera were also negative in the slide EIA (specificity 100%, 19/19). Sixty-eight samples of koala blood were collected by ear-prick using a sampling paper method and were assayed by both tests. Sensitivity of the slide EIA was 100% (15/15) and specificity of the test was 96.2% (51/53). To simplify the slide EIA for use as a practical screening test, a 3-point serum dilution series (1:100, 1:200, 1:400) was used. This 3-point slide EIA was compared with the CF test using sheep strain chlamydial antigen. Thirty-nine sera were assayed by both tests. The sensitivity of the 3-point method was 85.7% (6/7) and the specificity was 71.9% (23/32) as compared with the sheep antigen CF test.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Babesia bovis: evidence for selection of subpopulations during attenuation.

DNA probes were used to detect variation in subpopulations of virulent and serially passaged Babesia bovis. Two distinct patterns were evident after hybridization to genomic DNA; the first was a basic profile typical of virulent B. bovis and the second, a more variable array, was characteristic of B. bovis after various stages of attenuation. Tick transmission of avirulent B. bovis causes reversion to the virulent genomic pattern, suggesting that selective enrichment of a small residual subpopulation caused reversion to a virulent profile of subpopulations. Certain genomic fragments, predominant in either virulent or avirulent parasite forms, are putative "markers" or actual elements responsible for these biological characteristics.

Animals↗
PubMed1990

Study of virulence and vector transmission of Babesia bovis by use of cloned parasite lines.

Cloned lines of Babesia bovis were prepared from the avirulent vaccine strain, Ka, by an in vivo limiting dilution procedure. The virulence of these clones for adult Bos taurus cattle varied from completely avirulent to highly virulent. This suggests that the parent strain, Ka, is composed of a mixture of subpopulations of varied virulence. Passage of the avirulent clone K-19-47 in intact (nonsplenectomized) cattle resulted in its full reversion to virulence. This suggests that two mechanisms are operating to enable virulence to be a readily modified characteristic in this parasite: differential gene expression and phenotypic selection of subpopulations. A series of experiments demonstrated that all clones were non-tick transmissible. This lack of vector transmission was a stable characteristic and could not be altered by the passage of K-19-47 in intact cattle, despite the fact that passage in intact animals caused this clone to revert to the fully virulent phenotype. A mechanism is suggested for the virulence and vector transmission variations observed in B. bovis.

Animals↗
PubMed1989

Development of babesial vaccines.

The most widely used vaccine against cattle babesiosis was blood from a carrier animal. The development of a calf passage attenuated strain in Australia was a great improvement in vaccine safety and efficacy. The degree of immunity generated by this vaccine remains a standard against which to compare other vaccines. Vaccines derived from culture supernatants have also been developed. These have considerable value. To date no efficacious recombinant vaccine exists. A variety of antigens will probably be required in any synthetic vaccine. Study of clones derived from calf passage attenuated vaccines indicate that the vaccine contains a variety of parasites of differing virulence. The attenuation process enriches for the less virulent clones. The clones may also vary in their antigenic characteristics. To date no single clone gives immunity equal to the original uncloned preparation. An ideal vaccine should not be a living agent. It will probably contain a variety of antigens and may induce less strong immunity than the living vaccine but its greater safety, stability and ease of handling will more than compensate for this.

Animals↗
PubMed1989

Conserved DNA sequences in chlamydial plasmids.

Two 7.4-kb plasmids from Chlamydia psittaci have been cloned and characterized. These plasmids are quite distinct from the 6.2-kb C. psittaci and the C. trachomatis plasmids when compared by restriction endonuclease analysis. The plasmids show considerable cross-hybridization, with only a small region highly conserved and identified as a 4 X 22-bp tandemly repeated region. This sequence is identical in the two size categories of C. psittaci plasmids and differs from C. trachomatis plasmids by only 2 bp in the 22-bp motif. AT-rich clusters 5' to the repeat region which are present in C. trachomatis and Escherichia coli plasmids were absent from both classes of C. psittaci plasmids. Extensive regions are less highly conserved but show a sufficient degree of cross-hybridization to suggest that the plasmids are homologous.

Animals↗
PubMed1989

The pathogenesis of hypochromic anaemia in Saudi infants.

The pathogenesis of hypochromic anaemia was studied in 138 Saudi bedouin infants aged 9 months. Approximately 25 per cent had hypochromic anaemia, but less than 10 per cent had serum ferritin levels indicative of iron deficiency. A few infants had heterozygous beta-thalassaemia, but many infants with hypochromic anaemia had normal haemoglobin A2 levels together with serum ferritin levels above 20 micrograms/l. DNA analysis of cord blood taken from the hospital where the infants were born showed that the frequency of the single alpha-globin gene deletion type (-alpha 3.7) of alpha-thalassaemia is 0.13 in the bedouin population of Western Saudi Arabia. alpha-Thalassaemia probably accounts for much of the anaemia previously thought to be due to iron deficiency in Saudi infants. Studies of iron status and estimation of the frequency of genetic causes of hypochromic anaemia are important when assessing the need for widespread nutritional programmes to prevent iron deficiency and in the interpretation of reference ranges of red cell indices in populations from malarial areas.

Anemia, Hypochromic↗
PubMed1989

Growth of Babesia bovis parasites in stationary and suspension cultures and their use in experimental vaccination of cattle.

A combination of stationary culture and suspension culture was used to produce litre quantities of Babesia bovis parasites suitable for use as live vaccine. The Australian vaccine strain of B bovis, Ka, was maintained continuously in microaerophilus stationary phase (MASP) cultures, and for a short period in batch and flow-through spinner flask cultures. Although continuous culturing was not achieved in spinner flasks, the production of litre quantities of heavily parasitised erythrocytes was achieved more simply than by using MASP cultures. Ka strain parasites were maintained continuously in MASP culture for 174 days without altering their virulence or immunogenicity when compared to calf-derived parasites. Cultured parasites also survived storage at 4 degrees C for six days in basal medium, adding to their potential usefulness as a live vaccine in field situations.

Animals↗
PubMed1988

Comparison of DNA-spot hybridization, cell culture and direct immunofluorescence staining for the diagnosis of avian chlamydiae.

DNA-spot hybridization, cell culture and direct immunofluorescence staining were compared for the detection of avian Chlamydia psittaci strains in cell culture dilutions and in routine samples submitted for diagnosis. With dilutions of infected cell culture material, growth in BGM cells was by far the most sensitive technique, detecting 0.01 infected cells (20 elementary bodies) ml-1. DNA-spot hybridization and direct immunofluorescence staining were of approximately equal sensitivity, both detecting 16 infected cells (3.2 x 10(4) elementary bodies) per ml-1. When 27 avian liver and spleen samples were assayed, all 3 tests performed similarly (13 positive and 12 negative by all 3 tests). This suggests that in most avian samples presented for diagnosis, sufficient numbers of chlamydiae are present to allow any of the test to the be used. Thus, the direct immunofluorescence staining method is currently the test of choice for routine diagnosis since it is available in kit form, is relatively simple and quick to perform, and like DNA-spot hybridization, detects non-viable as well as viable organisms. However, if low levels of chlamydiae are to be effectively detected, such as in carrier birds or birds with recently acquired infections, then cell culture should be used.

Animals↗
PubMed1988

Comparison of Chlamydia psittaci isolates by restriction endonuclease and DNA probe analyses.

DNAs from eight Chlamydia psittaci isolates (koala conjunctivitis, avian psittacosis, avian ornithosis, ovine abortion, ovine polyarthritis, sporadic bovine encephalomyelitis, and feline conjunctivitis) and one Chlamydia trachomatis isolate (lymphogranuloma venereum) were compared by restriction endonuclease and DNA probe analyses. Digestion with HindIII yielded a series of discrete fragments which allowed the differentiation of most isolates. A gene probe, pFEN207, which encodes the chlamydia-specific component of the lipopolysaccharide group antigen was used in Southern hybridizations. The probe was chlamydia specific and hybridized to a single BamHI fragment and multiple HindIII fragments in each isolate. The variation in size of the hybridizing fragments allowed easy differentiation of the isolates and may eventually lead to a meaningful subgrouping of the diverse group of disease agents presently included in the species C. psittaci.

Animals↗