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Title: Antigenic ureaplasma variation and adaptation to ovine complement response following experimental intrauterine infection
Author: Ahmed, Shatha Thanoon
ISNI:       0000 0004 5368 6931
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2015
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Ureaplasma parvum is one of the smallest (genome <0.8 Mb), free living, self-replicating organisms identified. There are seven species of Ureaplasma but only two infect humans: Ureaplasma parvum (serovars 1, 3, 6 and 14) and Ureaplasma urealyticum (serovars 2, 4, 5, 7–13). While frequently thought to be a commensal, Ureaplasma spp. has been found to be the most frequently identified infectious organism associated with preterm delivery. The mechanisms it uses to escape recognition by the host immune system and establish long term chronic infections in the host are unknown. However, association of infection with pregnancy and preterm birth, which have lower immune response are well known and its infection of and induction of preterm birth leads to short and long term sequel. Objectives of the study Using a model of experimental infection, this thesis sought to investigate differences be-tween systemic Ureaplasma infection in mid-second trimester equivalent and early third trimester equivalent, and relate these differences to the stage of development of the com-plement system in preterm lambs. These studies required the development of a new assay to measure complement function in sheep. The ability of the infecting strain of Ureaplas-ma to adapt to the sheep immune system was also investigated, examined at varying lengths of infection. These studies required the use of a new in vivo catheterisation method to establish the kinetics of early experimental intrauterine Ureaplasma infection and to re-cover Ureaplasma from the amniotic fluid by amniocentesis at various times during chron-ic infection. Emerging resistance to sheep complement was investigated in these strains and related to coincident alterations in the major surface antigen (multiple banded antigen; MBA) and alterations in the whole genome sequence. These studies required the charac-terisation of new monoclonal antibodies that recognise the MBA and establishment of a synthetic MBA expressed in E.coli to aid in reagent characterisation. Materials and Methods: Different erythrocyte sources and sensitising antibodies were utilised to establish the guin-ea pig erythrocyte as the best target for measuring the 50% haemolytic activity in fetal and adult sheep sera. Flow cytometry methods were also used to establish endogenous anti-erythrocyte antibodies in adult, but not fetal sheep, revealing a failure of maternal IgG transfer across the placenta in contrast to humans. A bactericidal assay for the decrease in surviving Ureaplasma following incubation with sheep sera was utilised and modified conditions revealed the rate of killing and calcium-dependence of Ureaplasma killing for both fetal and adult sera. Experimental infection of singleton pregnant ewes by catheter or ultrasound guided needle, followed by caesarean delivery at either 94 or 125 days gestation (term=150 days) was central to all of the studies. Over 3 years, different infection duration (from 5 days to 6 weeks) and gestational age at infection (from 70 days to 116 days gesta-tional age) were investigated in this model. Western blot analysis was also a central meth-od for both examining the antibody response of foetus and ewe to Ureaplasma as well as examination of the size and number of MBA isoforms in evolving strains during chronic infection. The use of genomic analysis software Geneious (Biomatters ltd., New Zealand) was also utilised to compare the genomic sequences of Ureaplasma strains recovered by amniocentesis during chronic infection relative to the original infecting strain to determine number and site of genetic alterations with evolving serum resistance. Only the repeat re-gion of the MBA gene required investigation by PCR and traditional Sanger sequencing, due to limitations of Illumina sequencing methods and repeats greater than 150 bp. Results: The 50% complement haemolytic activity of preterm sheep foetuses was very poor at 95 day gestation and improved by 125 day gestation, but was still much lower than adult sera. This was reflected in both in vivo bacteraemia during experimental infection and in vitro examining Ureaplasma-cidal activity of sera. A direct correlation (R2=0.30; p<0.05) was found between haemolytic activity of sera and capacity to kill Ureaplasma in vitro. The strain utilised for experimental infection (HPA5) has a single low mass isoform of MBA consisting of 17 PAGKEQ repeats in the C-terminus. Following 5-7 days infection two of six of the infected animals began to show emergence of a second larger MBA isoform. The number and size of MBA isoforms continued to increase at 3 and 5 weeks, until recov-ered strains appeared to solely consist of strains with much larger MBA isoforms (72->120 k Da). The underlying mechanisms of increased MBA mass was found to be increasing number of PAGKEQ repeats from 17 to >200 repeats. Genomic analysis of recovered strains identified conserved single nucleotide polymorphisms in the two genes encoding the ammonium ion transport, but otherwise very few alterations were observed and no ob-vious candidate to explain increased serum resistance (other than increased MBA mass) were observed. For the most part, alterations occurred in polyA, polyT and polyAT inter-genic regions. Despite the apparent correlation between increasing complement resistance of recovered strains and increasing MBA number of repeats, transposon delivery and ex-pression of the MBA gene from a resistant recovered strain and the serum-sensitive origi-nal HPA5 strain did not transfer serum resistance in one experiment. Conclusions: Ureaplasma can change its surface epitopes continuously however, the ex-pected phase variation loss of MBA expression with development of anti-MBA antibodies was not observed. A strong selection for increased MBA mass was apparent with longer infections and this was coincident with evolution of serum resistance (which increased steadily after 1 week infection to high serum resistance after 5 weeks infection). The de-velopment of the complement system was directly related to the detection of Ureaplasma in the circulation of infected lambs, but even 6 weeks of chronic persistent Ureaplasma infection was found to induce an inconsistent and low antibody response, and only in the later gestational age foetuses. While increased MBA size appears to be coincident with evolved serum resistance, it must require either loss of the original MBA isoform (domi-nant serum susceptibility determinant) or other less obvious alterations in the genome of Ureaplasma.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: R Medicine (General)