Ureaplasma parvum Serovar 3 Multiple Banded Antigen Size Variation after Chronic Intra-Amniotic Infection/Colonization

James W Robinson,Ilias Nitsos,Alan H Jobe,John Newnham,Christine L Knox,Suhas G Kallapur,Diane Payton,Samantha J Dando,Graeme R Polglase,J Jane Pillow,Boris W Kramer

doi:10.1371/journal.pone.0062746

Abstract

Ureaplasma species are the microorganisms most frequently associated with adverse pregnancy outcomes. The multiple banded antigen (MBA), a surface-exposed lipoprotein, is a key virulence factor of ureaplasmas. The MBA demonstrates size variation, which we have shown previously to be correlated with the severity of chorioamnion inflammation. We aimed to investigate U. parvum serovar 3 pathogenesis in vivo, using a sheep model, by investigating: MBA variation after long term (chronic) and short term (acute) durations of in utero ureaplasma infections, and the severity of chorioamnionitis and inflammation in other fetal tissues. Inocula of 2×107 colony-forming-units (CFU) of U. parvum serovar 3 (Up) or media controls (C) were injected intra-amniotically into pregnant ewes at one of three time points: day 55 (69d Up, n = 8; C69, n = 4); day 117 (7d Up, n = 8; C7, n = 2); and day 121 (3d Up, n = 8; C3, n = 2) of gestation (term = 145–150d). At day 124, preterm fetuses were delivered surgically. Samples of chorioamnion, fetal lung, and umbilical cord were: (i) snap frozen for subsequent ureaplasma culture, and (ii) fixed, embedded, sectioned and stained by haematoxylin and eosin stain for histological analysis. Selected fetal lung clinical ureaplasma isolates were cloned and filtered to obtain cultures from a single CFU. Passage 1 and clone 2 ureaplasma cultures were tested by western blot to demonstrate MBA variation. In acute durations of ureaplasma infection no MBA variants (3d Up) or very few MBA variants (7d Up) were present when compared to the original inoculum. However, numerous MBA size variants were generated in vivo (alike within contiguous tissues, amniotic fluid and fetal lung, but different variants were present within chorioamnion), during chronic, 69d exposure to ureaplasma infection. For the first time we have shown that the degree of ureaplasma MBA variation in vivo increased with the duration of gestation.

Highlights

The Ureaplasma species, bacteria from the class Mollicutes, are the most prevalent, potentially pathogenic bacteria isolated from the urogenital tract of both men and women (40–80%) [1] and are the microorganisms most frequently associated with preterm birth [2,3,4]
Summary of Pathology Pathology results using the Redline classifications [27] summarised in Table 1, demonstrated that the severity of inflammation of the maternal and fetal chorioamnion membranes increased with the duration of ureaplasma colonization
The increased duration of exposure to intra-amniotic ureaplasmas was associated with an influx of macrophages and neutrophils within the maternal membranes of the chorioamnion when compared to the response within the control tissues (Maternal response Stage 2/Grade 1(b) – Table 1)

Summary

Introduction

The Ureaplasma species, bacteria from the class Mollicutes, are the most prevalent, potentially pathogenic bacteria isolated from the urogenital tract of both men and women (40–80%) [1] and are the microorganisms most frequently associated with preterm birth [2,3,4]. U. parvum is the most common species isolated from the genital tracts of men and women [1,9,10]. Ureaplasma colonization of the female upper genital tract in pregnancy is associated with preterm labor, preterm birth, perinatal morbidity and mortality, premature rupture of membranes [8,11,12] and is a major cause of histological chorioamnionitis [1,13,14]. Isolation of ureaplasmas from endotracheal secretions of newborns [16] shows that infection of the fetus can occur in utero or alternatively be acquired by vertical transmission at birth [17]

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