Genotyping of human and animal isolates of Giardia intestinalis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University

Giardia intestinalis is an important protozoan parasite that infects humans and animals. It has been suggested that cattle may be a major source of human Giardia infection so a dairy farming region of New Zealand was investigated. This thesis uses three molecular methods to genotype G. intestinalis isolates obtained from human and animal faecal specimens collected in the Waikato region of New Zealand, to determine if giardiasis is a zoonotic disease. Random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) fingerprinting techniques were initially assessed for their ability to genotype G. intestinalis isolates. "Clear cut" evidence of zoonosis could not be established by either method, due to a low sample number. To determine the stability of the G. intestinalis genome an axenic culture of G. intestinalis trophozoites was stressed with toxic levels of metronidazole and the survivors, following a number of passages, were examined using AFLP and RAPD analysis. The DNA fingerprints were compared to those of the original wild-type with the results being indicative of an unstable G. intestinalis genome. A third molecular method was employed, which amplifies a portion of the tandemly repeated ribosomal DNA (rDNA). Each cyst contains 512 head to tail tandem repeat copies of the rRNA gene made up of both conserved and variable regions. The use of nested primers increased the sensitivity and specificity of the PCR reaction allowing the amplification of a 505bp rDNA fragment. DNA sequence analysis and alignment of the amplified products facilitated the comparison of G. intestinalis isolates. The relationship of the sequence data was generated and displayed using Splitstree software indicating that zoonosis did occur

Whole-Genome Comparison of Two Campylobacter jejuni Isolates of the Same Sequence Type Reveals Multiple Loci of Different Ancestral Lineage

Campylobacter jejuni ST-474 is the most important human enteric pathogen in New Zealand, and yet this genotype is rarely found elsewhere in the world. Insight into the evolution of this organism was gained by a whole genome comparison of two ST-474, flaA SVR-14 isolates and other available C. jejuni isolates and genomes. The two isolates were collected from different sources, human (H22082) and retail poultry (P110b), at the same time and from the same geographical location. Solexa sequencing of each isolate resulted in 1.659 Mb (H22082) and 1.656 Mb (P110b) of assembled sequences within 28 (H22082) and 29 (P110b) contigs. We analysed 1502 genes for which we had sequences within both ST-474 isolates and within at least one of 11 C. jejuni reference genomes. Although 94.5% of genes were identical between the two ST-474 isolates, we identified 83 genes that differed by at least one nucleotide, including 55 genes with non-synonymous substitutions. These covered 101 kb and contained 672 point differences. We inferred that 22 (3.3%) of these differences were due to mutation and 650 (96.7%) were imported via recombination. Our analysis estimated 38 recombinant breakpoints within these 83 genes, which correspond to recombination events affecting at least 19 loci regions and gives a tract length estimate of 2 kb. This includes a 12 kb region displaying non-homologous recombination in one of the ST-474 genomes, with the insertion of two genes, including ykgC, a putative oxidoreductase, and a conserved hypothetical protein of unknown function. Furthermore, our analysis indicates that the source of this recombined DNA is more likely to have come from C. jejuni strains that are more closely related to ST-474. This suggests that the rates of recombination and mutation are similar in order of magnitude, but that recombination has been much more important for generating divergence between the two ST-474 isolates

Identification and characterization of proteins in amniotic fluid that are differentially expressed before and after antenatal corticosteroid administration

OBJECTIVE: We sought to examine changes in the intraamniotic proteomic environment after the administration of antenatal corticosteroids to women with impending preterm delivery. STUDY DESIGN: Amniotic fluid samples were collected at the time of clinically indicated amniocentesis before and within 7 days of administration of antenatal corticosteroids for impending preterm delivery (n = 12). Proteins differentially expressed before and after corticosteroids were identified by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. They were isolated, characterized, and quantified by fast protein liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digestion, immunodepletion assays, enzyme-linked immunosorbent assay, and electrospray ionization tandem mass spectrometry. RESULTS: Five protein peaks of interest were identified and characterized, all of which were significantly decreased after antenatal corticosteroid administration. These included 2 isoforms of transthyretin, albumin, prothrombin fragment 2, and lumican. CONCLUSION: Four proteins, identified and characterized in amniotic fluid, were differentially expressed with antenatal corticosteroid administration. These data may provide additional insight into the molecular mechanisms by which antenatal corticosteroids prevent neonatal complications.Park JS, 2008, REPROD SCI, V15, P457, DOI 10.1177/1933719108316909Mavrou A, 2008, J PROTEOME RES, V7, P1862, DOI 10.1021/pr700588uRomero R, 2008, J MATERN-FETAL NEO M, V21, P367, DOI 10.1080/14767050802045848Liu LY, 2007, CANCER SCI, V98, P1617, DOI 10.1111/j.1349-7006.2007.00576.xDasgupta SK, 2007, J THROMB THROMBOLYS, V24, P157, DOI 10.1007/s11239-007-0018-8Wu F, 2007, J BIOL CHEM, V282, P26409, DOI 10.1074/jbc.M702402200Maurya P, 2007, ANTICANCER RES, V27, P1247Escher N, 2007, NEOPLASIA, V9, P254, DOI 10.1593/neo.06805Vascotto C, 2007, J PROTEOME RES, V6, P160, DOI 10.1021/pr060315zGuerrier L, 2007, NAT PROTOC, V2, P831, DOI 10.1038/nprot.2007.114Michaels JEA, 2007, J PROTEOME RES, V6, P1277, DOI 10.1021/pr060543tRichardson BS, 2006, AM J OBSTET GYNECOL, V195, P1357, DOI 10.1016/j.ajog.2006.03.053Miguet L, 2006, J PROTEOME RES, V5, P2258, DOI 10.1021/pr060058yTsangaris GT, 2006, PROTEOMICS, V6, P4410, DOI 10.1002/pmic.200600085Gharraee Z, 2006, J INVEST MED, V54, P245, DOI 10.2310/6650.2006.05060ROBERTS D, 2006, COCHRANE DB SYST REV, V3, P4454, DOI DOI 10.1002/14651858.CD004454.PUB2Park SJ, 2006, PROTEOMICS, V6, P349, DOI 10.1002/pmic.200500084Kao WWY, 2006, EXP EYE RES, V82, P3, DOI 10.1016/j.exer.2005.08.012Cho S, 2005, PROSTAG OTH LIPID M, V78, P139, DOI 10.1016/j.prostaglandins.2005.06.003Kozak KR, 2005, PROTEOMICS, V5, P4589, DOI 10.1002/pmic.200500093Karcher R, 2005, AM J OBSTET GYNECOL, V193, P1680, DOI 10.1016/j.ajog.2005.03.080Fung ET, 2005, INT J CANCER, V115, P783, DOI 10.1002/ijc.20928Buhimschi IA, 2005, BJOG-INT J OBSTET GY, V112, P173, DOI 10.1111/j.1471-0528.2004.00340.xYeh LK, 2005, INVEST OPHTH VIS SCI, V46, P479, DOI 10.1167/iovs.04-1014Gravett MG, 2004, JAMA-J AM MED ASSOC, V292, P462, DOI 10.1001/jama.292.4.462McElrath TF, 2004, OBSTET GYNECOL, V103, P463ZHU XD, 2004, WORLD J GASTROENTERO, V10, P2327Kaplan LA, 2002, CLIN CHIM ACTA, V326, P61Chakravarti S, 2002, GLYCOCONJUGATE J, V19, P287Kim BJ, 2002, THROMB RES, V106, P81Oliveira FR, 2002, BRAZ J MED BIOL RES, V35, P215Hamilton JA, 2001, CELL MOL LIFE SCI, V58, P1491Bolt RJ, 2001, PEDIATR PULM, V32, P76Kallapur SG, 2001, AM J PHYSIOL-LUNG C, V280, pL527Shimoya K, 2000, HUM REPROD, V15, P2234CROWLEY P, 2000, COCHRANE DB SYST REV, V2, DOI DOI 10.1002/14651858Winn HN, 2000, J PERINAT MED, V28, P210Tan RC, 1999, AM J PHYSIOL-LUNG C, V277, pL1142Dolhnikoff M, 1998, AM J RESP CELL MOL, V19, P582Bry K, 1997, J CLIN INVEST, V99, P2992Jensen ON, 1997, ANAL CHEM, V69, P1706Yoon BH, 1996, OBSTET GYNECOL, V88, P1034Berman S, 1996, AM J OBSTET GYNECOL, V175, P73HanlonLundberg KM, 1996, OBSTET GYNECOL, V87, P128DEKOWSKI SA, 1995, PEDIATR RES, V38, P513GROVER J, 1995, J BIOL CHEM, V270, P21942CROWLEY PA, 1995, AM J OBSTET GYNECOL, V173, P3221995, AM J OBSTET GYNECOL, V173, P246LOLIS D, 1995, GYNECOL OBSTET INVES, V40, P231ROONEY SA, 1994, FASEB J, V8, P957TANEDA H, 1994, J BIOCHEM-TOKYO, V116, P589WINN HN, 1992, AM J PERINAT, V9, P326LIEBERMAN E, 1992, OBSTET GYNECOL, V79, P564FUNDERBURGH JL, 1991, J BIOL CHEM, V266, P24773GROSS I, 1990, AM J PHYSIOL, V259, pL337BALLARD PL, 1989, ENDOCR REV, V10, P165RATH W, 1984, Z GEBURTSH PERINATOL, V188, P218HEYES H, 1982, ARCH GYNECOL, V233, P7TORDAY JS, 1981, AM REV RESPIR DIS, V123, P205LIGGINS GC, 1972, PEDIATRICS, V50, P515LIGGINS GC, 1969, J ENDOCRINOL, V45, P515

Timing of Histologic Progression from Chorio-Deciduitis to Chorio-Deciduo-Amnionitis in the Setting of Preterm Labor and Preterm Premature Rupture of Membranes with Sterile Amniotic Fluid.

Histologic chorio-deciduitis and chorio-deciduo-amnionitis (amnionitis) in extra-placental membranes are known to represent the early and advanced stages of ascending intra-uterine infection. However, there are no data in humans about the time required for chorio-deciduitis to develop and for chorio-deciduitis without amnionitis to progress to chorio-deciduitis with amnionitis, and the effect of prolongation of pregnancy on the development of chorio-deciduitis and amnionitis in patients with preterm labor and intact membranes (PTL) and preterm premature rupture of membranes (preterm-PROM). We examined these issues in this study.The study population consisted of 289 women who delivered preterm (133 cases with PTL, and 156 cases with preterm-PROM) and who had sterile amniotic fluid (AF) defined as a negative AF culture and the absence of inflammation as evidenced by a matrix metalloproteinase-8 (MMP-8) level <23 ng/ml. We examined the association between amniocentesis-to-delivery interval and inflammatory status in the extra-placental membranes (i.e., inflammation-free extra-placental membranes, choroi-deciduitis only, and chorio-deciduitis with amnionitis) in patients with PTL and preterm-PROM.Amniocentesis-to-delivery interval was longer in cases of chorio-deciduitis with amnionitis than in cases of chorio-deciduitis only in both PTL (median [interquartile-range (IQR)]; 645.4 [319.5] vs. 113.9 [526.9] hours; P = 0.005) and preterm-PROM (131.3 [135.4] vs. 95.2 [140.5] hours; P<0.05). Amniocentesis-to-delivery interval was an independent predictor of the development of both chorio-deciduitis and amnionitis after correction for confounding variables such as gestational age at delivery in the setting of PTL, but not preterm-PROM.These data confirm for the first time that, in cases of both PTL and preterm-PROM with sterile AF, more time is required to develop chorio-deciduitis with amnionitis than chorio-deciduitis alone in extra-placental membranes. Moreover, prolongation of pregnancy is an independent predictor of the development of both chorio-deciduitis and amnionitis in cases of PTL with sterile AF

Presenting Twins Are Exposed to Higher Levels of Inflammatory Mediators than Nonpresenting Twins as Early as the Midtrimester of Pregnancy.

Presenting twins are less likely to develop respiratory complications than non-presenting twins. The precise reason for this difference is not well understood, although it is known that the presence of inflammation reduces the risk of respiratory morbidity at birth. To further investigate this association, we compared the concentrations of inflammatory biomarkers in mid-trimester amniotic fluid (AF) of asymptomatic twin pairs.The study population consisted of women with twin pregnancies who underwent mid-trimester amniocentesis (15-20 weeks) for routine clinical indications and delivered at term. AF was analyzed for pro-inflammatory cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IFN-γ, TNF-α), matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-12), and chemokines (Complement Factor D/Adipsin, Serpin E1/PAI-1, Adiponectin/Acrp30, CRP, CCL2/MCP-1, Leptin, Resistin) using Luminex Performance Assay multiplex kits. Data were analyzed using Wilcoxon signed rank test.A total of 82 twin pairs were enrolled. Mid-trimester AF concentrations of IL-8, MMP-8, CRP, MCP-1, leptin, and resistin were significantly higher in the presenting twin compared with the non-presenting twin (p<0.05 for each). Differences in AF concentrations of IL-8, MMP-8, and CRP persisted after adjustment for the fetal growth restriction at the time of birth and chorionicity.These data suggest that, as early as the mid-trimester, the presenting fetus in an otherwise uncomplicated twin pregnancy is exposed to higher levels of pro-inflammatory mediators (especially IL-8, MMP-8, and CRP) than its non-presenting co-twin. Whether this pro-inflammatory milieu reduces the risk of neonatal respiratory morbidity at birth or has other functional implications needs to be further evaluated

Histopathology of extra-placental membranes (chorio-decidua and amnion) (These images are based on the magnification setting X200).

<p>Hematoxylin and eosin stained histologic sections of extra-placental membranes (chorio-decidua and amnion) are shown for inflammation-free placenta (A, PTL; and B, preterm-PROM), choriodeciduitis only (C, PTL; and D, preterm-PROM) and chorio-deciduitis with amnionitis (E, PTL; and F, preterm-PROM). Neutrophils in the chorio-decidua are indicated with arrows in panel C and panel D (see insets of panels C-D), and arrows in panel E and panel F indicate neutrophilic infiltration in amnion (see insets of panels E-F).</p

Boletín de Segovia: Número 128 - 1879 octubre 27

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Frequency of inflammation in placental compartments according to amniocentesis-to-delivery interval.

<p>The frequency of chorio-deciduitis (A), amnionitis (B), funisitis (C), and chorionic plate inflammation (D) according to amniocentesis-to-delivery interval (i.e., ≤2 days, 2–7 days, and >7 days) is shown in patients with PTL and preterm-PROM. Frequency and P-values are shown.</p

Relationship of various independent variables with the development of chorio-deciduitis and amnionitis analyzed by overall logistic regression analysis in preterm-PROM with sterile amniotic fluid.

<p><i>preterm-PROM</i>, preterm premature rupture of membranes; <i>CI</i>, confidence interval.</p><p>Relationship of various independent variables with the development of chorio-deciduitis and amnionitis analyzed by overall logistic regression analysis in preterm-PROM with sterile amniotic fluid.</p

Relationship between the latency after ROM and chorioamnionitis in previous studies.

<p><i>GA</i>, gestational age; <i>NA</i>, not available; <i>ROM</i>, rupture of membranes; <i>AF</i>, amniotic fluid; <i>CA</i>, chorioamnionitis.</p><p>Relationship between the latency after ROM and chorioamnionitis in previous studies.</p