Phenotypic characteristics of differing genetic groups of Giardia duodenalis and their implication for species identification

Although it has long been acknowledged that the Giardia duodenalis group of isolates is very diverse and heterogeneous, we still lack comprehensive studies which compare groups of isolates from a selected geographical area on the basis of both genotypic and phenotypic characteristics. In the present study, twelve isolates (eleven from Western Australia, one from the USA) were selected based on the genetic diversity they exhibited upon isoenzyme analysis. Prior to any characterisation, the isolates were all cloned and a cloned cell line was subsequently selected at random for further characterisation. RAPD-PCR verified their distinctive groupings and showed that both parent and clone retained their basic classification and that none of the cloned cell lines had become cross contaminated despite more than three years of continuous in vitro cultivation. Isolates were first verified as being members of the duodenalis morphological group on the basis of Filice’s (1952) system which used the shape of the median body, to order isolates as belonging to one of three morphological groups. The usefulness of Filice’s (1952) system in further subdividing the duodenalis morphological group was also tested. Although his system holds true in differentiating between the three morphological groups of G. duodenalis, G. agilis and G. muris, it failed to resolve the complex of isolates constituting G. duodenalis any further. Morphological variation at the ultrastructural level has recently been reintroduced as a taxonomic tool in Giardia research and caudal flagellar and total trophozoite length were used to characterise the isolates chosen for the present study. Intraspecific variation within the G. duodenalis group was found to extend into the morphological characters tested here. Trophozoite length varied from 13.5|im (PIcl0) to 18.4|im (BAH 12cl4) and caudal flagellar length ranged from 4.6|im (PlclO) to 16.1pm (BAH 33c7). UPGMA analysis grouped isolates similarly to previously obtained isoenzyme electrophoretic data. Prior analysis of growth dynamics in isolates of G. duodenalis had shown significant differences between two genetically distinct isolates (Binz etal, 1992) and these results were confirmed and extended during the present study. Mean generation times obtained for the twelve isolates studied varied from 6.6 hours (PlclO) to 24.5 hours (BAH 33c7). Scheffe’s post hoc test identified four distinct groups among the isolates examined. These results have implications not only for the epidemiology of infection but also for the suggested artificial laboratory induced selection of some genotypes over others by the currently used in vitro culture system. At the commencement of this study only tentative data were available on the characteristics of the dual nuclei of Giardia trophozoites, and the DNA content of individual nuclei. Again, significant intraspecific variation was demonstrated among the isolates studied and DNA content was found to vary from 0.060pg per trophozoite (BAH 33c7) to 0.165pg per trophozoite (BAH 39c9), representing a 2.75 fold difference. Scheffe’s post hoc test identified six distinct groups. Although the data in context with previously published information permitted speculation about ploidy levels in G. duodenalis, the question of genome size could not be resolved. The extensive heterogeneity demonstrated across the characters examined in the present study reflected that observed in most previous studies on G. duodenalis. Isoenzyme electrophoretic data in particular led Meloni etal. (1988a) and Andrews et al. (1989) to speculate about the presence of a complex of cryptic species within the duodenalis morphological group. However, no study to date has addressed the problem of species identification within this heterogeneous assemblage of isolates, nor has any attempted to collect data to solve the question of how many species there are. The identification of species is a multi-step process and in the first instance requires the selection of an appropriate species concept and a case for the adoption of the evolutionary species concept was made. The delimitation of species within this framework requires organisms first to be grouped phylogenetically, preferably using DNA sequence or isoenzyme data, and these groups must then be ranked into the same or different species, measuring genetic differences in biologically meaningful characters. Taking into consideration all the data generated during the present study, the value of these genotypic and phenotypic characters in the identification of species within the G. duodenalis morphological group was examined. Growth dynamics and DNA content were found to be more useful in distinguishing groups of isolates than both trophozoite and caudal flagellar length. Although no species could be identified using the current set of characters, the study did demonstrate that some characters were more appropriate for delimiting species than others. Should the evolutionary species concept be adopted for the delimitation of species within the G. duodenalis morphological group, then there is a definite need to design studies in such a way that the data can be applied to the problem of species identification within the group, and biological characters of medical importance may provide the kind of data needed to rank groups of genetically differentiated isolates into species

Immunohistochemical Characterization of Connexin43 Expression in a Mouse Model of Diabetic Retinopathy and in Human Donor Retinas

Diabetic retinopathy (DR) develops due to hyperglycemia and inflammation-induced vascular disruptions in the retina with connexin43 expression patterns in the disease still debated. Here, the effects of hyperglycemia and inflammation on connexin43 expression in vitro in a mouse model of DR and in human donor tissues were evaluated. Primary human retinal microvascular endothelial cells (hRMECs) were exposed to high glucose (HG; 25 mM) or pro-inflammatory cytokines IL-1β and TNF-α (10 ng/mL each) or both before assessing connexin43 expression. Additionally, connexin43, glial fibrillary acidic protein (GFAP), and plasmalemma vesicular associated protein (PLVAP) were labeled in wild-type (C57BL/6), Akita (diabetic), and Akimba (DR) mouse retinas. Finally, connexin43 and GFAP expression in donor retinas with confirmed DR was compared to age-matched controls. Co-application of HG and cytokines increased connexin43 expression in hRMECs in line with results seen in mice, with no significant difference in connexin43 or GFAP expression in Akita but higher expression in Akimba compared to wild-type mice. On PLVAP-positive vessels, connexin43 was higher in Akimba but unchanged in Akita compared to wild-type mice. Connexin43 expression appeared higher in donor retinas with confirmed DR compared to age-matched controls, similar to the distribution seen in Akimba mice and correlating with the in vitro results. Although connexin43 expression seems reduced in diabetes, hyperglycemia and inflammation present in the pathology of DR seem to increase connexin43 expression, suggesting a causal role of connexin43 channels in the disease progression

Biomarkers for Diabetic Retinopathy – Could Endothelin 2 Be Part of the Answer? - Fig 1

<p><b>Edn2 (A) and Ednrb (B) fold changes in mRNA expression in young and mature retinae of wt, Akita, Kimba and Akimba mice</b>. Data represented are fold changes in expression normalised against Ppia as the housekeeping gene. Differential expression was determined using delta delta Ct according to Livak and Schmittgen [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160442#pone.0160442.ref023" target="_blank">23</a>]. Fold changes in expression in Akita, Kimba and Akimba retinae represent increases or decreases in mRNA expression levels compared to wt retinae. N = 4 per group; *p<0.05 compared to wt and Akita</p

Evidence of Müller cell gliosis in mature Akimba mice with a severe phenotype.

<p>In the mature wt retina GFAP expression was localised and restricted to the astrocytes that reside on the GCL (arrowheads, C-D). In Akimba mice, GFAP expression was observed as thick processes in the GCL and long radial processes in the INL/OPL, indicating Müller cell gliosis (G-H). Edn2 only co-localised with GFAP in Müller cell processes in the INL/OPL (arrows, H) but not in the astrocytes of the GCL. Sections were counterstained with DAPI (B, F). GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PIS/POS: photoreceptor inner and outer segments. Scale bar: 200μm</p

Cellular localisation of Edn2 in the young retina.

<p>Edn2 expression was localised to Müller cells as demonstrated by co-localisation with glutamine synthetase (GS), a Müller cell-specific enzyme, and photoreceptor inner and outer segments (PIS, POS) in wt, Akita, Akimba and Kimba retinae (C, G, K, O; arrows and arrowhead). In Akita, Edn2 expression was mostly in the GCL, where Müller cell endfeet reside, the outer plexiform layer (OPL) and the outer nuclear layer (ONL; E, G). In Akimba and Kimba, Edn2 expression was mostly in the inner retina, the GCL and the inner plexiform layer as well as the photoreceptor inner and outer segments (IPL, PIS, POS; J, K, N, O). Co-localisation of Edn2 and GS was most pronounced in the GCL and Müller cell processes in the IPL of Kimba mice (O). Scale bar: 100μm</p

Cellular localisation of Edn2 in the mature retina.

<p>Following 20 weeks of chronic hyperglycaemia in the mature Akita retina there was increased Edn2 staining in the IPL compared to young Akita retinae (F). Particularly in the mature Akimba retina GS staining was much reduced compared to the young retina, suggesting not only loss of photoreceptors (L) but also loss of Müller cells (I, arrowheads). Müller cell loss was also evident in mature Kimba retinae (M, arrowheads; O, arrows) compared to young Kimba retinae, but the loss was less pronounced. Scale bar: 100μm</p

Serum Edn2 concentration in young and mature wt, Akita, Kimba and Akimba mice.

<p>Serum concentrations did not differ between young wt, Akita, Kimba and Akimba mice (A). In wt mice, Edn2 serum concentration decreased with age. In mature mice (B), there was no significant difference in serum Edn2 concentration between wt, Akita and Kimba mice. However, circulating levels of Edn2 were significantly higher in mature Akimba mice (6.9pg/mg total retina protein) compared to wt (3.9pg/mg; p<0.01), Akita (3.8pg/mg; p<0.01) and Kimba (4.6pg/mg total retina protein; p<0.05) mice.</p

Physiologic and metabolic parameters of young and mature wt, Kimba, Akita and Akimba mice.

<p>Physiologic and metabolic parameters of young and mature wt, Kimba, Akita and Akimba mice.</p