Early local activation of complement in aqueous humour of patients with age-related macular degeneration.

OBJECTIVE: To investigate complement activation in aqueous humour of patients with early, intermediate and neovascular age-related macular degeneration (AMD). PATIENTS AND METHODS: Aqueous humour of 79 AMD patients (early, intermediate and neovascular) and 77 age-matched controls was prospectively collected. The levels of the complement protein 3 (C3), activation products complement factor 3a (C3a) and Ba, C3b/iC3b, complement factors B, H and I (CFB, CFH and CFI), and total protein concentration were measured. Data were modelled using covariate analysis to assess the impact of age and glaucoma status of patients and total protein concentration of samples on complement protein concentration across groups. RESULTS: C3a concentration was significantly increased in the aqueous humour of early (p = 0.016), intermediate (p = 0.003) and neovascular (p = 0.018) AMD patients, whilst C3 concentration was significantly increased in early AMD patients only (p = 0.019). Levels of CFB and CFH were significantly increased in the aqueous humour of neovascular AMD patients (p = 0.023 and p = 0.018, respectively). CONCLUSIONS: Our findings provide evidence for early local complement dysregulation in AMD patients, suggesting that complement pathway inhibition may be a clinically relevant intervention for early stages of AMD.

Genotypic Characterization of Cryptosporidium Species in Humans and Peri-Domestic Animals in Ekiti and Oyo States, Nigeria.

Cryptosporidiosis is one of the leading causes of diarrhea in humans and several other vertebrate species. Because surveys of Cryptosporidium genotypes from animals and humans living in the same region are rare, our understanding of the importance of zoonotic transmission in the epidemiology of cryptosporidiosis remains superficial. PCR was used to amplify a portion of the Cryptosporidium 18S small subunit ribosomal RNA gene from fecal DNA from humans and livestock living in Ekiti and Oyo states, Nigeria. PCR-positive samples were further analyzed using PCR targeting the heat-shock protein HSP-70, the actin, and the sporozoite glycoprotein gene gp60. A questionnaire was used to collect demographic information. Sixteen of 187 samples collected were Cryptosporidium 18S PCR positive. Of these, 5 samples originating from HIV-positive patients, 5 from otherwise healthy children, 2 from chickens, 3 from goats, and 1 from a dog were positive for at least 1 marker. Sequencing of the 18S rRNA amplicons revealed the presence of Cryptosporidium parvum in 2 HIV positive patients and in a child; the actin sequence confirmed the presence of this species. Two samples of HIV-positive patients amplified Cryptosporidium hominis 18S rRNA, one of them confirmed by the HSP-70, actin, and gp60 sequences. Cryptosporidium meleagridis was found in another HIV patient, while C. hominis was detected in 3 children (of which 2 were confirmed by gp60). Cryptosporidium muris was found in 1 child. In birds, we found C. meleagridis and, significantly, C. parvum, whereas we detected C. parvum and C. muris in 1 goat each. The only dog sampled was positive for Cryptosporidium canis. We conclude that, in the environment we surveyed, humans and animals are a potential part of the same transmission cycle. Measures to prevent zoonotic transmission should therefore be considered to reduce the prevalence of cryptosporidiosis.

Pneumo-thorax/mediastinum/(retro)peritoneum/ scrotum - a full house of complications following JET ventilation.

We present a patient who presented to our clinic with airway obstruction secondary to oropharygeal cancer. He underwent emergent tracheostomy with JET ventilation, the latter resulting in a "full house" of barotraumatic complications including pneumothorax, pneumomediastinum, pneumoperitoneum, pneumoretroperitoneum and pneumo-scrotum. Free air, while sometimes dramatic as in our case, need not always be a cause for alarm and can often be managed expectantly. Our patient was treated with only a chest drain and otherwise made an uneventful recovery.

A comparison of sequence and length polymorphism for genotyping Cryptosporidium isolates.

Simple sequence repeat markers have played an important role in elucidating the epidemiology of human and animal cryptosporidiosis. The drawback of sequence length polymorphisms is that nucleotide substitutions remain undetected. As some laboratories have opted for using length polymorphisms, while others have relied on sequencing, there is a need to compare both methods. We used a diversified set of unique length polymorphisms and matching nucleotide sequences to assess the ability of each genotyping protocol to discern clusters of related Cryptosporidium parvum isolates. We found a weak correlation between the two distance measures for individual markers. This analysis was extended to four-locus genotypes based on sequence length data or concatenated sequences from the same loci. We interrogated these data to assess whether one would reach the same conclusions regardless of the genotyping method. Clusters of isolates generated with the concatenated sequences were not observed with amplicon length, indicating that inferences on the structure of a Cryptosporidium population depend on the genotyping method. Moreover, isolate clusters derived from concatenated sequences were dependent on the algorithm used to calculate distances. These results emphasize the need for harmonizing genotyping tools, not only by selecting informative markers, but also by standardizing the entire genotyping method.

Genomics and population biology of Cryptosporidium species.

We describe recent advances in the genomics and population biology of Cryptosporidium parvum and C. hominis, the causative agents of cryptosporidiosis in humans and animals. Many basic aspects of the biology of Cryptosporidium species remain to be investigated and effective drugs to control cryptosporidiosis are not available. Sequencing and annotation of the genome of C. parvum and C. hominis has uncovered unique features of the metabolism of these species. The recently sequenced genome of the gastric species C. muris is providing new insights into the evolution of the genus. Cryptosporidian sequence information has facilitated the identification of polymorphic genetic markers. Genotyping of oocysts excreted by human and animal hosts using such markers has revealed many new species and genotypes, and is leading to a better understanding of the epidemiology of cryptosporidiosis.

High-throughput screening in suboptimal growth conditions identifies agonists of Giardia lamblia proliferation.

Giardia lamblia is one of the most prevalent parasites of mankind and is estimated to cause over 200 million infections per year. To screen chemical libraries for compounds that perturb trophozoite proliferation we adapted a conventional culture method to 384-well plates and identified numerous inhibitors. Here we used a modified assay to screen for compounds that promote trophozoite multiplication. Trophozoite growth was reduced by dilution of the culture medium and the growth period was extended to screen 2 compound libraries comprising 1500 compounds. A total of 4 agonists of trophozoite multiplication were identified. In the presence of one of these compounds, strychnine, enhanced growth was accompanied by unusual trophozoite morphology characterized by dividing trophozoites displaying more than the 2 nuclei per cell which are normally observed. The other agonists, although belonging to 2 distinct chemical groups, are known to affect isoprenylation, indicating a link between protein or lipid isoprenylation and growth in culture. Although inhibitors of isoprenylation are known to antagonize proliferation of mammalian cells, an agonistic effect of isoprenylation modulators has to our knowledge not been described previously. These observations illustrate the power of chemical genetics for identifying pathways controlling specific traits in G. lamblia.

Meta-analysis of a polymorphic surface glycoprotein of the parasitic protozoa Cryptosporidium parvum and Cryptosporidium hominis.

Due to its extensive polymorphism, a partial sequence of the Cryptosporidium surface glycoprotein gene gp60 has been frequently used as a genetic marker. I explored the global diversity of this protein, and compared its sequence diversity in Cryptosporidium parvum and Cryptosporidium hominis. In marked contrast to the geographical partition of C. parvum and C. hominis multi-locus genotypes, gp60 allelic groups showed no evidence of segregating in space, or of differing with respect to geographical diversity. Globally, genetic diversity of C. hominis gp60 exceeded that of C. parvum. Within C. parvum, gp60 alleles originating from human isolates were more diverse than those infecting ruminants. Phylogenetic analysis grouped gp60 sequences into a small number of relatively homogenous allelic groups, with only a small number of alleles having evolved independently. With the notable exception of a group of alleles restricted to humans, C. parvum alleles are found in ruminants and humans.

Cryptosporidium parvum DNA replication in cell-free culture.

The lack of robust methods for culturing Cryptosporidium parasites remains a major challenge and is hampering efforts to screen for anti-cryptosporidial drugs. In existing culture methods, monolayers of mammalian epithelial cells are inoculated with oocysts. The system supports an initial phase of asexual proliferation of the parasite. For reasons that are not clear, development rapidly declines within 2-3 days. The unexpected report of Cryptosporidium parvum culture in the absence of host cells, and the failure of others to reproduce the method, prompted us to apply quantitative PCR to measure changes in C. parvum DNA levels in cell-free cultures, and parasite-specific antibodies to identify different life cycle stages. Based on this approach, which has not been applied previously to analyze C. parvum growth in cell-free culture, we found that the concentration of C. parvum DNA increased by about 5-fold over 5 days of culture. Immuno-labeling of cultured organisms revealed morphologically distinct stages, only some of which reacted with Cryptosporidium-specific monoclonal antibodies. These observations are indicative of a modest proliferation of C. parvum in cell-free culture.

Genetic diversity and zoonotic potential of Cryptosporidium parvum causing foal diarrhea.

Cryptosporidium isolates from diarrheic foals in New Zealand (n = 9) were identified as C. parvum, subtyped at two polymorphic loci, and compared with human (n = 45) and bovine (n = 8) isolates. Foal C. parvum isolates were genetically diverse, markedly similar to human and bovine isolates, and carried GP60 IIaA18G3R1 alleles, indicating a zoonotic potential.

Host-shaped segregation of the Cryptosporidium parvum multilocus genotype repertoire.

Cattle are among the major reservoirs of Cryptosporidium parvum in nature. However, the relative contribution of C. parvum oocysts originating from cattle to human disease burden is difficult to assess, as various transmission pathways -- including the human to human route -- can co-occur. In this study, multilocus genotype richness of representative samples of human and bovine C. parvum are compared statistically using analytical rarefaction and non-parametric taxonomic richness estimators. Results suggest that in the time and space frames underlying the analysed data, humans were infected with significantly wider spectra of C. parvum genotypes than cattle, and consequently, a significant fraction of human infections may not have originated from the regional bovine reservoirs. This study provides statistical support to the emerging idea of the existence of distinct anthroponotic C. parvum cycles that do not involve cattle.

Adaptation of Cryptosporidium oocysts to different excystation conditions.

Within the genus Cryptosporidium 2 lineages have evolved, one adapted to the acid environment of the stomach and abomasum, the other comprising parasites that multiply in the small intestine. We tested whether the release of sporozoites from oocysts, a process known as excystation, is triggered by conditions which mimic the site of infection. Specifically, we exposed oocysts from gastric and intestinal Cryptosporidium species to acid conditions or to a neutral solution of taurocholic acid, at 37 degrees C. We found that oocysts from the gastric species C. muris and C. andersoni excysted in both conditions, whereas the intestinal species C. parvum and C. hominis did not respond to acid. When the effect of temperature alone was tested on C. muris and C. parvum, only oocysts from the former species excysted in significant numbers. Oocysts from intestinal species did not respond to temperature alone, nor to acidity. These observations are consistent with the need of gastric species to rapidly excyst and release the sporozoites upon ingestion, and indicate that Cryptosporidium oocysts have evolved to maximize delivery of sporozoites to the region of the gastro-intestinal tract where the parasite multiplies.

Preferential infection of dividing cells by Cryptosporidium parvum.

In spite of its limitations, the culture of Cryptosporidium parvum in monolayers of epithelial cells is a suitable model to study the interaction of this protozoan parasite with the host cell, to assay oocyst infectivity, and to screen drugs for anti-cryptosporidial activity. For unknown reasons, growth of Cryptosporidium in culture is limited in time and generally does not lead to the production of significant numbers of oocysts. In monolayers infected with high doses of oocysts, we observed that many cells remain uninfected, suggesting that some cells are less susceptible to the infection. Since C. parvum and the related species C. hominis lack many essential biosynthetic pathways, we tested whether the dependence of the parasite on host cell metabolites may favour the infection of cells in mitosis. The proportion of monolayer cells in stationary (G0/G1) phase and in mitosis (S/G2/M) was determined and the prevalence of infected cells in each subpopulation was quantified. Although C. parvum infects and develops in dividing and stationary cells, a significant preference for cells in S/G2/M phase was observed. Consistent with previous observations showing that C. parvum induces apoptosis in cell monolayers, infection was accompanied by a significant increase in the proportion of mitotic cells.

Trichomonad gastritis in rhesus macaques (Macaca mulatta) infected with simian immunodeficiency virus.

In a retrospective study, 51 cases of gastritis (14%) were identified from among 341 necropsies performed on simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) at the New England Primate Research Center from 1993 to 2001. Protozoa were seen in the stomach of 13 monkeys (25%) with gastritis. Two histopathologic manifestations of gastritis were observed: seven cases of lymphoplasmacytic gastritis with trichomonad trophozoites within lumens of gastric glands and four cases of necrosuppurative gastritis containing intralesional periodic acid-Schiff-positive protozoa; two cases of gastritis had morphologic features of both types of gastritis. In instances of necrosuppurative and combined lymphoplasmacytic and necrosuppurative gastritis, protozoa were 4-35 microm in diameter and round to tear-shaped. Because of the unusual morphology of the protozoa in these latter cases, transmission electron microscopy and polymerase chain reaction (PCR) were used to further identify these organisms. The protozoa were definitively identified as Tritrichomonas in all cases on the basis of ultrastructural characteristics (flagella and undulating membranes) and amplification of a 347-bp product of the 5.8S ribosomal RNA gene of Tritrichomonas foetus, Tritrichomonas suis and Tritrichomonas mobilensis by PCR using DNA extracted from stomach tissue. On the basis of these observations, we conclude that Tritrichomonas can be a significant cofactor in the development of necrosuppurative gastritis in SIV-infected rhesus macaques.

Genetic analysis of a Cryptosporidium parvum human genotype 1 isolate passaged through different host species.

Cryptosporidium parvum TU502, a genotype 1 isolate of human origin, was passaged through three different mammalian hosts, including humans, pigs, and calves. It was confirmed to be genotype 1 by PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene, direct sequencing of PCR fragments of the small subunit rRNA and beta-tubulin genes, and microsatellite analysis. This isolate was shown to be genetically stable when passaged through the three mammalian species, with no evidence of the emergence of new subpopulations as observed by a genotype-specific PCR assay. TU502 oocysts from different sources failed to infect gamma interferon knockout mice, a characteristic of genotype 1 isolates. The genotypic and phenotypic characterization of TU502 is significant since it is the isolate selected to sequence the genome of C. parvum genotype 1 and is currently used in several research projects including human volunteer studies.

Genotyping Cryptosporidium parvum by single-strand conformation polymorphism analysis of ribosomal and heat shock gene regions.

Polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism (SSCP) approaches utilizing nuclear DNA regions of the small subunit (SSU) of ribosomal RNA and heat shock protein 70 gene (HSP70) were established for genotyping Cryptosporidium parvum. The regions were amplified (individually or in a multiplex reaction) by PCR from DNA extracted from oocysts from ruminant or human hosts, then denatured and subjected to electrophoresis in a mutation detection enhancement (nondenaturing) gel matrix. Single-strand profiles produced in SSCP allowed the unequivocal identification/differentiation of the two common (human, 1 and cattle, 2) genotypes of C. parvum and the direct display of sequence variability within some samples, reflecting population variation. As these are considered among the most closely related genotypes (based on SSU and HSP70 sequence data), these findings and other preliminary results for C. felis (from cat) C. serpentis (from snake) and C. baileyi (from bird) indicate that the SSCP approaches established could be employed to identify any of the currently recognised genotypes and species of Cryptosporidium.

Host cell apoptosis impairs Cryptosporidium parvum development in vitro.

The absence of a self-sustaining in vitro propagation method for Cryptosporidium parvum is a major obstacle for research on this parasite. Conventional cell monolayers are unsuitable for long-term parasite propagation because the level of infection decreases over time and few oocysts, if any, are produced. The interaction between parasite and host cell was studied to identify factors limiting parasite development in vitro. Loss of substrate adherence and death of parasitized host cells was observed in 2 epithelial cell lines. Nuclear morphology, DNA laddering, annexin V binding, and terminal deoxytransferase-mediated dUTP nick end labeling indicated that host cell death occurred by apoptosis. At 6 hr postinfection, only a minority of infected cells remained in the monolayer, and few survived the initial phase of parasite development without losing adherence. Treatment of infected monolayers with caspase inhibitors drastically reduced cell detachment but failed to increase the number of parasites in monolayers. In contrast, cell cultures grown on laminin-coated plates showed a higher proportion of infected cells. These observations indicate that cell detachment and apoptosis in C. parvum-infected cell culture negatively affect parasite survival in vitro.

Extensive polymorphism in Cryptosporidium parvum identified by multilocus microsatellite analysis.

Restriction fragment length polymorphism and DNA sequence analysis discern two main types of Cryptosporidium parvum. We present a survey of length polymorphism at several microsatellite loci for type 1 and type 2 isolates. A total of 14 microsatellite loci were identified from C. parvum DNA sequences deposited in public databases. All repeats were mono-, di-, and trinucleotide repeats of A, AT, and AAT, reflecting the high AT content of the C. parvum genome. Several of these loci showed significant length polymorphism, with as many as seven alleles identified for a single locus. Differences between alleles ranged from 1 to 27 bp. Karyotype analysis using probes flanking three microsatellites localized each marker to an individual chromosomal band, suggesting that these markers are single copy. In a sample of 19 isolates for which at least three microsatellites were typed, a majority of isolates displayed a unique multilocus fingerprint. Microsatellite analysis of isolates passaged between different host species identified genotypic changes consistent with changes in parasite populations.

Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parvum.

Human cryptosporidiosis is attributed to two major Cryptosporidium parvum genotypes of which type 1 appears to be the predominant. Most laboratory investigations however are performed using genotype 2 isolates, the only type which readily infects laboratory animals. So far type 1 has only been identified in humans and primates. A type 1 isolate, obtained from an individual with HIV and cryptosporidiosis, was successfully adapted to propagate in gnotobiotic piglets. Genotypic characterization of oocyst DNA from this isolate using multiple restriction fragment length polymorphisms, a genotype-specific PCR marker, and direct sequence analysis of two polymorphic loci confirmed that this isolate, designated NEMC1, is indeed type 1. No changes in the genetic profile were identified during multiple passages in piglets. In contrast, the time period between infection and onset of fecal oocyst shedding, an indicator of adaptation, decreased with increasing number of passages. Consistent with other type 1 isolates, NEMC1 failed to infect mice. A preliminary survey of the NEMC1 genome covering approximately 2% of the genome and encompassing 200 kb of unique sequence showed an average similarity of approximately 95% between type 1 and 2 sequences. Twenty-four percent of the NEMC1 sequences were homologous to previously determined genotype 2 C. parvum sequences. To our knowledge, this is the first successful serial propagation of genotype 1 in animals, which should facilitate characterization of the unique features of this human pathogen.

beta-tubulin mRNA as a marker of Cryptosporidium parvum oocyst viability.

Determining the viability of waterborne Cryptosporidium parvum oocysts remains a technical challenge. rRNA and mRNA were evaluated in a reverse transcription (RT)-PCR assay as potential markers of oocyst viability. The rationale for this approach is the rapid turnover and postmortem decay of cellular RNA. The beta-tubulin mRNA and an anonymous mRNA transcript were chosen as potential markers because they are the only mRNA species in C. parvum known to possess introns. This feature facilitated the distinction between genuine RT-PCR products and PCR products originating from copurifying DNA. Prolonged incubation at room temperature of initially viable oocysts resulted in a gradual decrease in mRNA levels, which correlated with the loss of oocyst infectivity to neonatal mice. In contrast, oocysts stored at 4 degrees C for over 39 weeks maintained their infectivity and displayed no decrease in the level of beta-tubulin RT-PCR product. The postmortem decay of two mRNA species demonstrates that RT-PCR analysis can provide information on the viability of C. parvum oocysts. The methodological similarity between PCR detection and RT-PCR viability analysis could facilitate the development of a combined detection and viability assay.