Origin of the New World Plasmodium vivax: Facts and New Approaches.

Malaria is one of the most important human diseases throughout tropical and sub-tropical regions of the world. Global distribution and ample host range have contributed to the genetic diversity of the etiological agent, Plasmodium. Phylogeographical analyses demonstrated that Plasmodium falciparum and Plasmodium vivax follow an Out of Africa (OOA) expansion, having a higher genetic diversity in African populations and a low genetic diversity in South American populations. Modeling the evolutionary rate of conserved genes for both P. falciparum and P. vivax determined the approximate arrival of human malaria in South America. Bayesian computational methods suggest that P. falciparum originated in Africa and arrived in South America through multiple independent introductions by the transatlantic African slave trade; however, in South America, P. vivax could have been introduced through an alternate migratory route. Alignments of P. vivax mitogenomes have revealed low genetic variation between the South American and Southeast Asian populations suggesting introduction through either pre-Columbian human migration or post-colonization events. To confirm the findings of these phylogeographical analyses, molecular methods were used to diagnose malaria infection in archeological remains of pre-Columbian ethnic groups. Immunohistochemistry tests were used and identified P. vivax but not P. falciparum in histologically prepared tissues from pre-Columbian Peruvian mummies, whereas shotgun metagenomics sequencing of DNA isolated from pre-Columbian Caribbean coprolites revealed Plasmodium-homologous reads; current evidence suggests that only P. vivax might have been present in pre-Columbian South America.

Global phylogeography and evolution of chelonid fibropapilloma-associated herpesvirus.

A global phylogeny for chelonid fibropapilloma-associated herpesvirus (CFPHV), the most likely aetiological agent of fibropapillomatosis (FP) in sea turtles, was inferred, using dated sequences, through Bayesian Markov chain Monte Carlo analysis and used to estimate the virus evolutionary rate independent of the evolution of the host, and to resolve the phylogenetic positions of new haplotypes from Puerto Rico and the Gulf of Guinea. Four phylogeographical groups were identified: eastern Pacific, western Atlantic/eastern Caribbean, mid-west Pacific and Atlantic. The latter comprises the Gulf of Guinea and Puerto Rico, suggesting recent virus gene flow between these two regions. One virus haplotype from Florida remained elusive, representing either an independent lineage sharing a common ancestor with all other identified virus variants or an Atlantic representative of the lineage giving rise to the eastern Pacific group. The virus evolutionary rate ranged from 1.62×10(-4) to 2.22×10(-4) substitutions per site per year, which is much faster than what is expected for a herpesvirus. The mean time for the most recent common ancestor of the modern virus variants was estimated at 192.90-429.71 years ago, which, although more recent than previous estimates, still supports an interpretation that the global FP pandemic is not the result of a recent acquisition of a virulence mutation(s). The phylogeographical pattern obtained seems partially to reflect sea turtle movements, whereas altered environments appear to be implicated in current FP outbreaks and in the modern evolutionary history of CFPHV.

Enterophages, a group of phages infecting Enterococcus faecalis, and their potential as alternate indicators of human faecal contamination.

We have developed a method for the detection of viruses in environmental samples that we have called enterophages, that specifically infect Enterococcus faecalis. This method has allowed us to determine the prevalence and to study the ecology of this group of phages. The enterophages replicate at 37 degrees C, and at 41 degrees C. The presence of NaN(3) in the media inhibits the growth of background microbiota and allows an accurate, specific and rapid detection of these viruses. Enterophages were present in raw domestic sewage at lower concentrations (average 1.8 x 10(2) PFU/100 mL) than those of coliphages (average 1.7 x 10(5) PFU/100 mL). Phages were characterised by transmission electron microscopy showing icosahedral capsids, some with non-contractile tails as well as icosahedral non-tailed capsids. Different isolates had capsid sizes ranging from 20 nm to about 75 nm in diameter. These data describe a new group of phages that may serve as alternate indicators of human faecal pollution, especially in recreational waters. The ecology of these enterophages indicates that these may be strictly of human origin.

Evaluation of Bacillus subtilis and coliphage MS2 as indicators of advanced water treatment efficiency.

The assessment of water treatment facilities for their efficiency using alternate indicators is of paramount importance. Current methods for assessing efficiency are limited by the specific characteristics of the microorganisms, such as their different sensitivities to disinfectants. A pilot study was carried out to compare different treatment scenarios for the future upgrade of the Sergio Cuevas Water Treatment plant (the largest in the Caribbean) in San Juan, Puerto Rico. The treatment units under investigation included a coagulation-flocculation-sedimentation unit, dual-media filters, micro-filtration units, intermediate ozone injection and contact columns as well as a biological filtration unit. The plant was challenged at different stages of treatment with Bacillus subtilis spores and MS2 coliphages in an attempt to test them as possible alternate indicators of treatment plant performance. These organisms were chosen because of their resistance to disinfection and desiccation, their low analysis costs and ease of detection. The removal of spores and coliphages by each treatment unit tested was calculated by seeding a known concentration (5-7 log10) of spores and coliphages and following the removal or disinfection rates. The seeded indicators were detected using traditional culture techniques. Ballasted clarification was shown to be highly efficient at removing 99.1% (approximately 3 log10) of the spores and 85.1% (approximately 0.86 log10) of MS2. Ozone treatment inactivated 80.37% (approximately 1.4 log10) spores and 99.95% (approximately 3.07 log10) coliphages. The coliphage inactivation rate obtained confirmed data obtained by previous studies indicating that MS2 was less resistant to ozonation than B subtilis spores. The membrane technology had the best efficiency in terms of physical removal of spores achieving over 99.9% (> 3 log10) removal. Coliphage removal mechanisms remain to be determined and will be a future focus of the study. Preliminary results indicate that aerobic spores and coliphages may be useful as indicators to determine the efficiency of different drinking water treatment technologies.

Use of solid-phase PCR for enhanced detection of airborne microorganisms.

The solid-phase PCR (SP-PCR) was compared with a culture-based technique for the detection of aerosolized Escherichia coli DH1. Results with SP-PCR showed an increase in detection sensitivity over that of culture methods. Therefore, SP-PCR may be useful for the detection of airborne microorganisms which may be nonculturable because of aerosolization or sampling stress.

Extraction and purification of a catalase from Candida albicans.

Candida albicans yeast cells H317 were grown to mid-log phase, mechanically disrupted and the resulting crude extract clarified by centrifugation. This catalase rich fraction (1.26 x 10(-4) units/ml) was fractionated by liquid phase isoelectric focusing in a pH gradient ranging from 3 to 10 using the Rotofor Isoelectric Focusing Preparative Cell. After isoelectric separation, fractions containing catalase activity were focused between pH 6.7 and 9.3. Active fractions were pooled and re-focused. After the second fractionation, catalase activity increased to 1.52 x 10(-2) units/ml and was restricted to fractions ranging from pH 7.6 to 8.8. To this point a 121 fold purification was achieved. Native polyacrylamide gel electrophoresis analysis of active fractions revealed a band migrating between 272,000 and 132,000 daltons which showed catalase activity. Purification of C. albicans catalase will allow us to evaluate its potential role in protecting this opportunistic pathogen from products of the oxidative burst. Antibodies generated against the catalase provide means for the evaluation of neutralizing fungal defenses against products of the oxidative burst during phagocytosis.

Extraction and purification of a catalase from Candida albicans

Candida albicans yeast cells H317 were grown to mid-log phase, mechanically disrupted and the resulting crude extract clarified by centrifugation. This catalase rich fraction (1.26 x 10(-4) units/ml) was fractionated by liquid phase isoelectric focusing in a pH gradient ranging from 3 to 10 using the Rotofor Isoelectric Focusing Preparative Cell. After isoelectric separation, fractions containing catalase activity were focused between pH 6.7 and 9.3. Active fractions were pooled and re-focused. After the second fractionation, catalase activity increased to 1.52 x 10(-2) units/ml and was restricted to fractions ranging from pH 7.6 to 8.8. To this point a 121 fold purification was achieved. Native polyacrylamide gel electrophoresis analysis of active fractions revealed a band migrating between 272,000 and 132,000 daltons which showed catalase activity. Purification of C. albicans catalase will allow us to evaluate its potential role in protecting this opportunistic pathogen from products of the oxidative burst. Antibodies generated against the catalase provide means for the evaluation of neutralizing fungal defenses against products of the oxidative burst during phagocytosis

Solid-phase polymerase chain reaction: applications for direct detection of enteric pathogens in waters.

The techniques in current use for detection of pathogens in environmental samples are restricted to those organisms whose replication in either culture media or cell culture is feasible. These methods lack the selectivity and sensitivity necessary for their unequivocal detection and identification. We have developed an assay for the detection of bacterial cells in large volumes of water. Low concentrations of cells containing target sequences were concentrated on membrane filters and were subjected to amplification directly using a stepwise polymerase chain reaction. This procedure, together with nucleic acid probes, has enhanced the limit of detection to the level of a single bacterial cell. This technique could be used for the detection of any bacteria or virus in water or air.

Use of modified diatomaceous earth for removal and recovery of viruses in water.

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased the negative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostatic interactions were more important than hydrophobic interactions in virus adsorption to modified diatomaceous earth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferric chloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, and coxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of 100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceous earth with 3% beef extract plus 1 M NaCl (pH 9).

Survival and activity of Salmonella typhimurium and Escherichia coli in tropical freshwater.

The survival of Salmonella typhimurium LT2 and Escherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Numbers were determined by acridine orange staining and a Coulter counter. Population activity was determined by microautoradiography, cell respiration, frequency of dividing cells, and by nucleic acid composition. Numbers of Salm, typhimurium and E. coli decreased less than 1 log unit after 105 h as measured by direct count methods. Activity as measured by respiration, acridine orange activity, frequency of dividing cells, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 24 h, E. coli was more active than Salm. typhimurium, as measured by nucleic acid composition, and frequency of dividing cells. Both E. coli and Salm. typhimurium survived and remained active in this tropical rain forest watershed for more than 5 d, suggesting that Salm. typhimurium may be of prolonged public health significance once it is introduced into tropical surface waters. As E. coli was active and survived for a long time in this natural environment, it would seem to be unsuitable as an indicator of recent faecal contamination in tropical waters.

Novel method for monitoring genetically engineered microorganisms in the environment.

A method has been devised for directly detecting and monitoring genetically engineered microorganisms (GEMs) by using in vitro amplification of the target DNAs by a polymerase chain reaction and then hybridizing the DNAs with a specific oligonucleotide or DNA probe. A cloned 0.3-kilobase napier grass (Pennisetum purpureum) genomic DNA that did not hybridize to DNAs isolated from various microorganisms, soil sediments, and aquatic environments was inserted into a derivative of a 2,4-dichlorophenoxyacetic acid-degradative plasmid, pRC10, and transferred into Escherichia coli. This genetically altered microorganism, seeded into filter-sterilized lake and sewage water samples (10(4)/ml), was detected by a plate count method in decreasing numbers for 6 and 10 days of sample incubation, respectively. The new method detected the amplified unique marker (0.3-kilobase DNA) of the GEM even after 10 to 14 days of incubation. This method is highly sensitive (it requires only picogram amounts of DNA) and has an advantage over the plate count technique, which can detect only culturable microorganisms. The method may be useful for monitoring GEMs in complex environments, where discrimination between GEMs and indigenous microorganisms is either difficult or requires time-consuming tests.

An improved method for the concentration of rotaviruses from large volumes of water.

A microporous filter method for the concentration and reconcentration of rotaviruses from large volumes of water was developed. This method allows for the processing of large volumes of water with high recoveries of rotaviruses. Averages of 49% and 41% of seeded viruses were recovered from 20 and 400 l of water, respectively. Of the various eluents evaluated, a mixture of 10% tryptose phosphate broth and 3% beef extract at a pH value of 10.0 was found to give the greatest elution efficiency. The use of the positively charged 1MDS filters allowed for the processing of waters without the need for pH adjustments. An overall recovery of 35% was obtained from 400 l when hydroextraction was used as a second-step concentration procedure.

Survival and activity ofStreptococcus faecalis andEscherichia coli in tropical freshwater.

The survival ofStreptococcus faecalis andEscherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Densities were determined by acridine orange direct count and Coulter Counter. Population activity was determined by microautoradiography, cell respiration, and by nucleic acid composition. Densities ofS. faecalis andE. coli decreased less than 1 log unit after 105 hours as measured by direct count methods. Activity as measured by respiration, acridine orange activity, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 12 hours,E. coli was more active thanS. faecalis as measured by nucleic acid composition. In this tropical rain forest watershed,E. coli andS. faecalis survived and remained active for more than 5 days; consequently, both would seem to be unsuitable as indicators of recent fecal contamination in tropical waters.

In situ survival of plasmid-bearing and plasmidless Pseudomonas aeruginosa in pristine tropical waters.

Two rare wild-type strains of Pseudomonas aeruginosa were mixed in membrane diffusion chambers and then introduced into a natural freshwater environment for 72 h. The plasmid-containing strain (R serotype 15) and the plasmidless strain (H serotype 5) had initial bacterial densities of 2 x 10(5) cells per ml. Samples collected from the chambers were analyzed for viable and direct counts and for acquired-resistance frequencies. Suspected transconjugant-to-donor ratios ranged from 0.5 to 1.3; transfer percentages ranged from 13 to 70%. [3H]thymidine uptake indicated DNA synthesis in both strains as well as in transconjugants. These studies indicate that rare wild-type bacterial strains with large plasmid loads can survive as well as can bacteria with low plasmid loads when exposed to the in situ conditions of a tropical freshwater habitat. These results also suggest that genetic modification of indigenous microbiota through conjugation or transformation is feasible when rare wild-type strains or genetically engineered microorganisms are released in large numbers in tropical aquatic ecosystems.

Isolation of fecal coliforms from pristine sites in a tropical rain forest.

Samples collected from water accumulated in leaf axilae of bromeliads (epiphytic flora) in a tropical rain forest were found to harbor fecal coliforms. Random identification of fecal coliform-positive isolates demonstrated the presence of Escherichia coli. This bacterium was also isolated from bromeliad leaf surfaces. These data indicate that E. coli may be part of the phyllosphere microflora and not simply a transient bacterium of this habitat. The isolation of fecal coliforms from these sites was unexpected and raises questions as to the validity of using fecal coliforms as indicators of biological water quality in the tropics.

Simple field method for concentration of viruses from large volumes of water.

A pressure spray tank was adapted to supply positive pressure for processing water samples for concentrating viruses with microporous filters under field conditions. This low-cost system allows water to be processed in locations where electric current is not readily available or where light-weight portable equipment is required.