Species concepts and malaria parasites: detecting a cryptic species of Plasmodium.

Species of malaria parasite (phylum Apicomplexa: genus Plasmodium) have traditionally been described using the similarity species concept (based primarily on differences in morphological or life-history characteristics). The biological species concept (reproductive isolation) and phylogenetic species concept (based on monophyly) have not been used before in defining species of Plasmodium. Plasmodium azurophilum, described from Anolis lizards in the eastern Caribbean, is actually a two-species cryptic complex. The parasites were studied from eight islands, from Puerto Rico in the north to Grenada in the south. Morphology of the two species is very similar (differences are indistinguishable to the eye), but one infects only erythrocytes and the other only white blood cells. Molecular data for the cytochrome b gene reveal that the two forms are reproductively isolated; distinct haplotypes are present on each island and are never shared between the erythrocyte-infecting and leucocyte-infecting species. Each forms a monophyletic lineage indicating that they diverged before becoming established in the anoles of the eastern Caribbean. This comparison of the similarity, biological and phylogenetic species concepts for malaria parasites reveals the limited value of using only similarity measures in defining protozoan species.     

PCR detection of lizard malaria parasites: prevalence of Plasmodium infections with low-level parasitemia differs by site and season

Plasmodium-specific polymerase chain reaction (PCR) primers allowed detection of infections with very low-level parasitemia for 3 species of malaria parasites infecting Anolis lizards at 2 Caribbean sites, Puerto Rico and Saba, Netherlands Antilles. A verification study, using a single-tube nested PCR to eliminate contamination, showed that infections as low as 1 parasite per millions of erythrocytes could be detected by amplifying a 673 bp fragment of the cytochrome b gene. Very low-level parasitemia infections, subpatent under the microscope, were common in A. sabanus on Saba sites, with no significant seasonal difference (31% of infections appearing uninfected by microscopic examination in summer were found infected by PCR, 38% in winter). At the Puerto Rico site, the subpatent infections were also common in A. gundlachi, but were more prevalent in winter (53%) than in summer (17%). A similar high frequency of subpatent infections is known from studies on human and bird malaria, but a previous PCR-based study on a temperate lizard malaria system found few such low-level infections. Differences in the prevalence of subpatent infections by site and season suggest transmission biology may select for distinct life history strategies by the parasite.     

Effects of immigration on the prevalence of malaria in rural areas of the Amazon basin of Brazil

Epidemiological studies were conducted on malaria in three rural areas of the Amazon basin in the State of Rond?nia: the town of Costa Marques, Forte Principe da Beira (Fort), and an immigrant settlement in the nearby forest. These studies were instituted to document the malaria problem and to describe the role of immigration on its distribution and prevalence. Hospital records in the town show that the number of malaria cases increased five fold from 1983 to 1987 and that the predominant malaria parasite changed from Plasmodium vivax to P. falciparum. Increased malaria followed increased immigration and colonization of the forest. A series of epidemiologic studies suggested the linkage between malaria and immigration as the prevalence of malaria was 1-2% at the Fort, a stable community, 8-9% at Costa Marques, a growing community, and 14-26% in the new settlements in the forest.     

Regeneration filters in post-agricultural fields of Puerto Rico and Ecuador

To better understand how rainforest regenerates after agriculture I sampled the seed rain and seed bank, and set out seeds and seedlings, on microsites defined by distance to the forest in fields both in Puerto Rico and Ecuador. I found that (1) total seeds, species richness and life-form richness were twice as great in the Ecuador seed rain compared to Puerto Rico but Puerto Rico seeds were more evenly distributed among species and (2) total seedlings from the seed bank were similar between Puerto Rico and Ecuador, (3) the majority of seeds were lost to predation among all species and study sites, (4) seed disease was absent in P. aduncum and Miconia prasina, and no seeds germinated for Gonzalagunia spicata and P. riparia, (5) in Ecuador pathogenic disease claimed more seeds than germinated for all species, and Solanum ovalifolium was the only species that had seeds germinate but did not lose seeds to disease, (6) also in Ecuador, insect predation was significantly lower in the forest border for P. aduncum, and seed disease was significantly greater at the 10 m micro site for S. ovalifolium, (7) distance has a significant effect on seedling height and basal diameter, (8) losses of leaf area due to herbivory and pathogens were always low and (9) biomass and leaf specific mass were significantly reduced in the border and forest microsites. I conclude that Ecuador fields had more seeds, species, and life-forms than Puerto Rico fields, predation was the most severe post-dispersal seed filter in all fields, seeds that survived predation on Puerto Rico were lost either to disease or germination but to both mechanisms in Ecuador, all three seed mechanisms in Ecuador fields showed distance effects of seedling growth but not survivorship.     

Biology of the Mongoose (Herpestes javanicus) in a Rain Forest of Puerto Rico1

Ecological aspects of the mongoose (Herpestes javanicus) were studied in the Luquillo Mountains, a rain forest region in eastern Puerto Rico. Information was obtained by removal trapping of mongoose from grids placed in tree plantations, colorado and tabonuco forests. Trapping efficiency was two mongoose per 100 trap-days, suggesting mongoose abundance is low in wet montane forests of Puerto Rico. Sex ratio was biased (2.6:1) in favor of males. Body masses of male mongoose inhabiting rain forests of the Luquillo Mountains were larger than those in dry forests at Guánica (P < 0.0001). Stomach contents from 18 mongoose were examined, animal matter comprised 75 percent of the total food items encountered. Of these, 33 percent were from vertebrates. The food items most frequently encountered were lizards (Anolis spp.), centipedes (Scolopendra spp.), and cockroaches (Blatellidae).     

Blood values of free-ranging patas monkeys (Erythrocebus patas)

Free-ranging patas monkeys (Erythrocebus patas) from El Guayacán island, Puerto Rico, were surveyed to establish values for the hemogram, serum biochemicals, calcium, and phosphorus. Results were tabulated for males and nonpregnant/nonlactating, pregnant, and lactating females. A summary of blood values from previous studies on captive patas monkeys was also tabulated for comparison.     

Spatial Dispersion and Nonmigratory Spawning in the Bluehead Wrasse (Thalassoma bifasciatum)

Earlier studies of the behavior of the bluehead wrasse Thalassoma bifasciatum have shown it to be a migratory spawner with large terminal-phase males defending temporary spawning territories. We describe a variant social structure where fish occupy permanent home ranges, spawn within or near these home ranges, and are not territorial. Movements of identified terminal-phase and intermediate males and females were mapped in the backreef areas of three coral reefs in Puerto Rico. Locations of spawns were then compared with these home-range maps. All fish used most or all of their morning home range during the afternoon spawning period. Both terminal-phase males and females spawned within or near their home ranges. Males were relatively tolerant of other males during the afternoon spawning period: only one in five encounters between large males resulted in aggressive chases. The location of chases bore no relation to spawning sites, areas of morning foraging, or to home-range borders. A Monte Carlo computer simulation was used to measure home-range dispersion of terminal-phase and intermediate males at our main site. If males were territorial, we should expect their home ranges to be significantly overdispersed within the cumulative area they occupied. During the first year male home ranges were overdispersed but during the second year they were randomly dispersed, even though the same number of terminal-phase males occupied the same study site both years. Differences in social behavior between backreef areas and other areas reported in the literature, and found in other portions of our reefs, may be related to differences in feeding ecology between populations and between different portions of the same reef.     

Reproduction of the Bat Glossophaga commissarisi (Phyllostomidae: Glossophaginae) in the Costa Rican Rain Forest During Frugivorous and Nectarivorous Periods1

I studied the reproduction of the 9 g nectarivorous bat Glossophaga commissarisi (Phyllostomidae: Glossophaginae) in relation to its food choice in a Costa Rican lowland rain forest over more than 2 yr. Reproduction was bimodal with birth peaks in April/May and October/November. The first birth peak coincided with a period of frugivory, whereas the second occurred during a period of almost exclusive nectarivory. All adult females recaptured in consecutive reproductive periods were reproductively active on both occasions, indicating that individual G. commissarisi females regularly reproduce twice per year at the study site. Throughout the annual cycle the mean testis length of the males correlated best with the proportion of females lactating, suggesting a post partum estrous pattern. Animals became reproductively active at about 1 yr of age. Fecal samples showed that females consumed significantly more insects than males.     

Burden of Complicated Malaria in a Densely Forested Bastar Region of Chhattisgarh State (Central India)

Background

A prospective study on severe and complicated malaria was undertaken in the tribal dominated area of Bastar division, Chhattisgarh (CG), Central India, with an objective to understand the clinical epidemiology of complicated malaria in patients attending at a referral hospital.

Methods

Blood smears, collected from the general medicine and pediatric wards of a government tertiary health care facility located in Jagdalpur, CG, were microscopically examined for malaria parasite from July 2010 to December 2013. The Plasmodium falciparum positive malaria cases who met enrollment criteria and provided written informed consent were enrolled under different malaria categories following WHO guidelines. PCR was performed to reconfirm the presence of P.falciparum mono infection among enrolled cases. Univariate and multivariate logistic regression analysis was done to identify different risk factors using STATA 11.0.

Results

A total of 40,924 cases were screened for malaria. The prevalence of malaria and P.falciparum associated complicated malaria (severe and cerebral both) in the hospital was 6% and 0.81%, respectively. P.falciparum malaria prevalence, severity and associated mortality in this region peaked at the age of>4–5 years and declined with increasing age. P.falciparum malaria was significantly more prevalent in children than adults (P<0.00001). Among adults, males had significantly more P.falciparum malaria than females (P<0.00001). Case fatality rate due to cerebral malaria and severe malaria was, respectively, 32% and 9% among PCR confirmed mono P.falciparum cases. Coma was the only independent predictor of mortality in multivariate regression analysis. Mortality was significantly associated with multi-organ complication score (P = 0.0003).

Conclusion

This study has revealed that the pattern of morbidity and mortality in this part of India is very different from earlier reported studies from India. We find that the peak morbidity and mortality in younger children regardless of seasonality. This suggests that this age group needs special care for control and clinical management.     

Presence and seasonal prevalence of Plasmodium spp. in a rare endemic New Zealand passerine (tieke or Saddleback, Philesturnus carunculatus)

The conservation and management of Saddlebacks (Philesturnus carunculatus) and other New Zealand birds, currently relies on the translocation of individuals to predator-free sites. Avian malaria has been identified as one of the diseases to be tested for prior to translocations in New Zealand, with the aim of translocating disease-free individuals. We describe avian malaria lineages and their seasonal prevalence in 2007-2008 in Saddlebacks from Mokoia Island, a source of birds for translocations, and investigate their pathogenicity. Three lineages of avian malaria were found at low prevalence (≤10.6%) and parasitemia (all but one infection were below 1/10,000 erythrocytes), typical of chronic infections. Two lineages clustered with previously identified lineages of Plasmodium relictum and one with a lineage of Plasmodium (Huffia) elongatum. Prevalence of malaria infection was higher in the spring with no significant difference in prevalence between juvenile and adult birds. We found no effect of stress on infections or any indication of pathogenicity.     

Acaricide susceptibility ofAmblyomma variegatum (Acari: Ixodidae) from Puerto Rico and Guadeloupe

Base-line data on acaricide susceptibility of larvae and of larvae and nymphs of the tropical bont tick,Amblyomma variegatum (Fabricius), from Puerto Rico and Guadeloupe were collected on 14 and six compounds, respectively. Nymphs 7–8 weeks old from Puerto Rico and Guadeloupe, and larvae 3–4 weeks old from Guadeloupe, were exposed for 24 or 48 h to residues of acetone dilutions of technical-grade or commercial formulations of acaricides on disposable glass pipettes. Effectiveness of the acaricides in killing nymphs (lc ₅₀) from Guadeloupe and Puerto Rico ranged from 0.0001% for deltamethrin to 0.269% for malathion. For Puerto Rico, the susceptibility to toxaphene and amitraz, but not to dioxathion, increased with increasing age of nymphs. Nymphs from Guadeloupe were 9.5 times more susceptible to chlorpyrifos than nymphs from Puerto Rico. The susceptibility of larvae from Guadeloupe to acaricides tested ranged fromlc ₅₀ 0.000012% for deltamethrin to 0.0141% for ethion. There was no evidence of development of acaricide resistance in ticks from Guadeloupe or Puerto Rico.     

Infectivity of Plasmodium gallinaceum Sporozoites from Oocysts

SYNOPSIS. Infectivity of Plasmodium gallinaceum (Brumpt) sporozoites isolated from midguts and salivary glands of experimentally infected Aedes fluviatilis (Lutz) was studied. The 2 populations were compared at 7, 8, and 9 days postisolation from mosquitoes, which were maintained at 27 C ± 1C and ∼75% relative humidity. Infectivity of the parasites was evaluated by the length of the prepatent period of the infection in 2-week-old chicks inoculated intramuscularly. Infection was caused by 7-day-old sporozoites from salivary glands, but not from midguts. Older sporozoites induced infection in all the inoculated chicks. The results suggested a somewhat higher infectivity of the 8- and 9-day salivary-gland parasites than of the oocyst sporozoites. However, unlike sporozoites from mammalian malaria, oocyst sporozoites from avian malaria were highly infective at this age.     

Plasmodium relictum as a cause of avian malaria in wild-caught magellanic penguins (Spheniscus magellanicus)

Avian malaria (Plasmodium relictum) caused significant mortality in wild-caught Magellanic penguins (Spheniscus magellanicus) in 1986 at the Blank Park Zoo in Des Moines, Iowa (USA). In early winter, wild birds were captured off the southern coast of Chile and flown to Detroit, Michigan for a 38 day quarantine. After quarantine, 18 birds were dispersed to Lansing, Michigan, six to a facility in Maine, and 46 to Des Moines, Iowa. Upon arrival in Des Moines, several penguins became weak and inactive, had to be force-fed, and died after 2 days. Gross lesions at postmortem included splenomegaly, hepatomegaly, and pulmonary edema. Histopathological examination revealed numerous intraendothelial schizonts in spleen, lung, liver, heart and kidney. Schizonts were generally 16 to 28 micron by 11 to 16 micron and contained merozoites of two distinct sized (macromerozoites, nuclei 1.0 micron; micromerozoites, nuclei 0.5 micron). Based on the morphology of the abundant exoerythrocytic forms, a tentative diagnosis of avian malaria (Plasmodium sp.) was made. Subsequent transmission electron microscopic examination of schizonts in formalized tissue revealed merozoites with tear-shaped rhoptries. Antimalarial therapy was initiated early but deaths continued for 5 mo. Mortality, which eventually totaled 83%, occurred in three distinct waves, each separated by a hiatus of approximately 1 mo. Despite examinations of repeated blood smears, intraerythrocytic Plasmodium relictum was not detected until late in the outbreak. Diagnosis was based on morphologic characteristics including schizonts with eight to 12 merozoites/segmenter and round gametocytes that displaced and turned the infected erythrocyte nucleus. In addition to malaria, penguins showed evidence of aspergillosis, bacterial enteritis (Escherichia coli; Proteus sp.; and Edwardsiella sp.), and helminthiasis (Contracaecum sp. and Tetrabothrius sp.). Based on gross and histological lesions, disease prevalence in this group of penguins was malaria 58%, aspergillosis 61%, enteritis 60%, helminthiasis 26%. Epidemiologic investigation including group transport history, disease prevalence in co-quarantined birds not sent to Des Moines and climatological data implicated Des Moines as the likely site for initial exposure, although information is not conclusive. Stress and concurrent disease certainly contributed to the severe mortality in this group of penguins infected with P. relictum.     

INTRAPOPULATIONAL VARIATION IN LEAFLET MORPHOLOGY OF ZAMIA PUMILA L. IN RELATION TO MICROENVIRONMENT AND SEX

The cycad Zamia pumila L. was examined for differences in leaflet morphology between sun and shade plants and between males and females within a single population in northern Puerto Rico. Sun leaflets are significantly smaller than shade leaflets in length, width, and surface area; and sun leaflets have a higher length: width ratio than shade leaflets. Also, average densitythickness is greater for sun leaflets than for shade leaflets. Males and females are similar in leaflet size; but the females had a larger number of leaflets per leaf than males.     

Trends and scenarios of the carbon budget in postagricultural Puerto Rico (1936–2060)

Contrary to the general trend in the tropics, Puerto Rico underwent a process of agriculture abandonment during the second half of the 20th century as a consequence of socioeconomic changes toward urbanization and industrialization. Using data on land‐use change, biomass accumulation in secondary forests, and ratios between gross domestic product (GDP) and carbon emissions, we developed a model of the carbon budget for Puerto Rico between 1936 and 2060. As a consequence of land abandonment, forests have expanded rapidly since 1950, achieving the highest sequestration rates between 1980 and 1990. Regardless of future scenarios of demography and land use, sequestration rates will decrease in the future because biomass accumulation decreases with forest age and there is little agricultural land remaining to be abandoned. Due to high per‐capita consumption and population density, carbon emissions of Puerto Rico have increased dramatically and exceeded carbon sequestration during the second half of the 20th century. Although Puerto Rico had the highest percent of reforestation for a tropical country, emissions during the period 1950–2000 were approximately 3.5 times higher than sequestration, and current annual emission is almost nine times the rate of sequestration. Additionally, while sequestration will decrease over the next six decades, current socioeconomic trends suggest increasing emissions unless there are significant changes in energy technology or consumption patterns. In conclusion, socioeconomic changes leading to urbanization and industrialization in tropical countries may promote high rates of carbon sequestration during the decades following land abandonment. Initial high rates of carbon sequestration can balance emissions of developing countries with low emission/GDP ratio. In Puerto Rico, the socioeconomic changes that promoted reforestation also promoted high‐energy consumption, and resulted in a net increase in carbon emissions.     

Pilot study of mold populations inside and outside of Puerto Rican residences

Puerto Rico has the highest asthma prevalence in the US. In the states, mold exposures have been linked to the development and exacerbation of asthma. For a pilot study of mold populations in Puerto Rico, dust and air samples were collected in January 2013 inside and outside of residences (n = 10) in the north eastern quadrant of Puerto Rico. Inside and outside (protected from rain) settled dust was collected by wiping above floor or ground-level surfaces with a Swiffer? duster cloth. Air samples were collected in five of the residences inside and five matching outside onto Malt Extract Agar and also onto G25N Agar. The dust (5 mg) was analyzed using mold-specific quantitative PCR and mold contamination estimated using the Environmental Relative Moldiness Index (ERMI) metric. Air sampled (150 l) plates were incubated and resulting mold colonies enumerated. The ERMI values were significantly (p = 0.03) greater inside the homes than outside, 20.37 versus 12.55, respectively. Aspergillus penicillioides, A. sydowii, Chaetomium globosum, the Eurotium group and Penicillium crustosum and A. ustus were found in significantly greater numbers inside than outside Puerto Rican residences based on the Wilcoxon rank sum test. However, after correction for multiple comparisons using the Holms–Bonferroni test, only the Eurotium group remained significantly greater. Air samples suggested higher culturable concentrations of molds outside but the differences were not significantly different. Although this is a small study, it suggests that some people in Puerto Rico are exposed to significantly higher mold concentrations indoors than outdoors.     

Plasmodium ovale in Bangladesh: genetic diversity and the first known evidence of the sympatric distribution of Plasmodium ovale curtisi and Plasmodium ovale wallikeri in southern Asia

In spite of the high prevalence of malaria in Bangladesh and other southern Asian countries, there remains a substantial shortage of knowledge about the less common human malaria parasites. Recent studies indicate that Plasmodium ovale is made up of two species, namely Plasmodium ovale wallikeri and Plasmodium ovale curtisi. Genus- and species-specific nested PCR analyses of the ssrRNA gene was used to detect P. ovale infections among 2,246 diagnostic samples. Plasmodium ovale infections were further differentiated by nested PCR of the potra gene and multilocus sequence analysis of the cox1, porbp2 and the ssrRNA genes. Both P. ovale curtisi and P. ovale wallikeri occur sympatrically in the Chittagong Hill Tracts, Bangladesh and all patients presented with a mild or asymptomatic symptom complex at the time of diagnosis. The pathogens can be differentiated by nested PCRs targeting the ssrRNA and potra genes, and display dimorphism in multilocus sequence analyses. We believe that we report the first evidence of sympatric P. ovale curtisi and P. ovale wallikeri in southern Asia within a relatively confined study area of less than 5,000 km(2). High rates of mixed infections, the emergence of "new" human malaria parasite species and the evidence of zoonotic capability call for optimised diagnostic strategies for a new era of eradication.     

Chromosomal mapping of the host resistance locus to rodent malaria (Plasmodium yoelii) infection in mice

The disease outcome in malaria caused by the protozoan parasite Plasmodium is influenced by host genetic factors. To identify host genes conferring resistance to infection with the malaria parasite, we undertook chromosomal mapping using a whole-genome scanning approach in cross-bred mice. NC/Jic mice all died with high parasitemia within 8 days of infection with 1 x 10(5) parasitized erythrocytes. In contrast, 129/SvJ mice all completely excluded malaria parasites from the circulation and remained alive 21 days after infection. We performed linkage analysis in backcross [(NC/Jic x 129/SvJ)xNC/Jic] mice. The Pymr ( Plasmodium yoelii malaria resistance) locus was mapped to the telomeric portion of mouse Chromosome (Chr) 9. This locus controls host survival and parasitemia after infection. The Char1 locus ( P. chabaudi resistance locus 1), controlling host survival and peak parasitemia in P. chabaudi infection, was previously mapped to the same region. This host resistance locus mapping to Chr 9 may represent a ubiquitous locus controlling susceptibility to rodent malaria. Elucidation of the function of this gene will provide valuable insights into the mechanism of host defense against malaria parasite infection.     

Evaluation of the eradication program forAmblyomma variegatum (Acari: Ixodidae) on Puerto Rico

A cooperative effort between the United States Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, the Agricultural Research Service, and the Department of Agriculture, Commonwealth Government of Puerto Rico has been successful in eardicatingAmblyomma variegatum (Fabricius) from the islands of Puerto Rico and Vieques (an island municipality of Puerto Rico). Application of acaricides to livestock and dogs at 14-day intervals for an 18-month period eliminated foci of tick infestation on 188 farms in four different locations without additional aerial or ground treatment. Reasons for the success and some of the costs associated with the eradication program on Puerto Rico are presented.