Jefferson A. Vaughan

Comparison of PCR and microscopy for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand

ObjectiveThe main objective of this study was to compare the performance of nested PCR with expert microscopy as a means of detecting Plasmodium parasites during active malaria surveillance in western Thailand.MethodsThe study was performed from May 2000 to April 2002 in the village of Kong Mong Tha, located in western Thailand. Plasmodium vivax (PV) and Plasmodium falciparum (PF) are the predominant parasite species in this village, followed by Plasmodium malariae (PM) and Plasmodium ovale (PO). Each month, fingerprick blood samples were taken from each participating individual and used to prepare thick and thin blood films and for PCR analysis.ResultsPCR was sensitive (96%) and specific (98%) for malaria at parasite densities ≥ 500/μl; however, only 18% (47/269) of P. falciparum- and 5% (20/390) of P. vivax-positive films had parasite densities this high. Performance of PCR decreased markedly at parasite densities <500/μl, with sensitivity of only 20% for P. falciparum and 24% for P. vivax at densities <100 parasites/μl.ConclusionAlthough PCR performance appeared poor when compared to microscopy, data indicated that the discrepancy between the two methods resulted from poor performance of microscopy at low parasite densities rather than poor performance of PCR. These data are not unusual when the diagnostic method being evaluated is more sensitive than the reference method. PCR appears to be a useful method for detecting Plasmodium parasites during active malaria surveillance in Thailand.

Population dynamics of Plasmodium falciparum sporogony in laboratory-infected Anopheles gambiae.

The population dynamics of cultured Plasmodium falciparum parasites was examined during their sporogonic development in Anopheles gambiae mosquitoes. Estimates of absolute densities were determined for each life stage, and life tables were constructed for each of 38 experimental infections. Macrogametocyte and ookinete mortalities contributed equally to the overall mortality. On average, there was a 40-fold decrease in parasite numbers in the transition from the macrogametocyte to the ookinete stage, a 69-fold decrease in the transition from ookinete to oocyst stages, and a total net decrease in parasite numbers from macrogametocyte to oocyst stage of 2,754-fold (i.e., multiplicative). There was no relationship between macrogametocyte and ookinete densities due to the inherent variability in fertility among different gametocyte cultures. There was a curvilinear relationship (r2 = 0.66) between ookinete and oocyst densities. Above a threshold of about 30 ookinetes/mosquito, the oocyst yield per ookinete became increasingly greater with increasing ookinete density. There was a linear relationship (r2 = 0.73) between oocyst and sporozoite densities, with an average of 663 salivary gland sporozoites produced per oocyst. Sporozoite production per oocyst was not affected by oocyst density and virtually all oocyst infections resulted in sporozoite infections of the salivery glands. This quantitative study indicates that the sporogony of cultured P. falciparum in laboratory-infected A. gambiae is an inefficient process and that the ookinete is the key transitional stage affecting the probability of vector infectivity.

Anti-mosquito midgut antibodies block development of Plasmodium falciparum and Plasmodium vivax in multiple species of Anopheles mosquitoes and reduce vector fecundity and survivorship.

The mosquito midgut plays a central role in the sporogonic development of malaria parasites. We have found that polyclonal sera, produced against mosquito midguts, blocked the passage of Plasmodium falciparum ookinetes across the midgut, leading to a significant reduction of infections in mosquitoes. Anti-midgut mAbs were produced that display broad-spectrum activity, blocking parasite development of both P. falciparum and Plasmodium vivax parasites in five different species of mosquitoes. In addition to their parasite transmission-blocking activity, these mAbs also reduced mosquito survivorship and fecundity. These results reveal that mosquito midgut-based antibodies have the potential to reduce malaria transmission in a synergistic manner by lowering both vector competence, through transmission-blocking effects on parasite development, and vector abundance, by decreasing mosquito survivorship and egg laying capacity. Because the intervention can block transmission of different malaria parasite species in various species of mosquitoes, vaccines against such midgut receptors may block malaria transmission worldwide.

West Nile virus epizootiology, central Red River Valley, North Dakota and Minnesota, 2002-2005.

West Nile virus (WNV) epizootiology was monitored from 2002 through 2005 in the area surrounding Grand Forks, North Dakota. Mosquitoes were tested for infection, and birds were surveyed for antibodies. In 2003, WNV was epidemic; in 2004, cool temperatures precluded WNV amplification; and in 2005, immunity in passerines decreased, but did not preclude, WNV amplification.

SPOROGONIC DEVELOPMENT OF PLASMODIUM YOELII IN FIVE ANOPHELINE SPECIES

Sporogonic development of Plasmodium yoelii yoelii 17XNL was examined in 5 species of Anopheles mosquitoes; A. albimanus, A. dirus, A. freeborni, A. gambiae, and A. stephensi. The kinetics of ookinete formation differed among species. In A. freeborni, A. gambiae, and A. stephensi, mature ookinetes formed synchronously at 8 hr, then quickly subsided. In A. albimanus and A. dirus, ookinete formation was more protracted, and ookinete densities peaked from 12 to 24 hr. Losses in parasite abundance during the conversion of ookinetes to oocysts were similar between A. dirus and A. gambiae (55- and 41-fold losses, respectively) but were an order of magnitude less in A. stephensi (1.3-fold loss). Ookinete conversion to oocysts in A. albimanus was nil. Melanotic encapsulation of oocysts occurred in 25-30% of infected A. gambiae and A. dirus. Melanized parasites in A. gambiae at days 7-10 were small (10 microns diameter) and retort-shaped, whereas melanized parasites in A. dirus were generally as large as normal oocysts (60 microns) and many were incompletely melanized. Melanotic encapsulation did not occur in A. stephensi, A. freeborni, or A. albimanus. On day 16, sporozoites were present in the salivary glands of A. freeborni, A. gambiae, and A. stephensi, but only half of mosquitoes were mature oocysts also had gland infections. When present in the glands, sporozoites were successfully transmitted to mice via mosquito bite. Parasite populations were not normally distributed in any mosquito species but were adequately described by a negative binomial type of distribution.

Neorickettsial endosymbionts of the digenea: diversity, transmission and distribution.

Digeneans are endoparasitic flatworms with complex life cycles and distinct life stages that parasitize different host species. Some digenean species harbour bacterial endosymbionts known as Neorickettsia (Order Rickettsiales, Family Anaplasmataceae). Neorickettsia occur in all life stages and are maintained by vertical transmission. Far from benign however, Neorickettsia may also be transmitted horizontally by digenean parasites to their vertebrate definitive hosts. Once inside, Neorickettsia can infect macrophages and other cell types. In some vertebrate species (e.g. dogs, horses and humans), neorickettsial infections cause severe disease. Taken from a mostly parasitological perspective, this article summarizes our current knowledge on the transmission ecology of neorickettsiae, both for pathogenic species and for neorickettsiae of unknown pathogenicity. In addition, we discuss the diversity, phylogeny and geographical distribution of neorickettsiae, as well as their possible evolutionary associations with various groups of digeneans. Our understanding of neorickettsiae is at an early stage and there are undoubtedly many more neorickettsial endosymbioses with digeneans waiting to be discovered. Because neorickettsiae can infect vertebrates, it is particularly important to examine digenean species that regularly infect humans. Rapid advances in molecular tools and their application towards bacterial identification bode well for our future progress in understanding the biology of Neorickettsia.

Population dynamics of sporogony for Plasmodium vivax parasites from western Thailand developing within three species of colonized Anopheles mosquitoes

BackgroundThe population dynamics of Plasmodium sporogony within mosquitoes consists of an early phase where parasite abundance decreases during the transition from gametocyte to oocyst, an intermediate phase where parasite abundance remains static as oocysts, and a later phase where parasite abundance increases during the release of progeny sporozoites from oocysts. Sporogonic development is complete when sporozoites invade the mosquito salivary glands. The dynamics and efficiency of this developmental sequence were determined in laboratory strains of Anopheles dirus, Anopheles minimus and Anopheles sawadwongporni mosquitoes for Plasmodium vivax parasites circulating naturally in western Thailand.MethodsMosquitoes were fed blood from 20 symptomatic Thai adults via membrane feeders. Absolute densities were estimated for macrogametocytes, round stages (= female gametes/zygotes), ookinetes, oocysts, haemolymph sporozoites and salivary gland sporozoites. From these census data, five aspects of population dynamics were analysed; 1) changes in life-stage prevalence during early sporogony, 2) kinetics of life-stage formation, 3) efficiency of life-stage transitions, 4) density relationships between successive life-stages, and 5) parasite aggregation patterns.ResultsThere was no difference among the three mosquito species tested in total losses incurred by P. vivax populations during early sporogony. Averaged across all infections, parasite populations incurred a 68-fold loss in abundance, with losses of ca. 19-fold, 2-fold and 2-fold at the first (= gametogenesis/fertilization), second (= round stage transformation), and third (= ookinete migration) life-stage transitions, respectively. However, total losses varied widely among infections, ranging from 6-fold to over 2,000-fold loss. Losses during gametogenesis/fertilization accounted for most of this variability, indicating that gametocytes originating from some volunteers were more fertile than those from other volunteers. Although reasons for such variability were not determined, gametocyte fertility was not correlated with blood haematocrit, asexual parasitaemia, gametocyte density or gametocyte sex ratio. Round stages and ookinetes were present in mosquito midguts for up to 48 hours and development was asynchronous. Parasite losses during fertilization and round stage differentiation were more influenced by factors intrinsic to the parasite and/or factors in the blood, whereas ookinete losses were more strongly influenced by mosquito factors. Oocysts released sporozoites on days 12 to 14, but even by day 22 many oocysts were still present on the midgut. The per capita production was estimated to be approximately 500 sporozoites per oocyst and approximately 75% of the sporozoites released into the haemocoel successfully invaded the salivary glands.ConclusionThe major developmental bottleneck in early sporogony occurred during the transition from macrogametocyte to round stage. Sporozoite invasion into the salivary glands was very efficient. Information on the natural population dynamics of sporogony within malaria-endemic areas may benefit intervention strategies that target early sporogony (e.g., transmission blocking vaccines, transgenic mosquitoes).

Comparing the mitochondrial genomes of Wolbachia-dependent and independent filarial nematode species

BackgroundMany species of filarial nematodes depend on Wolbachia endobacteria to carry out their life cycle. Other species are naturally Wolbachia-free. The biological mechanisms underpinning Wolbachia-dependence and independence in filarial nematodes are not known. Previous studies have indicated that Wolbachia have an impact on mitochondrial gene expression, which may suggest a role in energy metabolism. If Wolbachia can supplement host energy metabolism, reduced mitochondrial function in infected filarial species may account for Wolbachia-dependence. Wolbachia also have a strong influence on mitochondrial evolution due to vertical co-transmission. This could drive alterations in mitochondrial genome sequence in infected species. Comparisons between the mitochondrial genome sequences of Wolbachia-dependent and independent filarial worms may reveal differences indicative of altered mitochondrial function.ResultsThe mitochondrial genomes of 5 species of filarial nematodes, Acanthocheilonema viteae, Chandlerella quiscali, Loa loa, Onchocerca flexuosa, and Wuchereria bancrofti, were sequenced, annotated and compared with available mitochondrial genome sequences from Brugia malayi, Dirofilaria immitis, Onchocerca volvulus and Setaria digitata. B. malayi, D. immitis, O. volvulus and W. bancrofti are Wolbachia-dependent while A. viteae, C. quiscali, L. loa, O. flexuosa and S. digitata are Wolbachia-free. The 9 mitochondrial genomes were similar in size and AT content and encoded the same 12 protein-coding genes, 22 tRNAs and 2 rRNAs. Synteny was perfectly preserved in all species except C. quiscali, which had a different order for 5 tRNA genes. Protein-coding genes were expressed at the RNA level in all examined species. In phylogenetic trees based on mitochondrial protein-coding sequences, species did not cluster according to Wolbachia dependence.ConclusionsThus far, no discernable differences were detected between the mitochondrial genome sequences of Wolbachia-dependent and independent species. Additional research will be needed to determine whether mitochondria from Wolbachia-dependent filarial species show reduced function in comparison to the mitochondria of Wolbachia-independent species despite their sequence-level similarities.

Plasmodium berghei ookinete densities in three anopheline species.

Plasmodium berghei ookinete kinetics and densities were examined in the blood meals of 3 species of Anopheles mosquitoes fed simultaneously from a gametocytemic mouse. Simple techniques were developed for estimating relative and absolute ookinete densities within individual mosquito blood meals. The kinetics of ookinete formation were similar in all 3 species, with peak ookinete densities occurring from 12 to 24 hr postingestion. Ookinete densities consistently were lower in Anopheles stephensi than in Anopheles albimanus or Anopheles freeborni and could not be accounted for by species differences in blood meal volumes or gametocyte densities. Likely explanations involve species differences in blood meal hemolysis or sampling error as the result of ookinete emigration from the blood meal.

Plasmodium falciparum: The population structure of mature gametocyte cultures has little effect on their innate fertility

In vitro cultured Plasmodium falciparum gametocytes were fed to Anopheles gambiae (G3) mosquitoes to identify parasite population characteristics useful for predicting successful mosquito infections. Parameters were collected from an initial study of 90 infections over a two year period and a second study of 55 infections over 12 weeks. Parasite isolate/clone was identified as the most reliable predictor of gametocyte infectiousness. Parameters such as gametocyte age structure (stage IV:V ratio), exflagellation rate and macrogametocyte maturity were not reliable for predicting infectiousness but were useful for monitoring overall culture maturity. Other variables such as gametocyte density, chronological age of the culture at the time of feed, gametocyte sex ratio, asexual parasitemia, and mixing cultures before mosquito feeding were not predictive. Thus, if a reliable parasite isolate or clone is used, there is no need to measure other characteristics of in vitro gametocyte populations because these will not significantly improve ones ability to predict oocyst infection rates.