Ingestion of Plasmodium falciparum sporozoites during transmission by anopheline mosquitoes.

We investigated the process of sporozoite transmission during blood feeding for Anopheles gambiae and An. stephensi experimentally infected with Plasmodium falciparum. When infective mosquitoes were fed 22-25 days postinfection on an anesthetized rat, sporozoites were detected in the midgut of 96.5% of 57 An. gambiae (geometric mean [GM] = 32.5, range 3-374) and in 96.2% of 26 An. stephensi (GM = 19.5, range 1-345). There were no significant differences between species either in salivary gland sporozoite loads or in the number of ingested sporozoites. There was a significant linear relationship between sporozoite loads and the numbers of ingested sporozoites for both An. gambiae (r = 0.38) and An. stephensi (r = 0.69). Subsequently, An. gambiae were tested for sporozoite transmission by allowing them to feed individually on a suspended capillary tube containing 10 microliters of blood. A total of 83.3% of 18 infective mosquitoes transmitted a GM of 5.9 (range 1-36) sporozoites. The same mosquitoes contained a GM of 23.4 (range 2-165) ingested sporozoites. The number of ingested sporozoites was related to sporozoite loads (r = 0.42) but not to the number of sporozoites ejected into capillary tubes. Ingested sporozoites remained in the midgut up to 10 hr after feeding. The comparable numbers of sporozoites ingested by infective mosquitoes in both experiments indicates that the actual number of sporozoites transmitted to the vertebrate host during blood feeding is significantly reduced by the blood ingestion process.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of Plasmodium falciparum sporozoites transmitted in vitro by experimentally infected Anopheles gambiae and Anopheles stephensi.

The frequency and numbers of Plasmodium falciparum sporozoites transmitted in vitro and corresponding sporozoite loads were determined for experimentally infected Anopheles gambiae and An. stephensi. Geometric mean (GM) sporozoite loads in three experiments ranged from 808 to 13, 905 for An. gambiae and from 6, 608 to 17, 702 for An. stephensi. A total of 44.1% of 68 infected An. gambiae and 49.2% of 63 infected An. stephensi transmitted sporozoites in vitro. The GM number of sporozoites transmitted was 4.5 for An. gambiae and 5.4 for An. stephensi. Overall, 86.9% of the mosquitoes transmitted from one to 25 sporozoites, and only 6.6% transmitted over 100 sporozoites (maximum = 369). Sporozoite loads were not a useful predictor of potential sporozoite transmission. Despite higher sporozoite loads, the numbers of sporozoites transmitted in vitro by the experimentally infected mosquitoes were similar to estimates obtained, using the same techniques, for naturally infected An. gambiae in western Kenya. The low but highly variable numbers of sporozoites transmitted in vitro by mosquitoes used in malaria vaccine challenge studies appears to be a reasonable simulation of natural sporozoite transmission.

Effects of ingested human anti-sporozoite sera on Plasmodium falciparum sporogony in Anopheles stephensi.

We investigated the effects of human anti-sporozoite antibodies on the sporogonic development of Plasmodium falciparum in Anopheles stephensi. Equal volumes of washed human erythrocytes and human sera from 1) volunteers with protective immunity induced by immunization with irradiated P. falciparum sporozoites, 2) the same volunteers before immunization, or 3) Kenyans exposed to natural sporozoite transmission, were fed to cohorts of P. falciparum-infected A. stephensi on either day 5, 8, or 11 after infection. A fourth group of infected mosquitoes from the same cohort were not refed. In two experiments, the effects of anti-sporozoite antibodies were evaluated by determining the infection rates and parasite densities for oocysts and salivary gland sporozoites. There was no evidence that anti-sporozoite antibodies had any effect on the development or intensity of P. falciparum infection in A. stephensi. However, accelerated oocyst maturation was associated with mosquitoes taking a second blood meal, independent of serum source. Salivary gland sporozoites from mosquitoes that fed on immune human sera contained bound human IgG, which was detectable by indirect immunofluorescence assay. The infectivity and transmission potential of human IgG-coated sporozoites is unknown.

Identification of malaria species by ELISA in sporozoite and oocyst infected Anopheles from western Kenya.

Enzyme-linked immunosorbent assays (ELISAs) for the circumsporozoite (CS) antigens of Plasmodium falciparum, P. malariae, and P. ovale were used to identify species of sporozoite and oocyst infections detected by dissection in Anopheles gambiae s.1. and An. funestus collected in western Kenya. ELISAs identified 92.5% of 1,113 salivary gland infections; Plasmodium species infections included 79.4% P. falciparum, 3.2% P. malariae, 1.7% P. ovale, and 2 or more Plasmodium species were detected in 15.7% of the Anopheles in which the species of parasite was identified. Identification was more likely with greater numbers of sporozoites observed in dissections, increasing from 65% ELISA positivity in mosquitoes with 1-10 sporozoites in their salivary glands to 96% in mosquitoes with over 1,000 sporozoites. ELISAs detected CS antigen in 66% of 294 Anopheles that by dissection had oocysts but uninfected salivary glands. Of 112 Anopheles with a single species of Plasmodium detected in the salivary glands, 29 (25.9%) had 1 or more additional species detected in the midgut, indicating a high potential for multiple infections. Similar proportions of Plasmodium species were found in An. gambiae s.1. and An. funestus.

Urinary neopterin in volunteers experimentally infected with Plasmodium falciparum.

To investigate the kinetics of monocyte/macrophage activation in falciparum malaria we determined urinary neopterin values serially in experimentally infected volunteers. Three subjects who had been immunized with irradiated sporozoites via mosquito bites served as controls. These individuals remained aparasitaemic, afebrile and without a rise in neopterin after challenge by infective mosquitoes. Four non-immune subjects developed Plasmodium falciparum parasitaemia, fever (3 of 4) and sharp rises in neopterin. Parasite densities reached 10-100 parasitized erythrocytes per microliter before elevations in temperature or neopterin levels were detected. Onset of fever preceded the rise in neopterin excretion by one day. Prompt chemotherapy was associated with the clearance of parasites from the blood and the return of temperature and neopterin levels to normal.

Effects of para-aminobenzoic acid, insulin, and gentamicin on Plasmodium falciparum development in anopheline mosquitoes (Diptera: Culicidae).

Anopheles gambiae Giles sensu lato (s.l.) and An. stephensi Liston were exposed as adults to para-aminobenzoic acid (PABA), human insulin, or gentamicin sulfate, an antimicrobial solution, to determine their effects on Plasmodium falciparum Welch development to the oocyst stage. For both mosquito species, concentrations of PABA from 0.001 to 0.05% had no effect on either oocyst infection rates or oocyst densities. At higher concentrations, PABA-fed mosquitoes had decreased oocyst infection rates when they were exposed after, but not before, experimental infections. Insulin-fed mosquitoes had significantly higher oocyst infection rates than controls and increased oocyst densities in both An. gambiae s.l and An. stephensi. The most dramatic impact on parasite development was observed for mosquitoes fed on gentamicin. For An. gambiae s.l. fed 0.05% gentamicin, oocyst infection rates were 3.7-fold higher than in controls, whereas oocyst densities were 5.7 times greater than in controls. Anopheles stephensi fed on diets of 0.1% gentamicin had 2.4-fold higher infection rates and increased oocyst densities. Concentrations of gentamicin above 0.1% had deleterious effects on mosquito survival and their ability to digest blood meals. These findings provide insight into how antibiotics, hormones, and metabolites may affect the development of P. falciparum in An. gambiae s.l. and An. stephensi.

Laboratory rearing techniques and adult life table parameters for Anopheles sergentii from Egypt.

Techniques are presented for maintaining colonies of Anopheles sergentii, an important malaria vector in Egypt. Larval development time and survival rates were determined for 3 rearing solutions and 4 temperatures. Under optimal conditions larval survival rates averaged 85%. Mean life expectancy at emergence for mated An. sergentii was 23.3 days under insectary conditions of 27 +/- 2 degrees C, 70-80% R.H. The net reproductive rate, mean generation time and instantaneous rate of increase were respectively, 45.8 females per female per generation, 29.7 days and 0.127. In the context of vector potential for malaria transmission, An. sergentii has a daily survivorship rate of 0.95.

Sporozoite transmission by Anopheles freeborni and Anopheles gambiae experimentally infected with Plasmodium falciparum.

A micro-membrane feeding technique was used to evaluate sporozoite transmission for Anopheles freeborni and An. gambiae experimentally infected with Plasmodium falciparum. From cohorts of infected mosquitoes with equivalent sporozoite loads, 75.9% of 29 An. freeborni transmitted a geometric mean (GM) of 4.9 sporozoites and 80% of 30 An. gambiae transmitted a GM of 11.3 sporozoites. Ingested sporozoites, in the blood meal immediately after feeding, were detected in 86.2% of 29 An. freeborni (GM = 9.0) and in 70% of 30 An. gambiae (GM = 44.1). Overall, sporozoites were transmitted and/or ingested by 90% of both species. Most infective mosquitoes transmitted < 1% of the total sporozoites in the salivary glands, and only up to 30% of the variation in transmission, ingestion, or total sporozoite output was related to sporozoite loads. The demonstration that An. gambiae transmitted more than twice as many sporozoites as An. freeborni is the first indication that vector species of anopheline mosquitoes differ in their innate potential for sporozoite transmission.

Identification and molecular analysis of the gene encoding Rickettsia typhi hemolysin.

Rickettsia typhi, the causative agent of murine typhus, grows directly within the host cell cytoplasm, accumulating a large number of progeny, and eventually lyses the cells. Typhus group rickettsiae (R. typhi and R. prowazekii) adhere to and lyse human and sheep erythrocytes. However, the molecular mechanism underlying erythrocyte lysis by R. typhi has not been defined. Here we describe the cloning and nucleotide sequence analysis of the gene (tlyC) encoding a hemolysin from R. typhi. DNA sequence analysis of R. typhi tlyC revealed an open reading frame of 912 bp, which encodes a protein of 304 amino acids with a predicted molecular mass of 38 kDa. To associate the R. typhi tlyC gene product with hemolytic activity, we performed complementation studies with hemolysin-negative Proteus mirabilis WPM111 (a HpmA(-) mutant of BA6163) transformed with R. typhi tlyC or R. typhi GFPuv-tlyC constructs. We demonstrated that the cloned tlyC gene conferred a hemolytic phenotype on an otherwise nonhemolytic mutant of P. mirabilis. The availability of the cloned R. typhi tlyC will permit further characterization and definition of its role in rickettsial virulence.

Rickettsia-macrophage interactions: host cell responses to Rickettsia akari and Rickettsia typhi.

The existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akari and -Rickettsia typhi interactions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated that R. akari and R. typhi caused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. Furthermore, macrophage infection with R. akari and R. typhi resulted in differential synthesis and expression of IL-beta and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-gamma), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed between R. akari- and R. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria.

Plasmodium falciparum: inhibition of sporogonic development in Anopheles stephensi by gram-negative bacteria.

We investigated the effects of bacteria on Plasmodium falciparum sporogonic development in Anopheles stephensi. Four gram-negative (Escherichia coli H243, E. coli HB101, Pseudomonas aeruginosa, and Ewingella americana) and two gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) bacterial strains were used in the study. Tenfold dilutions of bacteria suspended in phosphate-buffered saline were mixed with an infectious meal of gametocyte-enriched cultures and fed to adult mosquitoes. All gram-negative bacteria strains partially or completely inhibited oocyst formation at different concentrations. Additionally, geometric mean number of oocysts showed a correspondingly significant decrease with increasing bacterial concentration (P < 0.001). In contrast, gram-positive bacteria strains did not have any inhibitory effects on oocyst formation even at very high concentrations. Oocyst development was not affected by: (i) culture supernatants of E. americana, (ii) formalin-treated E. coli H243, (iii) lipopolysaccharide of E. coli J5 (mutant of 0111:B4). These studies show that gram-negative but not gram-positive bacteria affect sporogonic-stage development of P. falciparum in A. stephensi. Inhibition of parasite acquisition may be an attribute of specific or nonspecific cytoadherence properties of gram-negative bacteria to the parasites.