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PubMed1994

Vector-parasite interactions for vaccine development.

The ingestion of blood by arthropod vectors of disease can be exploited in order to either kill the vector or render it incapable of disease transmission. This paper examines some approaches to identifying target molecules of vector origin, against which immunisation could result in blocking parasite transmission. Manipulation of the blood meal of vectors through such techniques as membrane feeding can help identify true target sites for attack, but just as useful, can identify structures or molecules that play no significant role in parasite development. Examples, mostly derived from the interactions between the malaria parasite, Plasmodium, and the mosquito midgut, illustrate the real need to understand the multiple aspects of vector-parasite interactions before they can be exploited for control purposes. The approaches outlined are however applicable directly to any vector-borne disease. Careful examination of the parasite life cycle in the vector, and comparisons with other parasites, vectors, non-vector insects and analogous vertebrate systems (the latter being often relatively well advanced) can result in the identification of specific and definable interactions which can then be further developed for vaccine purposes.

Animals↗
PubMed1998

Chronosexuality of Plasmodium species of Central African Muridae.

A host harbouring many parasite species of the same genus is a phenomenon frequently observed in numerous parasitic infections. This is the case for the Plasmodium parasites of Muridae in Central Africa, where three different parasite species are found in the same rodent host species. It is highly likely that these three Plasmodium species are transmitted simultaneously by the same vector. We and others have shown that the maturation periods of the various asexual and sexual stages in the rodent, differ amongst the three parasites. In this article we propose that these differences are the product of complex adaptations which result, for all three Plasmodium species, in a maximum peak of infectivity to the insect vector occurring around 3 a.m., the period of highest activity of the nocturnal host rodent.

Animals↗
PubMed1971

The relationship between the structure and the activity of pyrethroids.

There is considerable scope for developing new non-persistent insecticides with little hazard for man and mammals by modifying the structures of the natural pyrethrins. New compounds already synthesized are more effective against some insect species than are the natural compounds, are even less hazardous to mammals, and do not need synergists to supplement their insecticidal action. Other examples show considerable insect species specificity. These compounds may help to control insect vectors when other insecticides are no longer effective because resistance has developed or because their residues can no longer be tolerated.

Animals↗
PubMed2003

Innovative tools for detection of plant pathogenic viruses and bacteria.

Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.

Bacteria↗
PubMed2004

Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major.

Antimicrobial peptides are major components of the innate immune response of epithelial cells. In insect vectors, these peptides may play a role in the control of gut pathogens. We have analyzed antimicrobial peptides produced by the sand fly Phlebotomus duboscqi, after challenge by injected bacteria or feeding with bacteria or the protozoan parasite Leishmania major. A new hemolymph peptide with antimicrobial activity was identified and shown to be a member of the insect defensin family. Interestingly, this defensin exhibits an antiparasitic activity against the promastigote forms of L. major, which reside normally within the sand fly midgut. P. duboscqi defensin could be induced by both hemolymph or gut infections. Defensin mRNA was induced following infection by wild-type L. major, and this induction was much less following infections with L. major knockout mutants that survive poorly in sand flies, due to specific deficiencies in abundant cell surface glycoconjugates containing phosphoglycans (including lipophosphoglycan). The ability of gut pathogens to induce gut as well as fat body expression of defensin raises the possibility that this antimicrobial peptide might play a key role in the development of parasitic infections.

Amino Acid Sequence↗
PubMed2002

[Metacyclogenesis: a basic process in the biology of Leishmania].

Metacyclogenesis is a process whereby Leishmania transforms from poorly infective procyclic promastigotes into highly infective metacyclic promastigotes. In nature, metacyclogenesis occurs in the insect vector. This transformation is accompanied by an increased ability to infect and survive in the vertebrate host, where the parasite is attacked by the host's immune system. Metacyclogenesis has also been shown to occur in axenic cultures of promastigotes. Morphological changes in size and shape, and length of flagellum were first associated with differentiation in the insect gut and in different phases of growth in culture. Later, the expression of molecules such as LPG and the surface protease gp63 were associated with this process. These two molecules were observed to undergo qualitative and quantitative modifications as the promastigotes differentiated from procyclic to metacyclic forms. Using cDNA subtractive hybridization-based methods or differential amplification, previously unknown genes tightly linked to metacyclogenesis have been identified. Gene products exclusively expressed in metacyclic promastigotes included a gene B product and Mat-1--a gene associated with metacyclogenesis. Other proteins, Meta-1, SHERP and HASP, were up-regulated during the metacyclic stage. The function and stage-regulated expression of these molecules and their relationship with infectivity are now under investigation.

Animals↗
PubMed2002

Proventriculus-specific cDNAs characterized from the tsetse, Glossina morsitans morsitans.

Peritrophic matrix (peritrophic membrane or PM) is an important structure in the gut of most insects at some stage in their development. It is composed of chitin, proteins and proteoglycans. Multiple roles for the PM ranging from partitioning of digestive enzymes and food to protection of gut epithelial cells from viral and parasitic invasion have been proposed. While most adult members of Diptera have a Type I PM synthesized in response to a blood meal, the medically and agriculturally important vector insect, tsetse has a sleeve-like Type II PM which is constitutively synthesized by cells in the proventriculus (cardia). Using a differential hybridization approach, we have identified three abundant cDNAs from a proventriculus cDNA library of GLOSSINA MORSITANS MORSITANS: GmPro1, GmPro2 and GmPro3. DNA sequence analysis indicates that GmPro1 and GmPro2 share similarities with the peritrophin-15 family of larval PM proteins, while GmPro3 is a member of the serine protease family. Northern analysis indicates that transcripts for all three cDNAs are preferentially expressed in the proventriculus tissue. The expression profile of these genes in response to the presence of trypanosome indicates that transcription of GmPro1 is increased in the presence of parasites (immune sensitive), while the other two are not affected. Western analysis using antibodies developed against the recombinant GmPro2 shows its primary localization in the gut to be within the peritrophic matrix structure. We discuss the molecular characteristics of these proventriculus specific cDNAs and their products as well as their potential role for vector control studies.

Animals↗
PubMed2001

Why Bacillus thuringiensis insecticidal toxins are so effective: unique features of their mode of action.

The spore-forming bacterium Bacillus thuringiensis produces intracellular inclusions comprised of protoxins active on several orders of insects. These highly effective and specific toxins have great potential in agriculture and for the control of disease-related insect vectors. Inclusions ingested by larvae are solubilized and converted to active toxins in the midgut. There are two major classes, the cytolytic toxins and the delta-endotoxins. The former are produced by B. thuringiensis subspecies active on Diptera. The latter, which will be the focus of this review, are more prevalent and active on at least three orders of insects. They have a three-domain structure with extensive functional interactions among the domains. The initial reversible binding to receptors on larval midgut cells is largely dependent upon domains II and III. Subsequent steps involve toxin insertion into the membrane and aggregation, leading to the formation of gated, cation-selective channels. The channels are comprised of certain amphipathic helices in domain I, but the three processes of insertion, aggregation and the formation of functional channels are probably dependent upon all three domains. Lethality is believed to be due to destruction of the transmembrane potential, with the subsequent osmotic lysis of cells lining the midgut. In this review, the mode of action of these delta-endotoxins will be discussed with emphasis on unique features.

Animals↗
PubMed1970

Preliminary studies on the use of ultra-low-volume applications of malathion for control of Aedes simpsoni.

In view of the threat of additional yellow fever epidemics in East Africa and recent successes in the use of malathion applied by the ultra-low-volume technique against insect vectors, field trials were initiated in November 1968 to test the efficacy of this method against Aedes simpsoni. Results of these trials show that in areas where Musa ensetta is the principal breeding site, the application of 20.2 US fl oz/acre (1474 ml/ha) of malathion was capable of reducing vector populations 93%-100%. Comparable applications of 6 US fl oz/acre (438 ml/ha) reduced populations by 76% and 89%. Since this study did not conclusively demonstrate that the 6 US fl oz/acre dosage rate was sufficient to obtain maximum population reduction, further studies should be initiated to obtain information on the optimal dosage to be employed against this species.Under the conditions of these trials, the use of a light, single-engined aircraft equipped with a rotary atomizer spray system and capable of operation from improvised fields was shown to be feasible against this vector species. It was further demonstrated that the canopy formed by M. ensetta does not present an impossible barrier to the penetration of the spray droplets produced by the ULV application technique.Observations made on the application methodology and on the biology of A. simpsoni show the versatility of this species and the need for further studies on the integration of spray applications with the peak activity periods of the insect.

Aedes↗
PubMed2004

A novel purine nucleoside transporter whose expression is up-regulated in the short stumpy form of the Trypanosoma brucei life cycle.

Purine nucleoside and nucleobase transporters play a vital role in the metabolism and survival of Trypanosoma brucei because this parasitic protozoan is unable to synthesize purines de novo and thus must acquire preformed purines from its hosts. These parasites express a variety of nucleoside and nucleobase permeases with diverse substrate specificities and distinct patterns of expression during the trypanosome life cycle. We report here that expression of the newly characterized T. brucei nucleoside transporter 10 gene (TbNT10) is up-regulated in the short stumpy form of the life cycle, the bloodstream form of the parasite that is pre-adapted for infection of the tsetse fly vector. Functional expression of TbNT10 in Saccharomyces cerevisiae reveals that the TbNT10 gene encodes an adenosine/guanosine/inosine transporter with apparent Km values of approximately 1 microM and hence is a high affinity purine nucleoside transporter. The restricted expression of TbNT10 during the life cycle suggests that the functional properties of this permease may be specialized to support development and growth of the differentiated short stumpy form or to promote the transformation of short stumpy to procyclic forms within the insect vector.

Amino Acid Sequence↗
PubMed1976

Human sleeping sickness in the Gboko endemic area of Nigeria.

Human infection with Trypanosoma gambiense in the Gboko endemic area was first reported in May, 1974 although T. gambiense sleeping sickness had been present there since the turn of the century. The disease is associated with the presence of the tsetse Glossina tachinoides and Glossina palpalis which is plentiful and widespread throughout the division as well as in thickets along the streams in the area. No successful attempt has been made to control the tsetse vector in the Division. The incidence and geographical distribution of cases of T. gambiense sleeping sickness in the Gboko area are described in this report. Cases were treated with Antrypol Tryparsamide mixture and Mel B. The highest number of cases of infection is usually picked up just before the start of the rains in early April. It is suggested that, for meaningful control of the disease a quick method should be devised to rid the area of the insect vector.

Humans↗
PubMed2002

[Definition of appropriate temperature and storage conditions in the detection of Leishmania DNA with PCR in phlebotomine flies].

For epidemiological studies and control programs of leishmaniasis, taxonomic identification of the etiologic agent of the disease in the insect vector is of critical importance. The implementation of molecular techniques such as the polymerase chain reaction (PCR) has permitted great advances in the efficacy and sensitivity of parasite identification. Previously, these investigations involved labor-intensive dissections and required expert personnel. The present work evaluates the effects of storage methods of phlebotomine samples in the optimization of PCR identification of Leishmania. Females of Lutzomyia longipalpis, from the colony of the Instituto Nacional de Salud, were experimentally infected with Leishmania chagasi (= L. infantum), from the upper Magdalena Valley (Quipile, Cundinamarca, Colombia). The infected insects were preserved in three solutions: 100% ethanol, 70% ethanol, and TE; subsamples of each class were stored at -80 degrees C, -20 degrees C and room temperature. To determine infection rates, samples were dissected and screened microscopically. Chelex 100 was used for extraction of total Leishmania DNA. For PCR amplification, the kinetoplastic minicircle DNA primers OL1 and OL2 of Leishmania were used, and the products were visualized by electrophoresis in 1% agarose gels. For each of the 3 storage conditions, amplifications were successful, producing a approximately 120 base pair product unique to Leishmania. The results demonstrated the advantage of PCR as a routine screening method for detecting infected flies in endemic foci of visceral leishmaniasis. Since storage method did not affect PCR amplification success, the most cost effective method -70% ethanol at room temperature--is the option recommended for storing entomological samples in vector incrimination studies.

Animals↗
PubMed1997

Site-directed mutagenesis of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti confers insecticide insensitivity.

Insecticide resistance is a serious problem facing the effective control of insect vectors of disease. Insensitive acetylcholinesterase (AChE) confers resistance to organophosphorus (OP) and carbamate insecticides and is a widespread resistance mechanism in vector mosquitoes. Although the point mutations that underlie AChE insensitivity have been described from Drosophila, the Colorado potato beetle, and house flies, no resistance associated mutations have been documented from mosquitoes to date. We are therefore using a cloned acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti as a model in which to perform site directed mutagenesis in order to understand the effects of potential resistance associated mutations. The same resistance associated amino-acid replacements as found in other insects also confer OP and carbamate resistance to the mosquito enzyme. Here we describe the levels of resistance conferred by different combinations of these mutations and the effects of these mutations on the kinetics of the AChE enzyme. Over-expression of these constructs in baculovirus will facilitate purification of each of the mutant enzymes and a more detailed analysis of their associated inhibition kinetics.

Acetylcholinesterase↗
PubMed2001

Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes.

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

Animals↗
PubMed1995

Molecular cloning of a serine proteinase inhibitor from Brugia malayi.

The antigens produced by the infective-stage larvae of filarial parasites are potentially important targets for a protective immune response. A major impediment to studies on the biochemistry and molecular biology of antigens from infective larvae is a lack of parasite material. By employing a reverse transcription PCR-based strategy which exploited the presence of a conserved 22-nucleotide spliced leader sequence present at the 5' end of a proportion of nematode transcripts, spliced leader-containing cDNAs were amplified from the late-vector-stage larvae of the filarial nematode Brugia malayi. A major 1.4-kb PCR product was cloned into pBluescript. One of the PCR cDNA clones (BmY8) contained a 1,287-bp insert that encoded the first member of the serine proteinase inhibitor (serpin) superfamily to be described from nematodes. Reverse transcription PCR analysis of RNA isolated from different developmental stages of the parasite showed that transcription of the B. malayi serpin (Bmserpin) begins between days 8 and 9 of larval development within the insect vector and continues through to the adult and microfilarial stages. In immunoblot analyses of B. malayi somatic extracts, the native protein was estimated to have a molecular weight of 44,000. In immunoblots using excretory-secretory products from infective- and fourth-stage larvae, a single band with an estimated molecular weight of 75,000 was detected. A quantitative analysis of somatic extracts demonstrated that infective-stage larvae contained 10- to 16-fold-more Bmserpin than adults or microfilariae. Bmserpin was immunogenic in gerbils and was recognized strongly by sera from immunized animals. Bmserpin, which has the potential for modifying host defense responses, may play an important role in parasite survival during the early phase of vertebrate-stage development.

Amino Acid Sequence↗
PubMedDate not supplied

Trypanosoma cruzi in the anal glands of urban opossums. I--Isolation and experimental infections.

Opossums (Didelphis marsupialis) captured in intensely urbanized areas of the city of Caracas, Venezuela, were found infected with Trypanosoma cruzi. The developmental cycle of trypomastigote-epimastigote-metacyclic infective trypomastigote, usually occurring in the intestine of the triatomine vector, was taking place in the anal odoriferous glands of the opossums. Material from the glands, inoculated in young, healthy opossums and white mice by different routes, subcutaneously, intraperitoneally, orally, and into the eye, induced T. cruzi infections in all animals. Parasitemia, invasion of cardiac and skeletal muscle, and intracellular multiplication of amastigotes were observed. Inoculation of metacyclics from anal glands, cultured in LIT medium, gave equivalent results. All opossums survived; all mice died. Excreta of opossums may thus transmit Chagas' disease by contamination, even in urban areas where insect vectors are not present.

Anal Canal↗
PubMed2006

What do community-based dengue control programmes achieve? A systematic review of published evaluations.

Owing to increased epidemic activity and difficulties in controlling the insect vector, dengue has become a major public health problem in many parts of the tropics. The objective of this review is to analyse evidence regarding the achievements of community-based dengue control programmes. Medline, EMBASE, WHOLIS and the Cochrane Database of Systematic Reviews were searched (all to March 2005) to identify potentially relevant articles using keywords such as 'Aedes', 'dengue', 'breeding habits', 'housing' and 'community intervention'. According to the evaluation criteria recommended by the Cochrane Effective Practice and Organisation of Care Review Group, only studies that met the inclusion criteria of randomised controlled trials (RCT), controlled clinical trials (CCT), controlled before and after trials (CBA) or interrupted time series (ITS) were included. Eleven of 1091 studies met the inclusion criteria. Of these, two were RCTs, six were CBAs and three were ITS. The selected studies varied widely with respect to target groups, intervention procedures and outcome measurements. Six studies combined community participation programmes with dengue control tools. Methodological weaknesses were found in all studies: only two papers reported confidence intervals (95% CI); five studies reported P-values; two studies recognised the importance of water container productivity as a measure for vector density; in no study was cluster randomisation attempted; and in no study were costs and sustainability assessed. Evidence that community-based dengue control programmes alone and in combination with other control activities can enhance the effectiveness of dengue control programmes is weak.

Community Health Services↗
PubMed1999

Promoter-directed expression of recombinant fire-fly luciferase in the salivary glands of Hermes-transformed Aedes aegypti.

Molecular genetic analyses of biological properties characteristic of insect vectors of disease, such as hematophagy and competence for pathogens, require the ability to isolate and characterize genes involved in these processes. We have been working to develop molecular approaches for studying the promoter function of genes that are expressed specifically in the adult salivary glands of the yellow fever mosquito, Aedes aegypti. Genomic DNA fragments containing cis-acting promoter elements from the Maltase-like I (MalI) and Apyrase (Apy) genes were cloned so as to direct the expression of the reporter gene, luciferase (luc). The function of the promoters was assayed transiently in cultured insect cells and by germ-line transformation of Ae. aegypti. MalI and Apy DNA fragments consisting of at least 650 nucleotides (nt) of DNA immediately adjacent to the 5'-end of the initiation codon of the mosquito genes directed constitutive expression of the luc reporter gene in cultured cells. When introduced into Ae. aegypti chromosomes, approximately 1.5 kilobases (kb) of each promoter were able to direct the predicted developmental-, sex- and tissue-specific expression of the reporter gene in patterns identical to those determined for the respective endogenous genes.

Aedes↗