A novel duplex real-time PCR permits simultaneous detection and differentiation of Borrelia miyamotoi and Borrelia burgdorferi sensu lato.

PURPOSE: For simultaneous detection of Borrelia miyamotoi (relapsing fever spirochete) and Borrelia burgdorferi sensu lato, we have developed a duplex real-time PCR targeting the flagellin gene (flaB; p41), a locus frequently used in routine diagnostic PCR for B. burgdorferi s.l. detection. METHODS: Primers and probes were designed using multiple alignments of flaB sequences of B. miyamotoi and B. burgdorferi s.l. species. The sensitivity and specificity of primers and probes were determined using serial dilutions (ranging from 10(4) to 10(-1)) of B. miyamotoi and B. burgdorferi s.l. DNA and of several species of relapsing fever spirochetes. Conventional PCR on recG and glpQ and sequencing of p41 PCR products were used to confirm the species assignment. RESULTS: The detection limit of both singleplex and duplex PCR was 10 genome equivalents except for B. spielmanii and two B. garinii genotypes which showed a detection limit of 10(2) genome equivalents. There was no cross reactivity of the B. miyamotoi primers/probes with B. burgdorferi s.l. DNA, while the B. burgdorferi s.l. primer/probe generated a signal with B. hermsii DNA. Out of 2341 Ixodes ricinus ticks from Germany and Slovakia that were screened simultaneously for the presence of B. miyamotoi and B. burgdorferi s.l., 52 were positive for B. miyamotoi and 276 for B. burgdorferi s.l., denoting an average prevalence of 2.2% for B. miyamotoi and 11.8% for B. burgdorferi s.l., and B. miyamotoi DNA was also detectable by PCR using artificial clinical samples. CONCLUSION: The duplex real-time PCR developed here represents a method that permits simultaneous detection and differentiation of B. burgdorferi s.l. and B. miyamotoi in environmental and potentially clinical samples.

Trypanosoma cruzi: effects of azadirachtin and ecdysone on the dynamic development in Rhodnius prolixus larvae.

The effects of azadirachtin and ecdysone on the Trypanosoma cruzi population in the Rhodnius prolixus gut were investigated. T. cruzi were rarely found in the gut compartments of azadirachtin-treated larvae. High parasite numbers were observed in the stomach of the control and ecdysone groups until 10 days after treatment and in the small intestine and rectum until 25 days after treatment. High percentages of round forms developed in the stomachs of all groups, whereas azadirachtin blocked the development of protozoan intermediate forms. This effect was counteracted by ecdysone therapy. In the small intestine and rectum, epimastigotes predominated for all groups, but more of their intermediates developed in the control and ecdysone groups. Azadirachtin supported the development of round forms and their intermediates into trypomastigotes. In the rectum, trypomastigotes did not develop in the azadirachtin group and developed much later after ecdysone therapy. The parallel between the effects of azadirachtin and ecdysone on the host and parasite development is discussed on the basis of the present results because ecdysone appears to act directly or indirectly in determining the synchronic development of T. cruzi forms from round to epimastigotes, but not metacyclic trypomastigotes, in the invertebrate vector.

A salivary serine protease of the haematophagous reduviid Panstrongylus megistus: sequence characterization, expression pattern and characterization of proteolytic activity.

A cDNA encoding a trypsin-like protease from the salivary glands of the haematophagous reduviid Panstrongylus megistus was cloned and sequenced. The deduced protein sequence showed similarities to serine proteases of other hemipterans but with substitutions in the catalytic triad and the substrate binding site. The expression of the gene increased more than sixfold after feeding. Saliva showed the highest proteolytic activity at neutral to slightly basic pH. Substrate and inhibitor profiles and zymography indicated the presence of a trypsin-like protease with preference for Arg and Lys at P1. Using chromatography, a fibrinolytic enzyme was purified whose sequence was identified by tandem mass spectrometry as that encoded by the cDNA.

Sequence characterization and expression patterns of defensin and lysozyme encoding genes from the gut of the reduviid bug Triatoma brasiliensis.

The cDNAs encoding an intestinal defensin (def1) and lysozyme (lys1) of the reduviid bug Triatoma brasiliensis have been amplified by PCR using specific oligonucleotide primers and 5'- and 3'-RACE, cloned and sequenced. The 576 bp clone has an open reading frame of 282 bp and encodes a pre-prodefensin with 94 amino acid residues, containing a putative signal and activation peptide cleavage site at Ser19 and Arg51, respectively. The genomic DNA contains a second defensin gene with similar characteristics, 88.3% identity and also one intron of 107 nucleotides. The 538 bp clone has an open reading frame of 417 bp, encoding a pre-lysozyme with 139 amino acid residues. The putative signal peptide is cleaved at alanine 18. Using whole mount in situ hybridization, high expression of both genes has been found, distributed uniformly throughout the entire cardia and the blood-storing stomach and to a much lower extent in the digesting small intestine. Using quantitative real-time PCR, the expression level of def1 was also shown to be very low in small intestine, rectum and salivary glands; in the stomach, expression was 500-2500 times higher than in the cardia and fat body. No expression of lys1 could be detected in the salivary glands and rarely a very low expression in the small intestine, rectum and fat body. Lys1 expression in the stomach was 60-300 times higher than in the cardia. Comparing the levels in unfed fifth instars and up to 15 days after feeding, a strong def1 induction was evident in the fat body at 15 days after feeding and in the stomach a maximum level of def1 and lys1 at 5 days after feeding.

The effect of azadirachtin on Blastocrithidia triatomae and Trypanosoma cruzi in Triatoma infestans (Insecta, Hemiptera).

The effect of azadirachtin on Blastocrithidia triatomae and Trypanosoma cruzi, which colonise the intestinal tract of the blood-sucking bug Triatoma infestans, was investigated. In established infections of controls without azadirachtin treatment, the small intestine of fifth-instar T. infestans contained up to 7 x l0(6) B. triatomae and the rectum 3 x 10(6). In comparison to this homoxenous flagellate, the population densities of T. cruzi in the respective regions were 99.3 and 76% lower. Treatment with azadirachtin (1 microg ml(-1)) via a blood meal and a concurrent infection with B. triatomae resulted in an increase of the population density (3 weeks p.i.), caused mainly by the mastigote stages in the rectum. In an established B. triatomae infection (12 weeks p.i.), feeding of azadirachtin did not affect the population density and composition. In an optimal T. cruzi-vector system, i.e. parasite and bug originate from the same locality, the treatment with azadirachtin at 20 weeks p.i. strongly reduced the population density in the small intestine of all bugs up to 100 days after treatment, but only in a minor percentage of the bugs in the rectum. Trypanosoma (cruzi incubated for up to 24 h in faeces of azadirachtin-treated bugs were not affected, indicating that the rectum of these bugs contained no toxic substances. The importance of these findings is that investigations of the mechanisms of action of azadirachtin offer a possibility to identify vector-derived compounds, which are necessary for the development of T. cruzi, thereby, giving us a possible new strategy to combat Chagas' disease.

Trypanosoma cruzi: skin-penetration kinetics of vector-derived metacyclic trypomastigotes.

In order to investigate the natural route of infection of nude and normal BALB/c mice with Trypanosoma cruzi via the skin, a drop of vector faeces/urine containing metacyclic trypomastigotes was placed onto the puncture site of a bite from Triatoma infestans. The periods of exposure, i.e. until removal of flagellates from the skin, and the time elapsed until surgical removal of the skin around the puncture were varied. After 15 min of exposure, T. cruzi developed in all nude mice without surgery, and in four of 10 mice if the puncture region of the skin was removed directly after exposure. In a shaved puncture region, 5 min of exposure were sufficient to infect all normal BALB/c mice without surgery and one of four mice with direct removal of the puncture region. Longer periods of exposure or time until removal of the skin only sometimes resulted in higher infection rates. Prepatent periods and the development of parasitaemia varied irrespective of the period of exposure or the period until skin removal at the puncture site. The importance of these findings is that they clearly prove that T. cruzi can rapidly invade the host via the puncture site of the bite of the vector and that at least some parasites are immediately transported away from this site.

Trypanosoma cruzi in the rectum of the bug Triatoma infestans: effects of blood ingestion by the starved vector.

To follow the developmental effects of feeding of the insect host after long starvation periods, the population density and composition of an established infection of Trypanosoma cruzi in the rectum of Triatoma infestans were determined 60 days after the last feeding (daf) and then at different intervals after feeding. The original population decreased and then increased up to the 10th daf. In starved bugs, about 30% were spheromastigotes (including intermediate forms), 20% epimastigotes, and 50% trypomastigotes, but one daf, these forms represented 2%, 70%, and 10%, respectively. In addition, one daf there were about 10% giant cells, i.e., a multiple division stage. In the following two days, this form represented on average 30-50% of the total population, but it then disappeared nearly completely. Thus, giant cells evidently develop by rapid growth of epimastigotes, if conditions become optimal after long starvation periods of the vector.

Effects of lignoids on a hematophagous bug, Rhodnius prolixus: feeding, ecdysis and diuresis.

The effects of lignoids on feeding, ecdysis and diuresis in fourth-instar larvae of Rhodnius prolixus (Hemiptera) were investigated. Up to 100 microg/ml burchellin, podophyllotoxin, pinoresinol, sesamin, licarin A, or nordihydroguaiaretic acid (NDGA) in the diet did not induce antifeedant effects. Pinoresinol and NDGA significantly inhibited ecdysis. In experiments in vivo, burchellin and podophyllotoxin reduced the production of urine after feeding. 5-Hydroxytryptamine (5-HT), a diuretic hormone, partially counteracted this effect of burchellin. In experiments in vitro, using isolated Malpighian tubules, (i) burchellin reduced diuretic hormone levels in the hemolymph but not the amount of diuretic hormone stored in the thoracic ganglionic masses (including axons), (ii) burchellin decreased the volume of urine secreted by isolated Malpighian tubules, and (iii) 5-HT could not overcome the effect of burchellin upon the Malpighian tubules. We conclude that burchellin interfered with the release, but not with the production of diuretic hormone by the thoracic ganglionic mass or induced an antidiuretic hormone and directly affected the Malpighian tubules.

Developmental time and mortality of larvae of Triatoma infestans infected with Trypanosoma cruzi.

Triatoma infestans were infected with Trypanosoma cruzi (zymodeme 1) at the first feed after eclosion from the egg; interstadial development times and mortality rates were then recorded until the imaginal moult and compared with those of uninfected controls. No retardation of development occurred in infected bugs and their mean total mortality rate (9%) was only slightly higher than that (6%) of uninfected controls (due solely to 4 additional deaths). This is the first demonstration, under optimal and standardized rearing conditions, that infection of T. infestans with T. cruzi has little or no effect on development times or mortality rates.

Direct transmission of Trypanosoma cruzi between vectors of Chagas' disease.

Trypanosoma cruzi was transmitted directly between triatomines by cannibalism or coprophagy. Different conditions involving cannibalism that excluded coprophagy were studied in Dipetalogaster maximus. Infections occurred if an uninfected donor bug sucked infectious blood and if this blood was taken up from the stomach by a cannibalistic bug. If the donor was infected and sucked uninfected blood afterwards, the source of the uninfected blood determined the transmission rate: If the uninfected blood originated from mice, many cannibalistic bugs became infected because complement factors from mouse blood did not lyse T. cruzi in the stomach of the bug. If the uninfected bloodmeal originated from chickens, cannibalistic bugs occasionally became infected, even though chicken blood is known to lyse all stages of T. cruzi in the stomach. Experiments on coprophagy provided the first conclusive demonstration that transmission of T. cruzi occurs between individual Triatoma infestans, as a result of coprophagic behaviour alone, and excluding the possibility of cannibalistic transmission.

Rapid isolation of metacyclic trypomastigotes of Trypanosoma cruzi from feces and urine of the vector.

Metacyclic trypomastigotes of Trypanosoma cruzi from infected Triatoma infestans were isolated and purified by an improved procedure. By using feces and urine of bugs the duration and the number of steps for purification were greatly reduced, thereby minimizing any possible effects on the flagellates. After DEAE-Sephacel column chromatography, a buffer supplemented with bovine serum albumin protected against the slight surface alterations of the trypomastigotes otherwise observed. About 10(7) metacyclic trypomastigotes (including 30% intermediate flagellates) were obtained from 100 bugs. The intermediate forms possessed the surface charge of trypomastigotes, and the kinetoplast was beside or posterior to the nucleus but not subterminal. Further increases in the number of bugs and the purified trypomastigotes were also possible with only slight increases in the time needed for isolation. In a comparison of complement-mediated lysis resistance and infectivity for mice, pre- and post-separation trypomastigotes showed no significant differences.

Colonization of the rectum of Triatoma infestans by Trypanosoma cruzi: influence of starvation studied by scanning electron microscopy.

The colonization of the different regions of the rectum of Triatoma infestans by a Trypanosoma cruzi strain (zymodeme I) originating from the same locality as the bugs was studied by scanning electron microscopy after different periods of starvation of the bugs. Throughout the first 16 weeks no changes in colonization pattern could be observed. Parasite density was always minimal at the midgut/rectal junction and highest on the rectal pads; it was at a similar level in the other three regions of the rectum. Twenty weeks after feeding, a proportion of the bugs had died and in the surviving larvae a decreasing colonization of the cuticle occurred. Nonetheless, despite other regions being flagellate-free, a residual T. cruzi population always remained attached to the rectal pads. No changes in the proportion of trypomastigotes to epimastigotes were observed as starvation progressed.

Modes of association of Trypanosoma cruzi with the intestinal tract of the vector Triatoma infestans.

The interface between Trypanosoma cruzi and two regions of the intestinal tract of reduviid bugs, the small intestine and the rectum, was investigated by electron microscopy. The mode of association of the trypanosomes with the midgut surface differs fundamentally from that in the rectum, the preferred site of colonisation by T. cruzi. The parasites caused no detrimental changes in the extracellular membrane layers, microvilli or epithelial cells. Parasites resided mainly at the border of the gut contents, also regularly showing parasite-parasite interdigitations. In regions in which the extracellular membrane layers were absent or only weakly developed, trypanosome bodies or flagella occasionally could be found inserted shallowly between the tips of the microvilli. Since there were usually no ultrastructural modifications of the cell body and/or flagellum associated with attachment, there is apparently no strong attachment of the flagellates to the wall of the midgut. In the rectal lumen the flagellates also interdigitated with each other and on the rectal wall T. cruzi was intimately attached to the rectal cuticle lining. At the attachment site flagella were enlarged and sometimes contained electron-dense, hemidesmosome-like material beneath the plasma membrane.

Binding of lectin-gold conjugates by two Trypanosoma cruzi strains in ampullae and rectum of Triatoma infestans.

The ampullae and recta of Triatoma infestans infected with Trypanosoma cruzi were incubated with lectin-gold conjugates of wheat germ agglutinin (WGA) and soy bean agglutinin (SBA) and investigated by electron microscopy. T. cruzi colonized the whole lumen of the ampullae. The rectal pads with their intensive intracellular membrane system and thin cuticle were often covered by a compact layer of flagellates, whereas the remaining rectal sac- recognizable by a thick layered cuticle - was colonized less densely. All freely accessible stages of T. cruzi strain 'Chile 5' (Zymodeme 1) showed a reaction with SBA-gold complexes but not with WGA, revealing that N-acetyl-galactosamine and/or galactose but no N-acetyl-glucosamine residues were present on the surface. T. cruzi strain 'Chile 7' (Zymodeme 2) also reacted with SBA. WGA-bovine serum albumin-gold conjugates bound strongly to the surface of epimastigotes of this strain, in variable amounts to stages in transition to trypomastigotes, but not to metacyclic trypomastigotes.

Improvement of techniques for age grading hematophagous insects: ovarian oil-injection and ovariolar separation techniques.

After injection of oil into the oviduct of hematophagous Diptera, the ovarioles become completely separated from each other, allowing examination of a large number of undamaged ovarioles, an important advantage over other techniques for accurately determining the physiological age of mosquitoes. The technique has been simplified and improved, especially by using a sodium chloride-glycerol-formaldehyde mixture for mounting preparations, which are more convenient and permanent for examination of the ovarioles. The difficulties of using the technique, their possible causes, and possibilities for overcoming them are described. Similar results can be obtained with an alternative technique, the ovariolar separation technique, using strongly diluted Carnoy's solution, which leaves ovaries in a fixed condition. Both techniques can be used for several hematophagous dipteran groups.

Development of Blastocrithidia triatomae (Trypanosomatidae) in Triatoma infestans after vitamin B-supplementation of the blood-diet of the bug.

Investigations of the interrelationships of the trypanosomatid Blastocrithidia triatomae and the reduviid bug Triatoma infestans indicated an interference of B. triatomae with bug symbionts or with their vitamin supply. For an initial study of this possibility, B. triatomae-infected bugs were fed two or three times at monthly intervals on defibrinated sheep blood with or without B-group vitamin supplementation (folic acid, nicotinic acid, pantothenic acid, pyridoxine, riboflavin, and thiamin). The total number of flagellates in the small intestine and the rectum of the bug were then determined with a hemocytometer. Vitamin B-supplementation supported the initial development of B. triatomae in the small intestine of most third and fourth instar larvae which had been infected in the first instar. In the small intestines of fifth instars and adults, both which had been infected as third instar larvae, most populations of B. triatomae were not affected by vitamin B-supplementation. Similarly, rectal populations in all bugs were never increased by supplementation.

Attachment of Blastocrithidia triatomae (trypanosomatidae) by flagellum and cell body in the midgut of the reduviid bug Triatoma infestans.

The pathology and attachment of Blastocrithidia triatomae in two regions of the midgut of the reduviid bug Triatoma infestans (the stomach and the small intestine) were investigated by electron microscopy. In both regions the extracellular-membrane layers and the apical microvilli were often reduced, and some cells of the intestinal wall were vacuolated. In the stomach two types of attachment occurred: Flagella with intraflagellar swellings lay over and between the apices of the microvilli of the stomach cells, and in microvilli-free regions the cell body of B. triatomae seemed to be anchored to the host cell by corrugations of both cells in the attachment zone, a hitherto unknown mode of attachment of trypanosomatids in the intestinal tract. In the small intestine flagellar expansions held several microvilli, and rarely an unaltered flagellum seemed to be anchored between the microvilli. In both midgut regions, the flagellum of some epimastigotes was inserted into the apex of an intestinal cell.

Development of Trypanosoma cruzi after starvation and feeding of the vector - a review.

Trypanosoma cruzi develops in the intestinal tract of reduviid bugs and may be affected by changes in the nutritional state of the vector. In regularly fed Triatoma infestans the population of T. cruzi in the rectum consists mainly of equal amounts of epi- and trypomastigotes. Starvation of the bug reduces the total number of flagellates and the number and percentage of trypomastigotes. The number and the percentage of drop-like forms and of resulting spheromastigotes, however, increases up to 30% 60 days after the last feeding (daf). Feeding of starved bugs (60 daf) reduces the original population density, which then increases again. In starved bugs 1 daf spheromastigotes (including intermediate forms) have almost disappeared and epimastigotes dominate. In addition "giant cells" (a multiple division stage) comprise about 10% of the population and in the following two days this form represents on average 30-50% of the total population, before disappearing nearly completely. Feeding the vector at 40 daf; a) induces the appearance of pure populations of trypomastigotes in immediately deposited drops of bug urine; b) induces metacyclogenesis in epimastigotes, and c) reduces metacyclogenesis in spheromastigotes. Incubating isolated recta together with the Malpighian tubules in Drosophila Ringer's solution and initiating the excretion with 5-hydroxy-tryptamine also induces metacylogenesis in epimastigotes.

Hydrophobic attachment of Trypanosoma cruzi to the rectal cuticle of Triatoma infestans and its influence on metacyclogenesis - a review.

Trypanosoma cruzi colonizes mainly the rectum of the vector, especially the rectal glands. We investigated the basic architecture of the rectal cuticle of Triatoma infestans and the mode of attachment of T. cruzi in the small intestine and the rectum. In addition, we determined the capacity of culture-derived epimastigotes to attach to artificial substrates and the influence of attachment on metacyclogenesis. After incubation of the rectum with wheat germ lectin (WGA) coupled to gold particles, the procuticle contained chitin, but the two layers of the epicuticle and the superficial layer bordering the rectal lumen did not. The specific fluorochrom Nile Red stained the entire rectal cuticle green, indicating the waxy composition of the superficial layer. In electron microscopic analysis the parasites were attached to the hydrophobic superficial wax layer but not to the epithelium of the midgut. In vitro culture-derived epimastigotes attached with a high affinity to all hydrophobic substrates tested, whereas hydrophilic substrates did not permit attachment. Emulsified hexadecane localized the attachment molecules to the terminal part of the flagellum. Inhibition of attachment by coating the culture tubes with hydrophilic agarose and constant agitation decreased the rate of epimastigote to trypomastigote transformation, whereas wax coating enhanced metacyclogenesis.