[Personal protective measures against mosquito and other arthropods bites]. / Mesures de protection personnelle contre les piqcûres de moustiques et d'autres arthropodes.

An increasing number of travellers cross international borders and is exposed to arthropod-borne diseases. Primary care physician should not only know and give advice on preventive measures, but also estimate the risk to an individual traveller. Personal protective measures are an important and sometimes the only way to prevent arthropod-borne diseases. Personal protective measures are maximised by using an integrated approach that includes physical (e.g. clothing, bed net) and chemical barriers (e.g. repellents, insecticides).

[Protection against insects]. / Schutz vor Insekten.

Successful protection against haematophagous insects and ticks, especially in areas where transmission of diseases occurs, requires a consistent application of a combination of appropriate measures. However, this can never substitute a chemoprophylaxis. Which measures have to be used depends on the circumstances under which they have to work. Indoor, physical means such as mosquito-screens on doors and windows, air-conditioners, and bed nets can be used to keep the insects away. These measures can be supplemented or supported by insecticides used as knock-down sprays, by electrical evaporation or for the treatment of screens and bed nets. In the field, if it is not possible to avoid mosquito-areas during phases of activity, appropriate clothing and repellents must provide the protection. Bright, wide pants and shirts of dense weaving covering as much skin as bearable should be preferred. Repellents are sprays, lotions, milks or creams which are evenly applied to the skin to prevent insects from biting. They contain synthetic or natural active substances of substantially varying effectiveness. The gold standard since about 60 years is diethylbenzamine (DEET). There are a few other active substances with a lower risk of side effects, however, combined with a lower effectiveness mainly on people with a high attractiveness for mosquitoes. Products containing an extract of Eucalyptus citriodora provide the best protection amongst those with natural active substances. Wearing bracelets or necklaces treated with repellents, acoustic devices (buzzers), electrocuters, topical or systemic Vitamin B1 or eating garlic are useless measures to prevent insects from biting.

Normal versus alpha-amanitin induced cellular dynamics of the midgut epithelium in female Aedes aegypti L. (Insecta, Diptera) in response to blood feeding.

In midgut epithelial cells (stomach) of untreated female A. aegypti an increase in the surface area of the rough endoplasmic reticulum (rer) and in the ratio of membrane-bound to free ribosomes is morphometrically measured during digestion of the first blood meal. This can be correlated with the synthesis and release of digestive proteases. The dynamics of the ribosomes in A. aegypti are similar to those in A. stephensi. 3 ng alpha-amanitin per mosquito prevent normal blood digestion, the proliferation of the rer and the increase in the ratio of bound to free ribosomes. On the other hand, some synthesis of new ribosomes takes place.

Role of ICAM-1 (CD54) in the development of murine cerebral malaria.

In susceptible mouse strains, infection of mice with Plasmodium berghei ANKA (PbA) results in a lethal complication, cerebral malaria. Cerebral malaria is due to the immune response induced by the parasite, which results in an increased production of TNF, known to increase the expression of adhesion molecules on the endothelia. To investigate the role of the adhesion molecule ICAM-1 (CD54), we infected wild-type (+/+) and ICAM-1-deficient (-/-) mice with PbA. While +/+ mice died 6-8 days after infection, -/- mice survived > 15 days. Parasitaemia was similar in +/+ and -/- mice. Serum TNF concentration was increased by the infection and was significantly higher in infected +/+ than in -/- mice. However, TNF mRNA levels in spleen, lungs, and brain were elevated in both infected +/+ and -/- mice. For IFN-gamma, serum levels were similar in both groups. A breakdown of the blood-brain barrier was evident in infected +/+ mice only. Interestingly, thrombocytopenia was profound in infected +/+, but practically absent in -/- mice. Moreover, macrophage sequestration was evident in brain venules and lung capillaries of +/+ mice and was significantly less important in the alveolar capillaries of infected -/- mice. In contrast, neutrophil sequestration in the lung was similar in both +/+ and -/- mice. Sequestration of parasitized red blood cells was significantly greater in the alveolar capillaries from +/+ than -/- mice. These results indicate that while the immune response is similar in both +/+ and ICAM-1(-/-) mice, the absence of mortality in ICAM(-/-) mice correlates with a decrease of macrophage and parasitized RBC trapping and a less severe thrombocytopenia.

Comparison of cytokine measurements using ELISA, ELISPOT and semi-quantitative RT-PCR.

We have investigated the correlation between results obtained by three different methods (semi-quantitative RT-PCR, ELISA and ELISPOT) used to measure cytokine expression by mouse leukocytes. The production of the cytokines tumour necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) and interleukin-4 (IL-4), was analysed with all three methods. In a simple experimental murine in vivo model of leukocyte stimulation, consisting of a single intravenous injection of anti-CD3 antibodies followed by a short incubation in vitro, the results obtained with spleen cells for each of the three cytokines differed greatly, depending on the method used. For TNF alpha, a significant increase in RNA was observed upon stimulation, whereas the number of spot-forming cells did not increase and the protein was not detectable in serum or in cell culture supernatants by ELISA. In vitro cultured splenocytes showed a strong correlation between all three methods for IFN gamma. Upon stimulation, the amount of RNA for IL-4 increased in parallel with the secretion of the cytokine and the number of spot-forming cells. However, high numbers of spot forming cells were observed in controls. We conclude that, depending on the specific aim of an investigation, combinations of different methods have to be chosen carefully in order to detect activation of leukocytes for cytokine expression.

Solubilization of epicuticular antigen from Dirofilaria immitis third-stage larvae.

The solubilization of epicuticle from third-stage (L3) Dirofilaria immitis larval cuticles was investigated. Cuticles collected after L3 had molted were incubated in 1.5% sodium dodecyl sulfate (SDS) at 37 degrees C with vigorous shaking. Solubilization of epicuticular layers was accomplished as demonstrated by electron microscopy. Diminished binding of an epicuticular specific monoclonal antibody (DIM-229) was seen when SDS-treated cuticles were compared to untreated cuticles in an indirect fluorescence antibody assay. Cuticles which were extracted further by boiling in 1.5% dithiothreitol (DTT) produced less protein than cuticles solubilized in SDS. Both extracts reacted with DIM-229 in an indirect enzyme-linked immunosorbent assay, indicating retention of antigenic reactivity of the solubilized epitope. SDS-polyacrylamide gel electrophoresis of SDS-derived antigens revealed, after silver staining, proteins from 12 to 77 kDa and only 1 band at 15 kDa for SDS-treated cuticles boiled in DTT. Western blot analyses of the extracts with DIM-229 were inconclusive.

Identification of an Onchocerca volvulus cDNA encoding a low-molecular-weight antigen uniquely recognized by onchocerciasis patient sera.

The primary structure of an immunodominant antigen of the filarial parasite, Onchocerca volvulus was deduced from cDNA sequence analysis. Using affinity-purified antibody from onchocerciasis patients from West Africa, we have isolated a cDNA clone from a lambda gt11 cDNA expression library derived from microfilariae-producing female O. volvulus. The open reading frame encodes 152 amino acids, and the deduced sequence predicts a Mr of 16,850 (consistent with the apparent Mr of 18,000 of the immunoprecipitated in vitro translated product). The primary translation product contains a putative signal peptide of 16 amino acids. The mRNA coding for this antigen has an estimated size of 950 nucleotides. Furthermore, immunoelectron microscopy established that the antigen encoded by this clone is present in the hypodermis, the cuticle, and in the uterus of the filarial worms. Since this antigen is recognized exclusively by sera from onchocerciasis patients, and not by other sera from patients infected by other filarial parasites, it may prove to be an especially valuable tool for improving the specific diagnosis of onchocerciasis.

Immunoelectron microscopic demonstration of pancreatic polypeptide in midgut epithelium of hematophagous dipterans.

Midguts of mosquitoes, Aedes aegypti and Anopheles stephensi, and of the tsetse fly, Glossina morsitans morsitans, as well as guinea pig pancreas, were prepared for electron microscopy by using low-temperature embedding in Lowicryl K4M. Rabbit antiserum to bovine pancreatic polypeptide (PP) crossreacted with secretory granules of pancreatic PP-producing cells and of the clear cells in mosquito gut. Rabbit antiserum to human somatostatin crossreacted with the control tissue, guinea pig pancreas D-cells, but not with the mosquito clear cells. None of the antisera used showed a distinct reaction with the endocrine-like cells of tsetse fly midgut. Positive reactions were revealed by gold as electron-dense marker. The gold particles were coated with protein A-gold or goat antibodies to rabbit immunoglobulin.

Immunopathological aspects of trypanosomal meningoencephalitis in vervet monkeys after relapse following Berenil treatment.

Four quarantined vervet monkeys were treated with intramuscular Berenil in patent CNS infection after experimental trypanosome inoculation with Trypanosoma brucei rhodesiense or T. brucei brucei. All four animals relapsed in the post-therapeutic survival time of 37 to 209 days when they had fully developed meningoencephalitis in histological sections with the presence of interstitial intracerebral trypanosomes, which were confirmed in two monkeys by electron microscopy. In both, sequential samples of the serum and cerebrospinal fluid were analysed for circulating immune complexes, immunoglobulins and albumin. From these results the intracerebral IgG synthesis and the impairment of the blood-brain-barrier were calculated, both being present in advanced infection. Circulating immune complexes were present in the serum, but could not be demonstrated in the cerebrospinal fluid. The monkey model therefore permits the study of various aspects of cerebral trypanosomiasis. Berenil treatment is inefficient in patent CNS infection and leads to a protracted, less virulent disease course with terminal meningoencephalitis and intracerebral "persister" trypanosomes. This drug-induced trypanosome shift with meningoencephalitis could be used for chemotherapeutic purposes to test new compounds in late stage disease.

Comparison of the cuticular structure of parasitic nematodes recognized by immunocytochemical and lectin binding studies.

The cuticle structure of some nematode species was studied by immunogold and lectin-gold techniques. The gold labelling made it possible to distinguish the cuticle layers by the distribution and/or the density of the marker. On the other hand, no labelling pattern was found which led to a clear grouping of the layers into larger 'zones', since there were no subunits consisting of more than one layer which reacted in a characteristic way as compared to the rest of the cuticle. The outer surface of the epicuticle of parasitic adult worms turned out to be highly inert; it did not react with any of the antibodies or lectins tested. The cuticle of parasitic nematodes seems to function as a protection against the host's defense mechanisms rather than as an interaction site. An immunogenic component on the surface was only found in infective larvae. All antibodies and lectins showed a preferential binding to the electron dense layers and fibrous structures (HPL/GalNAc, WGA/GlcNAc) or to the amorphous ground-substance (Con A/Glc, RCA I/Gal).

Cytochemical demonstration of lectin binding-sites in the cuticle and tissues of Acanthocheilonema viteae (Filarioidea).

The lectin-gold technique was used for the ultrastructural localization of lectin binding sites on thin sections of Lowicryl K4M embedded adult females, infective larvae and SDS-2-mercaptoethanol-insoluble cuticle components of Acanthocheilonema (Dipetalonema) viteae. Helix pomatia lectin (HPL) coupled to 14 nm gold particles, was used for the demonstration of N-acetyl-D-galactosamine-containing glycoconjugates. Triticum vulgaris (wheat germ) agglutinin (WGA) coupled to 10 nm gold particles after cross-linking to BSA or ovomucoid-gold after application of unlabeled WGA, demonstrated WGA binding sites (N-acetyl-D-glucosamine). With both lectins no surface labelling of the cuticle was observed, but subcuticular layers reacted positively. HPL-gold was bound to cuticular fibers, the matrix and to the electron dense layer within the cortical zone of the cuticle of female worms. WGA-gold complexes were bound mainly to the cuticle matrix and somatic tissues. The results support the hypothesis that tissue-dwelling parasitic nematodes have reduced their surface carbohydrates perhaps as a consequence of their parasitic life.

Electron-microscopic localization of Trypanosoma brucei gambiense transmitted by Glossina morsitans centralis in Microtus montanus.

In Microtus montanus infected with T. b. gambiense, electron microscopic examination of lymph nodes, spleen, liver, heart, choroid plexus and brain demonstrated extravascular populations of trypanosomes distributed throughout interstitial spaces, accompanied by a moderate cellular infiltration of plasma cells. The trypanosomes exhibited numerous profiles; some were dividing, others were in different stages of lysis, or phagocytosed. Penetration of trypanosomes into hepatocytes was observed. The present investigation indicated that trypanosomes migrated to the brain parenchyma from the Virchow-Robin spaces but could not confirm whether the parasites reached the Virchow-Robin spaces by traversing the ependymal cells lining the choroid plexus or by migrating through the endothelial cells of the cerebral blood vessels.

Distribution of repetitive and non-repetitive circumsporozoite protein epitopes on Plasmodium falciparum sporozoites and immunochemical characterization of human malaria antisera.

The presence and distribution of circumsporozoite protein (CSP) epitopes located in the repetitive and non-repetitive regions were studied in three Plasmodium falciparum strains, NF54, IFA5 and IFA6. It was found by immunofluorescence, Western blotting and immunoelectron microscopy that mAbs to epitopes of the repetitive domaine bound similarly to the CSP of all three strains. MAbs to epitopes of the flanking regions yielded either some strain differences (mAbs to the C-terminal end), or reacted only in immunofluorescence tests on whole sporozoites (mAbs to the N-terminal end). Human sera from an area endemic for malaria, two of them positive in ELISA on (NANP)40 and two negative, were tested for their reactivity with epitopes of the flanking regions of the CSP. The presence of antibodies to such epitopes could be demonstrated by Western blots and immunocytochemistry independent of the reactivity of the sera to recognize (NANP)40. All tested bound to salivary gland tissues but not to their secretory product in immunocytochemical experiments.

Surface coat synthesis and turnover from epimastigote to bloodstream forms of Trypanosoma brucei.

Monoclonal antibodies to metacyclic surface coat glycoproteins of Trypanosoma brucei brucei STIB 247LG were produced for a study of the synthesis of metacyclic variable surface glycoproteins (VSGs) within the salivary gland of Glossina morsitans morsitans, and of the first exchange of the surface glycoproteins after infection in mice. Immunofluorescence antibody tests and protein A-gold labelling revealed that the VSGs are continuously integrated into the whole surface of the trypanosome while it is still attached to the gland epithelium. A pool of 8 antibodies recognized about 50% of the metacyclic forms present in the saliva of an infected tsetse fly, which confirmed the heterogeneity of the metacyclic VSG-generation. The labelling experiments showed that the integration of the first VSG-generation into the surface of bloodstream forms takes place in the same way as in the metacyclics. This process started on day 3 after infection and was finished on day 6.

Demonstration of anti-cuticular antibodies by immuno-electron microscopy in sera of mice immunized with cuticular extracts and isolated cuticles of adult Dipetalonema viteae (Filarioidea).

The immunogold technique was used for the ultrastructural localization of antibody-binding sites on thin sections of Lowicryl K4M embedded adult females, infective larvae and pieces of adult cuticles of Dipetalonema viteae insoluble in SDS-2-ME. The antisera used were either produced against SDS-2-ME extracts of cuticles or the insoluble pellet after SDS-2-ME extraction. With both types of antisera a labelling of epitopes on fibers was achieved in intact cuticles. In isolated cuticles the corresponding structures were absent. The same sera crossreacted with the larval and microfilarial cuticle as well as with somatic structures of all three stages. The only serum against isolated cuticle, which did not recognize cuticle fibers also did not crossreact with somatic structures. The recognition of the electron dense cortical layer insoluble in SDS-2-ME depended on the number of immunizations. A labelling of the filarial surface was never achieved. A dense labelling of the apical membrane enfoldings of the hypodermis pointed to an involvement in the synthesis of the nematode cuticle. The rough endoplasmic reticulum in the apex of the epithelial cells of the uterus, the content of the nutrient channels, and the substance between eggshell and microfilariae crossreacted with most of the antisera. This led to the conclusion that these substances are partly produced by the uterus epithelium.

Interaction of monoclonal antibodies with cuticular antigens of filarial parasites, Brugia malayi and Wuchereria bancrofti.

Monoclonal antibodies (mAbs) have been prepared against excretory-secretory-metabolic (ESM) antigens of microfilariae (mf) of Wuchereria bancrofti (WbmfESM) and against third stage larvae (L3) of Brugia malayi (BmL3), and purified from ascites fluids with ammonium sulphate. Both antibodies were of the IgM type and did not react with phosphorycholine. The mAb against BmL3 (F46) reacted in ELISA with antigens of L3 of B. malayi, B. pahangi and W. bancrofti and of adults of B. malayi. The mAb raised against wbmfESM (F32) resembled F46 in this respect, though with a lower titer towards the antigens, and in addition reacted with the ESM-antigens of mf and of L3 of W. bancrofti. F46 was able to detect L3 antigens of filarial parasites in spiked serum samples with a detection limit of 8-16 ng in absolute amount. The antibody was found to label the cuticular portion of L3 and adults of the lymphatic parasites, and not the epicuticular surface, in immunoelectron microscopic studies. The antibody recognized a 36 kDa component of the beta-mercaptoethanol extracts of B. pahangi-adults in Western blot analysis.

Morphometric comparison of the midgut epithelial cells in male and female Aedes aegypti L. (Insecta, Diptera).

Midgut epithelial cells of male and female Aedes aegypti, 3 days after emergence, were compared morphometrically. The results of the present investigation concerning the female, are in good agreement with those of a previous study (Hecker et al., 1974), demonstrating that morphometric investigation of midgut epithelia in A. aegypti can successfully be reproduced, and that the mosquito strain used did not show quantitative morphological changes due to laboratory rearing. In males, the cells of the anterior (A) and posterior part (P, 'stomach') of the midgut differ in their quantitative composition. Higher values are found for the microvilli and for the basal labyrinth in the A-part. On the other hand a higher volume density of the mitochondria is present in the P-part. No significant differences are found in the A-part between males and females. Significant differences, however, are present in the P-part. Distinctly more rer in the female stomach can be correlated with the synthesis of enzymes for blood digestion, which are absent in the male. In addition, the more complex functions of the female P-part are also reflected by higher values for other organelles and membrane systems (e.g. mitochondria, basal labyrinth).

Functional morphology of the midgut of a sandfly as compared to other hematophagous nematocera.

The midgut epithelium of female Lutzomyia longipalpis was investigated by means of electron microscopic morphometry before and during blood digestion. Ultrastructure and cytological changes of the stomach cells upon blood feeding were generally similar to the ones described for Phlebotomus longipes (Gemetchu, 1974) and for mosquitoes (Hecker, 1977). In addition, the quantitative composition of the cells resembled the one of mosquitoes in many respects. Despite some morphological differences in the functional gut cytology, it can be admitted that, in general, digestive processes may run similarly in the midguts of sandflies and mosquitoes.

Transport of peroxidase through the midgut epithelium of Glossina M. Morsitans (diptera, glossinidae).

The peritrophic membrane (pm) of teneral female tsetse flies, Glossina morsitans morsitans, did not extend to the full length of the midgut 1-12 hr after emergence. The ingested blood did not reach the posterior part of the midgut (p-part), and the crop still contained food 12 hr after feeding. In these flies, the p-part contained the remains of the larval gut, the meconium, and bacteria. Ferritin molecules fed to tsetse females together with human serum were only found in the endoperitrophic space of the gut. This electron-dense tracer did not penetrate and cross the pm. On the other hand, ingested peroxidase passed the pm, and was transported through intercellular clefts, the basal labyrinth and the basal lamina to the hemolymph. This uptake was observed in the anterior part and to a smaller extent in the middle part of the midgut within 2 hr after feeding. Peroxidase was incorporated from the hemolymph into fat body cells, where it was found 2 hr and later after feeding. Pinocytosis of the tracer molecules, as an additional intracellular pathway to the intercellular route of transport, could not be demonstrated.

The role of the mosquito peritrophic membrane in bloodmeal digestion and infectivity of Plasmodium species.

Secretion and luminal formation of the peritrophic membrane (PM) were induced in female Anopheles stephensi and Aedes aegypti by feeding the mosquitoes on a warmed suspension of latex particles in Ringer's solution. The PM in A. stephensi was produced from apical secretion vesicles stored in the midgut epithelial cells and secreted into the lumen during feeding. In A. aegypti, the PM was formed de novo. When the latex feeding was followed 24 hr later by a meal of lyophilized pig blood, the 2 mosquito species exhibited very different modifications to their PM structure; in A. stephensi no PM was formed around the blood meal, whereas de novo synthesis of the PM in A. aegypti continued during the blood meal, with the resulting PM greatly thickened compared to the normal feeding. This artificial induction of PM formation was used as the basis to study the role of the PM in blood meal digestion and in infectivity of mosquitoes by the appropriate species of Plasmodium. The feeding of a latex suspension alone had no stimulatory effect on the 2 major midgut proteases, trypsin and aminopeptidase, in either species. After a blood meal alone, proteases rose to maximum activity at 30 hr and 24 hr after feeding in A. stephensi and A. aegypti, respectively. After double feeding, protease activities in both species were almost identical to those in blood-fed mosquitoes. Neither the absence of a PM (in A. stephensi) nor the presence of a thickened PM (in A. aegypti), therefore, has any effect on the ability of mosquitoes to digest a blood meal. Malaria infectivity, measured by oocyst counts, also was compared after normal and double feeding using infective blood meals. Infectivity of A. stephensi by Plasmodium berghei was unaffected by the presence or absence of the PM. The thickened PM produced by double feeding in A. aegypti caused a reduction of midgut infectivity by Plasmodium gallinaceum. These results suggest that the PM may act as a partial, but not an absolute, barrier to invasion of the midgut by the ookinete.