Mady Ndiaye

One-step RT-PCR for detection of Zika virus

BACKGROUND Zika virus (ZIKV) is an emerging mosquito-borne flavivirus circulating in Asia and Africa. Human infection induces an influenza-like syndrome that is associated with retro-orbital pain, oedema, lymphadenopathy, or diarrhea. Diagnosis of Zika fever requires virus isolation and serology, which are time consuming or cross-reactive. OBJECTIVE To develop a one-step RT-PCR assay to detect ZIKV in human serum. STUDY DESIGN An assay targeting the envelope protein coding region was designed and evaluated for its specificity, detection limit, repeatability, and capacity to detect ZIKV isolates collected over a 40-year period from various African countries and hosts. RESULTS The assays detection limit and repeatability were respectively 7.7pfu/reaction and 100% in serum and L-15 medium; none of 19 other flaviviruses tested were detected. CONCLUSIONS The assay is rapid, sensitive, and specific to detect ZIKV in cell culture or serum, but needs to be validated for diagnosis using clinical samples.

Gene Flow, Subspecies Composition, and Dengue Virus-2 Susceptibility among Aedes aegypti Collections in Senegal

Background Aedes aegypti, the “yellow fever mosquito”, is the primary vector to humans of the four serotypes of dengue viruses (DENV1-4) and yellow fever virus (YFV) and is a known vector of Chikungunya virus. There are two recognized subspecies of Ae. aegypti sensu latu (s.l.): the presumed ancestral form, Ae. aegypti formosus (Aaf), a primarily sylvan mosquito in sub-Saharan Africa, and Ae. aegypti aegypti (Aaa), found globally in tropical and subtropical regions typically in association with humans. The designation of Ae. aegypti s.l. subspecies arose from observations made in East Africa in the late 1950s that the frequency of pale “forms” of Ae. aegypti was higher in populations in and around human dwellings than in those of the nearby bush. But few studies have been made of Ae. aegypti s.l. in West Africa. To address this deficiency we have been studying the population genetics, subspecies composition and vector competence for DENV-2 of Ae. aegypti s.l. in Senegal. Methods and Findings A population genetic analysis of gene flow was conducted among 1,040 Aedes aegypti s.l. from 19 collections distributed across the five phytogeographic regions of Senegal. Adults lacking pale scales on their first abdominal tergite were classified as Aedes aegypti formosus (Aaf) following the original description of the subspecies and the remainder were classified as Aedes aegypti aegypti (Aaa). There was a clear northwest–southeast cline in the abundance of Aaa and Aaf. Collections from the northern Sahelian region contained only Aaa while southern Forest gallery collections contained only Aaf. The two subspecies occurred in sympatry in four collections north of the Gambia in the central Savannah region and Aaa was a minor component of two collections from the Forest gallery area. Mosquitoes from 11 collections were orally challenged with DENV-2 virus. In agreement with the early literature, Aaf had significantly lower vector competence than Aaa. Among pure Aaa collections, the disseminated infection rate (DIR) was 73.9% with a midgut infection barrier (MIB) rate of 6.8%, and a midgut escape barrier (MEB) rate of 19.3%, while among pure Aaf collections, DIR = 34.2%, MIB rate = 7.4%, and MEB rate = 58.4%. Allele and genotype frequencies were analyzed at 11 nuclear single nucleotide polymorphism (SNP) loci using allele specific PCR and melting curve analysis. In agreement with a published isozyme gene flow study in Senegal, only a small and statistically insignificant percentage of the variance in allele frequencies was associated with subspecies. Conclusions These results add to our understanding of the global phylogeny of Aedes aegypti s.l., suggesting that West African Aaa and Aaf are monophyletic and that Aaa evolved in West Africa from an Aaf ancestor.

Toxic effects of neem products ( Azadirachta indica A. Juss) on Aedes aegypti Linnaeus 1762 larvae

Treatment and comparative analysis of the properties of aqueous extracts of seed kernel of Azadirachta indica A. Juss (neem) was carried out on Aedes aegypti larvae. The aim of this work was to evaluate lethal effects of neem products (1% Suneem, formulated neem oil and neem powder) on A. aegypti larvae. Assays showed that A. indica was toxic to larvae of A. aegypti. For 1% Suneem, 1% formulated neem oil and neem powder, the lethal concentrations and lethal time at 50% (LC50 and LT50) for A. aegypti were 2 and 8 mg/l after 24 h and 3 mg/l after 120 h, respectively. Assays showed that Suneem and Formulated neem oil were more toxic to A. aegypti than Neem powder. Both products of the neem (A. indica, A. juss) have a remarkable influence on the development of A. aegypti larvae, causing an inhibition of nymphs and adults emergency. The Histopathological results revealed a serious damage on the epithelial columnar cells, a perturbation of alimentary flow, slightly hypertrophied cells, a beginning of vacuolisation on apical level, and a bursting of some cells in posterior part of the gut. However, nuclei, adipose tissue and muscles seem to keep normal appearance.

Survey of Anaplasmataceae bacteria in sheep from Senegal

PurposeThe authors studied the role of bacteria belonging to Anaplasmataceae family as the causes of acute illnesses of sheep in West Africa.MethodsWe examined and sampled 120 febrile sheep in two regions of Senegal for this study. The DNA extracted from these blood samples was tested by PCR using two pairs of primers (groEL-based and 16S rRNA gene-based).ResultsIn 52/120 samples, the microscopic examination revealed intraerythrocytic and/or intraphagocytic spherical inclusions. In 48/52 cases, we succeeded in identifying the bacterial agent: in 38 cases, it was Anaplasma ovis; in six cases, it was Ehrlichia ruminantium; in two cases, Anaplasma phagocytophilum; in one case, Anaplasma platys; and in one case, a yet uncultured Anaplasma sp. closely related to A. phagocytophilum.ConclusionsOur studies demonstrated the great variety of pathogenic bacteria from the Anaplasmataceae family in the blood of clinically ill sheep. A. ovis was identified unexpectedly often. For the first time, A. phagocytophilum was found in sub-Saharan Africa, and its further epidemiology may be now reconsidered. The roles of canine pathogen, A. platys, and yet undescribed Anaplasma sp. “Badiouré” in ovine pathology should be more closely studied.

Pathogenicity of the Fungus, Aspergillus clavatus, Isolated from the Locust, Oedaleus senegalensis, Against Larvae of the Mosquitoes Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus

Abstract The use of insect pathogenic fungi is a promising alternative to chemical control against mosquitoes. Among the Hyphomycetes isolated from insects for mosquito control, the genus Aspergillus remains the least studied. In September 2005, four fungi were isolated from the Senegalese locust, Oedaleus senegalensis Kraus (Orthoptera: Acrididae), collected in Dakar, Senegal. One of these fungi, identified as Aspergillus clavatus, Desmazières (Eurotiales: Trichocomaceae) was highly pathogenic against larvae of the mosquitoes Aedes aegypti L., Anopheles gambiae s.l. Giles and Culex quinquefasciatus Say (Diptera: Culicidae). An application of 1.2 mg/ml dry conidia yielded 100% mortality after 24 hours against both Ae. aegypti and Cx. quinquefasciatus while with An. gambiae it was 95%. With unidentified species in the genus Aspergillus, mortality after 24 h was <5% against all the larval species. Application of A. clavatus produced in a wheat powder medium using doses ranging between 4.3 to 21×107 spores/ml, caused 11 to 68% mortality against Cx. quinquefasciatus at 24h, and 37 to 100% against Ae. aegypti. Microscopic observations showed fungal germination on both Ae. aegypti and Cx. quinquefasciatus larvae. Histological studies revealed that A. clavatus penetrated the cuticle, invaded the gut and disintegrated its cells. Some Cx. quinquefasciatus larvae, treated with A. clavatus reached the pupal stage and produced infected adults. However, the infection was mainly located on the extremity of their abdomen. These results suggest that A. clavatus could be an effective tool to manage mosquito proliferation.

Aedes Species in Treeholes and Fruit Husks between Dry and Wet Seasons in Southeastern Senegal

ABSTRACT: During the dry season in February, 2010 and the wet season in September, 2011 we sampled mosquito larvae and eggs from treeholes of seven native hardwood species and the husks of Saba senegalensis in 18 sites in the PK-10 forest in southeastern Senegal. Larvae were reared to adults for species identification. In the dry season, we recovered 408 Aedes mosquitoes belonging to seven species. Aedes aegypti s.l. comprised 42.4% of the collection, followed by Ae. unilineatus (39%). In contrast to reports from East Africa, both Ae. aegypti aegypti and Ae. aegypti formosus were recovered, suggesting that both subspecies survive the dry season in natural larval habitats in West Africa. In the wet season, 455 mosquitoes were collected but 310 (68.1%) were the facultatively predaceous mosquito Eretmapodites chrysogaster. The remaining 145 mosquitoes consisted of ten Aedes species. Aedes aegypti s.l. comprised 55.1% of these, followed by Ae. apicoargenteus (15.2%) and Ae. cozi (11.7%). Similar to East Africa, most (90%) of Ae. aegypti s.l. in the wet season were subspecies formosus.

MATURATION OF MOSQUITO SPERMATOZOA DURING THEIR TRANSIT THROUGHOUT THE MALE AND FEMALE REPRODUCTIVE SYSTEMS

The testes, seminal vesicles and spermathecae of 22 species of mosquitoes belonging to the genera Aedes, Anopheles, Culex, Mansonia and Toxorhynchites are investigated under the electron microscope. Modifications of the acrosome and sperm wall occur during the transit of the spermatozoon from the lower region of the testes to the spermathecae throughout the seminal vesicles. The origin and fate of the cell coat and the possible roles of somatic cell layers both in the testes and the seminal vesicles are discussed.

Identification du virus Nkolbisson par microscopie électronique

Summary Electron microscopy examination revealed that Nkolbisson virus is a Rhabdoviridae . The virus was isolated in Cameroun and the Ivory Coast from different Culicidae species, some of which are anthropophilic. Recently the Nkolbisson virus was obtained in the Central African Republic from human serum.

Production of two entomopathogenic Aspergillus species and insecticidal activity against the mosquito Culex quinquefasciatus compared to Metarhizium anisopliae

ABSTRACT The spore productivity and insecticidal activity of two opportunistic insect pathogenic Aspergillus species (namely: Aspergillus clavatus Desmazieres and Aspergillus flavus Link (Ascomycota: Eurotiales, Trichocomaceae)) were compared to Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin (Ascomycota: Hypocreales, Clavicipitaceae) for mosquito (Diptera: Culicidae) control. The production of aerial spores on wheat bran and white rice was investigated in solid-, semi-solid-, and liquid-state media supplemented with a nutritive solution. Wheat bran-based media increased the spore yield in solid-state from three to sevenfold: A. clavatus produced 48.4 ± 5.2 and 15.7 ± 1.6 × 108 spores/g, A. flavus produced 22.3 ± 4.1 and 3.1 ± 2.5 × 108 spores/g, and M. anisopliae produced 39.6 ± 6.5 and 13.1 ± 2.6 × 108 spores/g of wheat bran or white rice, respectively. A. clavatus, A. flavus and M. anisopliae spores harvested from wheat bran-based solid-state media showed lethal concentrations (LC50) of 1.1, 1.8, and 1.3 × 108 spores/ml against Culex quinquefasciatus Say larvae in 72 h. Because A. clavatus and M. anisopliae displayed similar features when cultured under these conditions, our results suggest that insect pathogenic Aspergillus species may be as productive and virulent against mosquito larvae as a well-recognised entomopathogenic fungus.

Borrelia infection in small mammals in West Africa and its relationship with tick occurrence inside burrows.

Tick-borne relapsing fever (TBRF) is a zoonotic disease caused by several Borrelia species transmitted to humans by Ornithodoros tick vectors. In West Africa, Borrelia crocidurae is a common cause of disease in many rural populations. Small mammals act as reservoirs of infection. We report here the results of surveys that investigated the occurrence of B. crocidurae infection in rodents and insectivores from eight countries of West and Central Africa. Animals were identified at the species level and tested for Borrelia either by examination of thick blood film, intra-peritoneal inoculation of blood or brain tissues into laboratory mice, or by molecular techniques. A total of 4358 small mammals belonging to 38 species and 7 families were collected, including 3225 specimens collected in areas where the occurrence of Ornithodoros sonrai tick in rodent burrows was documented, and 1133 in areas where this tick was absent. In areas with O. sonrai, Borrelia infection was demonstrated in 287 of 3109 (9.2%) small mammals tested, and none was documented in 1004 animals tested from other areas. There was no relationship between the occurrence of Rhipicephalus, Hyaloma and Argas ticks in burrows and the distribution of Borrelia infection in small mammals. The 287 specimens infected by Borrelia belonged to 15 rodent and shrew species, including three Saharo-Sahelian species - Gerbillus gerbillus, Gerbillus occiduus and Gerbillus tarabuli - identified as reservoirs for TBRF with a distribution restricted to this area. In Sudan and Sudano-Sahelian areas, Arvicanthis niloticus, Mastomys erythroleucus and Mastomys huberti were the main reservoir of infection. Although most small mammals species collected had a large distribution in West and Central Africa, the fact that only animals collected in areas with O. sonrai were found infected suggest that this tick is the only vector of TBRF in rodents and insectivores in this part of Africa.