Assessments of the transmission of Onchocerca volvulus by Simulium sanctipauli in the Upper Denkyira District, Ghana, and the intermittent disappearance of the vector.

Following studies on the transmission of Onchocerca volvulus (Leuckart) by Simulium sanctipauli Vajime & Dunbar (Diptera, Simuliidae) in Upper Denkyira District in Ghana in 2001 and 2002 (Kutin et al., Med Vet Ent 18:167-173, 2004), further assessments were carried out in 2006 and 2013/2014 to determine whether transmission parameters had changed since community-directed ivermectin treatment (CDTI) began in 1999. There were no marked changes of the transmission intensities in 2006. Only slight, but non-significant, reductions were observed in infection rates of parous flies with larval stages (L1-L3) of O. volvulus from 44.1 % (of 1672 parous flies) in 2001/2002 to 42.1 % (506) in 2006 and from 6.5 to 5.9 % of flies carrying infective larvae in their heads. This suggested that there was an ongoing transmission in the area and the parasite reservoir in the human population was still high. Unexpectedly, further assessments conducted in October 2013 and March and October 2014 revealed that the vector S. sanctipauli had apparently disappeared and transmission had ceased, probably as a result of intensified gold mining activities along the rivers Ofin and Pra. The water of both rivers was extremely turbid, heavily loaded with suspended solids, probably preventing the development of blackfly larvae. Some breeding and biting of Simulium yahense Vajime & Dunbar was observed in a small tributary of the Pra, the Okumayemfuo, which is not affected by gold mining. However, the infection rate of flies was low, only 3.7 % of 163 parous flies were infected with first stage (L1) larvae of O. volvulus.

The sandflies (Diptera: Psychodidae, Phlebotominae) in military camps in northern Afghanistan (2007-2009), as identified by morphology and DNA 'barcoding'.

As part of a continuous, standardized programme of monitoring the Leishmania vectors in German military camps in northern Afghanistan between 2007 and 2009, a detailed taxonomic analysis of the endemic sandfly fauna, as sampled using light and odour-baited traps, was conducted. Of the 10 sandfly species that were recorded, six may serve as enzootic and/or zooanthroponotic vectors of parasites causing human leishmaniasis. The use of a simple DNA-'barcoding' technique based on the mitochondrial cyt b gene, to identify the collected sandflies to species level, revealed (1) a clear discrimination between the potential vector species, (2) clustering of species within most subgenera, and (3) particularly high heterogeneity within the subgenus Paraphlebotomus (Phlebotomus alexandri being grouped with Ph. papatasi rather than with other Paraphlebotomus species). The data also indicate a high level of genetic heterogeneity within the subgenus Sergentomyia but close similarity between Sergentomyia sintoni and Sergentomyia murgabiensis. The morphological similarity of many medically important sandflies can make species identification difficult, if not impossible. The new DNA-barcoding techniques may provide powerful discriminatory tools in the future.

A guide to the Simulium damnosum complex (Diptera: Simuliidae) in Nigeria, with a cytotaxonomic key for the identification of the sibling species.

Although approximately 40% of all the people blinded by Onchocerca volvulus are Nigerians, almost nothing was known about the various cytospecies of the blackfly vectors present in Nigeria until 1981. The activation of the Nigerian National Onchocerciasis Control Programme in 1986 (and that programme's initiation of mass distributions of ivermectin in 1991) provided a significant stimulus to understand the biology of the Nigerian vectors but the exploration of any possible differences between the cytospecies has been hampered by a lack of accessible taxonomic information. This review attempts to satisfy that need. There are nine different cytoforms reliably recorded from Nigeria (Simulium damnosum s.s. Nile form, S. damnosum s.s. Volta form, S. sirbanum Sirba form, S. sirbanum Sudanense form, S. soubrense Beffa form, S. squamosum A, S. squamosum B, S. squamosum C and S. yahense typical form), and three more are known from surrounding countries and might be reasonably expected to occur in Nigeria. All of these cytospecies are presumed to be vectors, although there have been almost no identifications of the vectors of O. volvulus in Nigeria. The biogeographical distribution of the cytoforms is broadly similar to that known in other parts of West Africa (although many of the cytoforms remain insufficiently studied). The physico-chemical hydrology of the Nigerian breeding sites of the cytospecies does not, however, correspond to that seen elsewhere in West Africa, and it is not clear whether this might be related to differences in the cytoforms. An illustrated cytotaxonomic key is presented to facilitate and encourage future studies.

Construction and characterisation of a BAC library made from field specimens of the onchocerciasis vector Simulium squamosum (Diptera: Simuliidae).

A Bacterial Artificial Chromosome (BAC) library was made from wild-caught Simulium squamosum, which is an important vector of human onchocerciasis. The library is composed of 12,288 BACs, with an average insert size of 128 kb, and is expected to contain ~1.54 GB of cloned DNA. Random BAC-end sequencing generated over 95 kb of DNA sequence data from which putative S. squamosum gene sequences and novel repetitive DNA families were identified, including DNA transposons, retrotransposons and simple sequence repeats (SSRs). The sequence survey also provided evidence of DNA of microbial origin, and dissection of sample blackflies indicated that some of those used to prepare the library were likely to be parasitized by the mermithid Isomermis lairdi. Hybridisations with a set of three independent blackfly single-copy genes and two Wolbachia genes suggest that the library provides around 13-fold coverage of the S. squamosum genome and about 12-fold coverage of its Wolbachia endosymbiont.

An 18S ribosomal DNA barcode for the study of Isomermis lairdi, a parasite of the blackfly Simulium damnosum s.l.

The mermithid parasite, Isomermis lairdi Mondet, Poinar & Bernadou (Nematoda: Mermithidae), is known to have a major impact on populations of Simulium damnosum s.l. Theobald (Diptera: Simuliidae) and on their efficiency as vectors of Onchocerca volvulus (Leuckart) (Nematoda: Filarioidea). However, the value of I. lairdi and other mermithid parasites as potential means of integrated vector control has not been fully realized. This is partly because traditional taxonomic approaches have been insufficient for describing and analysing important aspects of their biology and host range. In total, rDNA barcode sequences have been obtained from over 70 I. lairdi mermithids found parasitizing S. damnosum s.l. larvae in three different rivers. No two sequences were found to vary by more than 0.5%, and cytospecies identification of mermithid hosts revealed that I. lairdi with identical rDNA barcodes can parasitize multiple cytoforms of the S. damnosum complex, including S. squamosum (Enderlein). Phylogenetic analysis using a partial sequence from the 18S ribosomal DNA barcode, grouped I. lairdi in a monophyletic group with Gastromermis viridis Welch (Nematoda: Mermithidae) and Isomermis wisconsinensis Welch (Nematoda: Mermithidae).

Taxonomy and inventory of the cytospecies and cytotypes of the Simulium damnosum complex (Diptera: Simuliidae) in relation to onchocerciasis.

We provide an inventory of all named cytoforms of the Simulium damnosum complex (including those which are now considered invalid), along with all inversions that have been recorded (including synonyms and homonyms). There are 55 valid and distinct cytoforms known from the S. damnosum complex making it the largest sibling species complex of any vectors, and probably of any insect or other animal. All cytoforms are listed along with their fixed and diagnostic inversions and country distribution. There are 183 inversions known from the complex as a whole, of which 49% are fixed and/or diagnostic between cytoforms, and the fixed/diagnostic inversions seem to occur disproportionately on chromosome arm 2L.

Cloning and characterization of a species-specific repetitive DNA sequence from Onchocerca armillata.

Two clones, pOA1 and pOA5, have been isolated from a genomic DNA library prepared from pools of Onchocerca armillata adults in the plasmid vector pUC12. In dot-blot hybridisations, these two clones do not cross-hybridise significantly with total genomic DNA from O. volvulus, O. gutturosa, O. ochengi, O. gibsoni, O. lienalis, bovine, human, Culicoides nubeculosus, Simulium species or Brugia pahangi, but do hybridise with as little as 100 pg of DNA from two separate geographic isolates of O. armillata. The sequence of pOA1 and pOA5 has been determined and found to contain a repetitive DNA sequence 147 bp in length. These clones can be used as specific and sensitive DNA probes for the identification of O. armillata capable of identifying a single L3 larva.

Regional genetic variation in the major sperm protein genes of Onchocerca volvulus and Mansonella ozzardi (Nematoda: Filarioidea).

Onchocerca volvulus and Mansonella ozzardi are two human filarial parasites present in South and Central America. In the Brazilian Amazonia they are found in sympatry, and the lack of clear morphological diagnostic characters in the microfilariae hinders their identification. The major sperm protein (MSP) gene of both species has been sequenced and characterised to determine its potential as a molecular diagnostic character. The length of the MSP gene is different in each species, and this could be used to detect and differentiate them by running the polymerase chain reaction (PCR) product in an agarose gel. Two major gene groups were identified in O. volvulus with a genetic distance of 6% between them. In M. ozzardi only one major group of genes was observed. The high similarity between the protein amino acid sequence of both filarial species confirms that the MSP has been highly conserved through nematode evolution.

Characterisation of nuclear ribosomal DNA sequences from Onchocerca volvulus and Mansonella ozzardi (Nematoda: Filarioidea) and development of a PCR-based method for their detection in skin biopsies.

The internal transcribed spacer region (ITS1, 5.8S gene and ITS2) of the two filarial nematodes Onchocerca volvulus and Mansonella ozzardi was sequenced, and two species-specific primers designed in the ITS2 to develop a PCR-based method for their specific detection and differentiation. When used with a universal reverse primer, the two species-specific primers gave amplification products of different size, which were readily separated in an agarose gel. The PCR was tested on skin biopsies from 51 people from three localities in Brazil where M. ozzardi is present, and results have been compared with those of parasitological examination of blood. The species-specific PCR gave a higher percentage of detection of infection by M. ozzardi than the parasitological examination of blood. No infection with O. volvulus was detected by PCR. This PCR-based assay may assist in determining the nature of infection in areas where both filarial species exist in sympatry.

Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the semi-nested multiplex malaria PCR (SnM-PCR).

A simplified protocol for the identification of Plasmodium species by semi-nested multiplex polymerase chain reaction (SnM-PCR) in human blood samples is compared with microscopical examination of thin and thick blood films in 2 field trials in Côte d'Ivoire and Cameroon. Also, dried blood spots or liquid blood collected from Dutch soldiers returning from Goma, Zaire (n = 141), Angola (n = 40), and from Marechaussee (Dutch border police) returning from various parts of the world (n = 161) were examined, together with miscellaneous other material obtained from laboratories and hospitals. The method is based on features of the small subunit nuclear ribosomal ribonucleic acid (RNA) gene (ssrDNA), a multicopy gene which possesses both highly conserved domains and domains characteristic for each of the 4 human malaria parasites. The first reaction of the SnM-PCR includes a universal reverse primer with 2 forward primers specific for Plasmodium and mammals, respectively. The mammalian-specific primer was included as a positive control to distinguish uninfected cases from simple PCR failures. The second PCR reaction includes a Plasmodium-specific forward primer plus species-specific reverse primers for P. vivax, P. ovale, P. falciparum and P. malariae. The technique worked better with samples collected in the field as dried blood spots on filter paper and heparinized blood rather than with frozen pelleted blood; it was more sensitive and more specific than the standard microscopical examination.

Discovery of a new focus of human onchocerciasis in central Brazil.

An autochthonous case of human onchocerciasis was reported 13 years ago in the town of Minaçu, northern Goiás (Brazil), but a subsequent survey of the population using the traditional technique of examining skin biopsies with the light microscope failed to detect other cases. Recent surveys using more sensitive diagnostic techniques (serodiagnosis, DNA probes, Mazzotti test) that are detailed in this paper revealed the presence of other cases of the disease in Minaçu, the nearby town of Formoso and at the Buracão gold mine near Paranã. The data show that transmission of the disease has occurred to local people living in town and on farms and that gold miners (garimpeiros) are a likely source of infection.

Deforestation and the spatio-temporal distribution of savannah and forest members of the Simulium damnosum complex in southern Ghana and south-western Togo.

Spatio-temporal data on cytotaxonomic identifications of larvae of different members of the Simulium damnosum complex collected from rivers in southern Ghana and south-western Togo from 1975 until 1997 were analysed. When the data were combined, the percentages of savannah blackflies (S. damnosum sensu stricto and S. sirbanum) in the samples were shown to have been progressively increasing since 1975. The increases were statistically significant (P < 0.001), but the rates of increase were not linear. Further analyses were conducted according to the collection seasons and locations of the samples, to account for possible biases such as savannah flies occurring further south in the dry season or a preponderance of later samples from northern rivers having more savannah flies. These analyses showed that the increasing trend was statistically significant (P < 0.0001) only during the periods April to June and October to December. The presence of adult savannah flies carrying infective larvae (L3) indistinguishable from those of Onchocerca volvulus in the study zone was confirmed by examinations of captured flies. The percentages of savannah flies amongst the human-biting populations and the percentages with L3s in the head were higher during dry seasons than wet seasons and the savannah species were found furthest south (5 degrees 25'N) in the dry season. Comparisons of satellite images taken in 1973 and 1990 over a study area in south-western Ghana encompassing stretches of the Tano and Bia rivers demonstrated that there have been substantial increases in urban and savannah areas, at the expense of forest. This was so not only for the whole images but also for subsamples of the images taken at 1, 2, 4, 8 and 16 km distant from sites alongside the River Tano. At every distance from the river, the percentages of pixels classified as urban or savannah have increased in 1990 compared with 1973, while those classified as degraded or dense forest have decreased. The possibility that the proportionate increases in savannah forms of the vectors of onchocerciasis, and hence in the likelihood of the transmission of savannah strains of the disease in formerly forested areas, were related to the decreases in forest cover is discussed.

Behavioral assessment of visual deficits in the taiep mutant.

Taiep (tremor, ataxia, immobility, epilepsy, paralysis) mutants show a significant increase in myelin thickness from 10 to 30 days of age but then demonstrate a decrease in myelin thickness from 1 to 6 months. The severity of the demyelination in the optic nerve suggests that visual deficits may exist in the taiep mutants. Animals were trained on a discrimination task, in which responses to a light stimulus (the SD period) were reinforced on a fixed ratio (FR)-1 schedule, and responses in the absence of the light stimulus (the SΔ period) were not reinforced. Following training, the light intensity presented during the SD period was gradually reduced between sessions until -6.0 candela/m2 was reached. Both groups of animals - taiep mutants and control Sprague Dawley rats - successfully recognized and responded in the presence of the stimulus near perfectly by the final day of training, suggesting that taiep mutants demonstrated normal learning, at least under this paradigm. Despite the severe demyelination of the taiep optic nerve, no visual deficits were detected as both groups of animals performed similarly as the light intensity decreased. Though the myelin loss of the optic nerve may have negatively affected signal transduction, this did not result in an increase in visual threshold.

Investigations into the isolation of the Tukuyu focus of onchocerciasis (Tanzania) from S. damnosum s.l. vector re-invasion.

As part of the feasibility study for an onchocerciasis vector elimination project we investigated the isolation of the Tukuyu focus in Tanzania from possible vector re-invasion. This was achieved by examining the distribution of the Simulium damnosum complex vector cytospecies outside the focus to look for potential sources of re-invasion. Besides cytotaxonomic identifications of the aquatic stages, we applied morphotaxonomic and molecular techniques to identify S. thyolense and confirm it as the anthropophilic species in both the Tukuyu and the neighbouring Ruvuma foci. We detected significant differences in chromosome inversion frequencies between the Tukuyu populations and those breeding to the southwest in the adjacent Songwe river basin and in northern Malawi (where there is no man-biting and no onchocerciasis), suggesting that there is not normally a great deal of migration in either direction. By contrast, populations of S. thyolense from the Tukuyu and Ruvuma foci (150km southeast of Tukuyu) were much more similar in terms of their chromosomal polymorphisms, indicating a higher possibility of re-invasion, although migration is still restricted to some extent, as indicated by some differences in chromosome polymorphisms between the two foci. Future migratory events which might be associated with vector control operations can be monitored by vector cytospecies identification, the frequency of polymorphic inversions which characterise the different vector populations, and the identification of accompanying non-vector cytospecies (e.g. S. plumbeum and cytotype Kasyabone occur exclusively in the two foci, and hence their re-appearance in Tukuyu could have only one outside source). The morphology of the scutal pattern of neonate males may act as a quick test for vector species identification where chromosome squashes are unavailable.

Phylogenetically distinct Wolbachia gene and pseudogene sequences obtained from the African onchocerciasis vector Simulium squamosum.

Wolbachia are intracellular bacteria mostly found in a diverse range of arthropods and filarial nematodes. They have been classified into seven distinct 'supergroups' and other lineages on the basis of molecular phylogenetics. The arthropod-infecting Wolbachia are usually regarded as reproductive parasites because they manipulate their host species' sexing system to enhance their own spread, and this has led to their investigation as potential agents of genetic control in medical entomology. We report 12 partial Wolbachia gene sequences from: aspC, aspS, dnaA, fbpA, ftsZ, GroEL, hcpA, IDA, rpoB, rpe, TopI and wsp as well as a single ftsZ pseudogene sequence, which have all been PCR-amplified from Simulium squamosum (Diptera: Simuliidae). To our knowledge this is the first such report from Simuliidae. Uninterrupted open-reading frame sequences were obtained from all 12 genes, covering approximately 6.2kb of unique DNA sequence. Phylogenetic analyses with the different coding genes gave consistent results suggesting that the Wolbachia sequences obtained here do not derive from any of the known Wolbachia supergroups or lineages. Consistent with a unique genetic status for the S. squamosumWolbachia, the hypervariable regions of the Wolbachia-specific wsp gene were distinct from all previous records in both sequence and length. As well as potential implications for newly emerging Wolbachia-based disease control methods, the results may be relevant to some problems experienced in the laboratory colonisation of Simulium damnosum sensu lato and why it is such a diverse species complex.

Laser-assisted microdissection for the study of the ecology of parasites in their hosts.

The population biology of internal parasites is difficult to study because the adult parasites are often inaccessible, deep within the host's body. Developing stages, such as eggs in the faeces or larvae in the skin are more easily obtained, but are difficult to handle because they are often very small and with a tough cuticle. This has limited their use in molecular ecology for estimating population biology parameters of the adults (their parents). We have used Onchocerca ochengi (a filarial nematode parasite of cattle) to describe a novel and generally applicable method of easily and conveniently isolating individual larvae (microfilariae) from the host using laser-assisted microdissection. Furthermore, we have been able to improve the isolation of DNA by using the laser to bisect the larva to release DNA from the tissues enclosed within the parasite cuticle, and in this way we have achieved amplification of fragments over 1400 bp, and routinely PCR-amplified single-copy sequences from 5% of the DNA from a single larva (the equivalent of approximately 15 nuclei), and regularly from 0.5%.

The elimination of the vector Simulium neavei from the Itwara onchocerciasis focus in Uganda by ground larviciding.

The Itwara focus of onchocerciasis covers an area of approximately 600 km(2) in western Uganda about 20 km north of Fort Portal. The vector is Simulium neavei, whose larvae and pupae live in a phoretic association on freshwater crabs. The phoretic host in the Itwara focus is the crab Potamonautes aloysiisabaudiae. Before any onchocerciasis control, ATPs were estimated to reach between 4500 and 6500 infective larvae per person per year. S. neavei was found to be a very efficient vector with 40% of parous flies harbouring developing larvae of Onchocerca volvulus. After 4 years of community-based distribution of ivermectin transmission was still considerable and in 1995 monthly treatment of streams with the larvicide temephos commenced in the first of three sub-foci, and was gradually extended to the whole focus. Biting S. neavei disappeared from the first sub-focus (Itwara main) in June 1996, and the last infested crab was caught in November 1996. In the second sub-focus (Siisa) treatment commenced towards the end of 1995, and the last biting fly was caught in March 1997, but a deterioration in the security situation interrupted the programme (after only three treatments in the third sub-focus). Monthly treatments restarted in the second and third sub-foci (Aswa) in September 1998, and when the situation was reassessed in 2003 no biting flies were found anywhere, and the flies had not reinvaded the first sub-focus, but infected crabs were found in the second and third sub-foci. The last treatments were carried out in April-June 2003, and since then no infested crabs have been found. In summary, no S. neavei-infested crabs have been found anywhere in the focus since June 2003 and the vector is considered eliminated from that date. However, transmission had already been halted since February 2001, when the last biting flies had been collected. The parasite reservoir should die out in the human population by 2016.