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This qualitative study utilized video games and virtual reality in an eighth-grade public middle school media literacy elective classroom in which the head researcher was also the official teacher for the students in the study. The nineteen students and their teacher used video games in small groups for five consecutive weeks. The teacher and nineteen students recorded data about their thoughts, experiences, and learning as they played in the classroom. Students responded to open-ended reader-response questions about their experiences after playing video games each day for forty-five minutes. Students reflected about their experiences in a thought journal. The teacher responded to all the students’ thought journals each day, and after the halfway point of the study, students responded to each other’s thought journals. Students also co-created additional reader-response questions for their peers to answer after the halfway point in the study. The teacher kept an observation log as a participant observer who sat in to play, listen, and talk with the small groups during the study. The teacher hosted five research meetings in class during the study, and students got a chance to help analyze and make sense of the data with the teacher. At the end of the study, the small groups were interviewed about their experiences. The study found that video games can act as literary vehicles for learning. When small groups responded to video games as literature using reader-response, they primarily took an aesthetic stance on their literary experiences.

The production of ornamental and vegetable crops in greenhouses and other controlled environments is not a novel concept. There are, however, new challenges that face controlled environment plant production (CEPP) professionals and the industry as a whole. Some of the issues that face the industry today include the need to conserve water and energy, strong global competition, and the lack of skilled labor. The integration of new technology and procedures in CEPP can help to mitigate most of these concerns. The European CEPP industry has already begun to implement such changes with favorable results while the American industry has suffered due to the limited access of knowledge pertaining to these advancements. This project and study was conducted to create a method of teaching that can be integrated into university undergraduate learning and industry training programs where it is generally absent. The method chosen to address this task was the creation of online multimedia teaching modules available to the public. The modules created were “Introduction to the Floriculture Industry”, “Light in the Greenhouse”, “Greenhouse Water Movement and a case study on different treatments when growing bedding and potted crops in the greenhouse. These modules include curriculum items of interest in CEPP in the form of PowerPoint presentations, vocabulary lists, and discussion and assessment questions. Completed modules may be integrated into traditional classroom teaching, used as training videos for entry- iii level industry professionals, or used individually. The modules created were presented to university undergraduate students (n=12, n=17) at The Ohio State University. Each student completed a self-assessment before and after the introduction of each module and was asked to rate their confidence on core competencies covered by the modules. The mean competency scores for students for every module increased from the pretest to the post-test. Students’ competencies increased the most with the “Greenhouse Water Movement” module and the least with the “Light in the Greenhouse” (n=17) module. The results reflect that this method of teaching with multimedia modules has the potential to enrich learning and improve education CEPP in and out of the classroom

Strongyloidiasis, caused by the gut nematode Strongyloides stercoralis, and visceral leishmaniasis (VL), caused by the protozoa Leishmania donovani and L. infantum, are two potentially fatal parasitic diseases with wide global distribution and close association with poverty. Although both infections are treatable, it is imperative to validate cure after treatment. Available diagnostics for both infections have reasonable to high sensitivity for current infection but cannot easily distinguish cure. There is a need for diagnostic tests that are rapid, simple to use and deployable in field conditions to diagnose infection and validate cure. This project aimed to identify candidate coproantigens of S. stercoralis and urine antigens of L. donovani, and to investigate the utility of IgG1 serology for determining cure versus relapse after treatment for VL. For Strongyloides coproantigen discovery, open access 'omic' data were analysed using computational tools. For Leishmania urine antigen discovery, antibodies were used to capture parasite antigens from VL urine, which were then identified by mass spectrometry. For Leishmania IgG1 serology, paired sera from preand post-treatment (cured) VL were compared with relapse sera in ELISA and with novel IgG1 specific rapid diagnostic tests (RDTs). This work identified over 40 candidate coproantigens of Strongyloides that satisfied the required characteristics. Seven L. donovani proteins were identified in VL urine, within which 22 protein sequences were indicated as having high epitope potential and specificity to L. donovani. In VL serology, IgG1 was able to differentiate between cure and relapse of VL in both ELISA and RDT assays. With development and optimisation, the candidate antigens and IgG1 assays presented here have potential to contribute to disease control for these parasitic infections. The computational method of antigen selection used here for S. stercoralis can be applied to multiple parasitic helminth infections using the wealth of open access 'omic' data.

A three-part study was conducted on the anatomy of Notropis atherinoides, type species of Notropis; a cladistic analysis of the subgenus Notropis; and an investigation of the intrageneric and intergeneric relationships of Notropis. Seventy-seven species of Notropis and 38 species of 21 other American genera were examined.N. atherinoides has several systematically useful apomorphic characters. A posterior process occurs on the basihyal, a character state shared with other members of Notropis (s.s.), Hydrophlox, the N. texanus species group (sensu Swift), N. boops, N. xaenocephalus, N. blennius, N. greenei, N. volucellus, and N. spectrunculus, which defines these and related species within the genus. Within this group the subgenus Notropis is defined by a large endopterygoid, mouth size and position, and a scale morphology characterized by a well developed notch between the posterior and lateral margins.Four species groups are recognized within the subgenus: an atherinoides group of N. atherinoides, its hypothesized sister species, N. perpallidus and N. amabilis; the shumardi group of N. shumardi, N. candidus, N. jemezanus and N. oxyrhynchus; the photogenis group of N. photogenis, N. stilbius and N. amoenus; and the ariommus group of N. ariommus, N. scepticus, N. semperasper and N. telescopus.There appears to be a trichotomy in Notropis (s.l.) among (1) those groups, Cyprinella, Luxilus, Lythrurus and Pteronotropis, that develop specialized spawning characters and have a similar scale morphology; (2) those species having the posterior basihyal projection; and (3) those species having tooth reductions, elongated kinethmoid, and a deep notch in the supraethmoids and mesethmoid. Among the latter are N. heterolepis, N. dorsalis, N. longirostris, N. maculatus, N. volucellus, N. spectrunculus and other species. Notropis volucellus and N. spectrunculus share characters with both groups two and three.No synapomorphy was found to define Notropis (s.l.). However all species examined that are now included in Notropis (except N. boucardi) have an ovate ventral opening in the floor of the posterior myodome. It is a synapomorphy shared with Hybopsis (s.s.), Erimystax, Extrarius, Pimephales, Ericymba, Hybognathus, Phenacobius and Hybopsis labrosa.

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Despite four decades of control of Chagas disease in Venezuela, domestic infestations still persist and transmission of Trypanosoma cruzi may be increasing. This is in contrast to the Southern cone region where control has successfully eliminated domestic populations of the main vector Triatoma infestans over large areas. However unlike T. infestans, the main vector in Venezuela, Rhodnius prolixus, has a widespread silvatic distribution occurring primarily in palm trees, which are a ubiquitous feature of the Venezuelan landscape. The palm tree is an important part of campensino life and is maintained for fruit, shade and for use in house construction. Control failures may be due to reinvasion of houses by these prevalent silvatic populations. However, debate exists as to whether the silvatic populations are in fact Rhodnius robustus, a related species of minor epidemiological importance, and therefore no threat to control. With an estimated 800,000 people infected with T. cruzi in Venezuela and a further 3 million at risk of infection, an effective control programme is required, which necessitates that this relationship between silvatic and domestic populations is resolved. This study was undertaken in order to (1) confirm the identity of silvatic populations of Rhodnius in Venezuela and (2) determine if domestic and silvatic populations are isolated, thus to clarify the role of silvatic populations in maintaining house infestations. To achieve these aims field collected silvatic and domestic populations of Rhodnius from 5 States were analysed by genetic methods, direct sequencing (cytochrome b, D2) and microsatellites, and by geometric morphometric analysis. A total of 551 specimens from 31 localities in six Venezuelan states were analysed by direct sequencing of cytochrome b (cytb). Results confirmed the presence of R. prolixus in both silvatic and domestic ecotopes, dispelling the belief that all silvatic populations are R.robustus. Rhodnius robustus does however occur and was found in this study in the Andean State Trujillo. Here it was limited to the silvatic environment. This project found that silvatic and domestic populations of R. prolixus are not isolated, sharing 6 haplotypes, including four silvatic haplotypes also detected in domestic nymphs, indicating that silvatic specimens are capable of domestic colonisation. This was also confirmed from the analysis of adjacent domestic and silvatic populations in Portuguesa and Barinas State where population homogeneity was detected. Additionally cytb analysis identified an introgression event between Amazonian R. robustus and R prolixus, confirmed by incongruence of cytb and D2 nuclear characterisation. Phylogenetic analysis of specimens was also undertaken. To investigate further fine scale population heterogeneity a panel of microsatellite markers, hitherto unavailable, was developed for R prolixus, using an enrichment technique. A panel of 10 loci was available for analysis following PCR screening and linkage analysis. A total of 555 specimens were analysed from 33 populations . Microsatellite analysis also detected population homogeneity between ecotopes, including adjacent populations, indicating that silvatic populations are not isolated. Population heterogeneity was greater among localities in Portuguesa than Barinas, may be due to landscape variation. Differences between control programmes may also play a role. Geometric morphometries identified shape similarity between populations across all States. However, shape convergence by ecotope was detected and results indicated that morphometries might be of limited use for the analysis of populations of R prolixus, with the exception of post-control reinvasion\recrudescent studies. The three methods did not always concur precisely. However comparison was difficult due to detected introgression and shape convergence distorting, respectively, mitochondrial and morphometric analysis. Results indicated that a combined use of microsatellites and morphometries would be beneficial in the analysis of adjacent domestic and silvatic populations. A similar pattern of a lack of isolation between silvatic and domestic ecotopes was detected by both genetic methods, with limited morphometries overlap detected; broadly all three methods showed that populations from differing ecotopes are not isolated. This is also supported by a parallel project on risk-factor analysis. From this study it is clear that silvatic populations of R prolixus present an unquestionable threat to the successful control of Chagas disease in some endemic regions of Venezuela and, unlike the Southern cone, elimination of domestic infestations may not be possible in areas where silvatic R prolixus occur. A restructuring of the control programme in Venezuela is required to deal with this silvatic threat. This may include increased vigilance and methodical respraying and the incorporation of novel approaches to deal with silvatic invasion such as use of insecticide treated curtains; ultimately more investment in the improvement of the rural rancho is required.

Triatomine bugs (Hemiptera: Reduviidae: Triatominae) are the vectors of Chagas disease in South and Central America. Chagas disease predominantly affects poor rural communities with simply constructed housing susceptible to infestation by triatomines. Chagas disease is restricted to the Americas largely due to the limited distribution of triatomine bugs. The global diversity of triatomines is -130 species, of which only -10% are known to occur outside the Americas, one species (Triatoma rubrofasciata) is tropicopolitan, and the others are concentrated on the Indian subcontinent (Linshcosteus spp. ) and adjacent south east Asian island groups (Triatoma spp. ). The main objectives of this PhD programme were to: a) assess the facility of morphometric approaches (measurement and robust statistical analysis of morphological variation) in the study of population structure of vector species with proximal domestic and silvatic distributions to detect population structure and give information on the risk of reinvasion, b) study interspecific and higher taxonomic level relationships of New World and Old World triatomine bugs. To these ends geometric morphometric analyses were conducted in concert with molecular genetic analyses of mitochondrial and nuclear DNA sequences. The principal question being: Does the relatively low cost method of morphometrics reveal patterns consistent with population structure, as otherwise determined by more expensive molecular genotyping methods? Or are such patterns disrupted by environmental effects and intraspecific convergent/divergent morphological evolution? Combined morphometrics and molecular genetics were used to study vector populations in three of the countries that continue to be most affected by Chagas disease. In Venezuela and Ecuador Rhodnius species (R. prolixus and R. ecuadoriensis respectively) were studied, in areas where they occur in both domestic and silvatic environments, and in Paraguay T. infestans from a domestic and a putative silvatic focus. Head and wing morphometrics were compared to mitochondrial DNA sequence data to assess the population structure and disparity among domestic and silvatic samples in each case. The results presented suggest that head shape variation is subject to morphological plasticity and/or selective pressure and functional constraint and does not correlate well with the 11 Abstract phylogeny. However, in all examples, wing shape was found to be congruent with the phylogenetic patterns inferred from sequence analysis. Consequently, it is recommended that wing shape and not head shape be used in morphometric assessments of population dynamics. It is also asserted here that if population structure is suggested by morphometrics, it should be followed by robust population genetic analysis. As such, morphometrics could be used as a tool for broad surveillance to identify areas of concern. A further objective was to elucidate the broader phylogeny of Triatominae and their relationships with other reduviid subfamilies. To investigate the debated polyphyletic origin of the Triatominae molecular approaches were used. Combined head and wing morphometric and molecular genetic analyses of New World and Old World Triatominae have revealed patterns of convergent morphological evolution (among New World and Old World Triatoma) and striking examples of strongly divergent morphological evolution (between Old World Triatoma and Linshcosteus). Applying a molecular clock based on the rate of sequence divergence for a fragment of ribosomal DNA (D2-28S), calibrated to the fossil record and vicariant events (the divergence of ancestral lineages due to separation by topographical or ecological barriers) it has been possible to reconstruct a likely evolutionary history for the Triatominae and the Reduviidae as a whole. The weight of evidence presented supports a polyphylectic origin for blood-feeding for the Triatominae. The apparent independent development of blood feeding among the main lineages of the Triatominae represented by the genera Triatoma and Rhodnius highlights a fundamental biological difference among important vector species. This difference is likely to become evident in the eventual post genomic era in studies of vector/parasite interactions and it highlights the importance of sequencing genomes from different vector genera.

The aims of this PhD programme were: optimisation of conditions for clonal isolation of Trypanosoma cruzi from individual triatomine bugs and molecular characterisation of clonal isolates from field sites in Paraguay, with special consideration given to host/vector associations and transmission cycles, and further characterisation of putative parents and experimental hybrid progeny of T cruzi 1. with comparison of hybrid genotypes and field isolates from Paraguay. The development of an optimised solid medium plating technique allowed the resolution of mixed infections by the isolation of clonal colonies of T cruzi from single triatomine bugs and experimentally infected mice. Some nutrient plate types produced rapid growth with high plating efficiencies and longer colony viability in comparison with previous methods. Using this technique growth was also possible without the use of a CO2 incubator. Paraguayan isolates were characterised from mammal hosts, sylvatic T infestans, and domestic T infestans from several geographical locations. Isolates were characterised by phenotyping and by genotyping large and small subunit rRNA and mini-exon genes. Analysis enabled assignment of isolates into one of two major lineages (TCI or TC2), and further attribution to one of five subgroups of TC2 (a-e). In Paraguay isolates were all subgroups of TC2. We identified TC2b, TC2c. TC2d and Tc2e circulating in Paraguay. TC2b, TC2d and TC2e were present in domestic cycles (with a predominance of TC2e, 55.2%) and TC2c, TC2b and TC2d in sylvatic cycles. Two species of armadillo (Dasypus novemcinctus; TC2c, TC2d and Euphractus sexcinctus; TC2b, TC2d) were identified as sylvatic reservoirs of T cruzi. These findings accord with our theory that terrestrial armadillos and rodents may have an evolutionary association with TC2. Sylvatic colonies of the vector T infestans were identified from two locations in the Paraguayan Chaco. T cruzi infection in sylvatic T infestans was identified as TC2b. This vector was previously regarded as almost exclusively domestic. and this finding has significant implications for future vector surveillance programmes in Paraguay. By further characterisation of hybrid clones it has been proved for the first time that T cruzi has an extant capacity for genetic exchange. Two genetically transformed putative parental types and six double drug resistant clones, previously isolated by coinfection of mammal cells, were analysed by isoenzyme phenotype, random amplified polymorphic DNA (RAPDs), karyotype analysis and DNA sequencing. These results, together with parallel work performed within our group, suggest an unusual mechanism of genetic exchange involving fusion of parental genotypes and subsequent loss of alleles in polyploid progeny, in conjunction with homologous recombination, and uniparental inheritance of kinetoplast maxicircle DNA. There were parallels between experimentally generated hybrids and natural isolates. The implications of genome fusion and aneuploidy are profound and the mechanism is likely to be widespread providing a possible means of rapid speciation and evolution.

An estimate >11 million people are infected by Trypanosoma cruzi in Latin America. Chagas disease control programmes have been successful in the Southern Cone countries, and similar initiatives are underway in the Andean countries. In Ecuador prevalence is estimated as ~ 130000 people, with 2.5-3.8 million at risk; annual associated costs may reach >20 million US dollars. We studied ecological, genetic, and evolutionary aspects of Rhodnius ecuadoriensis, an important disease vector in western Ecuador and northwestern Peru. Sylvatic and synanthropic populations are sympatric in central Ecuador; only domestic-peridomestic colonies occur in the temperate Andean valleys of southern Ecuador and northern Peru. Both morphological-chromatic features and ecological-behavioural preferences seem to define a cline [sylvatic-large (north) - synanthropic-small (south)]. The ecology of domestic and sylvatic populations was studied using logistic regression. Phytelephas aequatorialis palms are the primary natural ecotope of R ecuadoriensis; sylvatic bugs tend to favour male palms with large amounts of decomposing organic material and located in cropland/pasture fields. Poor households with mud walls, tiled roofs and large numbers of chickens were more likely to be infested. Isometry-free morphometric analysis consistently achieved >90% correct discrimination of sylvatic vs. synanthropic populations, supporting the use of metric variables in surveillance of reinfestations; size-free analyses revealed substantial divergence of Peruvian bugs from La Libertad. Mitochondrial DNA sequence polymorphisms (cytochrome b gene, 663 basepairs) were analysed; ~4% sequence divergence scored between Ecuadorian and Peruvian populations suggested they are independent phylogroups. Haplotype diversity and relationships indicate central coastal Ecuador as the centre of dispersal of this species, with isolated domestic populations in dry Andean valleys. The phylogeny of this species was explored using morphometric and molecular approaches. The monophyly of the `Pacific Rhodnius lineage' (pallescens, colombiensis and ecuadoriensis) was confirmed, with the parapatric pallescens and colombiensis being very closely related; R. pictipes is the closest relative to this lineage among Amazonian species, with the robustus group forming a distinct, major Glade. Control of R ecuadoriensis can contemplate local eradication in dry Andean valleys (southern Ecuador and northern Peru); special attention should be paid to peridomestic populations, including improvement of poultry management (burning-replacing nests every 15-30 days). Long-term interruption of disease transmission would benefit from educational interventions increasing awareness about Chagas disease and from housing improvements targeting mud walls and timber-and-tile roofs. In central-northern western Ecuador peridomestic palm trees may be the origin of reinfestations; environmental management (removing dead fronds and fibres from peridomestic palms), and continuous community- based surveillance are recommended. A comprehensive control programme over 15 years would probably result in interruption of disease transmission, and could bring savings of about 20 US$ per each dollar invested.

The aim of this work was to provide further insight into the relationships between species of the Leishmania subgenus Viannia by examining inter- and intra-specific genetic diversity. Diversity among Viannia strains and stocks was investigated using biochemical and molecular techniques. Isoenzyme and microsatellite analyses were found to be the most discriminatory and generated results which could be interpreted genotypically. These techniques were used to study diversity in Viannia populations consisting of i) Brazilian L V. braziliensis stocks, ii) Nicaraguan stocks belonging to different Viannia species complexes, including putative species-complex hybrids, and iii) uncharacterized stocks from a new epidemic focus in Huanuco, Peru. LEA identified all stocks to at least the species level. Microsatellite analyses using Genescan® / Genotyper® and direct sequencing were found to be more discriminatory than EEA for all populations. The application of Genescan® / Genotyper® to the identification of alleles at these microsatellite loci has not been described previously. Phylogenetic analysis was carried out for each population using enzyme and microsatellite sequence data: phylogenies constructed from multilocus enzyme data were most accurate. Putative species heterozygotes between/,. V. braziliensis/L. V. panamensis and/,. V. braziliensis / L. V. peruviana were identified in the Nicaraguan and Huanuco populations, respectively, using IEA. Microsatellite analysis identified heterozygous stocks in all 3 populations. This approach also supported the hybrid status of the Nicaraguan and Huanuco stocks. Population genetic analysis of stocks from Huanuco provided statistical evidence for a limited degree of genetic recombination between stocks in this population. Results indicated, however, that clonal expansion was the predominant mode of replication. To explore the possibility of the occurrence of genetic recombination between species, genetic transformation experiments were initiated using putative parental stocks from the Nicaraguan population.

The Leishmania donovani complex comprises four described species: L. donovani, L. archibaldi, L. infantum and L. chagasi. L. chagasi is the only New World species and has been considered similar to L. infantum, although some authors insist on maintenance of its independent species status. L. donovani has at least two major epidemiological subgroups whose relationships are poorly understood. In this thesis, molecular biological techniques were used to investigate the taxonomy and phylogenetic relationships within the L. donovani complex, with isoenzyme analysis (lEA) as reference technique. Random amplification of polymorphic DNA (RAPD) was used to provide anonymous genetic markers which allowed overall comparisons of genomes. Selected target genes and intergenic regions were also amplified by the polymerase chain reaction (PCR), namely the major surface protease (msp or gp63), the mini-exon and the ribosomal internal transcribed spacer (ITS). PCR products of intergenic regions between msp genes (ITG/CS and ITG/L), mini-exon and ITS were analysed by restriction fragment length polymorphism (RFLP). Phylogenies generated from each of the methods were compared with that of IEA. L. infantum and L. chagasi were found to be synonymous, whilst L. donovani was found to be more polymorphic than L. infantum and a fourth possible species in the complex, L. archibaldi, was not supported. Six genetic groups of strains were identified in the L. donovani complex, based on all DNA based analyses, which agreed with IEA typing. Pooled data from RFLP and RAPD analyses generated robust phylogenies which were congruent with ITG/CS RFLP and msp DNA sequence based phylogenies, but not with lEA phylogenies. The evolutionary history of the L. donovani complex is analysed in the light of the present results. The diverse typing methods were also evaluated and genetic markers suggested, that are applicable to classification and typing of L. donovani species and strains.

Entamoeba histolytica is an invasive intestinal amoeba morphologically indistinguishable from Entamoeba dispar, a closely related organism that is not able to invade tissues. Differential diagnosis under conventional microscopy is therefore impossible. Reliable tools are needed for clinical diagnosis and for the reevaluation of the prevalence of infection with the invasive species worldwide. Monoclonal Antibody (MAb) 20/7D exhibited promising results when ascites was used to identify cultured isolates of E. histolytica by indirect immunofluorescence assays (IFA), and when used in a Faecal Antigen Capture Enzyme-Linked Immunosorbent Assay (FAC-ELISA) for laboratory diagnosis of amoebic dysentery and colitis. Here, further development of the assay was attempted to increase its sensitivity and use it for detection of asymptomatic carriers of E. histolytica. After purification and subsequent titration in ELISA, MAb 20/7D did not adequately distinguish between crude lysates of cultured E. histolytica and E. dispar trophozoites. MAb 20/7D reacted with a similar soluble antigen of E. histolytica and E. dispar, which confirmed previous observations in western blot analysis under non-reducing conditions. Therefore, the use of the FAC-ELISA for diagnosis in areas where E. dispar is endemic is probably not viable. A nucleic acid detection method was therefore developed. Polymerase Chain Reaction was used to amplify specific tandem sequences in the 24.5 Kb episome of E. histolytica and E. dispar. After PCR, internal sequences of digoxigenin-labelled PCR products were hybridized to specific biotin-labelled probes for E. histolytica or E. dispar and detected in Enzyme- Linked Immunosorbent Assay (ELISA). The Polymerase Chain Reaction Solution- Hybridisation Immunosorbent Assay (PCR-SHELA) was evaluated on samples from travellers returning from the tropics to Barcelona. The sensitivity and specificity were 98% and 100% respectively, when results were compared with microscopy. PCR-SHELA was also useful for differential diagnosis in cases of amoebic abscesses, amoebic dysentery, salmonellosis, ulcerative colitis and in asymptomatic carriage of E. histolytica. The new test gives sensitive and specific differentiation between E. histolytica and E. dispar in clinical specimens and it has proved successful in screening faecal samples in endemic areas for epidemiological purposes.

Visceral leishmaniasis (VL) is endemic in the semi-arid region of north-eastern Brazil. The causative agent is Leishmania chagasi (infantum). The domestic, peri-domestic and anthropophilic sandfly vector is Lutzomyia longipalpis. The domestic dog may be the principal reservoir of infection in endemic urban regions. The primary aim of this project was to study comparative diagnosis and transmissibility of natural and experimental canine VL in the city of T eresina, Piaui state, Brazil. Most (67 %) of human cases of VL in Teresina occurred in children under 5 years of age, adult VL predominated in males, and there was a seasonal increase in incidence of cases. No phenotypic diversity was found between L. chagasi isolates from dogs and humans, although limited comparisons were performed. Clinical diagnosis, parasitological diagnosis and serology were compared in a cohort of 209 dogs, comprised of both symptomatic and asymptomatic animals. Presence of clinical symptoms was not sufficiently sensitive to diagnose canine VL: only 42 % of dogs positive by reference standard criteria (RS positive) were symptomatic. Parasitology was less sensitive than serology for the diagnosis of canine VL and no parasitological test sho~ed more than 60 % sensitivity in comparison with RS criteria. The indirect fluorescent antibody test (IF A T) and DOT -enzyme linked imrnunosorbent assay (DOT -ELISA) were the most sensitive of the serological assays tested. The direct agglutination test (OAT) was highly specific but lacked sensitivity. Serum samples were more sensitive than filter paper blood spot samples. The Lmet2 DNA probe was generally less sensitive than traditional parasitological and serological methods for diagnosis of canine VL, although the probe was useful for screening sandflies for L. chagasi infections (below). The chance of demonstrating parasites in canine VL increased with the serological titre. Nevertheless, parasitoIogicalIy positive dogs could be found among those that were serologically negative. Large numbers of Lu. longipalpis were found in pigsties and chicken houses in the city of Teresina. The Lmet2 probe was shown to be effective for determining prevalence rates of L. chagasi infection in wild caught sandflies. Prevalence of natural infection in sandflies was particularly high when flies were caught in kennels where there were dogs with disseminated cutaneous infections. Experimental studies demonstrated that Lu. longipaJpis could be very readily infected with L. chagasi by feeding on dogs with canine VL and that transmissibility was associated with amastigote infection of the skin. Altered skin of symptomatic dogs was more infective than normal skin of symptomatic animals. Although symptomatic animals were more infective than asymptomatic animals, asymptomatic dogs with normal skin were still infective to large numbers of sandflies and asymptomatic dogs cannot, therefore, be excluded as a significant reservoir of infection. Transmission of experimental canine VL was demonstrated by a single infective sandfly bite. In a cohort of 25 experimental animals many dogs developed discrete, self-curing, cutaneous lesions, typical of cutaneous leishmaniasis. Seroconversion was the most sensitive test for canine VL, but seronegativity was not a reliable indicator of the absence of infection. Bone marrow positivity was only seen in dogs that were serologically positive. Apparent recovery from L. chagasi infection was seen, with serological reversIOn. Aminosidine, dependent on dose, duration of treatment and clinical status of the infected animal, was shown to be capable of producing clinical recovery and clinical cure in a small proportion of infected dogs, but could not be recommended as a systematic method of control. Single applications of ultra-low volume pyrethroid insecticide to individual animal pens was not effective for controlling Lu. iongipaipis. Nevertheless, pyrethroid insecticides had a high residual activity against Lu. iongipalpis when sprayed on to the walls of animal enclosures. Lambda cyhalothrine (ICON) was the most effective of three pyrethroid insecticides tested in the laboratory against Lu. iongipalpis. Overall, this project has produced unique observations on canine VL, supports the fundamental role of the dog as a reservoir host, and explains why culling of seropositive dogs is likely to have limited impact as a disease control strategy.

This study evaluated the status of malaria transmission in Serra do Navio region where a malaria control programme had been carried out for more than 20 years, which included the provision of a daily intake of chioroquinised salt. Malaria endemicity in the study areas [target area: Serra do Navio (SNV); control areas: Colonia Agua Branca (CAB), Porto Terezinha (PT) and Arrependido (ARR)] was established based on the spleen rate and prevalence of malaria parasites among the 2-9 years age group. SNV was defined as a non-endemic area for malaria since both rates were zero, while CAB and ARR were concluded to be mesoendemic areas since the rates were between 10 and 50%, and PT was considered hypoendemic area because both rates were under 10%. Antibody prevalence measured using IFAT with asexual form antigens of Plasmodium .falciparum and Plasmodium vivax and ELISA (asexual forms of P.falciparum, sporozoites of P.falciparurn, P.vivax type 1, P.vivax type 2, P.vivax type 3 and P.malariae/P.brasilianum) was determined for individuals from each study area. These results showed a high frequency of P.falciparum antibodies in all areas. The level of haptoglobin in 100 children from all these areas demonstrated the close relationship between hypohaptoglobinaemia and malaria in the control areas. Haskin's method , a qualitative method, was applied for testing chioroquine in urine. Therefore, chioroquine levels were measured in serum and urine using the ELISA test and the results were in agreement with the reports of the intake of chioroquine salt. The distribution and potentiality of the malaria vectors in all areas was determined. Fifteen anopheline species were identified among 3053 mosquitoes collected by human biting catches in the 4 study areas. 96.4% of the total mosquitoes caught belonged to 4 species, namely, An.albitarsis, An.braziliensis, An.nuñeztovari and An.triannulatus. An.darlingi which is considered the main vector of malaria in the Amazon region of Brazil was very scarce. Using ELISA for the detection of Plasmodium spp sporozoites a positivity rate of 0.799% (23/2876) was found covering six species: 15 An. albitarsis, 4 An. nuñeztovari, and 1 of each: An. braziliensis, An. triannulatus, An. oswaldoi and An.rangeli. 9 out 23 positive mosquitoes were infected with P. raalariae ; 9 with P. vivax- variant VK 247; 3 with P. falciparum and 2 with the classical P.vivax.