Thesis (PhD (Veterinary Tropical Diseases)--University of Pretoria, 2009. / Includes bibliographical references. Also available in print format.

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1989. / Includes bibliographical references. / Microfiche. / xii, 297 leaves, bound ill. 29 cm

Filarial infections (lymphatic filariasis and onchocerciasis) are amongst the major neglected tropical diseases, and together account for more than 120 million infections in tropical and subtropical regions. The gold-standard technique for the diagnosis of filariases relies on the detection of microfilariae (mf) either in blood smears (lymphatic filariasis) or in skin biopsies (onchocerciasis). The secretion of extracellular RNAs (exRNAs) by parasitic nematodes has opened new avenues for the development of novel biomarkers for helminthiases, including filariasis. However, rather little is known about the origin and regulation of these RNAs inside the nematodes. One outstanding question is whether the secretion of small RNAs is distinct across the developmental stages of parasitic nematodes. Similarly, it is not clear whether the secretion of miRNAs is affected by treatment with anthelminthic chemotherapy or their potential as biomarkers for infection. Litomosoides sigmodontis is a murine filarial nematode closely related to filarial nematodes of medical and veterinary importance, including Onchocerca spp. and Brugia spp. L. sigmodontis has been extensively used to decipher multiple aspects of filarial biology, including parasite development, vaccine, and host-pathogen interactions. Therefore, we decided to use this model to address fundamental questions regarding the secretion of small RNAs and their biomarker potential. Our in vitro studies demonstrate that some extracellular miRNAs are enriched in a sexand stage-specific manner in the Excretion/Secretion (ES) products from early larval and adult stages from the rodent filarial nematode Litomosoides sigmodontis. Moreover, our data demonstrates that the gravid adult female worms secrete a plethora of miRNAs enriched in the secretome of this developmental stage when compared to adult males or mf. Further characterization studies show that the miRNAs are likely to be secreted in association with extracellular vesicles (EVs), as previously reported for other parasitic nematodes, including the human pathogen Brugia malayi. Interestingly, Ivermectin, which is typically used to treat filarial infections, does not have consistent effects on the secretion of miRNAs by gravid adult female worms in vitro, requiring further in vivo experiments to determine the effect of IVM on detection of extracellular parasite-derived miRNAs. In vivo experiments, using murine models of infection with L. sigmodontis (gerbils and BALB/c mice), as well as human samples from patients infected with Onchocerca volvulus and cattle infected with Onchocerca ochengi, demonstrated the presence of filarial-derived miRNAs, including female-specific miRNA markers, in biofluids from infected hosts. Further statistical analysis showed that two parasite-derived miRNAs, miR-71 and miR-100d, can significantly discriminate infected animals from naïve controls with high sensitivity/specificity (> 80%/100%). The results presented in this PhD thesis provide an initial framework to understand the secretion of small RNAs throughout nematode development, the potential interactions between anthelminthic chemotherapy and small RNA trafficking and secretion, as well as the use of parasite-derived miRNAs for the development of a new generation of biomarkers for filarial infections.

Brugia malayi is one of three species of nematode known to cause lymphatic filariasis (LF) in humans. LF infects over 120 million people, causing debilitating disease. Various global programs have been launched in the past 20 years to eliminate LF. These programs have greatly scaled up the resources and efforts allocated to halting the transmission and reducing disease burden. Only a few drugs are used to treat LF, and resistance is thus a devastating possibility. Research aimed at identifying new drug targets could therefore prove essential in elimination of LF. Genetic manipulation of B. malayi has been limited to transient transfections. A transfection system allowing for stable integration of transgenic sequences into the nuclear genome of this parasite would enable more robust studies that could lead to identification of novel drug targets and vaccine candidates. The piggyBac (pB) transposon system has been successfully applied to develop a stable transfection system in a variety of species. This system involves two plasmids, a helper and a donor. The donor plasmid contains the target DNA and a selectable marker flanked by specific inverted terminal repeat (ITR) regions. The helper plasmid expresses the pB transposase that will catalyze the precise integration of any DNA report tools necessary to adapt the pB system in B. malayi. Three versions of the donor plasmid were constructed, each containing a Gaussia Luciferase (GLuc) selectable marker but differing only by the fluorescent protein expressed. The construct containing a YFP gene was used to transfect embryos via biolistics to test whether YFP and GLuc are expressed.

This study was undertaken to determine the identity of the causative agents of the filariases reported from northwestern tetraonids (Fowle, 1946; Babero, 1953), the incidence of filarial infections in the Tetraonidae of British Columbia, and the means by which these birds acquire their infections. More than 400 wild gallinaceous birds of 10 species and over 200 adult non-gallinaceous birds from 27 families were examined. Adults of the following 4 species of filariae (Onchocercidae: Splendidofilariinae) were collected from the Tetraonidae of B.C. and/or Alaska and are described herein: 1) Skriabinocta flexivaqinalis (Jones, 1961) n. comb.; 2) Splendidofilaria pectoralis new species; 3) Splendidofilaria "species A"; and 4) Splendidof il aria papil locerca (Lubimov, 1946;) Anderson and Chabaud, 1959. Descriptions are provided also for the microfilariae of S. flexivaqinalis. S. pectoralis and S. papillocerca, and for Microfilaria laqopodis (Haaland, 1928),Brinkmann, 1950, and Microfilaria "species B" from tetraonid hosts. Recent systems of filarioid classification are evaluated and some modifications proposed, including revival of the genus Skriabinocta Chertkova, 1946 to comprise S. petrowi Chertkova, 1946, S. flexivaginalis. S. chitwoodae (Anderson, 1961) n. comb,, S. striatospicula (Hibler, 1964) n. comb., and provisionally S. lienalis (Orloff, 1947) n. comb. On Vancouver Island, Microfilaria sp. B is hyperenzootic in blue grouse, with a significantly higher prevalence in adult males than in adult hens or in yearlings or chicks. Mf. sp. B is enzootic in Vancouver Island ruffed grouse. S. flexivaginalis is sporadic in British Columbia ruffed grouse. Splendidofilaria pectoralis is sporadic to enzootic in tetraonids from central B. C. and Alaska. Mf. lagopodis is.enzootic in willow ptarmigan of northern B. C. Microfilariae closely resembling Mf. flexivaginalis occasionally parasitize non-gallinaceous birds of Vancouver Island, but Mf. sp. B and S. pectoralis seem to be restricted totetraonid hosts. The larvae of S. flexivaginalis develop in the thoracic muscles of the ceratopogonid, Culicoides unicolor (Coq.) group. Those of Mf. sp. B develop in abdominal fat bodies of the simuliids, Simulium aureum Fries and Cnephia minus (D. and S.); the former being considerably more efficient as an intermediate host. Development of Mf. sp. B fails at temperatures which do not range above 62°F. Larval development is described and on the basis of larval characters some features of the adults of Mf. sp. B are predicted. Knowledge acquired on the biology of the tetraonid hosts, the incidence of the filariases, and the seasonal abundance of the vectors and the effects of various factors on their feeding activities, is integrated in a discussion of the epizootiology of Skri abinoct a flexivaqinalis and Microfilaria sp. B. / Science, Faculty of / Zoology, Department of / Graduate

Le Programme africain de lutte contre l'onchocercose (APOC) et le Programme mondial d'élimination de la filariose lymphatique (GPELF), basés respectivement sur des traitements de masse par ivermectine et par une combinaison d'ivermectine et d'albendazole, sont des succès indéniables. Cependant, en Afrique centrale, région endémique pour la loase, les opérations de lutte sont fortement entravées du fait de la survenue possible d'effets secondaires graves induits par l'ivermectine chez les individus présentant plus de 30.000 microfilaires de Loa loa par mL de sang. Les résultats remarquables de l'APOC ont conduit récemment le programme à afficher un objectif plus ambitieux que celui défini initialement (l'élimination de la maladie comme problème de santé publique) : celui d'une élimination de la transmission. Les objectifs d'élimination d'APOC et du GPELF rendent nécessaires la réalisation d'une cartographie de la filariose lymphatique et de l'onchocercose hypoendémique en Afrique centrale, et imposent de développer des stratégies de lutte alternatives dans les zones coendémiques avec la loase. Nos travaux apportent des éléments nouveaux concernant l'épidémiologie de la filariose lymphatique en Afrique centrale. La distribution très focale de la maladie et les facteurs de risque particuliers que nous avons identifiés devraient être pris en compte pour déterminer l'échelle utilisée pour la réalisation de la cartographie. Par ailleurs, nous avons observé que le test ICT utilisé pour cartographier la filariose lymphatique pouvait se positiver en cas de forte microfilarémie à Loa loa (réaction croisée), ce qui va constituer un défi important pour les programmes de lutte. Nos travaux ont aussi permis de développer des stratégies alternatives permettant de lancer des programmes de lutte dans des zones de coendémie avec L. loa. Ainsi, nous avons participé au développement d'une technique permettant de dépister (et d'exclure des traitements de masse) les individus à risque d'effets secondaires graves post-ivermectine. D'autre part, nous avons montré que des traitements semestriels par albendazole seul pourraient constituer une alternative, applicable en zone de loase, au traitement standard de la filariose lymphatique. Concernant le diagnostic, nous avons montré l'intérêt qu'il y avait à lire les tests de diagnostic de la filariose lymphatique de manière semi-quantitative. Enfin, concernant la loase, nous avons étudié les relations existant, au niveau individuel, entre la microfilarémie à Loa et les antécédents d'œdème de Calabar et de passage sous-conjonctival du ver adulte. / The African Program (APOC) and the (GPELF), relying on the mass drug administration of ivermectine or of a combination of ivermectin and albendazole, respectively, are undeniably successful. Nonetheless, in Central Africa where loiasis is endemic, control operations are hampered by the possible occurence of ivermectine-induced severe adverse events in individuals harbouring more than 30 000 microfilaria of Loa loa per mL whole blood. The outstanding results obtained by APOC have recently led the programme to adopt a more ambitious goal than originally defined (elimination of the disease as a public health concern) : eliminating transmission. The elimination objectives of APOC and of the GPELF arise the need for mapping lymphatic filariasis and hypoendemic onchocerciasis areas in Central Africa, and strenghten the need to develop control strategies for loiasis coendemic areas. Our researches bring new insight into the epidemiology of lymphatic filariasis in Central Africa. The highly focal distribution of the disease, and the risk factors that we identified should be accounted for when defining the scale used for mapping processes. Furthermore, we observed that the immunochromatographic card test (ICT) used for mapping lymphatic filariasis might display false positive result in case of high L. loa microfilarial density (cross reaction), which will constitute a challenge for control programmes. Our work also allowed to develop alternative strategies for launching control programmes in areas of coendemy with L. loa. We participated in the developement of a technique to test (and exclude from mass drug administration) the individuals at risk of post-ivermectin severe adverse events. Besides, we showed that a semiannual mass drug administration of albendazole alone was deemed to be a suitable alternative strategy to standard lymphatic filariasis treatment applicable in loiasis endemic areas. As for the diagnosis, we highlighted the interest of semi-quantitative reading of diagnostic tests for lymphatic filariasis. Eventually, we studied the relation between the microfilaremia of L. loa and the histories of Calabar oedema and of sub-conjonctival migration of the adult worm, at the individual level.

Introdução: Este trabalho tem por objetivo identificar focos de filariose linfática na amazônia ocidental brasileira e constatar a infecção natural pela W. bancrofti do mosquito vetor, o C. quinquefasciatus. O estudo abrangeu as cidades de Porto Velho (RO), Guajará Mirim (RO) e Humaitá (AM). Método: foi utilizada a técnica da gota espessa de sangue, colhida após 22h00, corada com Giemsa para avaliação humana, e a Reação em Cadeia da Polimerase (PCR) para pesquisa de DNA de W. bancrofti em mosquitos vetores. Foram analisadas e avaliadas amostras do sangue de moradores dos bairros mais antigos dessas cidades e escolares noturnas e captura de mosquitos. Resultados: O inquérito hemoscópico em moradores resultou em 935 indivíduos (54,4%) examinados de um total de 1.720 cadastrados. As pesquisas entre escolares noturnos envolveram 2.709 indivíduos (75,2%) de um total de 3.601. Todos negativos para a presença de microfilaremia. Foram coletadas 7.849 fêmeas de C. quinquefasciatus, todas apresentaram resultados negativos para DNA de W. bancrofti. / Introduction: This work is aimed to identify possible lymphatic filariasis foci in the Western Brazilian Amazonian. Porto Velho and Guajara-Mirim (Rondonia State) and Humaita (Amazonas State) were the study target area. Methods: Blood thick film method with samples collected from 10 PM to 1 AM were stained using Giemsa to evaluate human infection and PCR for W. bancrofti DNA in mosquitoes vectors. Samples from the neighborhoods of these areas and from students attending public nighttime classes in the cities referred to above were analyzed and evaluated, as well as mosquitoes captured in the houses. Results: 935 individuals (54.4%) out of a total of 1,720 individuals engaged. Sample with night students involving 2,709 individuals (75.2%) out of the total of 3,601 previously selected. No individual examined was positive for the presence of microfilariae in the blood stream. 7,849 female C.quinquefasciatus specimens were captured and after evaluated by using PCRmethod, all of them were found negative for W. bancrofti DNA.

Introdução: Este trabalho tem por objetivo identificar focos de filariose linfática na amazônia ocidental brasileira e constatar a infecção natural pela W. bancrofti do mosquito vetor, o C. quinquefasciatus. O estudo abrangeu as cidades de Porto Velho (RO), Guajará Mirim (RO) e Humaitá (AM). Método: foi utilizada a técnica da gota espessa de sangue, colhida após 22h00, corada com Giemsa para avaliação humana, e a Reação em Cadeia da Polimerase (PCR) para pesquisa de DNA de W. bancrofti em mosquitos vetores. Foram analisadas e avaliadas amostras do sangue de moradores dos bairros mais antigos dessas cidades e escolares noturnas e captura de mosquitos. Resultados: O inquérito hemoscópico em moradores resultou em 935 indivíduos (54,4%) examinados de um total de 1.720 cadastrados. As pesquisas entre escolares noturnos envolveram 2.709 indivíduos (75,2%) de um total de 3.601. Todos negativos para a presença de microfilaremia. Foram coletadas 7.849 fêmeas de C. quinquefasciatus, todas apresentaram resultados negativos para DNA de W. bancrofti. / Introduction: This work is aimed to identify possible lymphatic filariasis foci in the Western Brazilian Amazonian. Porto Velho and Guajara-Mirim (Rondonia State) and Humaita (Amazonas State) were the study target area. Methods: Blood thick film method with samples collected from 10 PM to 1 AM were stained using Giemsa to evaluate human infection and PCR for W. bancrofti DNA in mosquitoes vectors. Samples from the neighborhoods of these areas and from students attending public nighttime classes in the cities referred to above were analyzed and evaluated, as well as mosquitoes captured in the houses. Results: 935 individuals (54.4%) out of a total of 1,720 individuals engaged. Sample with night students involving 2,709 individuals (75.2%) out of the total of 3,601 previously selected. No individual examined was positive for the presence of microfilariae in the blood stream. 7,849 female C.quinquefasciatus specimens were captured and after evaluated by using PCRmethod, all of them were found negative for W. bancrofti DNA.

The mosquito Aedes polynesiensis, a member of the Aedes scutellaris complex, is the main vector in the South Pacific region of the Wuchereria bancrofti parasite, the causative agent of lymphatic filariasis (LF), and is also a major nuisance biter. Decades of Mass Drug treatment (MDA) have not been successful in elimination LF. Two non-vector species in the Ae. scutellaris complex were introgressed with Ae. polynesiensis to attempt to obtain lines that would produce cytoplasmic incompatibility (CI) with wild populations and/or LF-refractoriness. Despite selection of progeny from Brugia-challenged, non-infective females at each backcross, no refractory line was acquired. However, three lines from crosses between aposymbiotic Ae. polynesiensis and Ae. riversi displayed CI and male mating competiveness suitable for the purpose of population suppression using the incompatible insect technique (IIT). A population study was conducted of potential release sites and the evaluation of monitoring tools for Ae. polynesiensis on Moorea and Tetiaroa, French Polynesia. There was no evidence of active migration between selected islets on the atoll of Tetiaroa, suggesting it is a suitable site for field releases of CI males. The BioGents Sentinel trap was shown to be an efficient and convenient trap suitable for Ae. polynesiensis monitoring. The effects of temperature and larval density on life-table parameters relevant to IIT were examined, including: larval survivorship, developmental time to pupation, male to female ratio, male pupae yield, male size and adult male survival. These findings were used to design and conduct a 14-week field experiment testing CI male strain against an isolated population, using optimized rearing conditions. Approximately 8000 males were released weekly on motu Onetahi, Tetiaroa atoll. Significant sterility was induced by Wolbachia in the targeted female population, supporting the development and scale-up of this approach toward Ae. polynesiensis nuisance and LF transmission reduction.

Estudo preliminar da redução da microfilaremia in vivo de Mansonella ozzardi (Manson, 1897) com uso de ivermectina, utilizando a técnica de filtração em membrana de policarbonato, Lábrea, Amazonas, Brasil. Estudou-se a eficácia do uso de dose única de ivermectina 0,15 mg/kg de peso no tratamento de 74 pacientes com infecção por Mansonella ozzardi. Inicialmente foi realizado a coleta de sangue para o diagnóstico parasitológico utilizando a filtração de sangue em membrana de policarbonato e para análises bioquímicas e hematológicas. Foi realizado a quantificação de microfilárias antes e depois do tratamento seguindo-os até 1 ano. Foi observado uma redução estatisticamente significativa em relação à densidade de microfilárias (&#967;2 de Friedman = 159,00; valor-p < 0, 0001) após 1 ano do uso da medicação, e que não houve alterações laboratoriais e efeitos adversos que comprometessem o uso da ivermectina. Concluiu-se que o fármaco nesta dose é eficaz e seguro, e mantém o \"clearance\" de microfilaremia pelo menos 1 ano após o uso. / Preliminary study of the reduction of microfilaremia in vivo of Mansonella ozzardi (Manson, 1897) with use of ivermectin, using the blood filtration polycarbonate membrane technique, in Labrea, Brazilian Amazon, Brazil. The study focused in the efficacy and tolerability of a single-dose use of ivermectin 0.15 mg / kg body weight in the treatment of 74 patients infected by the filarial worm M. ozzardi. Before and after the parasitological diagnosis by a blood filtration polycarbonate membrane and treatment, anamnesis, clinical examination and blood collection for quantification of microfilariae, biochemical and hematological analysis were done, comparing the outcomes of patients the in first day and after 72 hours and at least following up the patients for 1 year. The study concluded that there was a statistically significant reduction in microfilariaemia density (&#967;2 Friedman = 159, 00; p-value <0, 0001) after 1 year of use of the medication, and that there were no laboratory abnormalities and clinical symptoms that compromised the use of ivermectin. In conclusion, the use of ivermectin is an effective microfilaricide and maintains a suppressive effect for at least 1 year and the adverse reactions are not an obstacle for the treatment.