Thesis (Ph. D.)--University of Wisconsin, 1955. / Typescript (carbon copy). eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 124-125).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1956. / Typescript. Abstracted in dissertation abstracts, v. 16 (1956) no. 2, p. 210-211. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The haptoglobin-haemoglobin receptor (HpHbR) is expressed by the African try- panosome, T. brucei, whilst in the bloodstream of the mammalian host. This allows ac- quisition of haem, but also results in uptake of trypanolytic factor 1, a mediator of in- nate immunity against non-human African trypanosomes. Here, the structure of HpHbR in complex with its ligand, haptoglobin-haemoglobin (HpHb), is presented, revealing an elongated binding site along the membrane-distal half of the receptor. A ~50° kink allows the simultaneous binding of two receptors to one dimeric HpHb, increasing the efficiency of ligand uptake whilst also increasing binding site exposure within the densely packed cell surface. The possibility of targeting this receptor with antibody-drug conjugates is ex- plored. The characterisation of the unexpected interaction between T. congolense HpHbR and its previously unknown ligand, haemoglobin, is also presented. This receptor is iden- tified as an epimastigote-specific protein expressed whilst the trypanosome occupies the mouthparts of the tsetse fly vector. An evolutionary pathway of the receptor is proposed, describing how the receptor has changed to adapt to a role as a bloodstream form-specific protein in T. brucei. Apolipoprotein L1 (ApoL1) is the pore-forming component of the trypanolytic factors. An expression and purification protocol for ApoL1 is presented here, and the functionality of the protein established. Initial attempts to characterise the pores and structure of ApoL1 are described.

Made available in DSpace on 2014-06-11T19:31:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T18:41:54Z : No. of bitstreams: 1 teixeira_mca_dr_jabo.pdf: 2885202 bytes, checksum: 80086a83bb46ca2cb05bc1ae5ea75a8d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Trypanosoma evansi é patogênico para a maioria dos animais, acometendo bovinos, bubalinos, caprinos, ovinos, suínos, cães, quatis, capivaras, camelos e outras espécies animais em áreas tropicais e subtropicais do globo terrestre sendo, no Brasil, a doença endêmica no pantanal mato-grossense. O presente estudo teve como fito principal estudar a evolução clínica, as alterações hematológicas, bioquímicas sérica, imunológicas e anatomopatológicas de bovinos infectados experimentalmente com T. evansi. Para tal, foram utilizados oito bovinos, clinicamente sadios e sorologicamente negativos para T. evansi. Três foram mantidos como testemunhos e cinco inoculados com T. evansi. Exames físicos, parasitológicos, hematimétricos e bioquímicos séricos (proteínograma, índice ictérico e glicose) e do líquido cefalorraquidiano foram realizados. Nos exames físicos realizados nos bovinos até 525° DAI não foi notada nenhuma anormalidade clínica com relação à temperatura retal, batimentos cardíacos, frequência respiratória, movimentos ruminais, aspectos de membranas mucosas (nasal, conjuntival, oral, vaginal e/ou prepucial) e dos linfonodos externos (mandibulares, maxilares, parotídeos, cervicais superficiais, sublíacos e mamários). A presença de tripomastigotas foi demonstrada através da prova biológica nos bovinos 01, 06 e 08 no15° DAI, bovinos 06 e 07 no 30° DAI, bovinos 01 e 06 no 45° DAI, bovino 06 no 60° DAI, bovino 01 no 75° DAI. As contagens de hemácias, os teores de hemoglobina e os volumes globulares dos bovinos, experimentalmente infectados, variaram dentro dos limites de normalidade para a espécie bovina. O VGM, HGM e CHGM, apresentam alterações pontuais. / Trypanosoma evansi are pathogenic to most of animals, affecting cattle, buffaloes, goats, sheep, pigs, dogs, coatis, capybaras, camels and other animals in tropical and subtropical areas of the globe, and, in Brazil, it causes an endemic disease in the Pantanal Mato Grosso. This study primarily aimed to study the clinical, hematological, biochemical, immunological and pathological alterations in cattle experimentally infected with T. evansi. For this purpose, we used eight animals, clinically healthy and serologically negative for T. evansi. Three animas were kept as evidence and five were inoculated with T. evansi. Physical, parasitological, hematological and serum biochemical (proteins, icteric index and glucose) and cerebrospinal fluid examination were performed. In the physical examination conducted in cattle up to 525th DAI were not observated any clinical abnormality in concerning rectal temperature, heart rate, respiratory rate, ruminal movements, aspects of the mucous membranes (nasal, conjunctival, oral, vaginal and / or specimen) and external nodes (mandibular, maxillary, parotid, superficial cervical, breast and sublíacos). The presence of trypomastigotes was demonstrated by bioassay in cattle 01, 06 and 08 no 15th DAI, cattle 06 and 07 at 30° DAI, cattle 01 and 06 on the 45 th DAI, cattle 06 in 60 th DAI, cattle 01 in 75 th DAI. Red blood cells counts, hemoglobin content and volume cell of experimentally infected cattle were within normal limits for the bovine species. The MCV, MHC and MCHC, showed specific changes. Physical examination of the cerebrospinal fluid did not show alterations in appearance and coloration. Morever, using the Giensa-stained blood smears, buffy coat technique (BCT) and mouse inoculation procedure were negative for T. evansi tripomastigote. Serum protein concentrations, identified 26 proteins with molecular weights ranging from 20 to 245 KD.

Two aspects of trypanosomiasis have been investigated in this study. First, molecular methods were applied to the diagnosis of T.evansi in camels in South Libya. The aim of the study was to determine if FTA card blood sampling and PCR amplification could detect parasites and this be used as tool for diagnosis and epidemiology. Targeted samples of 70 camels were identified on the basis of symptoms of infection and blood was collected on FTA cards. PCR primers and conditions for the amplification of T.evansi DNA were developed on the basis of the literature and a positive control clone grown in the laboratory. The assay found 84.3% of camel samples positive using TBR primers (177bp amplicon) and ITS nested primers (611-1513bp amplicons). This result demonstrated that Surra is endemic in this area, and that T.evansi was the species that was involved. The ITS and TBR loci in the parasites identified in Libya were almost identical to those previously reported in the genbank database, though with some polymorphisms. Dullness and emaciation were the clinical signs of camels infected by trypanosomes, and these two symptoms were significantly related to the 1200bp ITS nested PCR amplicon. These two symptoms can be thus used as a sign an initial diagnosis of T.evansi infection in camels. The second aspect of trypanosomiasis studied was the occurrence of endotoxaemia in infection. The first part of this research investigated endotoxin levels in clinical human African trypasnosomiasis using the Limulus Amoebocyte lysate assay. Endotoxin levels were significantly increased over control individuals in the plasma of T.b.rhodesiense patients. This endotoxaemia was unrelated to infection duration, parasitaemia or clinical stage but resolved after clearance of parasites by drug treatment. In the cerebrospinal fluid there was no significant difference in endotoxin level between early and late stage cases and no relationship to parasite loads. It is argued on the basis of the data that endotoxaemia in trypanosomiasis most likely results from increases in permeability of the gut to endotoxins from gram negative enter bacteria. This conclusion was further supported from a study using cell culture adapted T.brucei and secreted products which gave no evidence of any endotoxin activity. Also samples of an acute experimental mouse infection with T.brucei gave no endotoxin activity, suggesting that this phenomenon requires a more chronic infection in mice. No relationships were found between plasma or CSF endotoxin levels to neurological signs of infection. However the presence of a gross inflammatory clinical symptom, splenomegaly, was associated with endotoxaemia and the concentrations of 3 plasma cytokines associated with the immune response in trypanosome infection were associated with correlated to plasma endotoxin levels. In order to determine the nature of the endotoxin activity, a biosensor cell assay for LPS was used, based on human embryonic kidney cells transfected with TLR4/MD3 and a NF-κB induced alkaline phosphatase reporter gene. This assay revealed low or undetectable levels of LPS in clinical samples from T.b.rhodesiense patients, in mouse samples from T.b.brucei infections and in vitro cultured trypanosomes. This suggests that either the endotoxin activity detected using the LAL assay is an unconventional endotoxin signalling via a TLR4 independent pathway or that the human plasma was in some way toxic to the reporter cell and this requires further investigation. In conclusion, this study has provided the first clear evidence of an association of endotoxaemia and inflammatory responses in clinical African trypansomiasis and helps resolve the question of whether endotoxaemia is a parasite or host-microbiota related phenomenon.

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Yes / African trypanosomes, except Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause human African trypanosomiasis, are lysed by the human serum protein apolipoprotein L1 (ApoL1). These two subspecies can resist human ApoL1 because they express the serum resistance proteins T. b. gambiense glycoprotein (TgsGP) and serum resistance-associated protein (SRA), respectively. Whereas in T. b. rhodesiense, SRA is necessary and sufficient to inhibit ApoL1, in T. b. gambiense, TgsGP cannot protect against high ApoL1 uptake, so different additional mechanisms contribute to limit this uptake. Here we report a complex interplay between trypanosomes and an ApoL1 variant, revealing important insights into innate human immunity against these parasites. Using whole-genome sequencing, we characterized an atypical T. b. gambiense infection in a patient in Ghana. We show that the infecting trypanosome has diverged from the classical T. b. gambiense strains and lacks the TgsGP defense mechanism against human serum. By sequencing the ApoL1 gene of the patient and subsequent in vitro mutagenesis experiments, we demonstrate that a homozygous missense substitution (N264K) in the membrane-addressing domain of this ApoL1 variant knocks down the trypanolytic activity, allowing the trypanosome to avoid ApoL1-mediated immunity. IMPORTANCE. Most African trypanosomes are lysed by the ApoL1 protein in human serum. Only the subspecies Trypanosoma b. gambiense and T. b. rhodesiense can resist lysis by ApoL1 because they express specific serum resistance proteins. We here report a complex interplay between trypanosomes and an ApoL1 variant characterized by a homozygous missense substitution (N264K) in the domain that we hypothesize interacts with the endolysosomal membranes of trypanosomes. The N264K substitution knocks down the lytic activity of ApoL1 against T. b. gambiense strains lacking the TgsGP defense mechanism and against T. b. rhodesiense if N264K is accompanied by additional substitutions in the SRA-interacting domain. Our data suggest that populations with high frequencies of the homozygous N264K ApoL1 variant may be at increased risk of contracting human African trypanosomiasis. / This work, including the efforts of Stijn Deborggraeve, was funded by Research Foundation Flanders (1501413N). This work, including the efforts of Bart Cuypers, was funded by Research Foundation Flanders (11O1614N). This work, including the efforts of Jean-Claude Dujardin and Etienne Pays, was funded by Interuniversity Attraction Poles Program of Belgian Science Policy (P7/41). This work, including the efforts of Jean-Claude Dujardin, was funded by Flemish Ministry of Sciences (SOFI-B SINGLE). This work, including the efforts of Etienne Pays, was funded by EC | European Research Council (ERC) (APOLs 669007).

Over the past two decades movement of cattle towards the north of Uganda has enabled the Trypanosoma brucei rhodesiense focus in south-eastern Uganda to spread into previously unaffected districts. This thesis brings together important epidemiological data regarding the impact of mass cattle drug treatment on the point prevalence of several different species of trypanosome in a newly endemic area of human sleeping sickness. Crucially the findings illustrate mass drug treatment is effective in reducing the prevalence of T. b. rhodesiense in cattle, thus minimising the reservoir potential of these animals in the epidemiology of human disease. During 2006 a control programme was launched to halt the northward spread of this zoonotic parasite. This programme, entitled ‘Stamping Out Sleeping Sickness’ (SOS) proposed to reduce the prevalence of Human African Trypanosomiasis (HAT) in the newly affected districts by reducing the prevalence of this parasite in the main animal reservoir of infection – domestic cattle. Cattle were mass treated using trypanocides to clear infections. Previous work demonstrated the prevalence of T. brucei s. l. and T. b. rhodesiense in cattle was higher in the districts of Dokolo and Kaberamaido than in the other SOS intervention districts (Selby 2011). To determine whether animals in these areas were also exposed to pathogenic cattle trypanosomes samples were screened for the presence of T. vivax and T. congolense savannah using PCR. Chapter three of this thesis determined the prevalence of these trypanosomes in cattle in these districts. Before treatment had taken place the prevalence of T. vivax was 2% (4/200, 95% CI 3.57 – 0.12%) in Dokolo and 7.3% (21/310, 95% CI 10.17 - 4.24 %) in Kaberamaido. The prevalence of T. congolense savannah at baseline was 3.5% (7/200, 95% CI 7.08–1.42 %) in Dokolo and 9.1% (21/230, 95% CI13.6–5.7 %) in Kaberamaido. Monitoring was conducted three, nine and 18 months post treatment and both pathogens were detected at all time points. The impact the treatment had on point prevalence varied by trypanosome species and between the two districts. Several clusters of villages in Dokolo and Kaberamaido continued to report cases of HAT after the initial SOS intervention due in part to their proximity to livestock markets (Batchelor et al., 2009). In 2008 re-treatment of these ‘high risk’ areas was undertaken. Monitoring was performed before and six months after treatment. Cattle blood samples were collected at 20 village sites from ten ‘case-positive villages’ (from which human sleeping sickness cases had been reported six months prior to June 2007) and from ten ‘case-negative villages’ (no reported human sleeping sickness cases six months prior to June 2007). These samples were screened for all of the aforementioned trypanosomes using species specific PCR protocols. Chapter five details the results of this screening, and assessed whether re-treatment in Dokolo and Kaberamaido was effective in reducing the prevalence of trypanosomiasis. The re-treatment had a dramatic effect, significantly reducing the point prevalence of overall trypanosomiasis in the 20 villages screened from 38.1% (95% CI = 40.5 – 35.79%) at baseline to 26.9% (95% CI 28.96 – 24.97, p < 0.0001) at six months. Looking at each species separately, point prevalence of three out of four detected species of trypanosome fell significantly, including T. b. rhodesiense, which was reduced to 25% of its baseline prevalence. Finally the two SOS treatment cycles were compared both statistically and spatially with emphasis on trends at village level and the occurrence of mixed infections.