Studies on the Trypanocidal Drug, Homidium: Development and Use of ELISA for Its Detection and Quantification in Cattle

This thesis concerns the development, validation and use of enzyme- linked immunosorbent assays (ELISA) to determine homidium concentrations in sera of treated cattle. Previously published work with particular emphasis on control and specifically, chemotherapy of animal trypanosomiases are reviewed in Chapter One. This includes the development and use of trypanocidal drugs detailing previous analytical techniques used in the determination of drug levels in plasma/serum of treated cattle. Chapter 2 describes the general materials and methods used in the experiments reported in the later Chapters of this thesis. Chapter 3 describes the development of two highly sensitive homidium ELISA methods (detection limit 0.1 ng ml-1); Assay 1 an indirect competition assay and the Assay 2 a direct competition assay. Validation of the assays was carried out on serum samples obtained from treated Friesian calves. Using Assay 2, the serum homidium concentrations obtained following treatment of calves showed less variations between individual animals when compared to Assay L It was thereafter adopted for use in all subsequent experiments. Following the development and validation of the ELISA method, several experiments were carried out in cattle using homidium bromide at 1 mg kg-1 b.w. to establish baseline data on serum homidium concentrations and pharmacokinetics in cattle. Serum homidium concentrations and pharmacokinetics were determined following i.v. treatment of Friesian calves. No drug was detectable after approximately 17 days of treatment in four out of five and in 21 days in the remaining calf showing rapid elimination of the drug. Following the establishment of homidium pharmacokinetics in the Friesian cattle in work carried out in Scotland, the studies were extended to Boran (Bos indicus) cattle, a breed of cattle which is commonly kept in the trypanosomiasis endemic areas, in Kenya. Following i.m. treatment with homidium bromide at 1 mg k-1, serum homidium concentrations were determined. The results showed a wide variation in serum homidium concentrations between individual Boran cattle when compared to the Friesian. However, both groups showed similar the serum homidium concentration-versus-time profiles. The results of investigations into homidium as a chemotherapeutic drug are reported in Chapter 5. Two groups of five animals were inoculated with two different populations of T. congolense; one drug-sensitive (IL 1180) and the other drug-resistant (IL 3330). The animals were treated with homidium bromide at 1 mg kg-1 seven days following detection of trypanosomes. No trypanosomes were detected in the cattle infected with the drug-sensitive trypanosome population within 24 hours in four and 48 hours in of five cattle following treatment. The animals remained aparasitaemic up to the end of the observation period of 90 days post-treatment. Whist trypanosomes did not disappear from the circulation following treatment of cattle infected with drug- resistant trypanosome population, low serum homidium concentrations of between 0.1 and 0.3 ng ml-1 remained in the circulation for over 10 weeks following treatment. Both groups showed an increase in the rate of drug elimination within the first week of treatment which reverted back to normal following disappearance of trypanosomes from circulation of cattle treated with drug-sensitive trypanosome population. This accelerated rate of drug elimination was maintained in the presence of drug-resistant trypanosomes until no drug was detectable within 10 days of treatment. Investigations into homidium as a chemoprophylactic drug under controlled conditions are reported in Chapter 6. Following monthly trypanosome challenge of homidium-treated cattle using the same T. congolense populations mentioned above, trypanosomes were detected in blood of the five cattle challenged with the drug-sensitive trypanosome population after 120, 134, 137, 143 and 144 days following treatment. Homidium concentrations of between 0.1 and 0.3 ng ml-1 were detectable for over 10 weeks in circulation in four out of five cattle challenged with drug-sensitive trypanosome population. However, tiypanosomes were detected in the circulation of all the five cattle challenged with the drug-resistant trypanosome population eight to nine days following the first challenge at 30 days post-treatment. Serum homidium concentrations were undetectable within 13 days of challenge

Isolation and Cryopreservation of Trypanosomes and their Vectors for Research and Development in Resource‐ Constrained Settings

Biorepositories for biological samples have increasingly become very important in supporting biomedical research since the 1990s. The Kenya Trypanosomiasis Research Institute Cryo‐bank for trypanosomes and their vectors was established in the 1970s with the aim of providing research materials to scientists. Over 2000 trypanosome isolates have been collected and stored in dewars under liquid nitrogen. Recent collections include tsetse flies—vectors of human and animal trypanosomiasis. Challenges encountered include distances to remote field sites and impassable roads and the cost of collection, preparation, storage, and maintenance under resource‐constrained settings. Under these settings, the challenges can be overcome through strategic leadership that ensures availability and sustainability of resources, appropriate institutional policies, adoption of multidisciplinary approach where appropriate, working with different sectors such as human health, livestock, and wildlife, and environmental conservation in order to leverage on capacities in these sectors, and acknowledging the role of communities from which materials are collected

In vitro and in vivo anti-trypanosomal activities of methanol extract of Azadirachta indica stem-bark

Background: Current chemotherapeutic agents for the treatment of African trypanosomiasis have become largely ineffective, necessitating the search for alternative compounds. The objective of this study was to evaluate in vitro anti-trypanosomal activities of methanol extracts of parts of Azadirachta indica against Trypanosoma brucei rhodesiense, Trypanosoma brucei brucei and Trypanosoma evansi and establish the in vivo efficacy of the most active extract.Materials and methods: Maceration of powdered leaves, stem bark and root bark of the plant in methanol afforded three extracts. In vitro assays were carried out with the extracts on the three trypanosome strains in 96-well microtitre plates at concentration ranges of 4000 - 1000μg/ml. The most active extract was assayed in vivo using Trypanosoma brucei rhodesiense infected Swiss albino mice at doses of 100, 200 and 400 mg/kg body weight. Melarsoprol and suramin served as positive controls. The infected untreated group served as the negative control. Parasitaemia levels, packed cell volume, body weight changes and mean survival period of all groups were monitored throughout the experimental period.Results: Methanol extract of the stem bark of A.indica was most active in vitro against all the three trypanosome strains (MIC values of 9.93±1.88, 16.25±0.92 and 9.97±0.44μg/ml for T. b. rhodesiense, T. b. brucei and T. evansi, respectively). The extract showed optimum activity at 400 mg/kg and was comparable to the positive control groups. Parasitaemia levels were kept at a significantly low level (p < 0.05) by the extract compared to the negative control. Notably, there was no significant difference (p>0.05) in mean survival time of mice treated with the extract at 400 mg/kg and the positive controls.Conclusion: In vitro and in vivo anti-trypanosomal activities of the methanol extract of A. indica stem bark could be attributed to the presence of constituents of moderate polarity.Keywords: Anti-trypanosomal activity, Azadirachta indica, Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense, Trypanosoma evans

Tsetse fly (Glossina pallidipes) midgut responses to Trypanosoma brucei challenge

Abstract Background Tsetse flies (Glossina spp.) are the prominent vector of African trypanosome parasites (Trypanosoma spp.) in sub-Saharan Africa, and Glossina pallidipes is the most widely distributed species in Kenya. This species displays strong resistance to infection by parasites, which are typically eliminated in the midgut shortly after acquisition from the mammalian host. Although extensive molecular information on immunity for the related species Glossina morsitans morsitans exists, similar information is scarce for G. pallidipes. Methods To determine temporal transcriptional responses of G. pallidipes to Trypanosoma brucei brucei challenge, we conducted Illumina based RNA-seq on midgut organ and carcass from teneral females G. pallidipes at 24 and 48 h post-challenge (hpc) with T. b. brucei relative to their respective controls that received normal blood meals (without the parasite). We used a suite of bioinformatics tools to determine differentially expressed and enriched transcripts between and among tissues, and to identify expanded transcripts in G. pallidipes relative to their orthologs G. m. morsitans. Results Midgut transcripts induced at 24 hpc encoded proteins were associated with lipid remodelling, proteolysis, collagen metabolism, apoptosis, and cell growth. Midgut transcripts induced at 48 hpc encoded proteins linked to embryonic growth and development, serine endopeptidases and proteosomal degradation of the target protein, mRNA translation and neuronal development. Temporal expression of immune responsive transcripts at 48 relative to 24 hpc was pronounced, indicative of a gradual induction of host immune responses the following challenge. We also searched for G. m. morsitans orthologous groups that may have experienced expansions in the G. pallidipes genome. We identified ten expanded groups in G. pallidipes with putative immunity-related functions, which may play a role in the higher refractoriness exhibited by this species. Conclusions There appears to be a lack of strong immune responses elicited by gut epithelia of teneral adults. This in combination with a compromised peritrophic matrix at this stage during the initial phase of T. b. brucei challenge may facilitate the increased parasite infection establishment noted in teneral flies relative to older adults. Although teneral flies are more susceptible than older adults, the majority of tenerals are still able to eliminate parasite infections. Hence, robust responses elicited at a later time point, such as 72 hpc, may clear parasite infections from the majority of flies. The expanded G. m. morsitans orthologous groups in G. pallidipes may also be functionally associated with the enhanced refractoriness to trypanosome infections reported in G. pallidipes relative to G. m. morsitans

IN VITRO AND IN VIVO ANTI-TRYPANOSOMAL ACTIVITIES OF METHANOL EXTRACT OF AZADIRACHTA INDICA STEM-BARK

Background: Current chemotherapeutic agents for the treatment of African trypanosomiasis have become largely ineffective, necessitating the search for alternative compounds. The objective of this study was to evaluate in vitro anti-trypanosomal activities of methanol extracts of parts of Azadirachta indica against Trypanosoma brucei rhodesiense, Trypanosoma brucei brucei and Trypanosoma evansi and establish the in vivo efficacy of the most active extract. Materials and methods: Maceration of powdered leaves, stem bark and root bark of the plant in methanol afforded three extracts. In vitro assays were carried out with the extracts on the three trypanosome strains in 96-well microtitre plates at concentration ranges of 4000 - 1000μg/ml. The most active extract was assayed in vivo using Trypanosoma brucei rhodesiense infected Swiss albino mice at doses of 100, 200 and 400 mg/kg body weight. Melarsoprol and suramin served as positive controls. The infected untreated group served as the negative control. Parasitaemia levels, packed cell volume, body weight changes and mean survival period of all groups were monitored throughout the experimental period. Results: Methanol extract of the stem bark of A.indica was most active in vitro against all the three trypanosome strains (MIC values of 9.93±1.88, 16.25±0.92 and 9.97±0.44μg/ml for T. b. rhodesiense, T. b. brucei and T. evansi, respectively). The extract showed optimum activity at 400 mg/kg and was comparable to the positive control groups. Parasitaemia levels were kept at a significantly low level (p 0.05) in mean survival time of mice treated with the extract at 400 mg/kg and the positive controls. Conclusion: In vitro and in vivo anti-trypanosomal activities of the methanol extract of A. indica stem bark could be attributed to the presence of constituents of moderate polarity

We remember… Elders’ memories and perceptions of sleeping sickness control interventions in West Nile, Uganda

The traditional role of African elders and their connection with the community make them important stakeholders in community-based disease control programmes. We explored elders’ memories related to interventions against sleeping sickness to assess whether or not past interventions created any trauma which might hamper future control operations. Using a qualitative research framework, we conducted and analysed twenty-four in-depth interviews with Lugbara elders from north-western Uganda. Participants were selected from the villages inside and outside known historical sleeping sickness foci. Elders’ memories ranged from examinations of lymph nodes conducted in colonial times to more recent active screening and treatment campaigns. Some negative memories dating from the 1990s were associated with diagnostic procedures, treatment duration and treatment side effects, and were combined with memories of negative impacts related to sleeping sickness epidemics particularly in HAT foci. More positive observations from the recent treatment campaigns were reported, especially improvements in treatment. Sleeping sickness interventions in our research area did not create any permanent traumatic memories, but memories remained flexible and open to change. This study however identified that details related to medical procedures can remain captured in a community’s collective memory for decades. We recommend more emphasis on communication between disease control programme planners and communities using detailed and transparent information distribution, which is not one directional but rather a dialogue between both parties

Pathogenicity of bloodstream and cerebrospinal fluid forms of Trypanosoma brucei rhodesiense in Swiss White Mice

Trypanosoma brucei rhodesiense (T.b.r.), the causative agent of the East African form of human African trypanosomiasis (HAT), is capable of crossing the blood brain barrier and invade the central nervous system (CNS). However, it is not clear whether bloodstream forms (BSF) of T.b.rhodesiense differ in biological characteristics from the cerebrospinal fluid (CSF) forms. The present study was carried out to compare the pathogenicity of CSF and BSF of T.b. rhodesiense parasites in Swiss white mice following intraperitoneal inoculation with 106 trypanosomes. The parasites were tested for presence of the serum resistance associated (SRA) gene. Parasitaemia, body weight, packed cell volume (PCV) and survival of the mice was monitored daily until the experiment was terminated. Data was analyzed using general linear model. Both forms of parasite were positive for the SRA gene, and there was no significant difference in progression of parasitaemia, PCV values or survival of the mice. However, the weights of BSF infected mice initially dropped faster than those of CSF infected mice (P<0.001)

The pathogenicity of blood stream and central nervous system forms of Trypanosoma brucei rhodesiense trypanosomes in laboratory mice: a comparative study [version 2; peer review: 2 approved]

Background: Human African trypanosomiasis (HAT) develops in two stages namely early stage when trypanosomes are found in the blood and late stage when trypanosomes are found in the central nervous system (CNS). The two environments are different with CNS environment reported as being hostile to the trypanosomes than the blood environment. The clinical symptoms manifested by the disease in the two environments are different. Information on whether blood stream are pathologically different from CNS trypanosomes is lacking. This study undertook to compare the inter-isolate pathological differences caused by bloodstream forms (BSF) and central nervous system (CNS) of five Trypanosoma brucei rhodesiense (Tbr) isolates in Swiss white mice. Methods: Donor mice infected with each of the five isolates were euthanized at 21 days post infection (DPI) for recovery of BSF trypanosomes in heart blood and CNS trypanosomes in brain supernatants. Groups of Swiss white mice (n = 10) were then infected with BSF or CNS forms of each isolate and monitored for parasitaemia, packed cell volume (PCV), body weight, survivorship, trypanosome length, gross and histopathology characteristics. Results: Amplification of SRA gene prior to trypanosome morphology and pathogenicity studies confirmed all isolates as T. b. rhodesiense. At 21 DPI, CNS trypanosomes were predominantly long slender (LS) while BSF were a mixture of short stumpy and intermediate forms. The density of BSF trypanosomes was on average 2-3 log-scales greater than that of CNS trypanosomes with isolate KETRI 2656 having the highest CNS trypanosome density. Conclusions: The pathogenicity study revealed clear differences in the virulence/pathogenicity of the five (5) isolates but no distinct and consistent differences between CNS and BSF forms of the same isolate. We also identified KETRI 2656 as a suitable isolate for acute menigo- encephalitic studies

Pharmacology of DB844, an Orally Active aza Analogue of Pafuramidine, in a Monkey Model of Second Stage Human African Trypanosomiasis

Novel drugs to treat human African trypanosomiasis (HAT) are still urgently needed despite the recent addition of nifurtimox-eflornithine combination therapy (NECT) to WHO Model Lists of Essential Medicines against second stage HAT, where parasites have invaded the central nervous system (CNS). The pharmacology of a potential orally available lead compound, N-methoxy-6-{5-[4-(N-methoxyamidino) phenyl]-furan-2-yl}-nicotinamidine (DB844), was evaluated in a vervet monkey model of second stage HAT, following promising results in mice. DB844 was administered orally to vervet monkeys, beginning 28 days post infection (DPI) with Trypanosoma brucei rhodesiense KETRI 2537. DB844 was absorbed and converted to the active metabolite 6-[5-(4-phenylamidinophenyl)-furanyl-2-y​l]-nicotinamide(DB820), exhibiting plasma Cmax values of 430 and 190 nM for DB844 and DB820, respectively, after the 14th dose at 6 mg/kg qd. A 100-fold reduction in blood trypanosome counts was observed within 24 h of the third dose and, at the end of treatment evaluation performed four days post the last drug dose, trypanosomes were not detected in the blood or cerebrospinal fluid of any monkey. However, some animals relapsed during the 300 days of post treatment monitoring, resulting in a cure rate of 3/8 (37.5%) and 3/7 (42.9%) for the 5 mg/kg×10 days and the 6 mg/kg×14 days dose regimens respectively. These DB844 efficacy data were an improvement compared with pentamidine and pafuramidine both of which were previously shown to be non-curative in this model of CNS stage HAT. These data show that synthesis of novel diamidines with improved activity against CNS-stage HAT was possible.This investigation received financial support from the Bill and Melinda Gates Foundation through the Consortium for Parasitic Drug Development

Multiple evolutionary origins of Trypanosoma evansi in Kenya

Trypanosoma evansi is the parasite causing surra, a form of trypanosomiasis in camels and other livestock, and a serious economic burden in Kenya and many other parts of the world. Trypanosoma evansi transmission can be sustained mechanically by tabanid and Stomoxys biting flies, whereas the closely related African trypanosomes T. brucei brucei and T. b. rhodesiense require cyclical development in tsetse flies (genus Glossina) for transmission. In this study, we investigated the evolutionary origins of T. evansi. We used 15 polymorphic microsatellites to quantify levels and patterns of genetic diversity among 41 T. evansi isolates and 66 isolates of T. b. brucei (n = 51) and T. b. rhodesiense (n = 15), including many from Kenya, a region where T. evansi may have evolved from T. brucei. We found that T. evansi strains belong to at least two distinct T. brucei genetic units and contain genetic diversity that is similar to that in T. brucei strains. Results indicated that the 41 T. evansi isolates originated from multiple T. brucei strains from different genetic backgrounds, implying independent origins of T. evansi from T. brucei strains. This surprising finding further suggested that the acquisition of the ability of T. evansi to be transmitted mechanically, and thus the ability to escape the obligate link with the African tsetse fly vector, has occurred repeatedly. These findings, if confirmed, have epidemiological implications, as T. brucei strains from different genetic backgrounds can become either causative agents of a dangerous, cosmopolitan livestock disease or of a lethal human disease, like for T. b. rhodesiense