Molecular identification of bloodmeal sources and trypanosomes in Glossina spp., Tabanus spp. and Stomoxys spp. trapped on cattle farm settlements in southwest Nigeria.

The interactions of host, vector and parasite in bovine trypanosomiasis transmission cycles in southwest Nigeria are not yet well understood. Trypanosoma (Trypanosomatida: Trypanosomatidae) species infection prevalences and bloodmeal sources were determined in transmitting vectors of the genera Glossina (Diptera: Glossinidae), Tabanus (Diptera: Tabanidae) and Stomoxys (Diptera: Muscidae) collected using Nzi traps in cattle settlements in southwest Nigeria. Sequenced cytochrome B mitochondrial DNA segments obtained from vector digestive tracts identified bloodmeal sources from eight host species, namely human, cattle, hippopotamus, giraffe, gazelle, spotted hyena, long-tailed rat and one unidentified species. Overall, 71.1% [95% confidence interval (CI) 63.0-78.1], 33.3% (95% CI 21.9-47.0) and 22.2% (95% CI 16.2-29.9), respectively, of Glossina, Tabanus and Stomoxys flies were positive for trypanosomes. The observed trypanosome species were Trypanosoma vivax, Trypanosoma congolense, Trypanosoma brucei, Trypanosoma evansi, Trypanosoma simiae and Trypanosoma godfreyi. Trypanosome DNA was more prevalent in tsetse (34.8% Tr. vivax, 51.1% Tr. b. brucei, 5.2% Tr. congolense, 4.4% Tr. simiae and 24.4% mixed infections) than in other flies and the main determinants in all flies were seasonal factors and host availability. To the best of the present group's knowledge, this is the first report of Trypanosoma species in Tabanus and Stomoxys flies in Nigeria. It indicates that vector control programmes should always consider biting flies along with tsetse flies in the control of human and animal trypanosomiasis.

Current initiatives in One Health: consolidating the One Health Global Network.

The Global Response to Avian Influenza has led to a longer-term One Health movement, which addresses risks, including zoonoses, at the human-animal- environment interface, and requires the development of innovative partnerships at the political, institutional and technical levels. One Health is a sustainable and rational option when the cumulative effects of health hazards on food and economic security are considered, but demands long-term financial investment. Projections of growth in the demand for livestock production and consumption in Asia and Africa also call for effective One Health responses. However, an effective response also requires validated evidence of the socio-economic value that the One Health approach can provide. Implementing the One Health approach depends on forging strong links between human and animal health services, the environment and public policy. The authors present a list of some of the national and transnational partnerships established since 2006. Political support, good governance and effective policies and networks are crucial building blocks for One Health sustainability. The Global Response to Avian Influenza was initially established under the joint leadership of the European Union, the United States and the United Nations System Influenza Coordination Office. Since then it has supported numerous initiatives, including the World Health Organization (WHO)/Food and Agriculture Organization of the United Nations (FAO)/World Organisation for Animal Health (OIE) Global Early Warning System (GLEWS). Indeed, the Global Response to Avian Influenza paved the way for an unprecedented WHO/FAO/OIE tripartite partnership, which promoted the integration of foodborne, neglected zoonotic and tropical diseases within the One Health movement and led to the tripartite High-Level Technical Meeting of 2011 in Mexico. The One Health Global Network, which began as a proposition at an Expert Consultation in Winnipeg, Canada, in 2009, is now a reality. While its Global Guidance Group takes shape, the choice of soft-governance--an approach which relies more on information and advisory guidelines than on hierarchy and legislation, and which aims to steer local organisations rather than to control them--remains challenging. Nonetheless, the emergence of One Health as a professional and academic discipline, together with the growing references to a One Health culture, also offers new opportunities.

Latent Trypanosoma brucei gambiense foci in Uganda: a silent epidemic in children and adults?

Trypanosoma brucei gambiense sleeping sickness follows a long asymptomatic phase and persists in ancient foci from which epidemic clinical disease arises. A putative focus of T. b. gambiense infections has been identified, initially in mothers and young children, on the Lake Albert shoreline of Western Uganda leading to mass screening of 6207 individuals in September 2008. T. b. gambiense infections were identified by Card Agglutination Test for Trypanosomiasis (CATT) and sub-species-specific PCR although parasitological methods failed to confirm any patent trypanosome infections. In April 2009, CATT positives were re-visited; diagnosis of individuals by CATT and PCR was unstable over the two time points and parasites remained undetected, even using mini Anion Exchange Centrifugation Technique (mAECT). These observations suggest the possibility of a silent focus of disease, where all infected individuals are in a latent stage, and highlight our limited understanding of the local natural history and disease progression of T. b. gambiense in children and adults.

Population-dynamics focussed rapid rural mapping and characterisation of the peri-urban interface of Kampala, Uganda.

In developing countries, cities are rapidly expanding and urban and peri-urban agriculture (UPA) has an important role in feeding these growing urban populations; however such agriculture also carries public health risks such as zoonotic disease transmission. It is important to assess the role of UPA in food security and public health risks to make evidence-based decisions on policies. Describing and mapping the peri-urban interface (PUI) are the essential first steps for such an assessment. Kampala, the capital city of Uganda is a rapidly expanding city where the PUI has not previously been mapped or properly described. In this paper we provide a spatial representation of the entire PUI of Kampala economic zone and determine the socio-economic factors related with peri-urbanicity using a population-dynamics focussed rapid rural mapping. This fills a technical gap of rapid rural mapping and offers a simple and rapid methodology for describing the PUI which can be applied in any city in developing countries for wide range of studies.

Controlling sleeping sickness - a review.

Following a period characterized by severe epidemics of sleeping sickness, restoration of effective control and surveillance systems has raised the question of eliminating the disease from sub-Saharan Africa. Given sufficient political and financial support, elimination is now considered a reasonable aim in countries reporting zero or less than 100 cases per year. This success may lead health authorities across the affected region to downgrade the disease from 'neglected' to simply being ignored. In view of the significant levels of under-reporting of sleeping sickness mortality in rural communities, this could be a short-sighted policy. Loss of capacity to deal with new epidemics, which can arise as a consequence of loss of commitment or civil upheaval, would have serious consequences. The present period should be seen as a clear opportunity for public-private partnerships to develop simpler and more cost-effective tools and strategies for sustainable sleeping sickness control and surveillance, including diagnostics, treatment and vector control.

Clinical features associated with seroconversion to Anaplasma marginale, Babesia bigemina and Theileria parva infections in African cattle under natural tick challenge.

A longitudinal study was conducted in Southeast Uganda for 14 months on 640 Zebu cattle kept under natural tick challenge, with a view to identifying clinical features for prediction of seroconversion to Anaplasma marginale, Babesia bigemina and Theileria parva infections. Physical examination, condition scoring and tick counts were undertaken on all cattle every 4 weeks. In addition, 5300 sera were collected and analysed for antibodies against A. marginale, B. bigemina and T. parva infections using the enzyme-linked immunosorbent assay (ELISA). The major clinical features compiled included weight loss, fever (rectal temperature), anaemia (packed cell volume), pallor of mucous membranes, lymph node enlargement, staring coat, diarrhoea and lacrymation. The risk factors included tick challenge at village level, sex, age, Rhipicephalus spp. density and Boophilus spp. density on individual animals. Using a binary logistic regression model, the clinical features and risk factors were analysed. The results suggest that increasing rectal temperature was associated with increased probability for seroconversion to A. marginale, while high level of Rhipicephalus spp. density and increasing packed cell volume (PCV) were significantly associated with reduced probability of seroconversion. Although statistically significant, none of the factors had large effects, with odds ratios (OR) of 0.87, 1.15 and 0.98 for Rhipicephalus spp. density, rectal temperature and PCV, respectively. For B. bigemina infection, a high level of Boophilus spp. density, anaemia and staring coat were significantly associated with increased probability of seroconversion (OR 1.50, 1.78, 1.37, respectively). Presence of lacrymation and old age were associated with reduced probability of seroconversion (OR 0.52, 0.86 respectively). For T. parva infection, lymph node enlargement (OR 1.30) was associated with increased probability of seroconversion, while high Rhipicephalus spp. density and increasing packed cell volume (PCV) were associated with reduced probability of seroconversion (OR 0.68 and 0.98, respectively). In conclusion, presence and intensity of the respective tick vectors for tick-borne diseases, age and clinical features such as anaemia, fever, staring coat, lymph node enlargement and lacrymation are indicators for seroconversion to A. marginale, B. bigemina and T. parva infections in cattle. These indicators for seroconversion could be exploited in the development of decision support tools for clinical diagnosis of tick-borne diseases.

People, Patches, and Parasites: The Case of Trypanosomiasis in Zimbabwe.

Understanding the socio-ecology of disease requires careful attention to the role of patches within disease landscapes. Such patches, and the interfaces between different socio-epidemiological systems, we argue, have important implications for disease control. We conducted an interdisciplinary study over three years to investigate the spatial dynamics of human and animal trypanosomiasis in the Zambezi valley, Zimbabwe. We used a habitat niche model to identify changes in suitable habitat for tsetse fly vectors over time, and this is related to local villagers' understandings of where flies are found. Fly trapping and blood DNA analysis of livestock highlighted the patchy distribution of both flies and trypanosome parasites. Through livelihoods analysis we explored who makes use of what areas of the landscape and when, identifying the social groups most at risk. We conclude with a discussion of the practical implications, including the need for an integrated 'One Health' approach involving targeted approaches to both vector control and surveillance.

A burgeoning epidemic of sleeping sickness in Uganda.

The epidemic of Trypanosoma brucei rhodesiense sleeping sickness in eastern Uganda, which began in 1998 as a result of movements of the livestock reservoir of the parasite, has continued to spread. An additional 133 000 people have been put at risk of infection in Kaberamaido, another newly affected district. The few resources committed to control interventions in Soroti district have failed to contain the epidemic. The high prevalence of the parasite in cattle presents a significant risk for transmission to human beings and further spread of this neglected zoonotic disease. Targeted interventions are urgently needed to control epidemics and reduce the high mortality resulting from sleeping sickness.

Human African trypanosomiasis: Epidemiology and control.

Human African trypanosomiasis (HAT), or sleeping sickness, describes not one but two discrete diseases: that caused by Trypanosoma brucei rhodesiense and that caused by T. b. gambiense. The Gambian form is currently a major public health problem over vast areas of central and western Africa, while the zoonotic, Rhodesian form continues to present a serious health risk in eastern and southern Africa. The two parasites cause distinct clinical manifestations, and there are significant differences in the epidemiology of the diseases caused. We discuss the differences between the diseases caused by the two parasites, with an emphasis on disease burden, reservoir hosts, transmission, diagnosis, treatment and control. We analyse how these differences impacted on historical disease control trends and how they can inform contemporary treatment and control options. We consider the optimal ways in which to devise HAT control policies in light of the differing biology and epidemiology of the parasites, and emphasise, in particular, the wider aspects of control policy, outlining the responsibilities of individuals, governments and international organisations in control programmes.

Glossina fuscipes fuscipes in the trypanosomiasis endemic areas of south eastern Uganda: apparent density, trypanosome infection rates and host feeding preferences.

A study was undertaken in three districts in south eastern Uganda endemic for human and animal trypanosomiasis, to investigate the status of the vector tsetse fly population. Apparent density (AD) of tsetse was between 2 and 21 flies/trap/day across the three districts, with Glossinia fuscipes fuscipes identified as the predominant species. Trypanosomes were observed in G.f. fuscipes with an infection rate, as determined by microscopy, of 1.55% across the three studied areas. However, trypanosome infections were only identified in female flies giving an infection rate of 2.39% for the female tsetse when this sex was considered in isolation; no male flies were found to be infected. Bloodmeal analysis highlighted 3 principal vertebrate hosts, namely cattle, pigs and monitor lizards (Varanus niloticus). The implication of this, in relation to the cycle of transmission for human infective trypanosomes between domestic animals and man, is discussed.

Central point sampling from cattle in livestock markets in areas of human sleeping sickness.

We present the results of a study to determine the value of central point sampling in cattle markets as a means of estimating the trypanosomiasis (T. brucei s.l.) prevalence in the surrounding landscape in Uganda. We find that in the epidemic area studied, central point sampling is a good predictor of prevalence in surrounding villages, but not in endemic areas. We also find that animals infected with trypanosomiasis are more likely to be brought for sale in livestock markets in endemic areas; we discuss these results in relation to the prevention of the spread of sleeping sickness.

Programmed cell death in procyclic Trypanosoma brucei rhodesiense is associated with differential expression of mRNAs.

Procyclic Trypanosoma brucei rhodesiense have a cell death mechanism which can be activated by an external signal, the lectin ConA, in vitro. ConA has been shown to cause profound changes in cellular morphology and induce fragmentation of nuclear DNA in T.b. rhodesiense which are characteristic of apoptosis, a form of programmed cell death (PCD) in other eukaryotic cells. RNA analysis of trypanosomes induced to undergo PCD revealed that RNA remains intact up to 48 h into the process, a time when nuclear DNA fragmentation has already started. Using the randomly amplified differentially expressed sequences polymerase chain reaction method, ConA-induced cell death in T.b. rhodesiense is shown to be associated with differential expression of mRNAs, including up regulation of mRNAs late in the death process. The results demonstrate that trypanosomes actively participate in their own destruction through a PCD process and confirm that cell death in trypanosomes is associated with de novo gene expression.

Prohibitin and RACK homologues are up-regulated in trypanosomes induced to undergo apoptosis and in naturally occurring terminally differentiated forms.

Two genes have been identified as up-regulated late during ConA-induced apoptosis in procyclic form Trypanosoma brucei rhodesiense. The first represents a homologue of prohibitin, a proto-oncogene originally described in mammals and subsequently in yeast, which is involved in cell-cycle control and senescence. The Trypanosoma prohibitin homologue appears to contain within it a putative death domain. The second gene, homologous to a family of regulatory proteins which are receptors for activated protein kinase C (RACKs), is also shown to be up-regulated in terminally differentiated bloodstream form trypanosomes. These are the first endogenous genes to be identified as up-regulated in programmed cell death (PCD) in unicellular organisms.

Apoptosis in procyclic Trypanosoma brucei rhodesiense in vitro.

Apoptosis is a phenomenon previously associated exclusively with metazoan organisms. We show here that procyclic insect form Trypanosoma brucei rhodesiense, a protozoan parasite, when treated in vitro with concanavalin A displayed several features normally associated with apoptosis in metazoan cells. Lectin treatment induced cleavage of nuclear DNA into oligonucleosomal fragments, suggesting activation of an endogenous nuclease in the parasite. Treated trypanosomes, although agglutinated and non-motile, exhibited fluorescence after treatment with the vital stain fluorescein diacetate and retained (3)H-uridine indicating that their cell membranes remained intact during the period of DNA fragmentation. Electron micrographs showed characteristic morphology of cells undergoing apoptosis, including surface membrane vesiculation and migration of chromatin to the periphery of the nuclear membrane while mitochondria remained intact. These results suggest that treatment with concanavalin A triggers a cell death mechanism in T. b. rhodesiense similar to the process of apoptosis described in metazoa.

The neglected zoonoses--the case for integrated control and advocacy.

The neglected zoonotic diseases (NZDs) have been all but eradicated in wealthier countries, but remain major causes of ill-health and mortality across Africa, Asia, and Latin America. This neglect is, in part, a consequence of under-reporting, resulting in an underestimation of their global burden that downgrades their relevance to policy-makers and funding agencies. Increasing awareness about the causes of NZDs and how they can be prevented could reduce the incidence of many endemic zoonoses. Addressing NZDs by targeting the animal reservoir can deliver a double benefit, as enhanced animal health means a reduced risk of infection for humans, as well as improved livelihoods through increased animal productivity. Advocacy for NZD control is increasing, but with it comes a growing awareness that NZD control demands activities both in the short term and over a long period of time. Moreover, despite the promise of cheap, effective vaccines or other control tools, these endemic diseases will not be sustainably controlled in the near future without long-term financial commitment, particularly as disease incidence decreases and other health priorities take hold. NZD intervention costs can seem high when compared with the public health benefits alone, but these costs are easily outweighed when a full cross-sector analysis is carried out and monetary/non-monetary benefits--particularly regarding the livestock sector--are taken into account. Public-private partnerships have recently provided advocacy for human disease control, and could prove equally effective in addressing endemic zoonoses through harnessing social impact investments. Evidence of the disease burdens imposed on communities by the NZDs and demonstration of the cost-effectiveness of integrated control can strengthen the case for a One Health approach to endemic zoonotic disease control.

Sleeping sickness: a tale of two diseases.

Sleeping sickness presents clinically as two distinct diseases, reflecting the fact that two very different trypanosomes are responsible. The African Rift separating East and West Africa defines the distribution of the two diseases. In this review, Susan Welburn, Eric Fèvre, Paul Coleman, Martin Odiit and Ian Maudlin discuss the biology and distribution of these two diseases in relation to the evolution of hominids in Africa.

Lessons learned from the emergence of a new Trypanosoma brucei rhodesiense sleeping sickness focus in Uganda.

During the latter months of 1998, cases of sleeping sickness caused by Trypanosoma brucei rhodesiense presented in Soroti district, eastern Uganda, a region which had not previously experienced cases of the disease. Cattle are the main reservoir for T b rhodesiense, by contrast with sleeping sickness caused by Trypanosoma brucei gambiense in west Africa where there appears to be no epidemiologically significant animal reservoir. Several factors have been identified that interacted to produce ideal conditions for the establishment of a new disease focus. After a period of civil unrest, Soroti, which is within the tsetse belt, was repopulated by people and later, cattle. Both the cattle restocking and the subsequent trade in these cattle at a local cattle market had a role in the appearance of the disease. Recently, molecular biology techniques have become available for the detection and genotype identification of T b rhodesiense and thus it is now possible to distinguish human infective and non-infective trypanosomes in cattle. In light of these advances in identification and in both field and epidemiological techniques, successful disease control management has become an achievable goal and will require the collaboration and expertise of clinicians, veterinarians, epidemiologists and laboratory scientists.

The major cell surface glycoprotein procyclin is a receptor for induction of a novel form of cell death in African trypanosomes in vitro.

Bloodstream forms (BSF) and procyclic culture forms (PCF) of African trypanosomes were incubated with a variety of lectins in vitro. Cessation of cell division and profound morphological changes were seen in procyclic forms but not in BSF after incubation with concanavalin A (Con A), wheat germ agglutinin and Ricinus communis agglutinin. These lectins caused the trypanosomes to cease division, become round and increase dramatically in size, the latter being partially attributable to the formation of what appeared to be a large 'vacuole-like structure' or an expanded flagellar pocket. Con A was used in all further experiments. Spectrophotometric quantitation of extracted DNA and flow cytometry using the DNA intercalating dye propidium iodide showed that the DNA content of Con A-treated trypanosomes increased dramatically when compared to untreated parasites. Examination of these cells by fluorescence microscopy showed that many of the Con A-treated cells were multinucleate whereas the kinetoplasts were mostly present as single copies, indicating a disequilibrium between nuclear and kinetoplast replication. Immunofluorescence experiments using monoclonal antibodies (mAb) specific for paraflagellar rod proteins and for kinetoplastid membrane protein-11 (KMP-11), showed that the Con A-treated parasites had begun to duplicate the flagellum but that this had only proceeded along part of the length of the cells, suggesting that the cell division process was initiated but that cytokinesis was subsequently inhibited. Tunicamycin-treated wild-type trypanosomes and mutant trypanosomes expressing both high levels of non-glycosylated procyclins and procyclin isoforms with truncated N-linked sugars were resistant to the effects of Con A, suggesting that N-linked carbohydrates on the procyclin surface coat were the ligands for Con A binding. This was supported by data obtained using mutant parasites created by deletion of all three EP procyclin isoforms, two of which contain N-glycosylation sites, by homologous recombination. The knockout mutants showed reduced binding of fluorescein-labelled Con A as determined by flow cytometry and were resistant to the effects of Con A. Taken together the results show that Con A induces multinucleation, a disequilibrium between nuclear and kinetoplast replication and a unique form of cell death in procyclic African trypanosomes and that the ligands for Con A binding are carbohydrates on the EP forms of procyclin. The possible significance of these findings for the life cycle of the trypanosomes in the tsetse fly vector is discussed.

The endosymbionts of tsetse flies: manipulating host-parasite interactions.

Through understanding the mechanisms by which tsetse endosymbionts potentiate trypanosome susceptibility in tsetse, it may be possible to engineer modified endosymbionts which, when introduced into tsetse, render these insects incapable of transmitting parasites. In this study we have assayed the effect of three different antibiotics on the endosymbiotic microflora of tsetse (Glossina morsitans morsitans). We showed that the broad-spectrum antibiotics, ampicillin and tetracycline, have a dramatic impact on tsetse fecundity and pupal emergence, effectively rendering these insects sterile. This results from the loss of the tsetse primary endosymbiont, Wigglesworthia glossinidia, which is eradicated by ampicillin and tetracycline treatment. Using the sugar analogue and antibiotic, streptozotocin, we demonstrated specific elimination of the tsetse secondary endosymbiont, Sodalis glossinidius, with no observed detrimental effect upon W. glossinidia. The specific eradication of S. glossinidius had a negligible effect upon the reproductive capability of tsetse but did effect a significant reduction in fly longevity. Furthermore, elimination of S. glossinidius resulted in increased refractoriness to trypanosome infection in tsetse, providing further evidence that S. glossinidius plays an important role in potentiating trypanosome susceptibility in this important disease vector. In the light of these findings, we highlight progress made towards developing recombinant Sodalis strains engineered to avoid potentiating trypanosome susceptibility in tsetse. In particular, we focus on the chitinase/N-acetyl-D-glucosamine catabolic machinery of Sodalis which has previously been implicated in causing immune inhibition in tsetse.

Spatial and temporal risk factors for the early detection of Trypanosoma brucei rhodesiense sleeping sickness patients in Tororo and Busia districts, Uganda.

We have carried out a study of risk factors for early detection of Trypanosoma brucei rhodesiense sleeping sickness. Records of sleeping sickness patients from 1987 to 2001 from Tororo and Busia districts in Uganda were reviewed for their village of origin and clinical stage (early or late). All villages that reported sleeping sickness and fixed post-diagnostic sleeping sickness health units in Tororo and Busia districts were geo-referenced. The spatial distribution of early and late stage patient detection by health units was analysed using Geographical Information Systems (GIS). Of 1316 sleeping sickness patients admitted at the Livestock Health Research Institute and Busolwe hospitals and Lumino health centre from Tororo and Busia districts, 471 (35.8%) were early stage, 825 (62.7%) were late stage, while 20 (1.5%) were not staged. Five hundred and eighty-five (44.5%) came from within a 10 km radius of the reporting health units. After multivariate analysis, the proportion of early stage patients detected was found to be significantly associated with patients originating from within a 10 km radius of the health unit (P < 0.01), with adults (>19 years) (P < 0.01), and with annual parish incidence (P < 0.01). Application of GIS and the early to late stages ratio are an informative and powerful means of determining efficiency of surveillance of sleeping sickness.