Human African Trypanosomiasis (HAT), Nagana and the Tsetse Fly
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Human African Trypanosomiasis (HAT) and Nagana (the animal form of the disease) occur throughout the countries of sub-Saharan Africa. HAT is lethal if undetected and/or untreated. The disease is primarily found in poor, rural and remote populations. The early stages of disease cause headaches, fever, weakness, sweating, joint pain and stiffness. The advanced stages of disease result in psychiatric disorders, seizures, coma and death. Detection of the advanced form of the disease requires examination of cerebrospinal fluid obtained by spinal tap. Treatment of advanced HAT is performed with drugs developed in the early twentieth century. These drugs are very painful and carry a significant risk of death due to side effects. Nagana has a tremendous economic cost and prevents the development of livestock agriculture in affected areas.
HAT and Nagana are vectored by the Tsetse fly (Glossina sp.). Tsetse flies are obligate blood feeders. Both males and females blood feed from vertebrate hosts and are capable of acting as vectors. Tsetse has a unique reproductive biology in that they give birth to fully developed larvae. Pregnant females develop a single offspring at a time and provide all of its nourishment via an intrauterine lactation system.
Trypanosomes are the pathogenic agents that cause HAT. They are single celled eukaryotic organisms. The human form of the disease is caused by two different trypanosome species. Trypanosoma brucei gambiense is responsible for HAT within the countries of West Africa and results in a chronic version of the disease which can take months to years to progress. Trypanosoma brucei rhodesiense is responsible for HAT in East Africa and causes an acute form of the disease. The animal form of the disease (Nagana) is caused by Trypanosoma brucei brucei.
The trypanosomes are transmitted to humans and livestock through the bite of an infected tsetse fly. Flies inject metacyclic trypomastigotes into a vertebrate host where they enter the blood and transform into bloodstream trypomastigotes. The bloodstream trypomastigotes multiply within bodily fluids (blood, lymph and spinal fluid). Bloodstream trypomastigotes can be taken up by tsetse flies that bite and infected host. Once in the fly the bloodstream trypomastigotes transform into procyclic trypomastigotes. Procyclic trypomastigotes are a sexual form of the organism and multiply by binary fission. The procyclic trypomastigotes then migrate out of the blood meal and back up the digestive tract to the salivary glands where they become epimastigotes. The epimastigotes establish themselves and reproduce in the salivary glands. They then transform into metacyclic trypomastigotes which are injected when the fly bites a vertebrate host.
Vector population control is an effective strategy to disrupt HAT transmission. The low reproductive rate of tsetse results in low population numbers. Populations can be disrupted through trapping (as seen in the picture) and pesticide treatment of livestock. Tsetse traps are made with blue cloth which attracts the flies. Once in the trap the flies are either poisoned with pesticides or funneled into a collection chamber from which they cannot escape. Trapping and treatment programs require consistent effort and organization to be effective.
Tsetse fly physiology is very different from that of other flies. The most dramatic changes have occurred in tissues associated with reproduction. The oviduct which is used to deposit eggs in other flies has been adapted into a uterus capable of supporting a fully developed third instar larva. The larva is nourished by secretions from an accessory gland called the milk gland. The milk gland is a series of large bifurcating tubes which synthesize protein and incorporate fat to produce milk secretions. The milk nourishes the larva throughout the entirety of its development and is the only food the larva takes. The ovaries of tsetse are significantly reduced in comparison with other flies. Tsetse only generates a single egg at a time. In comparison, flies like mosquitoes can produce over one hundred eggs at a time during a reproductive cycle. Egg development alternates between the right and left ovaries from one reproductive cycle to the next.
The tsetse reproductive cycle begins with egg development, ovulation and fertilization. Sperm used for fertilization is stored in an organ called the spermathica which preserves sperm from a single mating for the entire lifespan of the fly. Embryonic flies develop within ~5 days and hatch within the uterus. The larva then lives within the uterus and develops through 3 larval instars. Intrauterine larval development takes ~5-6 days. At the end of the 3rd larval instar the female undergoes parturition (gives birth to the larva). The larval fly will burrow into the ground and pupate within 30 minutes of deposition. The pupa will develop for 3-4 weeks after which the adult fly will emerge.
Tsetse flies harbor three bacterial symbionts, Wigglesworthia, Sodalis and Wolbachia which contribute to the flies health, fertility and immunity. Wigglesworthia live in two populations within the fly. In the gut Wigglesworthia is found in an intracellular form in a tissue called the bacteriome. This population assists tsetse in metabolizing and supplementing the rich yet nutritionally limited diet of vertebrate blood. Elimination of these bacteria by antibiotic treatment results in flies that do not digest meals properly and become infertile. The exact contribution that these bacteria make remains unidentified. The second Wigglesworthia population lives in the extracellular lumen of the milk gland tubules. This population is transmitted from mother to intrauterine offspring within the milk secretions. Sodalis can be found throughout the tissues of the fly in both intracellular and extracellular situations. The presence of Sodalis is not yet associated with concrete benefits to the fly; however it may be associated with immune function and priming immunity during development. Sodalis is also passed from mother to offspring via the milk secretions. Wolbachia are found within the ovaries associated with developing eggs. These bacteria are found in the reproductive tract of many insects and are known to influence host reproductive outcomes by affecting male/female offspring ratios and by causing a phenomenon known as cytoplasmic incompatibility. Cytoplasmic incompatibility influences reproduction in such a way that matings between Wolbachia infected males and uninfected females results in infertility. Mating between infected females and uninfected males result in successful fertility and the production of Wolbachia infected offspring. These dynamics result in the bacteria being driven into the population. This mechanism is being considered as a way to drive beneficial characteristics (such as trypanosome resistance) into wild populations using lab developed bacterial strains.
Tsetse immune function is a complex system which facilitates the existence of the bacterial symbionts and actively fights against pathogenic infection. An important player in immune regulation in tsetse is a protein called PGRP-LB (Peptidoglycan recognition protein B). This protein functions as a buffer for the immune system. PGRP-LB binds the cell wall components of pathogens and degrades them. This prevents activation of antimicrobial gene expression when normal levels of symbiotic bacteria are present. Antimicrobial gene activation results from activation of the PGRP-LC protein which also binds pathogen cell wall components. However, upon binding, the PGRP-LC receptor protein signals to activate production of antimicrobial proteins.
When the fly is immune challenged by a pathogen (such as a trypanosome). The PGRP-B system is saturated resulting in the binding of pathogen cell wall molecules to the PGRP-C. This results in the upregulation of antimicrobial genes and synthesis of their respective proteins. These proteins are then secreted throughout the fly to assist in killing invading organisms. Tsetse flies have a natural resistance to trypanosome infection with only a small portion of flies becoming infected when challenged. Without this system much higher numbers of flies would be infective in the wild.
This system is dynamic and complex with multiple interactions occurring between the fly, the symbionts and invading parasites. Our aim is to understand the interactions between the various aspects of this system such that this knowledge can be applied to novel control measures to prevent HAT transmission.
One strategy towards HAT control aims to modify the Sodalis symbiont with a recombinant anti-trypanosome gene to boost the fly’s natural immune system. This strategy is termed Paratransgenesis, the indirect modification of an organism by alteration of its symbionts. The bacteria are cultured outside of the fly and transformed with DNA coding for an antitrypanosomal gene. These bacteria are then reintroduced into the fly to generate a line of highly resistant flies. Through the use of gene drive mechanisms such as cytoplasmic incompatibility this trait can then be pushed into natural populations to reduce or eliminate trypanosome infected flies.
