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1.  Illuminating the Prevalence of Trypanosoma brucei s.l. in Glossina Using LAMP as a Tool for Xenomonitoring 
PLoS Neglected Tropical Diseases  2016;10(2):e0004441.
As the reality of eliminating human African trypanosomiasis (HAT) by 2020 draws closer, the need to detect and identify the remaining areas of transmission increases. Here, we have explored the feasibility of using commercially available LAMP kits, designed to detect the Trypanozoon group of trypanosomes, as a xenomonitoring tool to screen tsetse flies for trypanosomes to be used in future epidemiological surveys.
Methods and Findings
The DNA extraction method was simplified and worked with the LAMP kits to detect a single positive fly when pooled with 19 negative flies, and the absolute lowest limit of detection that the kits were able to work at was the equivalent of 0.1 trypanosome per ml. The DNA from Trypanosoma brucei brucei could be detected six days after the fly had taken a blood meal containing dead trypanosomes, and when confronted with a range of non-target species, from both laboratory-reared flies and wild-caught flies, the kits showed no evidence of cross-reacting.
We have shown that it is possible to use a simplified DNA extraction method in conjunction with the pooling of tsetse flies to decrease the time it would take to screen large numbers of flies for the presence of Trypanozoon trypanosomes. The use of commercially-available LAMP kits provides a reliable and highly sensitive tool for xenomonitoring and identifying potential sleeping sickness transmission sites.
Author Summary
Recent control efforts have reduced the global incidence of Gambiense human African trypanosomiasis (HAT) to <5,000 cases per year, strengthening the prospect of eliminating the disease as a public health problem by 2020. To meet this goal, new methods for identifying transmission must be explored to provide a cost-effective way of identifying hotspots and areas of re-emergence; commercial loop-mediated isothermal amplification (LAMP) kits that detect the trypanosome subgenus, responsible for the two forms of sleeping sickness, have been developed. The LAMP kits were tested to assess their sensitivity, specificity and suitability as a method of screening the vector of the disease, Glossina, for Trypanozoon infection, in xenomonitoring campaigns. A simplified DNA extraction process that worked in conjunction with the LAMP kits on pooled samples demonstrated a faster method of processing large numbers of flies compared to other molecular tools. The kits performed well in our experiments and demonstrated the ability of detecting low levels of target DNA, equivalent to 0.1 trypanosome per ml. The lack of cross reaction with non-target species of trypanosomes makes the kits reliable in so far as they will only react with the Trypanozoon group of parasites of which the two human forms of the disease belong, however, further species-specific tests would need to be undertaken to identify HAT areas on selected samples.
PMCID: PMC4758712  PMID: 26890882
2.  Complete Genome Sequence of Leptospira interrogans Serovar Bratislava, Strain PigK151 
Genome Announcements  2015;3(3):e00678-15.
Leptospira interrogans serovar Bratislava infection occurs in multiple domestic and wildlife species and is associated with poor reproductive performance in swine and horses. We present the complete genome assembly of strain PigK151 comprising two chromosomes, CI (4.457 Mbp) and CII (358 kbp).
PMCID: PMC4481285  PMID: 26112787
3.  Tsetse GmmSRPN10 Has Anti-complement Activity and Is Important for Successful Establishment of Trypanosome Infections in the Fly Midgut 
The complement cascade in mammalian blood can damage the alimentary tract of haematophagous arthropods. As such, these animals have evolved their own repertoire of complement-inactivating factors, which are inadvertently exploited by blood-borne pathogens to escape complement lysis. Unlike the bloodstream stages, the procyclic (insect) stage of Trypanosoma brucei is highly susceptible to complement killing, which is puzzling considering that a tsetse takes a bloodmeal every 2–4 days. In this study, we identified four tsetse (Glossina morsitans morsitans) serine protease inhibitors (serpins) from a midgut expressed sequence tag (EST) library (GmmSRPN3, GmmSRPN5, GmmSRPN9 and GmmSRPN10) and investigated their role in modulating the establishment of a T. brucei infection in the midgut. Although not having evolved in a common blood-feeding ancestor, all four serpins have an active site sharing remarkable homology with the human complement C1-inhibitor serpin, SerpinG1. RNAi knockdown of individual GmmSRPN9 and GmmSRPN10 genes resulted in a significant decreased rate of infection by procyclic form T. brucei. Furthermore, recombinant GmmSRPN10 was both able to inhibit the activity of human complement-cascade serine proteases, C1s and Factor D, and to protect the in vitro killing of procyclic trypanosomes when incubated with complement-activated human serum. Thus, the secretion of serpins, which may be part of a bloodmeal complement inactivation system in tsetse, is used by procyclic trypanosomes to evade an influx of fresh trypanolytic complement with each bloodmeal. This highlights another facet of the complicated relationship between T. brucei and its tsetse vector, where the parasite takes advantage of tsetse physiology to further its chances of propagation and transmission.
Author Summary
Blood feeding arthropods are exploited by blood borne parasites as vectors of transmission. Trypanosoma brucei, a salivarian trypanosome species, must survive, migrate and differentiate in the tsetse until they become mature, mammalian-infective forms within the fly salivary glands. This constitutes a significant challenge to trypanosomes as the major parasite form colonising the tsetse midgut is sensitive to lysis by blood complement, which is introduced into the tsetse gut whenever the fly feeds. In this study, we show that T. brucei may avoid being eliminated by bloodmeal complement by benefitting from a complement-inhibiting enzyme secreted by the fly itself. We showed that this serine protease inhibitor (serpin) enzyme, Serpin10, can inactivate triggers of the complement cascade, protect tsetse-infective trypanosomes from complement lysis, and is important for trypanosome establishment in the tsetse midgut. Taken together, we propose that GmmSRPN10 may be part of a repertoire of complement-inhibiting proteins secreted by tsetse that are utilized by T. brucei to evade complement lysis in the tsetse midgut.
PMCID: PMC4287558  PMID: 25569180
4.  An Investigation into the Protein Composition of the Teneral Glossina morsitans morsitans Peritrophic Matrix 
Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), which is an acellular gut lining surrounding the blood meal. Crossing of this multi-layered structure occurs at least twice during parasite migration and development, but the mechanism of how trypanosomes do so is not understood. In order to better comprehend the molecular events surrounding trypanosome penetration of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism.
PMs from male teneral (young, unfed) flies were dissected, solubilised in urea/SDS buffer and the proteins precipitated with cold acetone/TCA. The PM proteins were either subjected to an in-solution tryptic digestion or fractionated on 1D SDS-PAGE, and the resulting bands digested using trypsin. The tryptic fragments from both preparations were purified and analysed by LC-MS/MS.
Overall, nearly 300 proteins were identified from both analyses, several of those containing signature Chitin Binding Domains (CBD), including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, 27 proteins from the tsetse secondary endosymbiont, Sodalis glossinidius, were also identified, suggesting this bacterium is probably in close association with the tsetse PM.
To our knowledge this is the first report on the protein composition of teneral G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology and may help identify potential molecular targets to block trypanosome development within the tsetse.
Author Summary
African trypanosomes are transmitted by the haematophagous tsetse vector. For transmission to occur, bloodmeal ingested trypanosomes must overcome numerous barriers imposed by the fly. The first obstacle is the crossing of peritrophic matrix (PM), a cell-free structure that protects the midgut epithelial cells from coming under attack by the hosts' digestive enzymes, aids in water retention and helps prevent harmful pathogens from establishing a systemic infection. Trypanosomes cross the tsetse PM at least twice in their development but how they do so remains to be elucidated. Despite being a recognised barrier to trypanosome infections, there is limited knowledge of the molecular components of the tsetse PM. In this study we identified nearly 300 PM proteins using two mass spectrometry approaches. Several of the identified components were peritrophins, which are a key group of glycoproteins essential for PM integrity. In addition, we detected proteins from Sodalis glossinidius, a commensal bacterium linked to increased susceptibility to trypanosome infection in tsetse. Our study provides the first comprehensive identification of proteins from the tsetse PM, which provides a starting point for research into potential targets for vector control.
PMCID: PMC3998921  PMID: 24763256
5.  Post Eclosion Age Predicts the Prevalence of Midgut Trypanosome Infections in Glossina 
PLoS ONE  2011;6(11):e26984.
The teneral phenomenon, as observed in Glossina sp., refers to the increased susceptibility of the fly to trypanosome infection when the first bloodmeal taken is trypanosome-infected. In recent years, the term teneral has gradually become synonymous with unfed, and thus fails to consider the age of the newly emerged fly at the time the first bloodmeal is taken. Furthermore, conflicting evidence exists of the effect of the age of the teneral fly post eclosion when it is given the infected first bloodmeal in determining the infection prevalence. This study demonstrates that it is not the feeding history of the fly but rather the age (hours after eclosion of the fly from the puparium) of the fly when it takes the first (infective) bloodmeal that determines the level of fly susceptibility to trypanosome infection. We examine this phenomenon in male and female flies from two distinct tsetse clades (Glossina morsitans morsitans and Glossina palpalis palpalis) infected with two salivarian trypanosome species, Trypanosoma (Trypanozoon) brucei brucei and Trypanosoma (Nannomonas) congolense using Fisher's exact test to examine differences in infection rates. Teneral tsetse aged less than 24 hours post-eclosion (h.p.e.) are twice as susceptible to trypanosome infection as flies aged 48 h.p.e. This trend is conserved across sex, vector clade and parasite species. The life cycle stage of the parasite fed to the fly (mammalian versus insect form trypanosomes) does not alter this age-related bias in infection. Reducing the numbers of parasites fed to 48 h.p.e., but not to 24 h.p.e. flies, increases teneral refractoriness. The importance of this phenomenon in disease biology in the field as well as the necessity of employing flies of consistent age in laboratory-based infection studies is discussed.
PMCID: PMC3210762  PMID: 22087240
6.  An insight into the sialome of Glossina morsitans morsitans 
BMC Genomics  2010;11:213.
Blood feeding evolved independently in worms, arthropods and mammals. Among the adaptations to this peculiar diet, these animals developed an armament of salivary molecules that disarm their host's anti-bleeding defenses (hemostasis), inflammatory and immune reactions. Recent sialotranscriptome analyses (from the Greek sialo = saliva) of blood feeding insects and ticks have revealed that the saliva contains hundreds of polypeptides, many unique to their genus or family. Adult tsetse flies feed exclusively on vertebrate blood and are important vectors of human and animal diseases. Thus far, only limited information exists regarding the Glossina sialome, or any other fly belonging to the Hippoboscidae.
As part of the effort to sequence the genome of Glossina morsitans morsitans, several organ specific, high quality normalized cDNA libraries have been constructed, from which over 20,000 ESTs from an adult salivary gland library were sequenced. These ESTs have been assembled using previously described ESTs from the fat body and midgut libraries of the same fly, thus totaling 62,251 ESTs, which have been assembled into 16,743 clusters (8,506 of which had one or more EST from the salivary gland library). Coding sequences were obtained for 2,509 novel proteins, 1,792 of which had at least one EST expressed in the salivary glands. Despite library normalization, 59 transcripts were overrepresented in the salivary library indicating high levels of expression. This work presents a detailed analysis of the salivary protein families identified. Protein expression was confirmed by 2D gel electrophoresis, enzymatic digestion and mass spectrometry. Concurrently, an initial attempt to determine the immunogenic properties of selected salivary proteins was undertaken.
The sialome of G. m. morsitans contains over 250 proteins that are possibly associated with blood feeding. This set includes alleles of previously described gene products, reveals new evidence that several salivary proteins are multigenic and identifies at least seven new polypeptide families unique to Glossina. Most of these proteins have no known function and thus, provide a discovery platform for the identification of novel pharmacologically active compounds, innovative vector-based vaccine targets, and immunological markers of vector exposure.
PMCID: PMC2853526  PMID: 20353571
7.  Tsetse EP Protein Protects the Fly Midgut from Trypanosome Establishment 
PLoS Pathogens  2010;6(3):e1000793.
African trypanosomes undergo a complex developmental process in their tsetse fly vector before transmission back to a vertebrate host. Typically, 90% of fly infections fail, most during initial establishment of the parasite in the fly midgut. The specific mechanism(s) underpinning this failure are unknown. We have previously shown that a Glossina-specific, immunoresponsive molecule, tsetse EP protein, is up regulated by the fly in response to gram-negative microbial challenge. Here we show by knockdown using RNA interference that this tsetse EP protein acts as a powerful antagonist of establishment in the fly midgut for both Trypanosoma brucei brucei and T. congolense. We demonstrate that this phenomenon exists in two species of tsetse, Glossina morsitans morsitans and G. palpalis palpalis, suggesting tsetse EP protein may be a major determinant of vector competence in all Glossina species. Tsetse EP protein levels also decline in response to starvation of the fly, providing a possible explanation for increased susceptibility of starved flies to trypanosome infection. As starvation is a common field event, this fact may be of considerable importance in the epidemiology of African trypanosomiasis.
Author Summary
In Africa, tsetse flies transmit the trypanosomes causing the devastating diseases sleeping sickness in man and nagana in domesticated animals. These diseases are major causes of underdevelopment in Africa. Paradoxically, most, but not all, flies are resistant to infection with trypanosomes, but we do not have a clear picture of how flies fight off trypanosomes. Here we show that a particular, tsetse-specific immune responsive protein called tsetse EP acts as a powerful antagonist of trypanosome establishment in the fly midgut. It is known that starvation of flies leads to an increase in their susceptibility to trypanosomes and this may be a considerable factor in the epidemiology of the disease in Africa. Here we demonstrate that starvation leads to a decrease in tsetse EP levels, which may explain how starvation of the fly works to increase its susceptibility.
PMCID: PMC2832768  PMID: 20221444
8.  Killing of Trypanosomatid Parasites by a Modified Bovine Host Defense Peptide, BMAP-18 
Tropical diseases caused by parasites continue to cause socioeconomic devastation that reverberates worldwide. There is a growing need for new control measures for many of these diseases due to increasing drug resistance exhibited by the parasites and problems with drug toxicity. One new approach is to apply host defense peptides (HDP; formerly called antimicrobial peptides) to disease control, either to treat infected hosts, or to prevent disease transmission by interfering with parasites in their insect vectors. A potent anti-parasite effector is bovine myeloid antimicrobial peptide-27 (BMAP-27), a member of the cathelicidin family. Although BMAP-27 is a potent inhibitor of microbial growth, at higher concentrations it also exhibits cytotoxicity to mammalian cells. We tested the anti-parasite activity of BMAP-18, a truncated peptide that lacks the hydrophobic C-terminal sequence of the BMAP-27 parent molecule, an alteration that confers reduced toxicity to mammalian cells.
Methodology/Principal Findings
BMAP-18 showed strong growth inhibitory activity against several species and life cycle stages of African trypanosomes, fish trypanosomes and Leishmania parasites in vitro. When compared to native BMAP-27, the truncated BMAP-18 peptide showed reduced cytotoxicity on a wide variety of mammalian and insect cells and on Sodalis glossindius, a bacterial symbiont of the tsetse vector. The fluorescent stain rhodamine 123 was used in immunofluorescence microscopy and flow cytometry experiments to show that BMAP-18 at low concentrations rapidly disrupted mitochondrial potential without obvious alteration of parasite plasma membranes, thus inducing death by apoptosis. Scanning electron microscopy revealed that higher concentrations of BMAP-18 induced membrane lesions in the parasites as early as 15 minutes after exposure, thus killing them by necrosis. In addition to direct killing of parasites, BMAP-18 was shown to inhibit LPS-induced secretion of tumour necrosis factor alpha (TNF-α), a cytokine that is associated with inflammation and cachexia (wasting) in sleeping sickness patients. As a prelude to in vivo applications, high affinity antibodies to BMAP-18 were produced in rabbits and used in immuno-mass spectrometry assays to detect the intact peptide in human blood and plasma.
BMAP-18, a truncated form of the potent antimicrobial BMAP-27, showed low toxicity to mammalian cells, insect cells and the tsetse bacterial symbiont Sodalis glossinidius while retaining an ability to kill a variety of species and life cycle stages of pathogenic kinetoplastid parasites in vitro. BMAP-18 also inhibited secretion of TNF-α, an inflammatory cytokine that plays a role in the cachexia associated with African sleeping sickness. These findings support the idea that BMAP-18 should be explored as a candidate for therapy of economically important trypanosome-infected hosts, such as cattle, fish and humans, and for paratransgenic expression in Sodalis glossinidius, a bacterial symbiont in the tsetse vector, as a strategy for interference with trypanosome transmission.
Author Summary
Protozoan parasites cause serious diseases in large areas of the tropics. Control of these diseases depends to a great extent on the use of therapeutic drugs, many of which are highly toxic. In addition, parasite resistance to several of the front-line drugs is increasing. Host defense peptides (HDP; formerly called antimicrobial peptides) have recently received attention as potential anti-parasite effector molecules. We earlier reported that one such peptide, bovine myeloid antimicrobial peptide (BMAP-27), is a potent inhibitor of the growth of trypanosomes and Leishmania in vitro. Here we report our studies on BMAP-18, a truncated form of BMAP-27, which showed reduced toxicity to mammalian and insect cells and yet retained its direct toxicity to parasites in vitro. BMAP-18 also strongly inhibited LPS-induced release of tumour-necrosis factor alpha (TNF-α) from human leukocytes, and thus has immunomodulatory activity. These findings suggest that BMAP-18 has potential as a therapeutic agent for treatment of infected animals or as an inhibitor of parasite transmission by their insect vectors. In anticipation of using BMAP-18 in vivo, we have also developed high affinity antibodies to BMAP-18 and have shown that these can be used, in conjunction with mass spectrometry, to detect the peptide in whole blood or plasma.
PMCID: PMC2628741  PMID: 19190729
9.  Infections with Immunogenic Trypanosomes Reduce Tsetse Reproductive Fitness: Potential Impact of Different Parasite Strains on Vector Population Structure 
The parasite Trypanosoma brucei rhodesiense and its insect vector Glossina morsitans morsitans were used to evaluate the effect of parasite clearance (resistance) as well as the cost of midgut infections on tsetse host fitness. Tsetse flies are viviparous and have a low reproductive capacity, giving birth to only 6–8 progeny during their lifetime. Thus, small perturbations to their reproductive fitness can have a major impact on population densities. We measured the fecundity (number of larval progeny deposited) and mortality in parasite-resistant tsetse females and untreated controls and found no differences. There was, however, a typanosome-specific impact on midgut infections. Infections with an immunogenic parasite line that resulted in prolonged activation of the tsetse immune system delayed intrauterine larval development resulting in the production of fewer progeny over the fly's lifetime. In contrast, parasitism with a second line that failed to activate the immune system did not impose a fecundity cost. Coinfections favored the establishment of the immunogenic parasites in the midgut. We show that a decrease in the synthesis of Glossina Milk gland protein (GmmMgp), a major female accessory gland protein associated with larvagenesis, likely contributed to the reproductive lag observed in infected flies. Mathematical analysis of our empirical results indicated that infection with the immunogenic trypanosomes reduced tsetse fecundity by 30% relative to infections with the non-immunogenic strain. We estimate that a moderate infection prevalence of about 26% with immunogenic parasites has the potential to reduce tsetse populations. Potential repercussions for vector population growth, parasite–host coevolution, and disease prevalence are discussed.
Author Summary
In many cases, parasites adapt to their hosts' biology over time and the extent of their harmful effects gradually diminishes. Insect-transmitted parasites such as African trypanosomes, however, are unusually pathogenic for their mammalian hosts because they rely on their invertebrate hosts for transmission to the next mammalian host. To ensure their maximum transmission, it is essential that parasite infections do not compromise insect host's fitness traits, including longevity and host-finding ability. Our results in tsetse indicate that, as theory predicts, trypanosome infections do not reduce host longevity. Instead, they divert host resources from reproduction and can reduce reproductive output by as much as 30%. Such loss of reproductive fitness occurs as a result of the induction of tsetse's immune responses. A closely related non-immunogenic parasite line does not induce host responses and does not compromise host fecundity. It is possible that host immune responses are needed in the case of the immunogenic line to control the parasite density to prevent excessive host damage. Because tsetse are viviparous and each adult female typically gives rise to only few progeny during their lifetime, even modest costs on reproduction can have a significant impact on host abundance. Our model predicts that if the prevalence of immunogenic parasite infections in tsetse populations reaches over 26%, they begin to have a negative impact on population growth rate. Infection rates as high as 30% have been reported with trypanosomes in the field. Our laboratory findings coupled with our modeling studies now provide a framework to investigate the status of co-infections, host immune activation processes, fecundity outcomes, transmission dynamics, and host virulence phenotypes in natural tsetse–trypanosome populations.
PMCID: PMC2265429  PMID: 18335067

Results 1-9 (9)