The effects of (2R,6R)-hydroxynorketamine on oxycodone withdrawal and reinstatement.

Despite the thousands of lives lost during the ongoing opioid crisis, a scarcity of new and effective clinical treatments for opioid use disorder (OUD) remains. To address this unmet need, some researchers have turned to dissociative and psychedelic drugs to treat multiple psychiatric conditions. In particular, low doses of ketamine have been shown to attenuate opioid withdrawal and drug use in clinical and preclinical studies. However, ketamine has misuse liability and dissociative side effects that may limit its widespread application as a treatment for OUD. More recently, (2R,6R)-hydroxynorketamine (HNK), a ketamine metabolite that lacks misuse potential, has gained attention for its effectiveness in depression and stress models. To uncover its role in OUD, we tested the time-dependent effects of (2R,6R)-HNK on oxycodone withdrawal and reinstatement of oxycodone conditioned place preference (CPP). In male and female oxycodone-dependent mice, we found that 24h pretreatment with (2R,6R)-HNK (10 or 30mg/kg, s.c.) reduced the frequency of withdrawal-like behaviors and global withdrawal scores during naloxone-precipitated withdrawal, whereas 1h pretreatment with (2R,6R)-HNK only reduced paw tremors and the sum of global withdrawal scores but not GWS Z-scores. In other experiments, both 1h and 24h pretreatment with (2R,6R)-HNK (30mg/kg, s.c.) blocked drug-induced reinstatement of oxycodone CPP. Finally, we found (2R,6R)-HNK (30mg/kg, sc) had no effect on locomotor activity and thigmotaxis. Together, these results indicate that acute (2R,6R)-HNK has efficacy in some preclinical models of OUD without producing locomotor or anxiety-like side effects.

The Trypanosoma brucei MISP family of invariant proteins is co-expressed with BARP as triple helical bundle structures on the surface of salivary gland forms, but is dispensable for parasite development within the tsetse vector.

Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomic analyses of saliva from T. brucei-infected tsetse flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of glycosylphosphatidylinositol (GPI)-anchored surface proteins herein named as Metacyclic Invariant Surface Proteins (MISP) because of its predominant expression on the surface of metacyclic trypomastigotes. The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are potentially extended above the metacyclic VSG coat, and thus could be tested as a transmission-blocking vaccine target. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both CRISPR-Cas9-driven knock out and RNAi knock down of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We suggest MISP may be relevant during trypanosome transmission or establishment in the vertebrate's skin.

Estimating the impact of Tiny Targets in reducing the incidence of Gambian sleeping sickness in the North-west Uganda focus.

BACKGROUND: Riverine species of tsetse (Glossina) transmit Trypanosoma brucei gambiense, which causes Gambian human African trypanosomiasis (gHAT), a neglected tropical disease. Uganda aims to eliminate gHAT as a public health problem through detection and treatment of human cases and vector control. The latter is being achieved through the deployment of 'Tiny Targets', insecticide-impregnated panels of material which attract and kill tsetse. We analysed the spatial and temporal distribution of cases of gHAT in Uganda during the period 2010-2019 to assess whether Tiny Targets have had an impact on disease incidence. METHODS: To quantify the deployment of Tiny Targets, we mapped the rivers and their associated watersheds in the intervention area. We then categorised each of these on a scale of 0-3 according to whether Tiny Targets were absent (0), present only in neighbouring watersheds (1), present in the watersheds but not all neighbours (2), or present in the watershed and all neighbours (3). We overlaid all cases that were diagnosed between 2000 and 2020 and assessed whether the probability of finding cases in a watershed changed following the deployment of targets. We also estimated the number of cases averted through tsetse control. RESULTS: We found that following the deployment of Tiny Targets in a watershed, there were fewer cases of HAT, with a sampled error probability of 0.007. We estimate that during the intervention period 2012-2019 we should have expected 48 cases (95% confidence intervals = 40-57) compared to the 36 cases observed. The results are robust to a range of sensitivity analyses. CONCLUSIONS: Tiny Targets have reduced the incidence of gHAT by 25% in north-western Uganda.

Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.

Trypanosoma brucei colonizes the tsetse gut via an immature peritrophic matrix in the proventriculus.

The peritrophic matrix of blood-feeding insects is a chitinous structure that forms a protective barrier against oral pathogens and abrasive particles1. Tsetse flies transmit Trypanosoma brucei, which is the parasite that causes human sleeping sickness and is also partially responsible for animal trypanosomiasis in Sub-Saharan Africa. For this parasite to establish an infection in flies, it must first colonize the area between the peritrophic matrix and gut epithelium called the ectoperitrophic space. Although unproven, it is generally accepted that trypanosomes reach the ectoperitrophic space by penetrating the peritrophic matrix in the anterior midgut2-4. Here, we revisited this event using fluorescence- and electron-microscopy methodologies. We show that trypanosomes penetrate the ectoperitrophic space in which the newly made peritrophic matrix is synthesized by the proventriculus. Our model describes how these proventriculus-colonizing parasites can either migrate to the ectoperitrophic space or become trapped within peritrophic matrix layers to form cyst-like bodies that are passively pushed along the gut as the matrix gets remodelled. Furthermore, early proventricular colonization seems to be promoted by factors in trypanosome-infected blood that cause higher salivary gland infections and potentially increase parasite transmission.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

BACKGROUND: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. RESULTS: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. CONCLUSIONS: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

Proline Metabolism is Essential for Trypanosoma brucei brucei Survival in the Tsetse Vector.

Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly's midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut.

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.

Tsetse Control and Gambian Sleeping Sickness; Implications for Control Strategy.

BACKGROUND: Gambian sleeping sickness (human African trypanosomiasis, HAT) outbreaks are brought under control by case detection and treatment although it is recognised that this typically only reaches about 75% of the population. Vector control is capable of completely interrupting HAT transmission but is not used because it is considered too expensive and difficult to organise in resource-poor settings. We conducted a full scale field trial of a refined vector control technology to determine its utility in control of Gambian HAT. METHODS AND FINDINGS: The major vector of Gambian HAT is the tsetse fly Glossina fuscipes which lives in the humid zone immediately adjacent to water bodies. From a series of preliminary trials we determined the number of tiny targets required to reduce G. fuscipes populations by more than 90%. Using these data for model calibration we predicted we needed a target density of 20 per linear km of river in riverine savannah to achieve >90% tsetse control. We then carried out a full scale, 500 km2 field trial covering two HAT foci in Northern Uganda to determine the efficacy of tiny targets (overall target density 5.7/km2). In 12 months, tsetse populations declined by more than 90%. As a guide we used a published HAT transmission model and calculated that a 72% reduction in tsetse population is required to stop transmission in those settings. INTERPRETATION: The Ugandan census suggests population density in the HAT foci is approximately 500 per km2. The estimated cost for a single round of active case detection (excluding treatment), covering 80% of the population, is US$433,333 (WHO figures). One year of vector control organised within the country, which can completely stop HAT transmission, would cost US$42,700. The case for adding this method of vector control to case detection and treatment is strong. We outline how such a component could be organised.

Complete Genome Sequence of Leptospira interrogans Serovar Bratislava, Strain PigK151.

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).

Costs of using "tiny targets" to control Glossina fuscipes fuscipes, a vector of gambiense sleeping sickness in Arua District of Uganda.

INTRODUCTION: To evaluate the relative effectiveness of tsetse control methods, their costs need to be analysed alongside their impact on tsetse populations. Very little has been published on the costs of methods specifically targeting human African trypanosomiasis. METHODOLOGY/PRINCIPAL FINDINGS: In northern Uganda, a 250 km2 field trial was undertaken using small (0.5 X 0.25 m) insecticide-treated targets ("tiny targets"). Detailed cost recording accompanied every phase of the work. Costs were calculated for this operation as if managed by the Ugandan vector control services: removing purely research components of the work and applying local salaries. This calculation assumed that all resources are fully used, with no spare capacity. The full cost of the operation was assessed at USD 85.4 per km2, of which USD 55.7 or 65.2% were field costs, made up of three component activities (target deployment: 34.5%, trap monitoring: 10.6% and target maintenance: 20.1%). The remaining USD 29.7 or 34.8% of the costs were for preliminary studies and administration (tsetse surveys: 6.0%, sensitisation of local populations: 18.6% and office support: 10.2%). Targets accounted for only 12.9% of the total cost, other important cost components were labour (24.1%) and transport (34.6%). DISCUSSION: Comparison with the updated cost of historical HAT vector control projects and recent estimates indicates that this work represents a major reduction in cost levels. This is attributed not just to the low unit cost of tiny targets but also to the organisation of delivery, using local labour with bicycles or motorcycles. Sensitivity analyses were undertaken, investigating key prices and assumptions. It is believed that these costs are generalizable to other HAT foci, although in more remote areas, with denser vegetation and fewer people, costs would increase, as would be the case for other tsetse control techniques.

Optimal strategies for controlling riverine tsetse flies using targets: a modelling study.

BACKGROUND: Tsetse flies occur in much of sub-Saharan Africa where they transmit the trypanosomes that cause the diseases of sleeping sickness in humans and nagana in livestock. One of the most economical and effective methods of tsetse control is the use of insecticide-treated screens, called targets, that simulate hosts. Targets have been ~1 m2, but recently it was shown that those tsetse that occupy riverine situations, and which are the main vectors of sleeping sickness, respond well to targets only ~0.06 m2. The cheapness of these tiny targets suggests the need to reconsider what intensity and duration of target deployments comprise the most cost-effective strategy in various riverine habitats. METHODOLOGY/PRINCIPAL FINDINGS: A deterministic model, written in Excel spreadsheets and managed by Visual Basic for Applications, simulated the births, deaths and movement of tsetse confined to a strip of riverine vegetation composed of segments of habitat in which the tsetse population was either self-sustaining, or not sustainable unless supplemented by immigrants. Results suggested that in many situations the use of tiny targets at high density for just a few months per year would be the most cost-effective strategy for rapidly reducing tsetse densities by the ~90% expected to have a great impact on the incidence of sleeping sickness. Local elimination of tsetse becomes feasible when targets are deployed in isolated situations, or where the only invasion occurs from populations that are not self-sustaining. CONCLUSION/SIGNIFICANCE: Seasonal use of tiny targets deserves field trials. The ability to recognise habitat that contains tsetse populations which are not self-sustaining could improve the planning of all methods of tsetse control, against any species, in riverine, savannah or forest situations. Criteria to assist such recognition are suggested.

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.

Explaining the host-finding behavior of blood-sucking insects: computerized simulation of the effects of habitat geometry on tsetse fly movement.

BACKGROUND: Male and female tsetse flies feed exclusively on vertebrate blood. While doing so they can transmit the diseases of sleeping sickness in humans and nagana in domestic stock. Knowledge of the host-orientated behavior of tsetse is important in designing bait methods of sampling and controlling the flies, and in understanding the epidemiology of the diseases. For this we must explain several puzzling distinctions in the behavior of the different sexes and species of tsetse. For example, why is it that the species occupying savannahs, unlike those of riverine habitats, appear strongly responsive to odor, rely mainly on large hosts, are repelled by humans, and are often shy of alighting on baits? METHODOLOGY/PRINCIPAL FINDINGS: A deterministic model that simulated fly mobility and host-finding success suggested that the behavioral distinctions between riverine, savannah and forest tsetse are due largely to habitat size and shape, and the extent to which dense bushes limit occupiable space within the habitats. These factors seemed effective primarily because they affect the daily displacement of tsetse, reducing it by up to ∼70%. Sex differences in behavior are explicable by females being larger and more mobile than males. CONCLUSION/SIGNIFICANCE: Habitat geometry and fly size provide a framework that can unify much of the behavior of all sexes and species of tsetse everywhere. The general expectation is that relatively immobile insects in restricted habitats tend to be less responsive to host odors and more catholic in their diet. This has profound implications for the optimization of bait technology for tsetse, mosquitoes, black flies and tabanids, and for the epidemiology of the diseases they transmit.

An investigation into the protein composition of the teneral Glossina morsitans morsitans peritrophic matrix.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Community acceptance of tsetse control baits: a qualitative study in Arua District, North West Uganda.

BACKGROUND: There is renewed vigour in efforts to eliminate neglected tropical diseases including sleeping sickness (human African trypanosomiasis or HAT), including attempts to develop more cost-effective methods of tsetse control. In the West Nile region of Uganda, newly designed insecticide-treated targets are being deployed over an area of ∼500 km(2). The operational area covers villages where tsetse control has not been conducted previously. The effectiveness of the targets will depend, in part, on their acceptance by the local community. METHODOLOGY/PRINCIPAL FINDINGS: We assessed knowledge, perceptions and acceptance of tsetse baits (traps, targets) in villages where they had or had not been used previously. We conducted sixteen focus group discussions with male and female participants in eight villages across Arua District. Discussions were audio recorded, translated and transcribed. We used thematic analysis to compare the views of both groups and identify salient themes. CONCLUSIONS/SIGNIFICANCE: Despite the villages being less than 10 km apart, community members perceived deployed baits very differently. Villagers who had never seen traps before expressed fear, anxiety and panic when they first encountered them. This was related to associations with witchcraft and "ghosts from the river" which are traditionally linked with physical or mental illness, death and misfortune. By contrast, villagers living in areas where traps had been used previously had positive attitudes towards them and were fully aware of their purpose and benefits. The latter group reported that they had similar negative perceptions when tsetse control interventions first started a decade ago. Our results suggest that despite their proximity, acceptance of traps varies markedly between villages and this is related to the duration of experience with tsetse control programs. The success of community-based interventions against tsetse will therefore depend on early engagements with communities and carefully designed sensitization campaigns that reach all communities, especially those living in areas new to such interventions.

Long-term effects and parental RNAi in the blood feeder Rhodnius prolixus (Hemiptera; Reduviidae).

RNA interference (RNAi) has been widely employed as a useful alternative to study gene function in insects, including triatomine bugs. However, several aspects related to the RNAi mechanism and functioning are still unclear. The aim of this study is to investigate the persistence and the occurrence of systemic and parental RNAi in the triatomine bug Rhodnius prolixus. For such, the nitrophorins 1 to 4 (NP1-4), which are salivary hemeproteins, and the rhodniin, an intestinal protein, were used as targets for RNAi. The dsRNA for both molecules were injected separately into 3rd and 5th instar nymphs of R. prolixus and the knockdown (mRNA levels and phenotype) were progressively evaluated along several stages of the insect's life. We observed that the NP1-4 knockdown persisted for more than 7 months after the dsRNA injection, and at least 5 months in rhodniin knockdown, passing through various nymphal stages until the adult stage, without continuous input of dsRNA. The parental RNAi was successful from the dsRNA injection in 5th instar nymphs for both knockdown targets, when the RNAi effects (mRNA levels and phenotype) were observed at least in the 2nd instar nymphs of the F1 generation. However, the parental RNAi did not occur when the dsRNA was injected in the 3rd instars. The confirmation of the long persistence and parental transmission of RNAi in R. prolixus can improve and facilitate the utilization of this tool in insect functional genomic studies.

Application of RNA interference in triatomine (Hemiptera: Reduviidae) studies.

Triatomines (Hemiptera: Reduviidae) are obligate hematophagous insects. They are of medical importance because they are vectors of Trypanosoma cruzi, the causative agent of Chagas disease in the Americas. In recent years, the RNA interference (RNAi) technology has emerged as a practical and useful alternative means of studying gene function in insects, including triatomine bugs. RNAi research in triatomines is still in its early stages, several issues still need to be elucidated, including the description of the molecules involved in the RNAi machinery and aspects related to phenotype evaluation and persistence of the knockdown in different tissues and organs. This review considers recent applications of RNAi to triatomine research, describing the major methods that have been applied during the knockdown process such as the double-stranded RNA delivery mechanism (injection, microinjection, or ingestion) and the phenotype characterization (mRNA and target protein levels) in studies conducted with the intent to provide greater insights into the biology of these insects. In addition to the characterization of insect biomolecules, some with biopharmacological potential, RNAi may provide a new view of the interaction between triatomine and trypanosomatids, enabling the development of new measures for vector control and transmission of the parasite.

Host and environmental reservoirs of infection for bovine digital dermatitis treponemes.

Bovine digital dermatitis (BDD) is a global infectious disease causing lameness of cattle and is responsible for substantial animal welfare issues and economic losses. The causative agents are considered to be spirochetal bacteria belonging to the genus Treponema, which have consistently been identified in BDD lesions worldwide. One potential means of controlling infection is the disruption of transmission; however, the infection reservoirs and transmission routes of BDD treponemes have yet to be elucidated. To address these issues, we surveyed for evidence of BDD treponeme presence in the dairy farm environment, in bovine tissues and in bovine gastrointestinal (GI) tract contents. A total of 368 samples were tested using PCR assays specific for each of three currently recognised, isolated phylotypes of BDD treponemes. All environmental samples, together with insects and GI tract content samples were negative for BDD treponeme DNA from the three phylotypes. However, we identified BDD treponemes in two non-pedal bovine regions: the oral cavity (14.3% of cattle tested) and the rectum (14.8% of cattle tested). Whilst only single phylotypes were detected in the oral cavity, two of the rectal tissues yielded DNA from more than one phylotype, with one sample yielding all three BDD treponeme phylotypes. Whilst it might be considered that direct skin to skin contact may be a major transmission route of BDD treponemes, further studies are required to characterise and determine the potential contribution of oral and rectal carriage to BDD transmission.