Distribution and abundance of trypanosome (subgenus Nannomonas) infections of the tsetse fly Glossina pallidipes in southern Africa

Over 10000 Glossina pallidipes tsetse flies were collected during two field studies in the Zambezi Valley, Zimbabwe and one in the Luangwa Valley, Zambia. These were screened for mature trypanosome infections and 234 dot-blot preparations were made of infected midguts, which were screened using DNA probes or PCR with primers specific to different species or types of the trypanosome subgenus Nannomonas. Over 70% of midgut infections were successfully identified as either Trypanosoma godfreyi, T. simiae or three types of T. congolense, savannah, riverine-forest and Kilifi. The relative abundance of species and types did not vary significantly between study locations, habitat, season or tsetse age or sex, although there were differences between DNA probe and PCR results. Mixed species and/or mixed type infections were common and were more often detected using PCR. The distribution of infections among flies was highly aggregated, but there was no tendency for multiple infections to accumulate in older flies, implying that sequential superinfection may be uncommon. Possible explanations for these patterns are discussed.     

A Proline Racemase Based PCR for Identification of Trypanosoma vivax in Cattle Blood

A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764–783 bp and 983–1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide.     

Seasonal variation of tsetse fly species abundance and prevalence of trypanosomes in the Maasai Steppe,Tanzania

Tsetse flies, the vectors of trypanosomiasis, represent a threat to public health and economy in sub‐Saharan Africa. Despite these concerns, information on temporal and spatial dynamics of tsetse and trypanosomes remain limited and may be a reason that control strategies are less effective. The current study assessed the temporal variation of the relative abundance of tsetse fly species and trypanosome prevalence in relation to climate in the Maasai Steppe of Tanzania in 2014–2015. Tsetse flies were captured using odor‐baited Epsilon traps deployed in ten sites selected through random subsampling of the major vegetation types in the area. Fly species were identified morphologically and trypanosome species classified using PCR. The climate dataset was acquired from the African Flood and Drought Monitor repository. Three species of tsetse flies were identified: G. swynnertoni (70.8%), G. m. morsitans (23.4%), and G.pallidipes (5.8%). All species showed monthly changes in abundance with most of the flies collected in July. The relative abundance of G. m. morsitans and G. swynnertoni was negatively correlated with maximum and minimum temperature, respectively. Three trypanosome species were recorded: T. vivax (82.1%), T. brucei (8.93%), and T. congolense (3.57%). The peak of trypanosome infections in the flies was found in October and was three months after the tsetse abundance peak; prevalence was negatively correlated with tsetse abundance. A strong positive relationship was found between trypanosome prevalence and temperature. In conclusion, we find that trypanosome prevalence is dependent on fly availability, and temperature drives both tsetse fly relative abundance and trypanosome prevalence.     

Identification of a 4-fluorobenzyl l-valinate amide benzoxaborole (AN11736) as a potential development candidate for the treatment of Animal African Trypanosomiasis (AAT)

Novel l-valinate amide benzoxaboroles and analogues were designed and synthesized for a structure-activity-relationship (SAR) investigation to optimize the growth inhibitory activity against Trypanosoma congolense (T. congolense) and Trypanosoma vivax (T. vivax) parasites. The study identified 4-fluorobenzyl (1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-l-valinate (5, AN11736), which showed IC₅₀ values of 0.15?nM against T. congolense and 1.3?nM against T. vivax, and demonstrated 100% efficacy with a single dose of 10?mg/kg against both T. congolense and T. vivax in mouse models of infection (IP dosing) and in the target animal, cattle, dosed intramuscularly. AN11736 has been advanced to early development studies.     

The diurnal activity, movement and trypanosome infection rates of Glossina fuscipes fuscipes （Diptera： Glossinidae） in Buvuma Island, Lake Victoria, Uganda

The diurnal activity patterns, trypanosome infection rates and movement of Glossinafuscipesfuscipes （Diptera： Glossinidae） were investigated in Buvuma Island, Lake Victoria, Uganda. Hourly trapping of tsetse flies was undertaken to determine their activity rhythm while a capture-mark-release-recapture method was conducted to assess the movement and dispersal of tsetse flies between lakeshore, hinterland and further inland sites along a transected area. Dissection of tsetse flies was also undertaken to determine the trypanosome infection rates in salivary glands, proboscis and mid-gut. Results indicated a bimodal diurnal activity profile for G. f fuscipes on the Island, both on the lakeshore and in the hinterland. Movement and dispersal of G. f fuscipes tsetse flies occurred between lakeshore, hinterland and further inland sites with a greater tendency of flies to move to the lakeshore. Trypanosome infection rates of 4.32% for Trypasoma vivax and 1.15% for 7. congolense were found in G. f. fuscipes.     

Molecular epidemiology of Animal African Trypanosomosis in southwest Burkina Faso

BackgroundAnimal African Trypanosomosis (AAT) is a parasitic disease of livestock that has a major socio-economic impact in the affected areas. It is caused by several species of uniflagellate extracellular protists of the genus Trypanosoma mainly transmitted by tsetse flies: T. congolense, T. vivax and T. brucei brucei. In Burkina Faso, AAT hampers the proper economic development of the southwestern part of the country, which is yet the best watered area particularly conducive to agriculture and animal production. It was therefore important to investigate the extent of the infection in order to better control the disease. The objective of the present study was to assess the prevalence of trypanosome infections and collect data on the presence of tsetse flies.MethodsBuffy coat, Trypanosoma species-specific PCR, Indirect ELISA Trypanosoma sp and trypanolysis techniques were used on 1898 samples collected. An entomological survey was also carried out.ResultsThe parasitological prevalence of AAT was 1.1%, and all observed parasites were T. vivax. In contrast, the molecular prevalence was 23%, of which T. vivax was predominant (89%) followed by T. congolense (12.3%) and T. brucei s.l. (7.3%) with a sizable proportion as mixed infections (9.1%). T. brucei gambiense, responsible of sleeping sickness in humans, was not detected. The serological prevalence reached 49.7%. Once again T. vivax predominated (77.2%), but followed by T. brucei (14.7%) and T. congolense (8.1%). Seven samples, from six cattle and one pig, were found positive by trypanolysis. The density per trap of Glossina tachinoides and G. palpalis gambiensis was 1.2 flies.Conclusions/SignificanceOverall, our study showed a high prevalence of trypanosome infection in the area, pointing out an ongoing inadequacy of control measures.     

Prevalence of trypanosomes,salivary gland hypertrophy virus and Wolbachia in wild populations of tsetse flies from West Africa

Background

Tsetse flies are vectors of African trypanosomes, protozoan parasites that cause sleeping sickness (or human African trypanosomosis) in humans and nagana (or animal African trypanosomosis) in livestock. In addition to trypanosomes, four symbiotic bacteria Wigglesworthia glossinidia, Sodalis glossinidius, Wolbachia, Spiroplasma and one pathogen, the salivary gland hypertrophy virus (SGHV), have been reported in different tsetse species. We evaluated the prevalence and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in four tsetse species (Glossina palpalis gambiensis, G. tachinoides, G. morsitans submorsitans, and G. medicorum) that were collected between 2008 and 2015 from 46 geographical locations in West Africa, i.e. Burkina Faso, Mali, Ghana, Guinea, and Senegal.

Results

The results indicated an overall low prevalence of SGHV and Wolbachia and a high prevalence of trypanosomes in the sampled wild tsetse populations. The prevalence of all three infections varied among tsetse species and sample origin. The highest trypanosome prevalence was found in Glossina tachinoides (61.1%) from Ghana and in Glossina palpalis gambiensis (43.7%) from Senegal. The trypanosome prevalence in the four species from Burkina Faso was lower, i.e. 39.6% in Glossina medicorum, 18.08%; in Glossina morsitans submorsitans, 16.8%; in Glossina tachinoides and 10.5% in Glossina palpalis gambiensis. The trypanosome prevalence in Glossina palpalis gambiensis was lowest in Mali (6.9%) and Guinea (2.2%). The prevalence of SGHV and Wolbachia was very low irrespective of location or tsetse species with an average of 1.7% for SGHV and 1.0% for Wolbachia. In some cases, mixed infections with different trypanosome species were detected. The highest prevalence of coinfection was Trypanosoma vivax and other Trypanosoma species (9.5%) followed by coinfection of T. congolense with other trypanosomes (7.5%). The prevalence of coinfection of T. vivax and T. congolense was (1.0%) and no mixed infection of trypanosomes, SGHV and Wolbachia was detected.

Conclusion

The results indicated a high rate of trypanosome infection in tsetse wild populations in West African countries but lower infection rate of both Wolbachia and SGHV. Double or triple mixed trypanosome infections were found. In addition, mixed trypanosome and SGHV infections existed however no mixed infections of trypanosome and/or SGHV with Wolbachia were found.

     

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.     

Sodalis glossinidius (Enterobacteriaceae) and Vectorial Competence of Glossina palpalis gambiensis and Glossina morsitans morsitans for Trypanosoma congolense Savannah Type

Sodalis glossinidius is an endosymbiont of Glossina palpalis gambiensis and Glossina morsitans morsitans, the vectors of Trypanosoma congolense. The presence of the symbiont was investigated by PCR in Trypanosoma congolense savannah type-infected and noninfected midguts of both fly species, and into the probosces of flies displaying either mature or immature infection, to investigate possible correlation with the vectorial competence of tsetse flies. Sodalis glossinidius was detected in all midguts, infected or not, from both Glossina species. It was also detected in probosces from Glossina palpalis gambiensis flies displaying mature or immature infection, but never in probosces from Glossina morsitans morsitans. These results suggest that, a) there might be no direct correlation between the presence of Sodalis glossinidius and the vectorial competence of Glossina, and b) the symbiont is probably not involved in Trypanosoma congolense savannah type maturation. It could however participate in the establishment process of the parasite.     

Sodalis glossinidius presence in wild tsetse is only associated with presence of trypanosomes in complex interactions with other tsetse-specific factors

Background

Susceptibility of tsetse flies (Glossina spp.) to trypanosomes of both humans and animals has been associated with the presence of the endosymbiont Sodalis glossinidius. However, intrinsic biological characteristics of the flies and environmental factors can influence the presence of both S. glossinidius and the parasites. It thus remains unclear whether it is the S. glossinidius or other attributes of the flies that explains the apparent association. The objective of this study was to test whether the presence of Trypanosoma vivax, T. congolense and T. brucei are related to the presence of S. glossinidius in tsetse flies when other factors are accounted for: geographic location, species of Glossina, sex or age of the host flies.

Results

Flies (n = 1090) were trapped from four sites in the Shimba Hills and Nguruman regions in Kenya. Sex and species of tsetse (G. austeni, G. brevipalpis, G. longipennis and G. pallidipes) were determined based on external morphological characters and age was estimated by a wing fray score method. The presence of trypanosomes and S. glossinidius was detected using PCR targeting the internal transcribed spacer region 1 and the haemolysin gene, respectively. Sequencing was used to confirm species identification. Generalised Linear Models (GLMs) and Multiple Correspondence Analysis (MCA) were applied to investigate multivariable associations. The overall prevalence of trypanosomes was 42.1%, but GLMs revealed complex patterns of associations: the presence of S. glossinidius was associated with trypanosome presence but only in interactions with other factors and only in some species of trypanosomes. The strongest association was found for T. congolense, and no association was found for T. vivax. The MCA also suggested only a weak association between the presence of trypanosomes and S. glossinidius. Trypanosome-positive status showed strong associations with sex and age while S. glossinidius-positive status showed a strong association with geographic location and species of fly.

Conclusions

We suggest that previous conclusions about the presence of endosymbionts increasing probability of trypanosome presence in tsetse flies may have been confounded by other factors, such as community composition of the tsetse flies and the specific trypanosomes found in different regions.

     

Ultrastructural Studies of Certain Aspects of the Development of Trypanosoma congolense in Glossina morsitans morsitans*

SYNOPSIS The course of Trypanosoma congolense infections in Glossina morsitans morsitans was followed by electron-microscopic examination of ultrathin sections of the guts and proboscises of infected flies. Guts dissected from flies 7 days after infection with culture procyclic forms of T. congolense had heavy trypanosome infections in the midgut involving both the endo- and ectoperitrophic spaces. Trypanosomes were also seen in the process of penetrating the fully formed peritrophic membrane in the central region of the midgut. By post infection day 21, trypanosomes had reached the proboscis of the fly and were found as clumps of epimastigote forms attached to the labrum by hemidesmosomes between their flagella and the chitinous lining of the food canal. Desmosome connections were observed between the flagella of adjacent epimastigotes. Flies examined at postinfection days 28 and 42 had, in addition to the attached forms in the labrum, free forms in the hypopharynx.     

Trypanosoma vivax GM6 Antigen: A Candidate Antigen for Diagnosis of African Animal Trypanosomosis in Cattle

Background

Diagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km² of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases.

Methodology/Principle Findings

the repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4).

Conclusion/Significance

this study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle.     

The internal transcribed spacer 1 (ITS-1), a controversial marker for the genetic diversity of Trypanosoma evansi

Seven Trypanosoma evansi isolates from China and a Trypanosoma congolense sp. gifted from Kenya were characterized genetically by the internal transcribed spacer 1 (ITS-1) of nuclear ribosomal DNA (rDNA). The ITS-1 rDNA with the length of 338–342 bp was amplified by polymerase chain reaction (PCR) and sequenced from individual isolates of T. evansi. Although sequence variation between T. evansi isolates from China only was 0.3–3.8%, the constructed phylogenetic tree based on the ITS-1 rDNA sequence by the method of neighbor-joining and maximum parsimony revealed the genetic diversity among T. evansi isolates from China. For T. congolense sp., the most phylogenetically related species was T. congolense IL1180. Although the sequence variation ranged 0.8–14.5% between T. congolense isolates, the phylogenetic tree can not reflected the genetic diversity among T. congolense isolates perhaps because of the fewer number of isolates and sequences. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.     

Metarhizium anisopliae infection reduces Trypanosoma congolense reproduction in Glossina fuscipes fuscipes and its ability to acquire or transmit the parasite

Background

Tsetse fly-borne trypanosomiasis remains a significant problem in Africa despite years of interventions and research. The need for new strategies to control and possibly eliminate trypanosomiasis cannot be over-emphasized. Entomopathogenic fungi (EPF) infect their hosts through the cuticle and proliferate within the body of the host causing death in about 3–14 days depending on the concentration. During the infection process, EPF can reduce blood feeding abilities in hematophagous arthropods such as mosquitoes, tsetse flies and ticks, which may subsequently impact the development and transmission of parasites. Here, we report on the effects of infection of tsetse fly (Glossina fuscipes fuscipes) by the EPF, Metarhizium anisopliae ICIPE 30 wild-type strain (WT) and green fluorescent protein-transformed strain (GZP-1) on the ability of the flies to harbor and transmit the parasite, Trypanosoma congolense.

Results

Teneral flies were fed T. congolense-infected blood for 2 h and then infected using velvet carpet fabric impregnated with conidia covered inside a cylindrical plastic tube for 12 h. Control flies were fed with T. congolense-infected blood but not exposed to the fungal treatment via the carpet fabric inside a cylindrical plastic tube. Insects were dissected at 2, 3, 5 and 7 days post-fungal exposure and the density of parasites quantified. Parasite load decreased from 8.7 × 10⁷ at day 2 to between 8.3 × 10⁴ and 1.3 × 10⁵ T. congolense ml^− 1 at day 3 post-fungal exposure in fungus-treated (WT and GZP-1) fly groups. When T. congolense-infected flies were exposed to either fungal strain, they did not transmit the parasite to mice whereas control treatment flies remained capable of parasite transmission. Furthermore, M. anisopliae-inoculated flies which fed on T. congolense-infected mice were not able to acquire the parasites at 4 days post-fungal exposure while parasite acquisition was observed in the control treatment during the same period.

Conclusions

Infection of the vector G. f. fuscipes by the entomopathogenic fungus M. anisopliae negatively affected the multiplication of the parasite T. congolense in the fly and reduced the vectorial capacity to acquire or transmit the parasite.

     

Mitonuclear genomics challenges the theory of clonality in Trypanosoma congolense: Reply to Tibayrenc and Ayala

We recently published the first genomic diversity study of Trypanosoma congolense, a major aetiological agent of Animal African Trypanosomiasis. We demonstrated striking levels of SNP and indel diversity in the Eastern province of Zambia as a consequence of hybridization between divergent trypanosome lineages. We concluded that these and earlier findings in T. congolense challenge the predominant clonal evolution (PCE) model. In a recent comment, Tibayrenc and Ayala claim that there are many features in T. congolense supporting their theory of clonality. While we can follow the reasoning of the authors, we also identify major limitations in their theory and interpretations that resulted in incorrect conclusions. First, we argue that each T. congolense subgroup should be analysed independently as they may represent different (sub)species rather than “near‐clades”. Second, the authors neglect major findings of two robust population genetic studies on Savannah T. congolense that provide clear evidence of frequent recombination. Third, we reveal additional events of introgressive hybridization in T. congolense by analysing the maxicircle coding region using next‐generation sequencing analyses. At last, we pinpoint two important misinterpretations by the authors and show that there are no spatially and temporally widespread clones in T. congolense. We stand by our earlier conclusions that the clonal framework is unlikely to accurately model the population structure of T. congolense. Other theoretical frameworks such as Maynard Smith's epidemic model may better represent the complex ancestry seen in T. congolense, where clones delimited in space and time arise against a background of recombination.     

Modeling the control of trypanosomiasis using trypanocides or insecticide-treated livestock

Background

In Uganda, Rhodesian sleeping sickness, caused by Trypanosoma brucei rhodesiense, and animal trypanosomiasis caused by T. vivax and T. congolense, are being controlled by treating cattle with trypanocides and/or insecticides. We used a mathematical model to identify treatment coverages required to break transmission when host populations consisted of various proportions of wild and domestic mammals, and reptiles.

Methodology/Principal Findings

An Ro model for trypanosomiasis was generalized to allow tsetse to feed off multiple host species. Assuming populations of cattle and humans only, pre-intervention Ro values for T. vivax, T. congolense, and T. brucei were 388, 64 and 3, respectively. Treating cattle with trypanocides reduced R ₀ for T. brucei to <1 if >65% of cattle were treated, vs 100% coverage necessary for T. vivax and T. congolense. The presence of wild mammalian hosts increased the coverage required and made control of T. vivax and T. congolense impossible. When tsetse fed only on cattle or humans, R ₀ for T. brucei was <1 if 20% of cattle were treated with insecticide, compared to 55% for T. congolense. If wild mammalian hosts were also present, control of the two species was impossible if proportions of non-human bloodmeals from cattle were <40% or <70%, respectively. R ₀ was <1 for T. vivax only when insecticide treatment led to reductions in the tsetse population. Under such circumstances R ₀<1 for T. brucei and T. congolense if cattle make up 30% and 55%, respectively of the non-human tsetse bloodmeals, as long as all cattle are treated with insecticide.

Conclusions/Significance

In settled areas of Uganda with few wild hosts, control of Rhodesian sleeping sickness is likely to be much more effectively controlled by treating cattle with insecticide than with trypanocides.     

Evaluation of the Loop Mediated Isothermal DNA Amplification (LAMP) Kit for Malaria Diagnosis in P. vivax Endemic Settings of Colombia

Background

Most commonly used malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections which are frequent in low transmission settings. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too laborious for field deployment. In this study, the applicability of a malaria diagnosis kit based on loop-mediated isothermal amplification (mLAMP) was assessed in malaria endemic areas of Colombia with Plasmodium vivax predominance.

Methodology/Principal Findings

First, a passive case detection (PCD) study on 278 febrile patients recruited in Tierralta (department of Cordoba) was conducted to assess the diagnostic performance of the mLAMP method. Second, an active case detection (ACD) study on 980 volunteers was conducted in 10 sentinel sites with different epidemiological profiles. Whole blood samples were processed for microscopic and mLAMP diagnosis. Additionally RT-PCR and nested RT-PCR were used as reference tests. In the PCD study, P. falciparum accounted for 23.9% and P. vivax for 76.1% of the infections and no cases of mixed-infections were identified. Microscopy sensitivity for P. falciparum and P. vivax were 100% and 86.1%, respectively. mLAMP sensitivity for P. falciparum and P. vivax was 100% and 91.4%, respectively. In the ACD study, mLAMP detected 65 times more cases than microscopy. A high proportion (98.0%) of the infections detected by mLAMP was from volunteers without symptoms.

Conclusions/Significance

mLAMP sensitivity and specificity were comparable to RT-PCR. LAMP was significantly superior to microscopy and in P. vivax low-endemicity settings and under minimum infrastructure conditions, it displayed sensitivity and specificity similar to that of single-well RT-PCR for detection of both P. falciparum and P. vivax infections. Here, the dramatically increased detection of asymptomatic malaria infections by mLAMP demonstrates the usefulness of this new tool for diagnosis, surveillance, and screening in elimination strategies.     

A Novel qPCR Assay for the Detection of African Animal Trypanosomosis in Trypanotolerant and Trypanosusceptible Cattle Breeds

This study was conducted to (i) determine the prevalence of African Animal Trypanosomosis (AAT) in tsetse challenged areas, (ii) compare conventional with qPCR detection systems and (iii) evaluate the host genetic background and biology as risk factors. AAT prevalence studies are often confronted with low levels of parasitaemia. Hence, we designed a novel qPCR assay using primers and species specific probes amplifying the Internal Transcribed Spacer 1 (ITS1) gene. Thereby all three AAT species could be detected simultaneously. 368 individuals from three cattle types (Baoulé, Zebu and hybrids) originating from 72 farms in Burkina Faso were analysed. Farmers were interviewed and morphometric measurements of the cattle taken. A chi-squared test and a logistic regression model were calculated to detect associations with infection. In our study, the overall rate of prevalence detected with the novel qPCR assay was 11.14%. Compared to conventional PCR we identified a concordance of 91.30%. We tested 41 animals positive for trypanosome DNA, five animals showed multiple infections. Zebus were twice as often infected (21.74%) compared to Baoulé (9.70%) and hybrids (9.57%). Trypanosoma vivax is the dominant species (9.24%), as compared to T. congolense (2.44%) and T. brucei (0.82%). The chi-squared tests linking the infection events to the breeds (Zebu vs. Baoulé and Zebu vs. hybrids) were on the border of significance. No significant association with other tested parameters could be detected. We introduce a novel qPCR technique for the fast, sensitive and simultaneous detection of the three AAT species. Our results suggest that associations with breed and infection exist since Zebu cattle are more likely to be infected compared to Baoulé and hybrids. Indigenous taurine cattle breeds, like the Baoulé, therefore provide a unique and valuable genetic resource.     

Development of Real Time PCR to Study Experimental Mixed Infections of T. congolense Savannah and T. b. brucei in Glossina morsitans morsitans

Tsetse flies are able to acquire mixed infections naturally or experimentally either simultaneously or sequentially. Traditionally, natural infection rates in tsetse flies are estimated by microscopic examination of different parts of the fly after dissection, together with the isolation of the parasite in vivo. However, until the advent of molecular techniques it was difficult to speciate trypanosomes infections and to quantify trypanosome numbers within tsetse flies. Although more expensive, qPCR allows the quantification of DNA and is less time consuming due to real time visualization and validation of the results. The current study evaluated the application of qPCR to quantify the infection load of tsetse flies with T. b. brucei and T. congolense savannah and to study the possibility of competition between the two species. The results revealed that the two qPCR reactions are of acceptable efficiency (99.1% and 95.6%, respectively), sensitivity and specificity and can be used for quantification of infection load with trypanosomes in experimentally infected Glossina morsitans morsitans. The mixed infection of laboratory Glossina species and quantification of the infection suggests the possibility that a form of competition exists between the isolates of T. b. brucei and T. congolense savannah that we used when they co-exist in the fly midgut.     

Trypanosome Diversity in Wildlife Species from the Serengeti and Luangwa Valley Ecosystems

Background

The importance of wildlife as reservoirs of African trypanosomes pathogenic to man and livestock is well recognised. While new species of trypanosomes and their variants have been identified in tsetse populations, our knowledge of trypanosome species that are circulating in wildlife populations and their genetic diversity is limited.

Methodology/Principal Findings

Molecular phylogenetic methods were used to examine the genetic diversity and species composition of trypanosomes circulating in wildlife from two ecosystems that exhibit high host species diversity: the Serengeti in Tanzania and the Luangwa Valley in Zambia. Phylogenetic relationships were assessed by alignment of partial 18S, 5.8S and 28S trypanosomal nuclear ribosomal DNA array sequences within the Trypanosomatidae and using ITS1, 5.8S and ITS2 for more detailed analysis of the T. vivax clade. In addition to Trypanosoma brucei, T. congolense, T. simiae, T. simiae (Tsavo), T. godfreyi and T. theileri, three variants of T. vivax were identified from three different wildlife species within one ecosystem, including sequences from trypanosomes from a giraffe and a waterbuck that differed from all published sequences and from each other, and did not amplify with conventional primers for T. vivax.

Conclusions/Significance

Wildlife carries a wide range of trypanosome species. The failure of the diverse T. vivax in this study to amplify with conventional primers suggests that T. vivax may have been under-diagnosed in Tanzania. Since conventional species-specific primers may not amplify all trypanosomes of interest, the use of ITS PCR primers followed by sequencing is a valuable approach to investigate diversity of trypanosome infections in wildlife; amplification of sequences outside the T. brucei clade raises concerns regarding ITS primer specificity for wildlife samples if sequence confirmation is not also undertaken.