Calculation of the ELISA's cut-off based on the change-point analysis method for detection of Trypanosoma cruzi infection in Bolivian dogs in the absence of controls.

In ELISAs, sera of individuals infected by Trypanosoma cruzi show absorbance values above a cut-off value. The cut-off is generally computed by means of formulas that need absorbance readings of negative (and sometimes positive) controls, which are included in the titer plates amongst the unknown samples. When no controls are available, other techniques should be employed such as change-point analysis. The method was applied to Bolivian dog sera processed by ELISA to diagnose T. cruzi infection. In each titer plate, the change-point analysis estimated a step point which correctly discriminated among known positive and known negative sera, unlike some of the six usual cut-off formulas tested. To analyse the ELISAs results, the change-point method was as good as the usual cut-off formula of the form "mean + 3 standard deviation of negative controls". Change-point analysis is therefore an efficient alternative method to analyse ELISA absorbance values when no controls are available.

Comparison of transmission parameters between Anopheles argyritarsis and Anopheles pseudopunctipennis in two ecologically different localities of Bolivia.

BACKGROUND: Anopheles (Anopheles) pseudopunctipennis is a recognized malaria vector in the slopes of the Andes of Bolivia. There, other species might be involved in malaria transmission and one candidate could be Anopheles argyritarsis. Although it is generally admitted that this species is not a malaria vector in the neotropical region, its potential role in transmission is still controversial and this situation has to be cleared, at least for Bolivia. Comparing the vectorial efficiency of An. pseudopunctipennis with that of An. argyritarsis could solve the question. METHODS: The two species were sampled throughout Bolivia to estimate their degree of co-existence in their distribution range. Vectorial efficiencies of the two species were compared in two ecologically different localities where the species were sympatric by analysing their vectorial capacities and components (i e, human biting rates, human biting index, survival, durations of the gonotrophic cycle and extrinsic cycle), and the entomological inoculation rates (EIR). Mosquitoes were sampled monthly during more than one year in the two localities. A monthly sample consisted in hourly captures in four houses (inside and outside) in each locality, during four consecutive nights. Climatic variables (temperature, humidity, potential evapo-transpiration and precipitations) were recorded to better understand variability in the entomological parameters. Relationships were analysed using multivariate methods. RESULTS: Anopheles pseudopunctipennis and An. argyritarsis are "altitude" species, sharing the same geographical distribution range in the Andes of Bolivia. No Plasmodium parasite was identified in An. argyritarsis and estimates of the vectorial capacity indicated that it is not a malaria vector in the two studied localities, unlike An. pseudopunctipennis which showed positive EIRs. This latter species, although not a very good malaria vector, exhibited better life traits values and better behavioural characteristics in favour of transmission as compared to An. argyritarsis. CONCLUSIONS: In the Andes of Bolivia, above 1000 m of altitude, An. pseudopunctipennis is likely to be the only malaria vector. There, it is present almost everywhere and priority control effort should be directed toward this species. Below 1000 m of altitude, vector incrimination should also be focused on other sympatric species (likely not An. argyritarsis) that might be locally important. From the present study, candidates would be among Anopheles rangeli, Anopheles triannulatus s.l., Anopheles trinkae, Anopheles nuneztovari s.l., Anopheles oswaldoi s.l. and Anopheles benarrochi s.l.

Deep learning and wing interferential patterns identify Anopheles species and discriminate amongst Gambiae complex species.

We present a new and innovative identification method based on deep learning of the wing interferential patterns carried by mosquitoes of the Anopheles genus to classify and assign 20 Anopheles species, including 13 malaria vectors. We provide additional evidence that this approach can identify Anopheles spp. with an accuracy of up to 100% for ten out of 20 species. Although, this accuracy was moderate (> 65%) or weak (50%) for three and seven species. The accuracy of the process to discriminate cryptic or sibling species is also assessed on three species belonging to the Gambiae complex. Strikingly, An. gambiae, An. arabiensis and An. coluzzii, morphologically indistinguishable species belonging to the Gambiae complex, were distinguished with 100%, 100%, and 88% accuracy respectively. Therefore, this tool would help entomological surveys of malaria vectors and vector control implementation. In the future, we anticipate our method can be applied to other arthropod vector-borne diseases.

Insecticide resistance of Triatoma infestans (Hemiptera, Reduviidae) vector of Chagas disease in Bolivia.

OBJECTIVE: To define the insecticide resistance status of Triatoma infestans to deltamethrin (pyrethroid), malathion (organophosphate) and bendiocarb (carbamate) in Bolivia. METHODS: Fifty populations of T. infestans were sampled in Bolivian human dwellings. Quantal response data were obtained by topical applications of 0.2 µl of insecticide-acetone solutions on nymphs N1 of the F1 generations. For most populations, dose-mortality relationships and resistance ratios (RR) were analysed. Discriminating concentrations were established for each insecticide with a susceptible reference strain and used on the other field populations. A tarsal-contact diagnostic test using insecticide impregnated papers was designed to rapidly identify deltamethrin-resistant populations in the field. RESULTS: Discriminating concentrations for topical applications were 5, 70 and 120 ng active ingredient per insect for deltamethrin, bendiocarb and malathion, respectively. The diagnostic concentration for deltamethrin was 0.30% for the 1-h exposure by tarsal contact. All populations sampled in human dwellings exhibited significant levels of resistance to deltamethrin, from 6 to 491 and varied among regions. Resistant populations did not recover complete susceptibility to deltamethrin when the synergist piperonyl butoxide (PBO) was used. None of the sampled populations exhibited significant resistance to bendiocarb (all RR(50) < 1.8) or malathion (all RR(50) < 2.2). CONCLUSION: In Bolivia, most 'domestic' T. infestans populations are resistant to deltamethrin. Because insecticide vector control is the only selection pressure, resistance likely originates from it. Switching from pyrethroids to organophosphates or carbamates could be a short-term solution to control this vector, but other alternative integrated control strategies should also be considered in the long term.

A physiological time analysis of the duration of the gonotrophic cycle of Anopheles pseudopunctipennis and its implications for malaria transmission in Bolivia.

BACKGROUND: The length of the gonotrophic cycle varies the vectorial capacity of a mosquito vector and therefore its exact estimation is important in epidemiological modelling. Because the gonotrophic cycle length depends on temperature, its estimation can be satisfactorily computed by means of physiological time analysis. METHODS: A model of physiological time was developed and calibrated for Anopheles pseudopunctipennis, one of the main malaria vectors in South America, using data from laboratory temperature controlled experiments. The model was validated under varying temperatures and could predict the time elapsed from blood engorgement to oviposition according to the temperature. RESULTS: In laboratory experiments, a batch of An. pseudopunctipennis fed at the same time may lay eggs during several consecutive nights (2-3 at high temperature and > 10 at low temperature). The model took into account such pattern and was used to predict the range of the gonotrophic cycle duration of An. pseudopunctipennis in four characteristic sites of Bolivia. It showed that the predicted cycle duration for An. pseudopunctipennis exhibited a seasonal pattern, with higher variances where climatic conditions were less stable. Predicted mean values of the (minimum) duration ranged from 3.3 days up to > 10 days, depending on the season and the geographical location. The analysis of ovaries development stages of field collected biting mosquitoes indicated that the phase 1 of Beklemishev might be of significant duration for An. pseudopunctipennis. The gonotrophic cycle length of An. pseudopunctipennis correlates with malaria transmission patterns observed in Bolivia which depend on locations and seasons. CONCLUSION: A new presentation of cycle length results taking into account the number of ovipositing nights and the proportion of mosquitoes laying eggs is suggested. The present approach using physiological time analysis might serve as an outline to other similar studies and allows the inclusion of temperature effects on the gonotrophic cycle in transmission models. However, to better explore the effects of temperature on malaria transmission, the others parameters of the vectorial capacity should be included in the analysis and modelled accordingly.

Optimization of a semi-nested multiplex PCR to identify Plasmodium parasites in wild-caught Anopheles in Bolivia, and its application to field epidemiological studies.

Without an adequate DNA extraction protocol, the identification of Plasmodium species in whole mosquitoes by PCR is difficult because of the presence of reaction inhibitors from the insects. In this study, eight DNA extraction protocols were tested, from which a chelex-based protocol was selected. Then a semi-nested multiplex PCR technique that detects and distinguishes among the four human Plasmodium species in single mosquitoes and in pools of up to 100 mosquitoes was optimized. The technique was used to detect P. vivax in wild-caught Anopheles pseudopunctipennis from a village in the Andean valleys of Bolivia in May 2003. The prevalence of infection was 0.9%. This is the first direct evidence of P. vivax transmission by this vector in this country. The extraction and PCR technique presented here can be useful to: (1) estimate Plasmodium prevalence in Anopheles populations in low prevalence areas where large numbers of individual mosquitoes would need to be processed to obtain a reliable estimate; (2) incriminate Anopheles species as malaria vectors; (3) identify all the circulating Plasmodium species in vectors from an area; (4) detect mixed infections in mosquitoes; and (5) detect mosquitoes with low-level parasite infections.

Host choice and human blood index of Anopheles pseudopunctipennis in a village of the Andean valleys of Bolivia.

BACKGROUND: The Human Blood Index (HBI, proportion of bloodmeals of a mosquito population obtained from man) is relevant to epidemiological assessment and to the modification of measures to interrupt malaria transmission since the vectorial capacity of the vector varies as the square of the HBI. Anopheles pseudopunctipennis is a main malaria vector in South America. Unfortunately, few data exist concerning HBI values in its range of distribution and none from Bolivia where this species is considered as an important malaria vector in the central Andes. METHODS: The host choice of An. pseudopunctipennis has been studied in Mataral, a characteristic village of the central Andes of Bolivia. Mosquito host feeding preference experiments (equal accessibility to host in homogenous environment) were monitored using baited mosquito nets in latin square designs. Host feeding selection experiments (natural feeding pattern in heterogeneous environment) was measured by bloodmeal analysis, using ELISA to determine the origin of blood. Mosquito bloodmeals were collected on various occasions, using various techniques in a variety of sampling sites. A survey of the possible blood sources has also been carried out in the village. Data were analysed with the forage ratio method. RESULTS: An. pseudopunctipennis chooses amongst hosts. Sheep, goats, donkeys and humans are the preferred hosts, while dogs, pigs and chicken are rarely bitten. An. pseudopunctipennis has an opportunistic behaviour, in particular within the preferred hosts. The HBI in Mataral is approximately 40% and in the central Andes, may range from 30-50%, in accordance to other findings. A high proportion of mixed meals were encountered (8%), and cryptic meals are likely more numerous. There was no difference amongst the HBI from parous and nulliparous mosquitoes. CONCLUSION: Forage ratio analysis is a powerful tool to interpret mosquito host choices. However, refinements in sampling strategies are still needed to derive accurate and precise HBIs that could be computed to compare or follow epidemiological situations. The low antropophily of An. pseudopunctipennis, associated with changing environmental conditions, leads to unstable malaria (Plasmodium vivax) transmission in the central Andes. The opportunistic behaviour of this vector may be used to attract mosquitoes to insecticide. Zooprophylaxis is a promising alternative control strategy.

Laboratory colonization of Anopheles pseudopunctipennis (Diptera: Culicidae) without forced mating.

Anopheles pseudopunctipennis is one of the main malaria vectors in the Andean regions of South America. Few experimental data exist on this species because it is not very available in laboratories due to its eurygamic status that makes colony maintenance difficult. Indeed, individuals do not mate in the confined space of insectary cages. To avoid this problem, forced artificial mating can be used. However, this technique is time consuming, requires a well-trained technician, and is inadequate for easy mass production, which is sometimes necessary for certain experimental works. This study presents a technique based on exposure of adult mosquitoes to a blue stroboscopic light for 20 min during several nights, which encourages them to copulate naturally under laboratory conditions. After some generations, a self-free-mating strain was obtained. The technique is simple, inexpensive and is probably effective whatever the An. pseudopunctipennis strain considered.

Bias due to methods of parasite detection when estimating prevalence of infection of Triatoma infestans by Trypanosoma cruzi.

The study aimed to quantify the bias from parasite detection methods in the estimation of the prevalence of infection of Triatoma infestans by Trypanosoma cruzi, the agent of Chagas disease. Three common protocols that detect T. cruzi in a sample of 640 wild-caught T. infestans were compared: (1) the microscopic observation of insect fecal droplets, (2) a PCR protocol targeting mini-exon genes of T. cruzi (MeM-PCR), and (3) a PCR protocol targeting a satellite repeated unit of the parasite. Agreement among protocols was computed using Krippendorff Kα. The sensitivity (Se) and specificity (Sp) of each protocol was estimated using latent class models. The PCR protocols were more sensitive (Se > 0.97) than microscopy (Se = 0.53) giving a prevalence of infection of 17-18%, twice as high as microscopy. Microscopy may not be as specific as PCR if Trypanosomatid-like organisms make up a high proportion of the sample. For small T. infestans, microscopy is not efficient, giving a prevalence of 1.5% when PCR techniques gave 10.7%. The PCR techniques were in agreement (Kα = 0.94) but not with microscopy (Kα never significant with both PCR techniques). Among the PCR protocols, the MeM-PCR was the most efficient (Se=1; Sp=1).

Experimental control of Triatoma infestans in poor rural villages of Bolivia through community participation.

BACKGROUND: Triatoma infestans is the main vector of Chagas disease in the southern cone countries. Present control strategies based on indoor and outdoor residual insecticide spraying are not sufficient to control disease transmission, particularly in Bolivia. Techniques based on the management of the human environment may be good alternatives or supplements. METHODS: Social and entomological surveys were carried out in four villages of Bolivia situated in the dry inter-Andean Valleys and the Chaco region. Risk factors for house infestation by T. infestans were identified, and an eco-health intervention based on education and community participation was carried out to reduce the risks of house infestation. It consisted of implementing simple and low cost vector control techniques such as coating of mud walls, cleaning activities and removal of poultry that enter rooms to lay eggs. RESULTS: The eco-health intervention significantly reduced the number of infested bedrooms, the mean abundance of T. infestans in bedrooms and beds, especially in the Chaco region. Mud wall coating was well accepted and could be proposed as a supplementary tool to the National Program of Chagas Disease Control to enhance the effects of insecticide sprayings. CONCLUSIONS: Even if cleaning activities were still neglected, community participation proved to be effective in reducing house infestation.

Age-grading and growth of Wuchereria bancrofti (Filariidea: Onchocercidae) larvae by growth measurements and its use for estimating blood-meal intervals of its Polynesian vector Aedes polynesiensis (Diptera: Culicidae).

Growth in length and width of Wuchereria bancrofti (Filariidea: Onchocercidae) larvae developing in its Polynesian vector Aedes polynesiensis (Diptera: Culicidae) was analysed using a mathematical approach to objectively extract patterns. L1 had a U-shaped growth in length, while widths followed an S-shaped function. L2 had an S-shaped growth in length and width. Growth in length of L3 was also S-shaped, while widths had an asymptotic size following a period of rapid shrinkage. The greatest difference between length and width was in stage 3 where the length was over 75 times greater than the width. The ratio of length to width was approximately 50 for microfilariae and only 10 for the L1 ('sausage') stage. Characteristic mean length (and width) were approximately 280(7) microm for microfilariae, approximately 181 microm for L1 at their smallest, and approximately 1584(22) microm for L3 infective larvae. There was a great increase in length during stage 2 from approximately 322(27) to approximately 982(31) microm. Stage duration decreased with increasing temperature while growth rate increased, giving steeper growth curves. There was no effect of temperature on size, except for L3, which were shorter when mosquitoes were reared at higher temperature. It appears that larval growth is a continuous process from microfilariae to the young L3 stage, and continuously modifies the larval parasite aspect, even within each stage. Thus, information on larval shape may be used as an age indicator and in some cases, may give an estimation on time elapsed since infection of the vector. An important demographic parameter used in most mathematical models describing transmission of parasites by insect vectors is the length of the gonotrophic cycle of the vector, i.e. the time interval between two successive blood-meals. Usual methods for computing such a parameter are based on mark-recapture techniques. However, reliable estimates need substantial capture rates, which are not always possible. This paper presents another approach in which marked mosquitoes are those naturally infected by W. bancrofti. For one mosquito, the time since infection is simply the age of the developing larval parasite. Our method first expresses the age of larval parasite as a fraction of total development time (from microfilariae entering the vector to L3 larvae) using a regression model based on measurements of the parasite's length and width. This fraction of development is then converted to a chronological age since infection, using a back-calculation procedure involving ambient temperatures and growth rates of W. bancrofti larvae in the vector. The method is applied to wild caught Ae. polynesiensis in French Polynesia to compute the length of the gonotrophic cycle. This mosquito species comes to bite approximately 3, 6-7 and 9 days after a first infectious blood-meal. Then the length of the gonotrophic cycle may be of 3-4 days.

Dosage-dependent effects of permethrin-treated nets on the behaviour of Anopheles gambiae and the selection of pyrethroid resistance.

BACKGROUND: The evolution and spread of pyrethroid resistance in Anopheles gambiae s.s, the major malaria vector in sub-Saharan Africa, is of great concern owing to the importance of pyrethroid-treated nets in the WHO global strategy for malaria control. The impact of kdr (the main pyrethroid-resistance mechanism) on the behaviour of An. gambiae is not well understood. The objective of this study was to determine whether high or low doses of permethrin differ in their resistance-selection effects. METHODS: The effect of permethrin treatment was assessed under laboratory conditions using the tunnel test technique against susceptible, heterozygous and homozygous genotypes. Experimental huts trials were then carried out in Benin to assess the level of personal protection conferred by nets treated with a variety of permethrin concentrations and their impact on the selection for kdr allele. RESULTS: Tunnel tests showed that nets treated with permethrin at 250 and 500 mg/m2 induced higher mortality and blood feeding reduction among susceptible and heterozygous (RS) females as compared to the lower concentration (100 mg/m2). The experimental hut trials showed that the best personal protection was achieved with the highest permethrin concentration (1,000 mg/m2). Mosquito genotyping revealed a non-linear relationship in the survival of kdr susceptible and resistant genotypes with permethrin dosage. Higher dosages (> or =250 mg/m2) killed more efficiently the RS genotypes than did lower dosages (50 and 100 mg/m2). CONCLUSION: This study showed that nets treated with high permethrin concentrations provided better blood feeding prevention against pyrethroid-resistant An. gambiae than did lower concentrations. Permethrin-treated nets seem unlikely to select for pyrethroid resistance in areas where the kdr mutation is rare and present mainly in heterozygous form.

Evaluation of insecticide impregnated baits for control of mosquito larvae in land crab burrows on French Polynesian atolls.

Land crab burrows are larval mosquito habitats of major significance in the Pacific region. They are constituted by a sinuous tunnel leading to a chamber in contact with the water table, where mosquito larvae proliferate. Controlling larvae in these sites is difficult, because the configuration of burrows prevents the use of standard techniques. An experiment was carried out in French Polynesia to control Aedes polynesiensis Marks and Culex spp. breeding in burrows of the land crab Cardisoma carnifex (Herbst). The technique was based on the crab's behavior, which involves the crab carrying food into its burrow. It was shown that appetizing baits impregnated with an insecticide were carried by crabs into the flooded chamber of their burrows. A field treatment of burrows was carried out by sowing insecticide impregnated baits on the ground. The treatment coverage was almost perfect and the easy implementation of the technique enabled large areas to be treated in a short time. The bait was developed by compacting various flours, which easily incorporate a large variety of insecticide formulations. Although the baits can be easily stocked, a reliable insecticide is still to be found. The results indicate that our technique could be a method of choice for treating crab burrows.

Integrated control of peridomestic larval habitats of Aedes and Culex mosquitoes (Diptera: Culicidae) in atoll villages of French Polynesia.

An integrated larval mosquito control program was carried out in Tiputa village on Rangiroa atoll of French Polynesia. Mosquito abundance before and after treatment was compared with the abundance in an untreated village. Mosquito larval habitats consisted of large concrete or polyurethane cisterns, wells, and 200-liter drums. Depending on the target species, larval habitat category, its configuration, and purpose (drinking consumption or not), abatement methods consisted of sealing the larval habitats with mosquito gauze, treating them with 1% Temephos, covering the water with a 10-cm thick layer of polystyrene beads or introducing fish (Poecillia reticulata Rosen & Bailey). All premises of the chosen village were treated and a health education program explained basic mosquito ecology and the methods of control. A community health agent was trained to continue the control program at the end of the experiment. Entomological indices from human bait collections and larval surveys indicated that mosquito populations were reduced significantly, compared with concurrent samples from the untreated control village, and that mosquito control remained effective for 6 mo after treatment. Effects of the treatment were noticed by the inhabitants in terms of a reduction in the number of mosquito bites. In the Polynesian context, such control programs may succeed in the long-term only if strong political decisions are taken at the village level, if a community member is designated as being responsible for maintaining the program, and if the inhabitants are motivated sufficiently by the mosquito nuisance to intervene.

Niche invasion, competition and coexistence amongst wild and domestic Bolivian populations of Chagas vector Triatoma infestans (Hemiptera, Reduviidae, Triatominae).

The model developed for human parasites by Bottomley et al. (2007) has been adapted to the dynamics of triatomines to better understand the processes of niche invasion, competition among species and coexistence. In Bolivia, both wild and domestic populations of Triatoma infestans exist. Their ecological niches are normally separated and the two populations do not interbreed, behaving as two distinct species. However, it has been suggested that the two populations may compete, highlighting therefore the potential risk of wild populations invading human dwellings. The model revealed the importance of the basic reproduction rates R0 of triatomine colonies for the risk of invasion. This depends not only on life traits such as survival and fecundity, but also on (1) the density-dependence phenomenon that limits triatomine establishment, (2) on house exposure to infection and (3) on the correlation between house susceptibility to domestic T. infestans and house susceptibility to wild T. infestans. Competition and coexistence amongst the two groups of T. infestans may occur under particular conditions, but are very unlikely.

Development of Exon-Primed Intron-Crossing (EPIC) PCR primers for the malaria vector Anopheles pseudopunctipennis (Diptera: Culicidae).

Using the Anopheles gambiae Giles genome as a template, we designed, screened and identified 14 novel Exon-Primed Intron-Crossing (EPIC) PCR primer pairs for Anopheles pseudopunctipennis Theobald 1901, a major vector of human Plasmodium sp. in South America. These primers were designed to target the conserved regions flanking consecutive exons of different genes and enabled the amplification of 17 loci of which nine were polymorphic. Polymorphisms at these loci ranged from two to four alleles. Intron length polymorphism analysis is a useful tool, which will allow the study of the population structure of this mosquito species, which remains poorly understood.

Calculation of the ELISA’ s cut-off based on the change-point analysis method for detection of Trypanosoma cruzi infection in Bolivian dogs in the absence of controls

In ELISAs, sera of individuals infected by Trypanosoma cruzi show absorbance values above a cut-off value. The cut-off is generally computed by means of formulas that need absorbance readings of negative (and sometimes positive) controls, which are included in the titer plates amongst the unknown samples. When no controls are available, other techniques should be employed such as change-point analysis. The method was applied to Bolivian dog sera processed by ELISA to diagnose T. cruzi infection. In each titer plate, the change-point analysis estimated a step point which correctly discriminated among known positive and known negative sera, unlike some of the six usual cut-off formulas tested. To analyse the ELISAs results, the change-point method was as good as the usual cut-off formula of the form “mean + 3 standard deviation of negative controls”. Change-point analysis is therefore an efficient alternative method to analyse ELISA absorbance values when no controls are available.

Anopheles of Bolivia: new records with an updated and annotated checklist.

Anopheles squamifemur has been identified from CDC light trap collections carried out at Arca de Israel, a small community located in the extreme north-east of Bolivia (Pando Department) on the banks of the river Madera, on the border with Brazil. Anopheles costai and An. forattinii have been identified in place of An. mediopunctatus which has been removed from the Bolivian list of Anopheles species. The first identification of An. trinkae in Bolivia by Dr. J.C. Lien in 1984 is cleared. The presence of An. deaneorum in Bolivia has been confirmed by our mosquito captures carried out in Guayaramerín (Pando Department, north-east of Bolivia), a border city separated from the type locality of An. deaneorum, the Brasilian city of Guajara-Mirin, by the large Mamoré River. These new findings increase to 43 the total number of known Anopheles species for Bolivia for which an updated and partially annotated checklist is given.

A "repertoire for repertoire" hypothesis: repertoires of type three effectors are candidate determinants of host specificity in Xanthomonas.

BACKGROUND: The genetic basis of host specificity for animal and plant pathogenic bacteria remains poorly understood. For plant pathogenic bacteria, host range is restricted to one or a few host plant species reflecting a tight adaptation to specific hosts. METHODOLOGY/PRINCIPAL FINDINGS: Two hypotheses can be formulated to explain host specificity: either it can be explained by the phylogenetic position of the strains, or by the association of virulence genes enabling a pathological convergence of phylogenically distant strains. In this latter hypothesis, host specificity would result from the interaction between repertoires of bacterial virulence genes and repertoires of genes involved in host defences. To challenge these two hypotheses, we selected 132 Xanthomonas axonopodis strains representative of 18 different pathovars which display different host range. First, the phylogenetic position of each strain was determined by sequencing the housekeeping gene rpoD. This study showed that many pathovars of Xanthomonas axonopodis are polyphyletic. Second, we investigated the distribution of 35 type III effector genes (T3Es) in these strains by both PCR and hybridization methods. Indeed, for pathogenic bacteria T3Es were shown to trigger and to subvert host defences. Our study revealed that T3E repertoires comprise core and variable gene suites that likely have distinct roles in pathogenicity and different evolutionary histories. Our results showed a correspondence between composition of T3E repertoires and pathovars of Xanthomonas axonopodis. For polyphyletic pathovars, this suggests that T3E genes might explain a pathological convergence of phylogenetically distant strains. We also identified several DNA rearrangements within T3E genes, some of which correlate with host specificity of strains. CONCLUSIONS/SIGNIFICANCE: These data provide insight into the potential role played by T3E genes for pathogenic bacteria and support a "repertoire for repertoire" hypothesis that may explain host specificity. Our work provides resources for functional and evolutionary studies aiming at understanding host specificity of pathogenic bacteria, functional redundancy between T3Es and the driving forces shaping T3E repertoires.

GASAT: a genetic local search algorithm for the satisfiability problem.

This paper presents GASAT, a hybrid algorithm for the satisfiability problem (SAT). The main feature of GASAT is that it includes a recombination stage based on a specific crossover and a tabu search stage. We have conducted experiments to evaluate the different components of GASAT and to compare its overall performance with state-of-the-art SAT algorithms. These experiments show that GASAT provides very competitive results.