A multiplex-PCR assay for identification of the quarantine plant pathogen Xanthomonas axonopodis pv. phaseoli.

In this study we developed an algorithm to screen for all exact molecular signatures of the quarantine pathogen Xanthomonas axonopodis pv. phaseoli (Xap), based on available data of the presence or absence of virulence-associated genes. The simultaneous presence of genes avrBsT and xopL is specific to Xap. Therefore we developed a multiplex PCR assay targeting avrBsT and xopL for the molecular identification of Xap. The specificity of this multiplex was validated by comparison to that of other molecular identification assays aimed at Xap, on a wide collection of reference strains. This multiplex was further validated on a blind collection of Xanthomonas isolates for which pathogenicity was assayed by stem wounding and by dipping leaves into calibrated inocula. This multiplex was combined to the previously described X4c/X4e molecular identification assay for Xap. Such a combination enables the molecular identification of all strains of Xanthomonas pathogenic on bean. Results also show that assay by stem wounding does not give reliable results in the case of Xap, and that pathogenicity assays by dipping should be preferred.

Synergism between permethrin and propoxur against Culex quinquefasciatus mosquito larvae.

To see if synergism occurs between carbamate and pyrethroid insecticides, we tested permethrin and propoxur as representatives of these two classes of compounds used for mosquito control. Larvicidal activity of both insecticides was assessed separately and together on a susceptible strain of the mosquito Culex quinquefasciatus (Diptera: Culicidae) by two methods. When mixed at a constant ratio (permethrin : propoxur 1 : 60 based on LC50) and tested at serial concentrations to plot dose/mortality regression, significant synergy occurred between them (co-toxicity coefficient = 2.2), not just an additive effect. For example, when the mixture gave 50% mortality, the same concentrations of permethrin and propoxur alone would have given merely 2 x 1% mortality. When a sublethal dose (LC0) of permethrin or propoxur was added to the other (range LC10-LC95), synergism occurred up to the LC80 level. Synergistic effects were attributed to the complementary modes of action by these two insecticide classes acting on different components of nerve impulse transmission. Apart from raising new possibilities for Culex control, it seems appropriate to consider using such mixtures or combinations for insecticide-treated mosquito nets in situations with insecticide-resistant Anopheles malaria vectors.

Control of the Aedes vectors of the dengue viruses and Wuchereria bancrofti: the French Polynesian experience.

In most of the 130 islands of French Polynesia, the stenotopic mosquitoes Aedes aegypti (the main local vector for the viruses causing dengue) and Aedes polynesiensis (the main local vector of Wuchereria bancrofti) share many breeding sites in water containers such as discarded cans, coconut shells, buckets and water-storage pots and drums. In addition to selective application of insecticides, non-polluting methods of controlling these mosquitoes have been evaluated during the last decade in two main ecological situations: (1) villages, where Aedes breeding sites are typically peridomestic; and (2) flooded burrows of land crabs, the major source of Ae. polynesiensis throughout the South Pacific region. Large-scale trials of biological control agents, such as mosquito fish (Gambusia affinis and Poecilia reticulata) and copepods (Mesocyclops aspericornis), and of integrated-control strategies have demonstrated the efficacy of certain techniques and control agents against the target Aedes populations in some village situations. Generally, mechanical methods (the use of layers of polystyrene beads against mosquito larvae and pupae, and screening against adult mosquitoes) were more efficient than use of the biological control agents. By integrating several methods of control, mosquito densities (as measured by human-bait collections and larval surveys) were reduced significantly compared with the results of concurrent sampling from untreated villages, and control remained effective for months after the interventions ceased. In land-crab burrows, the first attempts to control Aedes larvae used bacterial agents (Bacillus thuringiensis) and predatory copepods gave disappointing results. Mesocyclops aspericornis could be an effective control agent if the burrows were constantly flooded, but most burrows dry out and refill periodically, so copepod populations do not survive. As it proved difficult to reach all corners of the long sinuous burrows with any control agent, larvicidal (chlorpyrifos-methyl) baits were developed for foraging crabs to carry into their burrows. This novel technique proved to be effective and could become the method of choice for treating crab burrows. Further research is underway to find the optimum (biological or chemical) larvicidal ingredient for the crab bait. Despite the ecological and logistical challenges of controlling the Aedes vectors of the dengue viruses and W. bancrofti in so many scattered islands, the French Polynesian experience indicates that relatively simple methods can be integrated and applied effectively and economically. Operationally, however, success also depends on a strong political commitment and on at-risk communities that are sufficiently motivated to maintain a good level of Aedes control.

Ambient temperature effects on the extrinsic incubation period of Wuchereria bancrofti in Aedes polynesiensis: implications for filariasis transmission dynamics and distribution in French Polynesia.

Temperature effects on development of the human filarial parasite Wuchereria bancrofti (Cobbold) (Filaridea: Onchocercidae) in the main Pacific vector Aedes polynesiensis Marks (Diptera: Culicidae) are analysed in relation to ambient climatic conditions. A statistical model of the extrinsic cycle duration as a function of temperature is described and used to distinguish three patterns of W. bancrofti transmission dynamics: continuous, fluctuating and discontinuous, occurring from north to south geographically among French Polynesian archipelagos. In the northerly Marquesas Islands (8-11 degrees S) filariasis transmission is continuous and very active, facilitated by perennially high temperatures combined with constantly high rates of man-vector contact. In the southerly Australes Islands (21-28 degrees S) filariasis transmission is seasonally discontinuous and, during the cooler months (May-September), the model predicts virtually no transmission because the cycle duration exceeds the life expectancy of the vector. In the Society Islands (16-18 degrees S), between the Marquesas and Australes, transmission is predicted to be intermediate as expected from their latitude, with seasonally fluctuating transmission potential. In the Tuamotu Islands (also geographically intermediate: 14-23 degrees S), with theoretically perennial transmission potential, transmission occurs only intermittently, being limited by other human and environmental factors whereby man-vector contact is confined to seasonal agricultural situations. Generally, among French Polynesian archipelagos where Aedes polynesiensis is the vector, the transmission potential for W. bancrofti and resulting disease manifestations of lymphatic filariasis in humans are correlated with ambient temperature due to the degree of southern latitude.

Spectrofluorometers are not adequate for aging Aedes and Culex (Diptera: Culicidae) using pteridine fluorescence.

Attempts were made to use pteridine fluorescence levels as a tool for aging individual Aedes polynesiensis Marks and Culex quinquefasciatus Say mosquitoes. Fluorescent pigments were extracted and quantified with techniques and apparatus (i.e., a spectrofluorometer) that can be used in developing countries or in field laboratories, and that has already given consistent results in similar studies on other insect groups. However, for Ae. polynesiensis as well as Cx. quinquefasciatus, individual fluorescence measurements were mostly below the spectrofluorometer white noise level. With batches of Aedes or Culex of the same age, significant fluorescence levels were recorded but not in relation to their calendar ages. The low content of pteridines in mosquitoes suggests that standard spectrofluorometry is not sufficiently sensitive for such studies.

Density of larval Culicoides belkini (Diptera:Ceratopogonidae) in relation to physicochemical variables in different habitats.

Immature density and population size of the biting midge Culicoides belkini (Wirth & Arnaud) were estimated for habitats on Moorea Island, French Polynesia, by means of random, 2- and 3-stage sampling designs. Samples were taken in March 1993 from 5 strata of a large larval habitat: a sandy-mud surface of approximately 5,000 m2 (stratum 1) in which approximately 12,000 land crab burrows (stratum 2) were counted, a small pond surrounded by approximately 300 m2 of muddy bank (stratum 3), and a high organic muddy area (Kopara) of approximately 1,200 m2 (stratum 4) with approximately 3,500 crab burrows (stratum 5). Larval density was usually higher in the mud of crab burrows, especially those in the Kopara stratum. Larval density was significantly lower in the sediment of the sandy area as compared with pond banks or Kopara surface. The sampling designs and techniques were logistically adequate, statistically relevant, and were recommended for future studies on C. belkini larval density. Larval habitats were characterized by means of multivariate analysis. Comparison of larval densities with selected environmental variables indicated that larvae density was higher in wet sediments with high levels of organic matter (approximately 8% of dry weight of sediment) and low salinity (approximately 0.5-1.5% NaCl equivalents). These variables were considered significant if larval control by means of habitat modification has to be achieved. Nevertheless, C. belkini can tolerate a broad spectrum of variation in the other environmental variables measured and breed in a variety of ecological situations. Therefore, it has a high potential for colonizing new habitats.

Temperature thresholds and statistical modelling of larval Wuchereria bancrofti (Filariidea:Onchocercidae) developmental rates.

Developmental rates for Wuchereria bancrofti larvae maturing in the vector Aedes polynesiensis were estimated by analysing stage-frequency data consisting of counts of larval stages in mosquitoes reared at 20, 22.5, 25, 27.5, 30 and 32 degrees C. Base temperatures (i.e. low temperature thresholds) for W. bancrofti development were estimated by the x-intercept method and the model of Lactin et al. (1995). Resulting values were similar with both methods and were approximately 12.5 degrees C for microfilariae (mf) in thorax, approximately 17 degrees C for L1, 15.5 degrees C for L2 and 16.5 degrees C for L3. Upper thresholds estimated by the Lactin et al. model were 29.3 degrees C for mf, 29.1 degrees C for L1, 32.2 degrees C for L2 and 31.5 degrees C for L3. In addition, an original method was devised for computing the L3 upper threshold, by modelling L3 length shrinkage with temperature. It gave a value of 31.4 degrees C. At 32 degrees C, L2 and L3 stages exhibited altered morphology, larvae being shorter and wider than expected. The model of Lactin et al. described adequately the non-linear relationship between developmental rate and temperature, but a linear degree-day approach may be applied for temperatures below 27-28 degrees C.

Behavior of Wuchereria bancrofti (Filariidea: Onchocercidae) infective larvae in the vector Aedes polynesiensis (Diptera:Culicidae) in relation to parasite transmission.

In French Polynesia, Aedes polynesiensis Marks is the major vector of human filariasis caused by subperiodic Wuchereria bancrofti (Cobbold). Factors affecting transmission of infective larvae from vector to humans were assessed. The 66-100% loss of infective larvae during a blood meal was independent of the initial vector parasite burden. Infective larvae were able to migrate to the mouthparts during a blood meal. Blood feeding by mosquitoes to repletion was one important aspect in the escape of larvae. Infective larvae were not transmitted (lost) without a blood meal and may move in the vector's body. Ae. polynesiensis may retain infectivity potential during several days and may infect several hosts during one blood meal session or on several gonotrophic cycles. In terms of parasite transmission, these findings were interpreted as efficient adaptations of the filaria to the Ae. polynesiensis biting habits.

A polymerase chain reaction assay to determine infection of Aedes polynesiensis by Wuchereria bancrofti.

The sensitivity of a previously described polymerase chain reaction (PCR) assay was improved to detect a single mosquito, infected by as few as 1-2 microfilariae of Wuchereria bancrofti, among 20-50 uninfected mosquitoes. Wild-caught Aedes polynesiensis were used to compare assessment of infection by dissection of individuals with the PCR assay of pools of mosquitoes. The PCR assay was at least as sensitive as dissection for detection of mosquitoes infected with W. bancrofti.

Wuchereria bancrofti (Filariidea: Dipetalonematidae) and its vector Aedes polynesiensis (Diptera: Culicidae) in a French Polynesian village.

In March 1991, a study on Wuchereria bancrofti (Cobbold, 1887) infection rates in its vector, Aedes polynesiensis Marks, was carried out in a village of French Polynesia. Our data were collected 10 yr after the suspension of human mass chemoprophylaxis and served as a baseline for pending ivermectin treatment scheduled in 1991-1993. In total, 1,789 biting females were collected, of which 1,740 were dissected and 1,183 (68%) were parous. Among these, 106 (8.96%) were infected with W. bancrofti and 34 (2.87%) harbored infective L3 larvae. The mean number of larvae per infected mosquito was 2.69, and the mean number of L3 larvae per L3 positive mosquito was 1.44. The Ae. polynesiensis biting index was 4.7 bites per 15 min, but varied significantly among habitats. The highest parous biting rates occurred in fields and peridomestic gardens and the lowest was close to houses. The proportion of parous infected and infective mosquitoes was higher in peridomestic habitats (0.25 infective bites per 15 min) than in domestic habitats (0.09) or in fields (0.11).

Wuchereria bancrofti infection in human and mosquito populations of a Polynesian village ten years after interruption of mass chemoprophylaxis with diethylcarbamazine.

In 1991, a study on Wuchereria bancrofti microfilariae (mf) and infection rates was carried out in the human and mosquito populations of a Polynesian village where, 10 years before, the mf prevalence rate was 6.4% and twice-yearly mass treatment with 3 mg/kg of diethylcarbamazine (DEC) was interrupted. Venous blood samples were collected from 575 (97%) individuals aged 15 years or more, of whom 122 (21.4%) were mf positive. The mf carrier prevalence rate was 27.4% in males, significantly higher than that of 14% in females; it increased from 7-12% in the youngest age group (15-19 years) to 40-50% in the oldest (> or = 60 years) for both males and females. 387 mosquito collections were performed and 1748 female Aedes polynesiensis were dissected, of which 1176 were parous. Among the latter, 114 (9.7%) were infected with Wuchereria bancrofti larvae at L1, L2 or L3 stages. The mean number of larvae per mosquito was 2.46 (range 1-15). Of the 114 infected mosquitoes, 30 harboured L3 larvae, giving a 2.55% infective rate; the mean number of L3 larvae per mosquito was 1.15 (range 1-2). Such findings indicate that the interruption of systematic twice-yearly mass treatment with DEC (3 mg/kg) has resulted, after 10 years, in a substantial increase of microfilarial prevalence in humans, and in high infection rates in mosquitoes.

Release of Mesocyclops aspericornis (Copepoda) for control of larval Aedes polynesiensis (Diptera: Culicidae) in land crab burrows on an atoll of French Polynesia.

On Tereia Island, Rangiroa Atoll, 14,321 land crab burrows were treated with the copepod Mesocyclops aspericornis from January to June 1986, to control larvae of Aedes polynesiensis marks. In October 1987, the entire island of Tereia was retreated (17,300 burrows), and the neighboring island, Voisin, was left untreated as a control. From 5 to 15 mo after treatment, burrows with M. aspericornis contained an average of 2 Ae. polynesiensis immatures compared with 97 immatures from untreated burrows. Long-term larval control was successful in low-lying areas where burrows remained wet or were reflooded. Although there may have been other contributing factors, the major reason for lack of persistence of M. aspericornis in burrows over the entire island appeared to be poor resistance to desiccation. From all treated burrows in October 1987, M. aspericornis subsequently was found in 89.5, 39.1, and 24.1% of burrows sampled 5, 8, and 15 mo after treatment, respectively. The broad-scale results for Tereia indicated that there was no reduction of adult biting indices when compared with Voisin. Mark-release experiments on four occasions indicated that Ae. polynesiensis had a limited flight range and that the probability for interisland movement was low.

Biological control of Culicidae with the copepod Mesocyclops aspericornis and larvivorous fish (Poeciliidae) in a village of French Polynesia.

The copepod Mesocyclops aspericornis Daday and the larvivorous fishes Gambusia affinis (B. & G.) and Poecilia reticulata R. & B., were released into mosquito breeding sites in Tuherahera village, Tikehau atoll, French Polynesia, to control larvae of Aedes aegypti (L.), Ae.polynesiensis Marks, Culex annulirostris Skuse and Cx quinquefasciatus Say. Treatments were completed within a week, in January 1990. Fish quickly eliminated mosquito larvae from the open breeding sites (ponds, wells). The impact of copepods in water tanks, drums and covered wells was inconsistent, apparently depending on the availability of microfaunal diet for growth of copepod nauplii. As the biting rate of adult Ae.aegypti seemed to be unaffected by the biological control of larvae, this village-scale experiment was judged to be unsuccessful as a means of vector control.

[Ivermectin and the reduction of the rate of infection of simuliids in a forest focus of human onchocerciasis]. / Ivermectine et modalités de la réduction de l'infection des simulies dans un foyer forestier d'onchocercose humaine.

Eight patients from a forest onchocercian area "Grandes rivières", in Ivory Coast, were treated with a single oral dose of ivermectin (12 mg). The density of dermic microfilariae was estimated at days 0, 7 and 180; the mean numbers of ingested microfilariae and of developing larvae in the vectors S. soubrense-S. sanctipauli, engorged on the treated patients were recorded. Comparisons were made with non treated patients, having a similar density of microfilariae than the 8 treated patients at day 7. Results confirm the reduction induced by ivermectin of the dermic microfilarial density and the resulting reduction of the infection of the simuliids. Furthermore 7 days after treatment, a new phenomenon is demonstrated: for a similar dermic microfilarial density, simuliids take up a number of microfilariae 100 times lower from treated than from untreated patients. To explain this phenomenon, it is suggested that ivermectin induces a change in the microfilarial distribution in the layers of the dermis. Six months after treatment, this low uptake of microfilariae by the vectors had disappeared, and the infection rate of the engorged similiids was much higher than at day 7 although the dermic microfilarial density was similar.