Laboratory and field evaluation of an oviposition trap for Culex quinquefasciatus(Diptera: Culicidae)

An ovitrap (BR-OVT) based on physical and chemical stimuli for attracting gravid Culex quinquefasciatus (Diptera: Culicidae) females was developed and evaluated under laboratory and field conditions. Attractants were assayed using alternative chamber bioassays prior to being used in the BR-OVT oviposition trap. A significant preference of gravid females for sites containing conspecific egg rafts was observed, as a response to the natural oviposition pheromone, as well as for sites treated with the synthetic pheromone erythro-6-acetoxy-5-hexadecanolide. Five- to 20-day old grass infusion was strongly attractive to gravid females for laying eggs. On the other hand, entomopathogenic Bacillus sphaericus (Bs) did not influence the choice of an oviposition site when used in combination with grass infusion and can therefore be used as a larvicide in ovitraps. Results from field trials showed that the BR-OVT with grass infusion and with or without Bs works as a preferred oviposition site for Cx. quinquefasciatus. The BR-OVT was more effective for egg collection when placed indoors and comparison with the number of egg rafts laid in cesspits over 40 days indicates that this very simple ovitrap may be a useful tool for monitoring populations of the most important of the vectors of bancroftian filariasis.

Ribotyping and virulence markers of Yersinia pseudotuberculosis strains isolated from animals in Brazil

Ribotyping and virulence markers has been used to investigate 68 Yersinia pseudotuberculosis strains of serogroups O:1a and O:3. The strains were isolated from clinical material obtained from healthy and sick animals in the Southern region of Brazil. Ribotypes were identified by double digestion of extracted DNA with the restriction endonucleases SmaI and PstI, separation by electrophoresis and hybridization with a digoxigenin-labeled cDNA probe. The presence of the chromosomal virulence marker genes inv, irp1, irp2, psn, ybtE, ybtP-ybtQ, and ybtX-ybtS, of the IS100 insertion sequence, and of the plasmid gene lcrF was detected by polymerase chain reaction. The strains were grouped into four distinct ribotypes, all of them comprising several strains. Ribotypes 1 and 4 presented distinct profiles, with 57.3% genetic similarity, ribotypes 2 and 3 presented 52.5% genetic similarity, and genetic similarity was 45% between these two groups (1/4 and 2/3). All strains possessed the inv, irp1, and irp2 genes. Additionally, strains of serogroup O:1a carried psn, ybtE, ybtP-ybtQ, ybtX-ybtS, and IS100. As expected lcrF was only detected in strains harboring the virulence plasmid. These data demonstrate the presence of Y. pseudotuberculosis strains harboring genotypic virulence markers in the livestock from Southern Brazil and that the dissemination of these bacteria may occur between herds.

Impact of moisture on survival of Aedes aegypti eggs and ovicidal activity of Metarhizium anisopliae under laboratory conditions

The effect of relative humidity (43%, 75%, 86% and > 98%) on Aedes aegypti eggs treated with Metarhizium anisopliae or water only was tested for up to a six months exposure at 25ºC. Survival of larvae inside eggs was clearly affected by the lowest humidity (43%) tested, and eclosion diminished at all humidities after increasing periods of exposure. M. anisopliae showed to have a strong ovicidal activity only at humidity close to saturation. No difference of activity was found between conidia and hyphal bodies tested. This fungus affected larvae inside eggs and has potential as a control agent of this important vector in breeding sites with high moisture.

The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria

Malaria remains an important health problem in tropical countries like Brazil. Thrombocytopenia is the most common hematological disturbance seen in malarial infection. Oxidative stress (OS) has been implicated as a possible mediator of thrombocytopenia in patients with malaria. This study aimed to investigate the role of OS in the thrombocytopenia of Plasmodium vivax malaria through the measurement of oxidant and antioxidant biochemical markers in plasma and in isolated platelets. Eighty-six patients with P. vivax malaria were enrolled. Blood samples were analyzed for total antioxidant and oxidant status, albumin, total protein, uric acid, zinc, magnesium, bilirubin, total thiols, glutathione peroxidase (GPx), malondialdehyde (MDA), antibodies against mildly oxidized low-density lipoproteins (LDL-/nLDL ratio) and nitrite/nitrate levels in blood plasma and GPx and MDA in isolated platelets. Plasma MDA levels were higher in thrombocytopenic (TCP) (median 3.47; range 1.55-12.90 µmol/L) compared with the non-thrombocytopenic (NTCP) patients (median 2.57; range 1.95-8.60 µmol/L). Moreover, the LDL-/nLDL autoantibody ratio was lower in TCP (median 3.0; range 1.5-14.8) than in NTCP patients (median 4.0; range 1.9-35.5). Finally, GPx and MDA were higher in the platelets of TPC patients. These results suggest that oxidative damage of platelets might be important in the pathogenesis of thrombocytopenia found in P. vivax malaria as indicated by alterations of GPx and MDA.

Life cycle, feeding and defecation patterns of Rhodnius ecuadoriensis (Lent & León 1958) (Hemiptera: Reduviidae: Triatominae) under laboratory conditions

Rhodnius ecuadoriensis is the second most important vector of Chagas Disease (CD) in Ecuador. The objective of this study was to describe (and compare) the life cycle, the feeding and defecation patterns under laboratory conditions of two populations of this specie [from the provinces of Manabí (Coastal region) and Loja (Andean region)]. Egg-to-adult (n = 57) development took an average of 189.9 ± 20 (Manabí) and 181.3 ± 6.4 days (Loja). Mortality rates were high among Lojan nymphs. Pre-feeding time (from contact with host to feeding initiation) ranged from 4 min 42 s [nymph I (NI)] to 8 min 30 s (male); feeding time ranged from 14 min 45 s (NI)-28 min 25 s (male) (Manabí) and from 15 min 25 s (NI)-28 min 57 s (nymph V) (Loja). The amount of blood ingested increased significantly with instar and was larger for Manabí specimens (p < 0.001). Defecation while feeding was observed in Manabí specimens from stage nymph III and in Lojan bugs from stage nymph IV. There was a gradual, age-related increase in the frequency of this behaviour in both populations. Our results suggest that R. ecuadoriensis has the bionomic traits of an efficient vector of Trypanosoma cruzi. Together with previous data on the capacity of this species to infest rural households, these results indicate that control of synanthropic R. ecuadoriensis populations in the coastal and Andean regions may have a significant impact for CD control in Ecuador and Northern Peru.

Experimental infection of dogs with a Brazilian strain of Rickettsia rickettsii: clinical and laboratory findings

The bacterium Rickettsia rickettsii is the etiological agent of an acute, severe disease called Rocky Mountain spotted fever in the United States or Brazilian spotted fever (BSF) in Brazil. In addition to these two countries, the disease has also been reported to affect humans in Mexico, Costa Rica, Panama, Colombia and Argentina. Like humans, dogs are also susceptible to R. rickettsii infection. However, despite the wide distribution of R. rickettsii in the Western Hemisphere, reports of R. rickettsii-induced illness in dogs has been restricted to the United States. The present study evaluated the pathogenicity for dogs of a South American strain of R. rickettsii. Three groups of dogs were evaluated: group 1 (G1) was inoculated ip with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and the control group (G3) was infested by uninfected ticks. During the study, no clinical abnormalities, Rickettsia DNA or R. rickettsii-reactive antibodies were detected in G3. In contrast, all G1 and G2 dogs developed signs of rickettsial infection, i.e., fever, lethargy, anorexia, ocular lesions, thrombocytopenia, anemia and detectable levels of Rickettsia DNA and R. rickettsii-reactive antibodies in their blood. Rickettsemia started 3-8 days after inoculation or tick infestation and lasted for 3-13 days. Our results indicate that a Brazilian strain of R. rickettsii is pathogenic for dogs, suggesting that canine clinical illness due to R. rickettsii has been unreported in Brazil and possibly in the other South American countries where BSF has been reported among humans.

Influence of temperature and humidity on the biology of Triatoma mexicana (Hemiptera: Reduviidae: Triatominae) under laboratory conditions

Several biological parameters related to the Triatoma mexicana life-cycle were evaluated in this study. Three cohorts were maintained under different combinations of temperature and relative humidity (RH): 25ºC/50% RH; 25ºC/75% RH; and 30ºC/75% RH. Observed hatching rates varied from 49-57.5% whereas the average time of hatching varied from 19.5-22.7 days. In the three cohorts studied, the mean time-lapse between presentation of the blood meal and the beginning of feeding was less than 5 min in all instars; the mean feeding time was longer than 10 min in all the instars; the post-feed defecation delay was over 10 min in all the instars. Less than 50% of nymphs in each cohort completed the cycle and the average time from 1st instar nymph to adult was more than 255 days for the three cohorts. The number of blood meals before molt at each nymphal instar varied from 1-9. Our results appear to indicate a lack of influence of temperature and RH on the biological parameters of T. mexicana that were studied, which could reflect the adaptation capacity of this species. We also conclude that T. mexicana can not be considered an effective transmitter of Trypanosoma cruzi to human populations in areas where this species is currently present.

Laboratory evaluation of the effects of triflumuron on the development of Rhodnius prolixus nymph

Studies were carried out to evaluate the efficacy of the growth regulator, triflumuron (TFM) (Starycide® sc 480 Bayer), for disrupting the development of Rhodnius prolixus fifth-instar nymph by oral, topical or continuous treatment. All treatments were able to induce high levels of mortality, delay development and molt inhibition. Oral treatment induced molt inhibition in all insects that survived at doses of 0.25, 0.50 and 5.0 mg/mL of a blood meal. The highest levels of both mortality in 24 h and molt inhibition were always observed after topical treatment. The lowest doses needed to obtain considerable biological effects were always observed after continuous treatment. In this way, the highest levels of mortality within 30 days were detected after continuous treatment, which also induced an extended inter-molting period, a lower number of over-aged nymphs and the highest level of molting in nymphs that survived. Moreover, the effects of TFM on insects were often displayed in a dose response manner. These results indicate that TFM acts as a potent growth inhibitor of R. prolixus nymphs and has the potential to be used in integrated vector control programs against hematophagous triatomine species.

Response of Triatoma infestans to pour-on cypermethrin applied to chickens under laboratory conditions

This article reports the effects of a pour-on formulation of cypermethrin (6% active ingredient) applied to chickens exposed to Triatoma infestans, the main vector of Chagas disease in rural houses of the Gran Chaco Region of South America. This study was designed as a completely random experiment with three experimental groups and five replicates. Third instar nymphs were fed on chickens treated with 0, 1 and 2 cc of the formulation. Nymphs were allowed to feed on the chickens at different time intervals after the insecticide application. Third-instar nymphs fed on treated chickens showed a higher mortality, took less blood during feeding and had a lower moulting rate. The mortality rate was highest seven days after the insecticide solution application and blood intake was affected until 30 days after the application of the solution.

Influence of blood meal and mating in reproduction patterns of Triatoma brasiliensis females (Hemiptera: Reduviidae) under laboratory conditions

The influence of blood meal and mating on Triatoma brasiliensis (Neiva) female fecundity, fertility, life-span and the preoviposition period were investigated under laboratory conditions. Nourishment increased fecundity, fertility and adult lifespan, whereas mating increased fecundity, fertility and decreased the preoviposition period. Females also required more than one mating to reach their full reproductive potential. Results indicate that both nourishment and mating are important in T. brasiliensis proliferation. Such information will help towards developing effective control strategies of this vector of Chagas disease.

Tracing lineage by phenotypic and genotypic markers in Salmonella enterica subsp. enterica serovar 1,4,[5],12:i:- and Salmonella Typhimurium isolated in state of São Paulo, Brazil

Fifty-three Salmonella 1,4,[5],12:i:- and 45 Salmonella Typhimurium strains were characterised using phage typing, plasmid profiles and pulsed-field gel electrophoresis (PFGE) for comparison. The majority of the strains were subdivided into definitive type (DT) 41 (22.6%) and DT 193 (18%) and the 60-MDa plasmid was detected in 94.3% and 84.4% of strains, respectively. Genetic diversity was observed among all strains and 90% presented a > 70% similarity through PFGE analysis. These results suggest a close relationship between Salmonella 1,4,[5],12:i:- and Salmonella Typhimurium at the serotype level.

Microsatellite markers for population genetic studies of the blowfly Chrysomya putoria (Diptera: Calliphoridae)

The investigation of the genetic variation and population structure of Chrysomya species is of great interest for both basic and applied research. However, very limited genetic information is available for this genus across its geographical distribution. Here, we describe 12 polymorphic microsatellite loci isolated from Chrysomya putoria with expected heterozygosities ranging from 0.1402-0.8312. These markers are of potential applied interest for forensic entomologists and for the characterisation of the genetic structure of C. putoria from recently colonised regions, with great promise for understanding the colonisation dynamics and spread of the genus Chrysomya in the New World.

Occurrence of hybrids and laboratory evidence of fertility among three species of the Phyllosoma complex (Hemiptera: Reduviidae) in Mexico

In seven studied communities of Western Mexico, triatomine specimens were sympatrically collected, some with atypical morphological characteristics in contrast to pure specimens, which were presumed to be hybrids. More than 200 specimens of Meccus pallidipennis and Meccus longipennis with brown-yellow markings on dorsal connexival segments were collected in Ahuacapán and Quitupan. In La Mesa, more than 60 specimens similar to Meccus picturatus in most morphological characteristics (including size) were collected, although they presented a largely yellowish corium like M. pallidipennis. Interfertility was proven between all of the studied wild hybrid specimens, as well as between all the experimental laboratory hybrids. Two different phenotypes (M. picturatus and M. longipennis) were obtained from crosses between M. picturatus x M. picturatus and M. longipennis x M. longipennis from the three studied localities in state of Nayarit as from La Mesita. Results support the hypothesis that the subspecific ranking of those triatomines may, therefore, be more appropriate because reproductive isolation has not been developed and complete interbreeding was recorded.

Clinical and laboratory status of patients with chronic Chagas disease living in a vector-controlled area in Minas Gerais, Brazil, before and nine years after aetiological treatment

Twenty-eight Chagas disease patients (CD), 22 with the indeterminate clinical form (IND) and six with the cardiac or digestive form (CARD/DIG), were treated with benznidazole and underwent clinical and laboratorial analysis before (IND and CARD/DIG) and nine years after [patients after treatment (CDt), patients with the indeterminate clinical form at treatment onset (INDt) and with the cardiac or digestive form at treatment onset (CARD/DIGt)] treatment. The data demonstrate that 82.1% of CDt patients (23/28) remained clinically stable and 95.4% of the INDt (21/22) and 33.3% of the CARD/DIGt (2/6) patients showed unaltered physical and laboratorial examinations. The clinical evolution rate was 2%/year and was especially low in INDt patients (0.5%/year) relative to CARD/DIGt patients (7.4%/year). Positive haemoculture in treated patients was observed in 7.1% of the cases. None of the INDt (0/21) and 33.3% of the CARD/DIGt (2/6) patients displayed positive cultures. The PCR presented a positive rate significantly higher (85.2%, 23/27) than haemoculture and two samples from the same patient revealed the same result 57.7% of the patients. Conventional serology-ELISA on 16 paired samples remained positive in all individuals. Semi-quantitative ELISA highlighted significant decreases in reactivity, particularly in INDt relative to IND. Non-conventional serology-FC-ALTA-IgG, after treatment, showed positive results in all sera and 22 paired samples examined at seven and nine years after treatment, demonstrated significantly lower reactivity, particularly in INDt patients. This study was retrospective in nature, had a low number of samples and lacked an intrinsic control group, but the data corroborate other results found in the literature. The data also demonstrate that, even though a cure has not been detected in the none-treated patients, the benefits for clinical evolution were selectively observed in the group of INDt patients and did not occur for CARD/DIGt patients.

Life cycle of Triatoma ryckmani (Hemiptera: Reduviidae) in the laboratory, feeding patterns in nature and experimental infection with Trypanosoma cruzi

A cohort initiated with 121 eggs, yielding 105 first instar nymphs (eclosion rate: 86.78%), allowed us to observe the entire life cycle of Triatoma ryckmani under laboratory conditions (24ºC and 62% relative humidity), by feeding them on anesthetized hamsters. It was possible to obtain 62 adults and the cycle from egg to adult took a mean of 359.69 days with a range of 176-529 days (mortality rate of nymphs: 40.95%). Mean life span of adults was of 81 days for females and 148 days for males. The developmental periods of 4th and 5th nymphs were longer than those of the other instars. This suggests that young siblings have a better chance of taking a hemolymph meal from older ones, in order to survive during fasting periods during prolonged absences of vertebrate hosts from natural ecotopes. The stomach contents of 37 insects showed blood from rodents (15 cases), lizards (7 cases), birds (6 cases) and insect hemolymph (7 cases). Out of 10 insects fed by xenodiagnosis on a Trypanosoma cruzi infected mouse, all but one became infected with the parasite.