Evaluation by ELISA of Anisakis simplex Larval Antigen Purified by Affinity Chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of 1/400 and 1 µg/ml of antigenic concentration.

Domiciliation Trend of Panstrongylus rufotuberculatus in Colombia

The present paper presents evidence of the domiciliation of Panstrongylus rufotuberculatus in La Gardenia, Colombia through the collection of 2 unhatched eggs, 81 nymphs and 10 adults (4 males and 6 females), from 2 rural houses. The transmission risk indicators of Trypanosoma cruzi by P. rufotuberculatus in La Gardenia, were: domiciliary infestation 7.5%, density 2.35, colonization 66.6%, overcrowding 31.33, natural infection 4.6%, and relative infection 2.5%. These results and findings in Peru and Argentina, show that P. rufotuberculatus has a potential success in domiciliation and could some day become an alternate vector of American trypanosomiasis.

Oviposition Attractancy of Bacterial Culture Filtrates: response of Culex quinquefasciatus

Oviposition attractants could be used for monitoring as well as controlling mosquitoes by attracting them to lay eggs at chosen sites. In the present study, culture filtrates of seven bacterial species were tested for their attractancy against gravid females of Culex quinquefasciatus. When their oviposition active indices (OAI) were studied, the culture filtrates of Bacillus cereus and Pseudomonas fluorescens exhibited oviposition attractancy (OAI = >0.3) at 100 ppm and the OAI were respectively 0.70 and 0.47. Culture filtrates of B. thuringiensis var. israelensis (wild type), B. t. var. israelensis (mutant) and B. sphaericus showed attractancy at 2000 ppm with OAI of respectively 0.71, 0.59 and 0.68. However, the OAI of B. megaterium as well as Azospirillum brasilense was 0.13 (at 2000 ppm), which was less than 0.3 required to be considered them as attractants. When the oviposition attractancy of the bacterial culture filtrates were compared with that of a known oviposition attractant, p-cresol (at 10 ppm), the culture filtrates of B. t. var. israelensis (wild type) and B. cereus were found to be more active than p-cresol, respectively with 64.2 and 54.3% oviposition.

Aspects of the Maintenance of the Life Cycle of Fasciola hepatica in Lymnaea columella in Minas Gerais, Brazil

Fascioliasis is a parasitic disease of domestic ruminants that occurs worldwide. The lymnaeid intermediate hosts of Fasciola hepatica include Lymnaea columella, which is widely distributed in Brazil. A colony of L. columella from Belo Horizonte, MG, was reared in our laboratory to be used in studies of the F. hepatica life cycle, the intermediate host-parasite relationship and development of an anti-helminthic vaccine. In the first experiment 1,180 snails were exposed to miracidia of F. hepatica eggs removed from the biliary tracts of cattle from the State of Rio Grande do Sul. In the second and third experiments the snails were exposed to miracidia that had emerged from F. hepatica eggs from Uruguay, maintained in rabbits. The rates of infection in the first, second and third experiments were 0, 42.1 and 0% respectively. Over 15,806 metacercariae were obtained and stored at 4ºC. Four rabbits weighing 1.5 kg each were infected with 32-44 metacercariae and two with 200. Three rabbits begin to eliminate eggs of the parasite in the feces from 84 days after infection onwards. The biological cycle of F. hepatica in L. columella and the rabbit was completed within 124 days.

Variation of the Oviposition Preferences of Aedes aegypti in Function of Substratum and Humidity

Two Aedes aegypti (L.) populations were studied in the laboratory regarding the preference for three types of breeding sites, i.e., flasks containing only water, flasks with a plant and flasks with a stick. Each of these breeding units was placed in one cage and the choice of the oviposition sites was determined for individual females and three females per experimental unit at two humidity levels. Preference for ovipositing on the water surface was observed and varied according to experimental unit and humidity. Mean hatching of eggs in water surface was 46.6%. Experiments with three females showed a more marked difference than when only one female was used. Inter and intrapopulation variability regarding oviposition sites was observed. The discrimination between the different oviposition substrates, hatching in water surface and its implication for mosquito control are discussed.

Biology of Triatoma klugi Carcavallo, Jurberg, Lent & Galvão 2001 (Heteroptera: Reduviidae) under Laboratory Conditions: Effects of Distinct Blood Sources and Susceptibility to Trypanosoma cruzi and Trypanosoma rangeli

The life cycle of Triatoma klugi Carcavallo, Jurberg, Lent & Galvão 2001 was compared under laboratory conditions using two groups of the F1 generation obtained from field-collected bugs. Among the 100 nymphs weekly fed on mice (Group A) or chicken (Group B), 77% of Group A and 67% of Group B reached the adult stage, and the mean time from the first nymphal stage to adult was 190.08 ± 28.31 days and 221.23 ± 40.50, respectively. The average span in days for each stage per group and the number of blood meals required for each stage were also evaluated. The overall mortality rate was 23% and 33% for Groups A and B, respectively. The mean number of eggs laid per month in a three-month period was of 56.20, 51.70 and 73.20 for Group A, and 64.50, 53.50 and 38.71 for Group B. Despite the blood source, comparative analysis revealed no statistically significant differences in the life cycle of T. klugi under laboratory conditions. Infection rates over 60% were observed for both Trypanosoma cruzi strains tested. Even revealing high infection rates of the hemolymph by T. rangeli strains, T. klugi revealed no salivary gland infections and was not able to transmit the parasite.

Anopheles albitarsis Embryogenesis: Morphological Identification of Major Events

Anopheles albitarsis embryogenesis was analyzed through confocal microscopy of clarified eggs. Using Drosophila melanogaster as reference system, the major morphogenetic events (blastoderm, gastrulation, germ band extension, germ band retraction, dorsal closure) were identified. The kinetics of early events is proportionally similar in both systems, but late movements (from germ band retraction on) progress slower in An. albitarsis. Major differences in An. albitarsis related to D. melanogaster were: (1) pole cells do not protrude from the blastoderm; (2) the mosquito embryo undergoes a 180º rotation movement, along its longitudinal axis; (3) the head remains individualized throughout embryogenesis; (4) extraembryonary membranes surround the whole embryo. A novel kind of malaria control is under development and is based on the use of genetically modified mosquitoes. Phenotypic analysis of the embryonic development of mutants will be imposed as part of the evaluation of effectiveness and risk of employment of this strategy in the field. In order to accomplish this, knowledge of the wild type embryo is a prerequisite. Morphological studies will also serve as basis for subsequent development biology approaches.

Experimental Neuroschistosomiasis: Inadequacy of the Murine Model

Neuroschistosomiasis is rarely observed in human pathology, but it is of considerable importance. To investigate its pathogenesis, consequences and response to treatment, an experimental model would be desirable, but is not yet available, in spite of a few indications of a suitable mouse model in the literature. Severe, recent and late Schistosoma mansoni infections in outbred and inbred strains of mice revealed widespread distribution of parasite eggs in several organs, but only exceptionally did eggs reach the encephalus, thus revealing the inadequacy of the mouse as an experimental model for neuroschistosomiasis.

Redescription of Travassiella avitellina Rego & Pavanelli, 1987 (Proteocephalidea: Monticelliidae, Zygobothriinae), a parasite of Paulicea luetkeni (Siluriformes) from South America

The genus Travassiella Rego & Pavanelli, 1987 comprises only one species, T. avitellina Rego & Pavanelli, 1987; characterized by these authors on the basis of absence of vitelline follicles. In this study, the presence of cortical vitelline follicles in this species is confirmed after de Chambrier and Vaucher (1999); the redescription was performed for the first time based on type and new material collected from Argentina, Brazil and Paraguay. Travassiella is confirmed as a valid genus, and compared to other genera of the subfamily Zygobothriinae. T. avitellina is characterized by: (1) the presence of gland cells posteromedially to suckers; (2) the particular distribution of vitelline follicles, forming lateral arches; (3) uterine primordium cortical, growing into medulla and forming a sac-like uterus; (4) eggs, irregularly oval, outer envelop with excrescences and two digitate lappets in one of the poles.

Eye colour as a genetic marker for fertility and fecundity of Triatoma infestans (Klug, 1834) Hemiptera, Reduviidae, Triatominae

Eye colour of Triatoma infestans is controlled at a single autosomal locus, with black-eye as the dominant gene and red-eye as the recessive. Inheritance of these characters follows a classical Mendelian system, enabling eye colour to be used as a marker for studies of mating frequency. We found no significant differences in oviposition rates and egg hatching rates irrespective of parental phenotypes. Different mating schedules between red-eye and black-eye parents showed that eye colour did not affect mating competence. Females mated with a single male or with different males together or in succession produced similar numbers of fertile eggs, with the eye colour of the offspring reflecting exposure to the different males. We conclude that although a single mating can provide sufficient sperm for the whole reproductive life of the female, multiple matings can result in balanced assortative sperm usage from the spermatheca.

Temperature influence on embryonic development of Anopheles albitarsis and Anopheles aquasalis

Temperature influence on the embryonic development of Anopheles aquasalis and An. albitarsis was investigated. At 26ºC, 75% and 60% of respectively An. aquasalis and An. albitarsis eggs hatched, with one peak of eclosion, between the 2nd and 3rd day after oviposition. At 20 ± 2ºC, around 66-70% of An. aquasalis eggs hatched, with one eclosion peak, on the 5th day. On the other hand, An. albitarsis eclosion at 21 ± 2ºC decreased to 10-22%, with two eclosion peaks, on the 4th-5th day and on the 9th-12th day. These data indicate a stronger temperature influence over An.albitarsis than over An. aquasalis embryos.

Schistosomiasis mansoni in areas of low transmission: epidemiological characterization of Venezuelan foci

Severe schistosomiasis is a rare event in Venezuela nowadays, after a successful national campaign by the Schistosomiasis Control Program. Unfortunately, this program has practically disappeared, and snail surveillance in field is not a priority, anymore. Thus, schistosomiasis has become a neglected disease in this country. However, surveys in different populations from the endemic area have shown particular epidemiological features described herein. In five communities we evaluated 2,175 persons and searched for the presence of Biomphalaria glabrata snails. Some markers were used for classifying schistosomiasis foci: mean age of the persons with Schistosoma mansoni eggs in the stools, serological tests, presence of B. glabrata snails, and intensity of infection. Places without B. glabrata snails and with few schistosomiasis cases were defined as "past transmission sites"; a site with abundant snails but few cases was defined as "potential risk"; "new transmission" foci were characterized by the presence of infected snails and young people passing eggs in the stools. A "re-emergent" focus has shared these last features, showing in addition a place where schistosomiasis had been reported before. Recent evidences of active transmission with the increasing dispersion of B. glabrata snails, point out the necessity for the re-establishment of the Schistosomiasis Control Program in Venezuela.

Schistosomiasis mansoni in Bananal (State of São Paulo, Brazil): III. Seroepidemiological studies in the Palha District

The Palha district, municipality of Bananal, State of São Paulo, Brazil, had 10.3% cases of Schistosoma mansoni diagnosed from 1994 to 2000 by coproscopy: about three times the municipality average. The immunofluorescent antibody test was used to assess gut-associated IgM antibody titers of samples from 452 inhabitants. It disclosed 129 (28.5%) positive cases. Subjects were classified according to age, sex, birthplace, and period of residence. Titers varied from 8 to 4,096 (geometric mean: 170.2). Seropositives were aged from 6 to 69 years (average: 24.5), 75% of them aged 34 or less, predominantly males (78 or 60.5%). Of all subjects, 65.7% were born and had been living in Bananal since; 24.2% came from neighboring municipalities and are residing in Bananal from two months to 89 years (average: 22.7 years). Further Kato-Katz coproscopy from 97 seropositives (geometric mean titer, 619) revealed S. mansoni eggs in 11 subjects (11.3%). Serology was deemed useful in screening subjects to be further investigated by coproscopy, considering that blood collection had better acceptance than supplying fecal samples. Higher than average serological titers may indicate new cases in endemic areas. Longitudinal studies associated with epidemiological investigation, including titer evolution are advised, as isolated data are difficult to interpret.

Intestinal fibrovascular nodules caused by Schistosoma mansoni infection in Calomys callosus Rengger, 1830 (Rodentia: Cricetidae): a model of concomitant fibrosis and angiogenesis

Human schistosomiasis develops extensive and dense fibrosis in portal space, together with congested new blood vessels. This study demonstrates that Calomys callosus infected with Schistosoma mansoni also develops fibrovascular lesions, which are found in intestinal subserosa. Animals were percutaneously infected with 70 cercariae and necropsied at 42, 45, 55, 80, 90 and 160 days after infection. Intestinal sections were stained for brightfield, polarization microscopy, confocal laser scanning, transmission and scanning electron microscopies. Immunohistological analysis was also performed and some nodules were aseptically collected for cell culture. Numerous intestinal nodules, appearing from 55 up to 160 days after infection, were localized at the interface between external muscular layer and intestinal serosa, consisting of fibrovascular tissue forming a shell about central granuloma(s). Intranodular new vessels were derived from the vasculature of the external vascular layer and were positive for laminin, chondroitin-sulfate, smooth muscle alpha-actin and FVIII-RA. Fibroblastic cells and extracellular matrix components (collagens I, III and VI, fibronectin and tenascin) comprised the stroma. Intermixed with the fibroblasts and vessels there were variable number of eosinophils, macrophages and haemorrhagic foci. In conclusion, the nodules constitute an excellent and accessible model to study fibrogenesis and angiogenesis, dependent on S. mansoni eggs. The fibrogenic activity is fibroblastic and not myofibroblastic-dependent. The angiogenesis is so prominent that causes haemorrhagic ascites.

Parasitological characteristics of Schistosoma mansoni infection in swiss mice with underlying malnutrition

The effects of a protein-restricted diet (8% protein, 81% carbohydrate and 11% lipids) on Schistosoma mansoni infectivity, fecal egg excretion and intestinal egg distribution in Swiss (SW) mice were studied. Pregnant mice received a deficient diet from the middle of gestation until delivery. Seven-days-old mice were exposed to 50 cercariae (BH strain, Brazil). Offspring mice had a free access to the deficient diet since lactation until adulthood. The controls were fed with a commercial mice diet. A parasitological examination was performed between six and eight weeks post-infection while both groups were necropsied one week later. Mice on the experimental diet showed a significant loss in body weight. There was no significant difference (p > 0.05) in pre-patent period, kinetics of egg excretion and worm recovery from mice on either diet. Significant differences (p < 0.05) were found concerning to the percentage of deposited eggs in the distal segment of the small intestine from hosts on the experimental diet.Our data suggest that experimental malnutrition induced for a long term has no detrimental effect on the acute schistosomiais infection in SW mice.