Anopheles gambiae eicosanoids modulate Plasmodium berghei survival from oocyst to salivary gland invasion

Eicosanoids affect the immunity of several pathogen/insect models, but their role on the Anopheles gambiae response to Plasmodium is still unknown. Plasmodium berghei-infected mosquitoes were injected with an eicosanoid biosynthesis inhibitor, indomethacin (IN), or a substrate, arachidonic acid (AA), at day 7 or day 12 post-infection (p.i.). Salivary gland invasion was evaluated by sporozoite counts at day 21 p.i. IN promoted infection upon sporozoite release from oocysts, but inhibited infection when sporozoites were still maturing within the oocysts, as observed by a reduction in the number of sporozoites reaching the salivary glands. AA treatment had the opposite effect. We show for the first time that An. gambiae can modulate parasite survival through eicosanoids by exerting an antagonistic or agonistic effect on the parasite, depending on its stage of development.

Aflatoxin effetc on the oocysts morphometry and contribution on the morphology of Eimeria bateri Bhatia, Pandey and Pande, 1965 from the japanese quail Coturnix japonica in Brazil / Efeito da aflatoxina sobre a morfbmetria dos oocistos e contribuição para a morfologia de Eimeria bateri Bhatia, Pandey and Pande, 1965 da Codorna japonesa Coturnix japonica no Brasil

The purpose of this study was to characterize Eimeria bateri oocysts and to evaluate the aflatoxin effect in the morphometry of sporulated oocysts in Japanese quails infected naturally. Of a total of 50 quails naturally infected by E. bateri were randomly divided into two groups with 25 birds each. In one of them, quails were orally administered with aflatoxin in dose of 0.04 mg/kg body weight previously. Both experimental groups shed E. bateri oocysts. These oocysts were subspherical to ellipsoidal, 25.1 x 18.9 Lim, with bi-layered wall. Micropyle and residuum were absent, but one or more polar granules were present. Sporocysts elongate ovoid, 12.5 x 7.4 μm. Stieda and substieda bodies were present. Sporocyst residuum was dispersed and sporozoites presented a nucleus and a refractile body. Histograms confirmed the presence of a single species, E. bateri. Linear regression proved that E. bateri oocysts are polymorphic, due, basically, to shape of these oocysts. The comparative morphometry between two experimental groups demonstrated that the aflatoxin influenced significantly in the E. bateri oocysts.

O objetivo deste estudo foi caracterizar os oocistos de Eimeria bateri e avaliar o efeito da aflatoxina na morfometria destes oocistos em codornas japonesas naturalmente infectadas. Cinqüenta codornas naturalmente parasitadas por E. bateri foram separadas aleatoriamente em dois grupos com 25 aves cada. Um dos grupos foi intoxicado experimentalmente com aflatoxina, por via oral, na dose de 0,04 mg/kg de peso vivo. Os dois grupos experimentais eliminaram oocistos de E. bateri nas fezes. Esses oocistos foram de subesféricos a elipsóides, 25,1 x 18,9 Lm, com parede dupla. A micrópila e o resíduo estavam ausentes, mas um ou vários grânulos polares estavam presentes. Esporocistos ovóides alongados, 12,5 x 7,4 L m. Os corpos de Stieda e substieda estavam presentes. O resíduo do esporocisto estava disperso e os esporozoítas apresentaram um núcleo e um corpo refráctil. Os histogramas confirmaram a presença de uma única espécie, E. bateri. A regressão linear comprovou que os oocistos de E. bateri são polimórficos, devido, basicamente, à forma desses oocistos. A morfometria comparativa entre os dois grupos experimentais, demonstrou que a aflatoxina influiu significativamente nos oocistos de E. bateri.

[ effect of administration of anti-coccidial drugs on oocyst shedding and performance in experimental coccidiosis in broiler chickens]

Coccidiosis is one of the major parasitic diseases of poultry. In this study, to compare the effects of coccidiostatic drugs on fecal oocyst shedding and body weight gain of coccidi-infected broiler chickens, 180 one day old Ross 308 broiler chicks were randomly assigned to four treatments. Each treatment contained 3 replicates of 15 chickens. Treatments 1 and 2 were fed diets supplemented with 200ppm Diclazuril and 500ppm Salinomycin, respectively. Treatments 3 and 4 were designated as positive and negative control, received no coccidiostate. Chickens in treatment 1, 2 and 3 were inoculated with a suspension containing four Eimeria species. Frequency of excreted oocyst obtained from feces samples during 7-13 days post -challenged was carried out. Body weight, body weight gain, feed conversion ratio and mortality rate were evaluated weekly. The results revealed that coccidiostatic drugs decreased oocyst per gram of feces significantly in 7-13 days post inoculation [p<0.05]. The highest mean of body weight was related to negative control followed by chickens treated with Diclazuril. The lowest FCR was belonged to negative control followed by chickens treated with Diclazuril. It could be concluded that coccidiostate -supplemented diets in Eimeria infected groups shed less [P<0.05] oocyst than control-infected chickens and improved production performance in coccidian-infected broiler chicks

Activity of praziquantel on in vitro transformed Schistosoma mansoni sporocysts

Praziquantel (PZQ) is effective against all the evolutive phases of Schistosoma mansoni. Infected Biomphalaria glabrata snails have their cercarial shedding interrupted when exposed to PZQ. Using primary in vitro transformed sporocysts, labeled with the probe Hoechst 33258 (indicator of membrane integrity), and lectin of Glycine max (specific for carbohydrate of N-acetylgalactosamine membrane), we evaluated the presence of lysosomes at this evolutive phase of S. mansoni, as well as the influence of PZQ on these acidic organelles and on the tegument of the sporocyst. Although the sporocyst remained alive, it was observed that there was a marked contraction of its musculature, and there occurred a change in the parasite's structure. Also, the acidic vesicles found in the sporocysts showed a larger delimited area after contact of the parasites with PZQ. Damages to the tegument was also observed, as show a well-marked labeling either with Hoechst 33258 or with lectin of Glycine max after contact of sporocysts with the drug. These results could partially explain the interruption/reduction mechanism of cercarial shedding in snails exposed to PZQ.

Evaluation of herbal coccidiostat 'Coxynil' in broiler.

Anticoccidial efficacy of "Coxynil" a polyherbal preparation was tested against Eimeria tenella in broilers. Body weight of birds challenged with E. tenella in Coxynil treated groups was higher as compared to Coxynil untreated. Oocyst out put, lesion score, HI titres against New Castle disease virus were significantly higher in Coxynil supplemented groups in comparison to Coxynil un-supplemented groups. Examination of ceaca of the birds, revealed that the Coxynil interfered with life cycle of coccidia. The typical second generation schizonts were absent in ceacal section of Coxynil treated groups. The results indicate that Coxynil is effective herbal coccidiostat.

Giardiacidal activity of lemon juice, vinifer and vinegar on Giardia intestinalis cysts.

The giardiacidal efficacy of simple disinfecting materials, ie lemon juice, vinifer, and vinegar, for uncooked foods with Giardia cysts was investigated to help travelers in Giardia-endemic areas. The cysts were obtained from stools of individuals with Giardia intestinalis infection by modified sucrose gradient procedure. A pooled batch of 3 x 10(4)/ml Giardia cysts was made from all specimens. The cysts were kept at 4 degrees C until use. Before each experiment, the number of cysts was determined by hemocytometer. Two sets of Eppendorf tubes were used for the experiments, one set at 4 degrees C and one at 24 degrees C. One thousand microliters each of lemon juice, vinifer, or vinegar was poured into each tube, and 1,000 microl of Giardia cysts were added. Variables were disinfectant materials, temperature, and time of exposure. Cyst viability 140 was determined by eosin inclusion procedure. Viability of at least 250 cysts in each tube at 0, 0.5, 1, 2 and 3 hours after the beginning of the experiments was determined. The mean giardiacidal activity at 4 degrees C after 3 hours for lemon juice, vinifer, and vinegar was 18.9, 12.8, and 28.4%, and at 24 degrees C, 28.3, 16.2, and 40.6%, respectively. In conclusion, the giardiacidal activity of vinegar was more than the other materials, and as exposure time and temperature increased, giardiacidal activity also increased; the highest giardiacidal activity of vinegar was at 3-hours exposure at 24 degrees C.

Técnicas de purificación y ruptura de quistes de Giardia spp / Purification and breaking techniques of cysts of Giardia spp

El objetivo de este trabajo fue optimizar y evaluar las técnicas de purificación, aislamiento y ruptura de quistes de Giardia spp a partir de heces formoladas para la obtención de ADN. La materia fecal filtrada fue sometida a 3 técnicas de purificación, utilizando soluciones de formol-éter, sacarosa y formol-éter más sacarosa. La solución de sacarosa permitió aislar los quistes con menos detritos. Los quistes purificados fueron tratados con 3 técnicas para la ruptura de los mismos: shock osmótico y calor, degradación química y shock térmico, acción enzimática y efecto mecánico. Solamente con la técnica de shock térmico, acción enzimática y efecto mecánico se observaron bandas fluorescentes en geles de agarosa. Los resultados de este trabajo permiten contar con una metodología de rutina, simple, que podría ser usada en los pasos previos a la técnica de PCR para la genotipificación de este parásito.

The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.

Comparison of Cryptosporidium parvum development in various cell lines for screening in vitro drug testing.

This study describes the development of Cryptosporidium parvum in MDCK, MA-104, Hep-2 and Vero cell lines. Differences in susceptibility, infectivity, and the methodology of excystation were determined. Various solutions were considered to determine the factors which enhanced the excystation (eg with and without sodium hypochlorite, trypsin or sodium taurocholate). It was shown that the sporozoites could be excysted in media either with or without trypsin and sodium taurocholate, but the number of sporozoites in the latter solution was less than the former one. Only oocysts digested by sodium hypochlorite and trypsin can enter the culture cells. Numerous meronts and oocysts were demonstrated and persisted for 9 days. Asexual stages were not observed in MA-104. Only few oocysts could be detected 1-3 days post-inoculation. There was a significant difference between the number of oocysts, which invaded MDCK, MA-104, and Hep-2 cells. MDCK gave the highest susceptibility to oocyst invasion among the three cell lines and asexual stages were also found. Among the 25 isolates, which had been cultivated, 23 isolates could infect MDCK and Hep-2. Only 2 isolates could not infect the MDCK cell. These 2 isolates could infect the Vero cell and yielded high numbers of trophozoites. Praziquantel (PZQ), doxycycline, and paromomycin (PRM) were tested on the infecting parasites. The drugs were added either with the inoculum or 24 hours after inoculation. None of them was effective, including PRM, which had been previously reported as effective.

Viability of preserved Cryptosporidium baileyi oocysts

The present study was undertaken to determine the viability and infectivity of oocysts of Cryptosporidium baileyi that had been stored from 1 to 40 months at 4 degrees C preserved in 2.5% potassium dichromate solution. Oocysts of C. baileyi were purified from the feces of experimentally infected chickens using discontinuous sucrose gradients. Subsequently, the purified oocysts were suspended in 2.5% potassium dichromate solution at a concentration of 1 x 10 (7) organism/ml, and their viabilities were assessed by nucleic acid staining, histologic examination, and infectivity to 2-day-old chickens. All chickens inoculated with oocysts that had been stored for 1-18 months developed patent infections, while chickens infected with older oocysts remained uninfected. Between 5.8% and 82.2% of the oocysts, stored at 4 degrees C in 2.5% potassium dichromate solution, were found to be viable, as determined by nucleic acid staining. Parasite colonization in the bursa of Fabricius was detected in the microvillus border of bursal epithelium. The finding that C. baileyi oocysts remain infective to chickens for at least 18 months offers important time-saving advantages to investigators who frequently require large numbers of oocysts.