Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

Ticks, ivermectin, and experimental Chagas disease

Following an infestation of dogticks in kennels housing dogs used for long-term studies of the pathogenesis of Chagas disease, we examined the effect of ivermectin treatment on the dogs, ticks, trypanosome parasites, and also on triatomine vectors of Chagas disease. Ivermectin treatment was highly effective in eliminating the ticks, but showed no apparent effect on the dogs nor on their trypanosome infection. Triatominae fed on the dogs soon after ivermectin treatment showed high mortality, but this effect quickly declined for bugs fed at successive intervals after treatment. In conclusion, although ivermectin treatment may have a transient effect on peridomestic populations of Triatominae, it is not the treatment of choice for this situation. The study also showed that although the dogticks could become infected with Trypanosoma cruzi, this only occurred when feeding on dogs in the acute phase of infection, and there was no evidence of subsequent parasite development in the ticks.

Mast cell changes in experimental diabetes: focus on attenuation of allergic events

The prevalence of atopic diseases and diabetes is increasing worldwide though the concurrence of these pathologies in individual patients is found less frequent than it would be predicted. Moreover, co-existence of diabetes and allergy is generally marked by attenuation of their respective symptoms, and effective treatment of one disease exacerbates the other. This review gives an update of the state-of-the-art concerning the intercurrence of allergy and diabetes, particularly focusing on the consequences to the allergen-evoked vascular and cellular changes. It is proposed that the reduction in mast cell numbers and reactivity may be a pivotal mechanism behind the mutual exclusion phenomenon.

Single and concomitant experimental infectionsby Endotrypanum spp. and Leishmania (Viannia) guyanensis (Kinetoplastida: Trypanosomatidae) in the Neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae)

Lutzomyia longipalpis females received single and mixed infections with Endotrypanum and Leishmania. Two biological parameters were analyzed: the percentage of infected females and the distribution of flagellates in the gut of the females. The principal comparisons were performed between (1) two strains of Endotrypanum, (2) cloned versus primary sample of one strain of Endotrypanum, (3) Endotrypanum versus Leishmania guyanensis, and (4) the pattern of flagellates behaviour by optical microscopy in females with single or mixed infection versus the identification of parasites isolated from digestive tracts by isoenzyme electrophoresis. Flagellates of Endotrypanum showed distinct patterns of infection suggesting that there is variation between and within strains. The distribution of Endotrypanum and L. guyanensis differed significantly in relation to the colonization of the stomodeal valve. In co-infection with L. guyanensis, a large number of flagellates were seen to be plentifully infecting the stomodeal valve in significantly more specimens than in females infected by Endotrypanum only. However, the electrophoretic profiles of isoenzymes of parasites recovered from all co-infected specimens corresponded to Endotrypanum. This suggests that the mere correlation sand fly infection-biochemical analysis of isolates may induce parasitological incorrect consideration.

Cross-immunoreactivity between anti-potato apyrase antibodies and mammalian ATP diphosphohydrolases: potential use of the vegetal protein in experimental schistosomiasis

We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models.

Aluminum hydroxide associated to Schistosoma mansoni 22.6 kDa protein abrogates partial protection against experimental infection but not alter interleukin-10 production

The need to develop a vaccine against schistosomiasis led several researches and our group to investigate proteins from Schistosoma mansoni as vaccine candidates. Sm22.6 is a protein from S. mansoni that shows high identity with Sj22.6 and Sh22.6 (79 and 91%, respectively). These proteins are associated with high levels of IgE and protection to reinfection. Previously, we have shown that Sm22.6 induced a partial protection of 34.5% when used together with Freund's adjuvant and produced a Th0 type of immune response with interferon-g and interleukin-4. In this work, mice were immunized with Sm22.6 alone or with aluminum hydroxide adjuvant and high levels of IgG, IgG1, and IgG2a were measured. Unfortunately, no protection was detected. Since IL-10 is a modulating cytokine in schistosomiasis, we also observed a high level of this molecule in splenocytes of vaccinated mice. In conclusion, we did not observe the adjuvant effect of aluminum hydroxide associated with rSm22.6 in protective immunity.

Experimental transmission of the parasitic flagellates Trypanosoma cruzi and Trypanosoma rangeli between triatomine bugs or mice and captive neotropical bats

Trypanosoma cruzi and Trypanosoma rangeli-like trypanosomes have been found in a variety of neotropical bat species. In this study, bats (Artibeus lituratus, Carollia perspicillata, Desmodus rotundus, Glossophaga soricina, Molossus molossus, Phyllostomus hastatus) were maintained under controlled conditions, and experiments were conducted to determine how they might become infected naturally with trypanosomes. All bats were first screened for existing infections by hemoculture and the examination of blood smears, and only apparently uninfected animals were then used in the experiments. Proof was obtained that the triatomine bug Rhodnius prolixus would readily feed upon some of the bats, and two species became infected after being bitten by bugs infected with T. rangeli. Some bats also became infected by ingesting R. prolixus carrying T. cruzi, or following subcutaneous or intragastic inoculation with fecal suspensions of R. prolixus containing T. cruzi. P. hastatus became infected after ingesting mice carrying T. cruzi. All of the bats studied inhabit roosts that may be occupied by triatomine bugs and, with the exception of D. rotundus, all also feed to at least some extent upon insects. These findings provide further evidence of how bats may play significant roles in the epidemiology of T. cruzi and T. rangeli in the New World tropics.

Resistance to experimental autoimmune encephalomyelitis development in Lewis rats from a conventional animal facility

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the brain and spinal cord that is mediated by CD4+ T lymphocytes specific to myelin components. In this study we compared development of EAE in Lewis rats from two colonies, one kept in pathogen-free conditions (CEMIB colony) and the other (Botucatu colony) kept in a conventional animal facility. Female Lewis rats were immunized with 100 µl of an emulsion containing 50 µg of myelin, associated with incomplete Freund's adjuvant plus Mycobacterium butyricum. Animals were daily evaluated for clinical score and weight. CEMIB colony presented high EAE incidence with clinical scores that varied from three to four along with significant weight losses. A variable disease incidence was observed in the Botucatu colony with clinical scores not higher than one and no weight loss. Immunological and histopathological characteristics were also compared after 20 days of immunization. Significant amounts of IFN-gamma, TNF-alpha and IL-10 were induced by myelin in cultures from CEMIB animals but not from the Botucatu colony. Significantly higher levels of anti-myelin IgG1 were detected in the CEMIB colony. Clear histopathological differences were also found. Cervical spinal cord sections from CEMIB animals showed typical perivascular inflammatory foci whereas samples from the Botucatu colony showed a scanty inflammatory infiltration. Helminths were found in animals from Botucatu colony but not, as expected, in the CEMIB pathogen-free animals. As the animals maintained in a conventional animal facility developed a very discrete clinical, and histopathological EAE in comparison to the rats kept in pathogen-free conditions, we believe that environmental factors such as intestinal parasites could underlie this resistance to EAE development, supporting the applicability of the hygiene hypothesis to EAE.

Biological and genetic aspects of experimental hybrids from species of the Phyllosoma complex (Hemiptera: Reduviidae: Triatominae)

The present work is a thorough investigation of the degree of reproductive isolation between Meccus mazzottii and Meccus longipennis, Meccus picturatus, Meccus pallidipennis and Meccus bassolsae, as well as between M. longipennis and M. picturatus. We examined fertility and segregation of morphological characteristics in two generations of hybrids derived from crosses between these species. The percentage of pairs with (fertile) offspring was highest in the set of crosses between M. longipennis and M. picturatus, and lowest between M. mazzottii and M. picturatus. Most first-generation (F1) individuals from crosses involving M. mazzottii were morphologically similar to this species, while only F1 x F1 progeny of parental crosses between M. mazzottii and M. longipennis had offspring second generation that looked like M. mazzottii. The results indicate that different degrees of reproductive isolation apparently exist among the species of the Phyllosoma complex examined in this study. The biological evidence obtained in this study does not support the proposal that M. longipennis and M. picturatus are full species. It could indicate on the contrary, that both could be considered as subspecies of a single polytypic species. On the other hand, biological evidence supports the proposal that M. mazzottii is a full species.

Oxamniquine, praziquantel and lovastatin association in the experimental Schistosomiasis mansoni

The activity of lovastatin associated with oxamniquine or praziquantel against schistosomiasis mansoni was evaluated in mice infected with Schistosoma mansoni. Forty days after infection, mice were treated with lovastatin, 400 mg/kg for five consecutive days by oral route, and on the last day of this sequence with 50 mg/kg oxamniquine or with 200 mg/kg praziquantel, both by oral route, single dose. Fifteen days later, the animals were perfused in parallel with an untreated control group. Studies were carried out in vitro, using lovastatin in culture medium containing S. mansoni worms proceeding from experimentally infected mice. In the in vivo trials, the association of lovastatin with oxamniquine or praziquantel did not show any additive action, but there were oogram changes when lovastatin was associated with oxamniquine. In vitro lovastatin was able to interrupt the maturation of S. mansoni eggs, which remained at the 1st or 2nd stages, depending on the dose used. The total number of morphologically dead eggs found in culture of worms exposed to 2 µg/ml or 4 µg/ml concentrations of lovastatin was significantly higher than the number of viable eggs. Using the probe Hoescht 33258 it was observed that 70% of the eggs considered morphologically viable in the treated groups (against 16% in the control group) were labeled, indicating that the majority of the viable eggs had membrane permeability increased due to lovastatin action.

Experimental infection parameters in Galea spixii (Rodentia: Caviidae) with Leishmania infantum chagasi

In order to better understand the epidemiological transmission network of leishmaniasis, an endemic disease in Northeast Brazil, we investigated the susceptibility of Spix yellow-toothed cavies (Galea spixii) to the Leishmania infantum chagasi parasite. Nine cavies were experimentally infected, separated into three groups and monitored at 30, 90 and 180 days, respectively. Amastigotes were identified in the spleen slides of two cavies killed 180 days after infection. Antibodies against the L. i. chagasi were identified in one of the cavies. This demonstrates that G. spixii is in fact capable of maintaining a stable infection by L. i. chagasi without alterations in biochemical and hematological parameters of the host and without perceivable micro and macroscopic lesions.

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.

Association of oxamniquine praziquantel and clonazepam in experimental Schistosomiasis mansoni

The antischistosomal activity of clonazepam, when administered alone or in association with oxamniquine and praziquantel, was experimentally evaluated in mice infected with Schistosoma mansoni. The animals were treated 45 days post-infection with a single dose, by oral route, according to three treatment schedules: clonazepam 25 mg/kg and sacrificed 15 min, 1h or 4 h after treatment; clonazepam 1.0, 2.5 or 10.0 mg/kg and sacrificed 15 days post-treatment or with the dose of 10 mg/kg in association with oxamniquine 50 mg/kg or praziquantel 200 mg/kg, single dose, orally, every schedule with a control group. The efficacy of the drugs in vivo was assessed by means of worm counts and their distribution in mesentery and liver, mortality and oogram changes. In the chemotherapeutic schedules used, clonazepam did not present antischistosomal activity and the result of the association of this drug with oxamniquine or praziquantel was not significantly different from the one obtained when these two last drugs were administered alone. In the in vitro experiments, the worms exposed to 0.6 mg/mL clonazepam remained motionless throughout the 8-day-period of observation, without egg-laying, whereas the worms of the control group showed normal movements, egg-laying and hatching of miracidia on the last day of observation. The results obtained in the present study confirm the action of clonazepam on S. mansoni adult worm, in vitro, causing total paralysis of males and females. However, no additive or synergistic effects were observed when clonazepam were used in association with oxamniquine or praziquantel.

Experimental reinfection of BALB/c mice with different recombinant type I/III strains of Toxoplasma gondii: involvement of IFN-³ and IL-10

To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-³ and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-³ were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

Failure to demonstrate experimental vertical transmission of the epidemic strain of Chikungunya virus in Aedes albopictus from La Réunion Island, Indian Ocean

Aedes albopictus was responsible for transmission in the first outbreak of chikungunya (CHIK) on La Réunion Island, Indian Ocean, in 2005-2006. The magnitude of the outbreak on this island, which had been free of arboviral diseases for over 30 years, as well as the efficiency of Ae. albopictus as the main vector, raises questions about the maintenance of the CHIK virus (CHIKV) through vertical transmission mechanisms. Few specimens collected from the field as larvae were found to be infected. In this study, Ae. albopictus originating from La Réunion were orally infected with a blood-meal containing 10(8) pfu/mL of the CHIKV epidemic strain (CHIKV 06.21). Eggs from the first and second gonotrophic cycles were collected and raised to the adult stage. The infectious status of the progeny was checked (i) by immunofluorescence on head squashes of individual mosquitoes to detect the presence of viral particles or (ii) by quantitative RT-PCR on mosquito pools to detect viral RNA. We analysed a total of 1,675 specimens from the first gonotrophic cycle and 1,709 from the second gonotrophic cycle without detecting any viral particles or viral RNA. These laboratory results are compared to field records.