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Background: The reintroduction of sylvatic yellow fever in the state of São Paulo after about six decades was confirmed in the Northwestern region in 2000, where in 2008 there also occurred an important epizootic. The purpose of this study was to investigate the feeding habits of culicids potentially involved in the sylvatic transmission of the virus in this region., Methods: Specimens were collected in 24 forested localities at ground level with hand nets and mouth aspirators. Collections were made quarterly between October 2006 and July 2008 during daylight hours. Blood-meal identification was carried out in mosquitoes of the tribes Aedini, Mansoniini and Sabethini. The biotin/avidin sandwich ELISA was employed to determine six source types: bird, bovine, equine, rat, human and monkey., Results: A total of 24,879 females of the three tribes were obtained, 245 (0.98%) of which were engorged. The presence of three different blood sources per engorged female was the predominant situation, and included 35.10% of the total of samples processed. Samples with two or four different sources were represented by 25.31% and 25.71%, of the specimens, respectively, while just 9.39% had only one type and 1.22%, five different sources. Aedes scapularis, Ae. serratus (Group), Psorophora albigenu and Ps. ferox were the most abundant species and accounted for about 95% of the engorged specimens. Of the principal vector species, Haemagogus janthinomys/capricornii was found with bird, bovine and primate blood. These sources were predominant and alternated top ranking as the most frequent source according to the mosquito species and collection site. In general, primate blood was the most prevalent source., Conclusions: The human population of the region visits this ecotone frequently, which indicates the need for the periodical assessment of vaccination coverage against yellow fever. The frequency of non-human primate blood source in mosquito species that show minor vector importance in yellow fever virus transmission deserves attention. The eclectic feeding habits and some aspects of the interactions between potential vectors and reservoirs of yellow fever may be associated with the habitat fragmentation characteristic of the region. We recommend that further studies on the capacity and vector competence be performed on secondary vectors in extra-Amazonian region.

Introduction: The aim of this study was to identify the blood feeding sources of Nyssomyia intermedia (Ny. intermedia) and Nyssomyia neivai (Ny. neivai), which are Leishmania vectors and the predominant sandfly species in the Ribeira Valley, State of São Paulo, Brazil, an endemic area for cutaneous leishmaniasis., Methods: Specimens were captured monthly between February 2001 and December 2003 on a smallholding and a small farm situated in the Serra district in the Iporanga municipality. The blood meals of 988 engorged females were tested using the avidin-biotin immunoenzymatic enzyme-linked immunosorbent assay (ELISA). Seven blood meal sources were investigated: human, dog, chicken, bovine, pig, horse and rat., Results: The results showed that among the females that fed on one or more blood sources, the respective percentages for Ny. intermedia and Ny. neivai, respectively, were as follows: human (23% and 36.8%), pig (47.4% and 26.4%), chicken (25.7% and 36.8%) and dog (3.9% and 0%), and the differences in the blood sources between the two species were statistically significant (p = 0.043)., Conclusions: Both species had predominant reactivity for one or two blood sources, and few showed reactivity indicating three or four sources. Many different combinations were observed among the females that showed reactivity for more than one source, which indicated their opportunistic habits and eclecticism regarding anthropic environmental conditions.

Sand flies were captured weekly with CDC light traps from December 2003 to November 2005 in three areas of Campo Grande, in the Brazilian state of Mato Grosso do Sul. These areas incorporated two patches of remnant forest and five houses. The blood meals of engorged female sand flies were identified using the avidin-biotin system of immunoenzymatic ELISA capture. Most (327/355) of the females analysed were Lutzomyia longipalpis, of which 66.4% reacted with human blood, 64.8% with that of birds and 8.9% with that of dogs. Females that had taken human blood predominated in the residential areas and two forest patches. The following combinations of blood were also detected for L. longipalpis in some of the samples analysed: bird+human (43.4%), bird+human+dog (6.1%). The combination bird+human+dog+pig was also found for Nyssomyia whitmani. Dogs and pigs appear to have little attractiveness for L. longipalpis. The results obtained demonstrate the eclecticism and high anthropophily of L. longipalpis and raise new questions with regard to the importance of dogs in VL epidemiology and the possible role of man as a source of infection for sand flies.

Lutzomyia longipalpis and Lutzomyia almerioi, phlebotomine species from the fauna of Serra da Bodoquena, in the State of Mato Grosso do Sul, Brazil, have been studied, particularly due to the fact of their abundance and occurrence, the Guaicurus settlement, focus of human tegumentary and canine visceral leishmaniasis. In researches that are being carried out in this settlement for identifying the vectors of these parasitosis, 83 engorged females belonging to the species Lutzomyia longipalpis and Lutzomyia almerioi were captured with automatic light traps from 2002 up to 2004 in the peridomiciliary environment of the Guaicurus settlement (hennery). The aim of this study was the investigation on bird feeding habit of females of both the phlebotomine species by the enzyme-linked immunosorbent assay technique, comparing the reactivity during the period from 2002 up to 2004. Of the 57 samples of Lutzomyia longipalpis and 26 of Lutzomyia almerioi that have been tested, 72% from Lutzomyia longipalpis and 96% from Lutzomyia almerioi were reactive, which justifies the feeding habit study in the region as a prevention measure and the institution of an epidemiological survey.

Bloodmeals taken by insects constitute an important parameter for clarifying aspects of the transmission of zoonoses, including leishmaniases. Immunological assays can be used to investigate the attraction of vectors to animals, which may be hosts of these parasitoses. The objective of this study was to standardize a sandwich enzyme-linked immunosorbent assay and titer samples with different time periods of digestion, in laboratory-bred Lutzomyia longipalpis fed on rats. In the light of the high sensitivity that the biotin-avidin method permits, the technique provided at least ninety repeat tests for each sample and identified recent bloodmeals taken by these insects. Bloodmeals were detectable up to 12 and 24 h after blood ingestion, and a significant difference between these titers was observed.

The aim of this study was to evaluate some immunological patterns involved in natural and acquired resistance against MHV3 using the original model of genetically modified lines of mice selected for high (HIII) and low (LIII) antibody responsiveness. As previously shown, a lower pre-existing anti-MHV antibody level was found in susceptible HIII mice as compared to resistant LIII mice. Mortality rates of the F1 (H x L) hybrids and F2 and backcross segregants reflected co-dominance of both characters and the survivors had higher preexisting anti-MHV antibody titers. The present data show that both lines had the potential to synthesize antibodies and that the resistance acquired by the susceptible HIII mice paralleled the antibody synthesis. Nevertheless, higher antibody titers were necessary to confer resistance in HIII mice than in LIII ones. When compared to uvMHV3, a single immunization with a related infectious MHV strain induced a higher antibody synthesis and led the HIII mice to resist the MHV3 challenge. A direct correlation between the antibody level and resistance to infection was always observed in HIII mice. Although mounting a Th2 response as indicated by IgG1 responses, they were also able to readily synthesize large amounts of IgG2a antibodies after immunization or during infection, reflecting a Th1 response. The transfer of anti-MHV antibodies to susceptible HIII mice was capable of conferring resistance to MHV3, providing the antibodies were present before virus infection and in large amounts. The resistance and the survival time of these animals increased with the level of antibody administered. If these direct and clear data suggest that HIII mice can acquire resistance through antibodies, the basis of the resistance of the resistant LIII mice may rely on mechanisms less dependent on antibodies.

Eight capuchin monkeys (Cebus apella) were vaccinated against rabies with an inactivated suckling mouse brain vaccine (SMBV). Three 1-ml doses of 2% brain tissue suspension were given by i.m. injection at 0, 30, and 60 days. Blood samples were collected at 0, 30, 60, 90, 150, 210, 240, 300, and 365 days and were tested by simplified fluorescence inhibition to titer-neutralizing antibodies. All of the animals developed neutralizing antibodies with titers >0.5 IU/ml after vaccination, but the immune response persisted for only 122.3 +/- 32.6 days. The SMBV was able to induce immune response in the capuchin monkeys, but protection was short-lived.

The human anti-rabies pre-exposure treatment currently used in Brazil, employing a 1-ml dose of suckling mouse brain vaccine (SMBV) administered on days 0, 2, 4 and 28, was compared to an alternative treatment with two 1 ml-doses on day 0, and one 1 ml-dose injected on days 7 and 21. The latter induced higher virus-neutralizing antibody (VNA) titers on day 21. Both Brazilian rabies vaccines produced with PV or CVS rabies virus strains were tested. Two additional volunteer vaccine groups, receiving the pre-exposure and the abbreviated post-exposure schedules recommended by the WHO using cell-culture vaccine (CCV) produced with PM rabies virus strain, were included as reference. The VNA were measured against both PV and CVS strains on days 21, 42 and 180 by the cell-culture neutralization microtest. The PV-SMBV elicited higher seroconversion rates and VNA by day 21 than the CVS-SMBV. Both, however, failed to induce a long-term immunity, since VNA titers were < 0.5 IU/ml on day 180, regardless of the schedule used. Cell-culture vaccine always elicited very high VNA on all days of collection. When serum samples from people receiving mouse brain tissue were titrated against the PV and CVS strains, the VNA obtained were similar, regardless of the vaccinal strain and the virus used in the neutralization test. These results contrast with those obtained with sera from people receiving PM-CCV, whose VNA were significantly higher when tested against the CVS strain.

Both fixed and street rabies virus when cultivated in McCoy cells caused cytopathic changes 24 to 72 h after infection, depending on the multiplicity of infection. The cytopathic effect (CPE) was easily recognizable and resembles that induced by other members of the Rhabdovirus group, such as vesicular stomatitis virus, in several cell cultures. Higher titers of the Pasteur strain (PV) of fixed rabies virus were found in supernatants of McCoy cells when compared to those in VERO cells. The virus titer increased with the number of passages attaining a high titer after three passages. Rabies antigens were detected by direct immunofluorescence labeling in most McCoy cells of the infected culture, and specific antibodies neutralized the virus growth and CPE. There was also inhibition by treatment of the cells with human interferon (HuIFN) -alpha or -gamma, but not by murine interferon (MuIFN) -alpha, -beta or -gamma. Rabies-infected McCoy cell cultures may provide a useful assay system, based on the induction of CPE, the high virus production and the sensitivity to IFN.

After infection with the Pasteur strain of fixed rabies virus, the onset of disease, mortality, interferon (IFN) synthesis and interaction of the virus with macrophages were investigated in high (HI) and low (LI) antibody responder lines of mice. The HI mice were shown to be more resistant than the LI mice, and resistance was age-dependent, since mice from both mouse lines were fully susceptible up to 2 weeks of age. IFN synthesis studies of the serum indicated that, after rabies infection, HI mice produced a slightly higher amount of IFN, which was determined to be predominantly IFN-gamma. In the brains of LI mice, only IFN-alpha/beta was found, in contrast to the mixture of IFN-alpha/beta and IFN-gamma observed in the brains of HI mice. Although macrophages from the two mouse lines expressed the same degree of extrinsic activity, their intrinsic activities were quite different; the LI mice showed a greater ability to uptake and process the virus or ingest C3 (IgM) sheep red blood cells. The present findings attribute the higher antibody response and IFN-gamma synthesis observed in HI mice during rabies infection to slower processing of the rabies antigen in their macrophages, thus conferring upon them a greater ability to present it to the immune system, leading to a higher degree of resistance to rabies infection.

In this paper we describe a methodology for the preparation of the Pasteur strain of fixed rabies virus in BHK-21 clone 13 cells and also its use for the production of antisera in horses. The methodology showed here is simple, rapid, facilitates the attainment of high protective titers, and the antisera produced are of high quality.