Molecular characterization and confocal laser scanning microscopic study of Pygidiopsis macrostomum (Trematoda: Heterophyidae) parasites of guppies Poecilia vivipara.

Pygidiopsis macrostomum and Ascocotyle (Phagicola) pindoramensis (Digenea: Heterophyidae) parasitize guppies as intermediate hosts and, respectively, fish-eating mammals or birds as definitive hosts. Heterophyids have zoonotic potential, and molecular studies associated with morphological and ecological aspects have helped to clarify their taxonomy and phylogeny. Poecilia vivipara naturally parasitized by metacercariae of both species (100% prevalence) exhibit no external signs of parasitism. In this work, four new sequences of P. macrostomum (18S rDNA, 28S rDNA and ITS2 rDNA) and one new sequence of A. (P.) pindoramensis (mtDNA cox-1) are presented. Phylogeny reconstructions linked P. macrostomum to other heterophyids, but the separation of the Heterophyidae and Opisthorchiidae remains unclear. Additionally, we used indirect immunocytochemistry and the phalloidin-fluorescence techniques allied with confocal laser scanning microscopy to describe muscular and neuronal structures of P. macrostomum. A complex arrangement of muscular fibres is associated with the tegument, suckers, gut and reproductive system. Radial fibres around the ventral sucker are thick, branched and extend to the body wall. High-resolution confocal imaging revealed a typical digenean muscular arrangement and important heterophyid morphological traits. These data will support future control measures to reduce the parasitism in guppies reared in fish farming systems, especially for aquarium and experimental purposes.

Rickettsia felis in the Americas.

The authors describe their work in the Americas in Rickettsia felis cases in humans and the presence of Rickettsia felis in vectors.

Revisiting brazilian spotted fever focus of Caratinga, Minas Gerais State, Brazil.

We revisited a Brazilian spotted fever focal area in Minas Gerais state, Brazil, in 2002, and performed a serologic survey in dogs and cats. The results of this survey are compared with the survey made 10 years before. The possible efficacy of vector control measures adopted in this area and the role of dogs and horses as sentinels of infection by Rickettsia are discussed.

Old and new human rickettsiosis in Minas Gerais state, Brazil.

The authors describe cases of Brazilian spotted fever (BSF) diagnosed in Minas Gerais state, Brazil, by the Minas Gerais Public Health Laboratory, Ezequiel Dias Foundation from 1995 to 2004. In addition they present three cases of human Rickettsia felis rickettsiosis from Minas Gerais diagnosed in France in 1999, and the first two suspected cases of human monocytic ehrlichiosis (HME) diagnosed in Ezequiel Dias Foundation in 2001. In both cases a differential diagnose was made with BSF.

Exoantigens of an attenuated strain of Babesia bovis used as a vaccine against bovine babesiosis.

Bovine babesiosis caused by Babesia bovis remains a significant constraint to beef and milk cattle production throughout the world. Exoantigens released by the parasites in culture supernatants are a potential source of antigen to induce protective immunity. An attenuated strain of B. bovis from Brazil, catalogued as BbUFV1, was maintained in vitro by the MASP method, and exoantigen-containing supernatant fluids were collected daily to form a pool representing a 72-h culture cycle for preparation of the vaccine. Exoantigen concentration was estimated using a two-site EIA. Three groups of susceptible non-splenectomised male Bos taurus cattle, 14 months old, were used. Group A (vaccinated) received two subcutaneous immunizations with a 21-day interval of B. bovis supernatant, content 6500 EIA units of exoantigens plus 1.5 mg saponin, and Group B (adjuvant control) received two injections of adjuvant alone. Four weeks after the second immunization, Groups A, B and C (control) were challenged intravenously with 10(8) virulent parasites of a heterologous B. bovis strain. The results showed that exoantigens present in in vitro cultures can induce a high degree of protection against virulent heterologous challenge exposure. In Group A only one animal showed discrete parasitaemia; all developed a fever and slight decreases in PCV, with a rapid return to normal values. One animal of Group B died; the survivors showed fever, anaemia and parasitaemia. All animals of Group C died between 7 and 13 days after challenge. Vaccination elicited both humoral and cell-mediated immune responses. In Group A, after the challenge, the maximum antibody titer was 12,800. When vaccinated, cattle were tested at the moment of challenge for B. bovis-specific cell-mediated immunity by the monocytemigration inhibition test. A mean inhibition index of 60 +/- 0.33 was observed. Preliminary Western blot analysis of the immunogen revealed at least four proteins of molecular weight ranging between 30 and 160 kDa.

Antigenic profile of a pure isolate of Anaplasma marginale of Brazilian origin, using a western blot technique.

Anaplasma marginale initial bodies of the Brazilian isolate AUFV1 were purified from infected erythrocytes using a combination of lysis, ultrasonic disruption and differential centrifugation. Initial bodies were solubilised with a buffer containing protease inhibitors and non-ionic detergents. Immunochemical analysis by the Western blot technique revealed at least five proteins with apparent molecular weights (MW) of 105, 100, 97, 87 and 38 kDa when homologous sera were used as primary antibodies. Sera from cattle from Mato Grosso do Sul State in Brazil revealed five proteins of 105, 100, 87, 38 and 25 kDa; other heterologous sera obtained from Illinois, USA, bound to four antigens with MW of 105, 100, 87 and 38 kDa, the latter being stronger and broader than the others. No bands were observed in the non-infected erythrocyte control when the different A. marginale sera or antibodies against Babesia bovis and Babesia bigemina were used. Antibodies from cattle infected with the A. marginale isolates bound to proteins of 105, 100, 87 and 38 kDa, indicating that there are at least four peptides common to the isolates. The major surface proteins, designated MSP-1, MSP-2 and MSP-3, are also present in the Brazilian isolate AUFV1 and the sample from the National Research Centre of Beef Cattle in Mato Grosso do Sul State. One practical consideration of this study is the possibility of cross-protection between different Anaplasma isolates including some from Brazil.

Babesia bovis: infectivity of an attenuated strain of Brazilian origin for the tick vector, Boophilus microplus.

In order to verify the infectivity and transovarian transmission of one strain of Babesia bovis of Brazilian origin, attenuated by repeated passages in splenectomized calves to the biological tick vector, Boophilus microplus, an inoculation of that strain was performed in hemoparasite-free calves infested with hemoparasite-free ticks. Subsequently, engorged female ticks were collected to examine hemolymph and progeny for presence of developing stages of Babesia bovis. Using an optical microscope, intrahemocytic merozoites were observed in female hemolymph on the second and fourth days, respectively, after ticks dropped from calves. By histological observation of F1 larvae, after more than 24 h of adherence on calves, sporokinetes and small merozoites were present in the samples of hemolymph. These results confirmed the maintenance of infectivity by the vaccine strain attenuated after 26 passages for tick Boophilus microplus.