Adivaldo Henrique da Fonseca

Effect of vaccination with a recombinant Bm86 antigen preparation on natural infestations of Boophilus microplus in grazing dairy and beef pure and cross-bred cattle in Brazil

Current methods for the control of the cattle tick Boophilus microplus infestations are not effective and the parasite remains a serious problem for the cattle industry in tropical and sub-tropical areas. Recent advances have introduced the possibility for the immunological control of the parasite through the use of recombinant vaccines. Recently, it was shown that the recombinant vaccine Gavac (Heber Biotec S.A.) is able to control B. microplus populations in artificially infected grazing dairy cattle in Cuba. To assay the effect of the vaccine on a different B. microplus strain and under different ecological conditions, we conducted a trial in Brazil on grazing dairy and beef pure and cross-bred cattle under natural infestation conditions. A farm in the northeast of the state of Sao Paulo was selected and two groups of animals per breed were included in the experiment and were maintained grazing on separate but similar pastures. For each breed, one group was vaccinated with the vaccine Gavac and the second group was not vaccinated and was employed as a control. In vaccinated cattle, during 36 weeks of experiment, the average infestation rate was maintained below 78 ticks per animal while average infestation peaks (mean +/- S.E.) of 144 +/- 44 ticks per animal (for dairy cross-bred cattle) and 195 +/- 42 ticks per animal (for beef cross-bred cattle) were recorded in the control groups. Tick infestation rates showed statistical significant differences (p = 0.04) between both experimental groups throughout the experiment. These results clearly showed, as in the Cuban study, that the vaccine controlled tick numbers in successive generations in the field.

Coexistence of antibodies to tick-borne agents of babesiosis and Lyme borreliosis in patients from Cotia county, State of São Paulo, Brazil

This paper reports a case of coinfection caused by pathogens of Lyme disease and babesiosis in brothers. This was the first case of borreliosis in Brazil, acquired in Cotia County, State of S o Paulo, Brazil. Both children had tick bite history, presented erythema migrans, fever, arthralgia, mialgia, and developed positive serology (ELISA and Western-blotting) directed to Borrelia burgdorferi G 39/40 and Babesia bovis antigens, mainly of IgM class antibodies, suggestive of acute disease. Also, high frequencies of antibodies to B. bovis was observed in a group of 59 Brazilian patients with Lyme borreliosis (25.4%), when compared with that obtained in a normal control group (10.2%) (chi-square = 5.6; p < 0.05). Interestingly, both children presented the highest titers for IgM antibodies directed to both infective diseases, among all patients with Lyme borreliosis.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.

Effects of homeopathy in mice experimentally infected with Trypanosoma cruzi.

AIM The aim of this study was to evaluate the action of homeopathic treatment on mice experimentally infected with Trypanosoma cruzi. METHODS Eighty adult male C57BL/6 inbred mice were randomly allocated to five groups treated with biotherapy (nosode) of T. cruzi 12dH (12x) pre- and post-infection; Phosphorus 12dH post-infection; infected control treated with control solution and uninfected control. The biotherapy was prepared by the Costa method from the blood of mice experimentally infected with the Y strain of T. cruzi. Phosphorus was used because of its clinical and reportorial similarity to Chagas disease. T. cruzi (10(4)) sanguineous forms were inoculated intraperitoneally per animal. Parasitaemia was monitored, leukocyte and serological responses were evaluated at 0, 7, 14 and 42 days after infection. The prepatent and patent periods of parasitaemia, maximum of parasitaemia, day of maximum parasitaemia and mortality rates were compared between groups. RESULTS A significantly shorter period of patent parasitaemia was observed in the group treated with the biotherapy before infection (p<0.05) than in the other groups. This group also had the lowest parasitaemias values at 9, 13, 15 (p<0.05), 17 (p<0.05), 22, 24 and 28 days, a lower rate of mortality and a significant increase of lymphocytes compared to the infected control group. The Phosphorus group had the longest period of patent parasitaemia, higher maximum parasitaemia, and a significant reduction of lymphocyte numbers, but no mortality. The infected control group had the highest mortality rate (not statistically significant), and the highest IgG titres at 42 days post-infection (p<0.05). CONCLUSIONS The results suggest that pre-treatment with biotherapy modulates host immune response to T. cruzi, mainly during the acute phase of the infection. Phosphorus shows an action on the pathogenicity by T. cruzi infection. Homeopathic treatment of T. cruzi infection should be further investigated.

Frequency of antibodies to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burdgorferi in cattle from the northeastern region of the state of Pará, Brazil

Babesiosis, anaplasmosis, and trypanosomosis are relevant diseases, potentially causing morbidity in cattle, leading to economic losses. Borreliosis is import as a potential zoonosis. The objective of this study was to determine, by indirect enzyme-linked immunosorbent assay (ELISA), the frequency of seropositive cattle to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burgdorferi in cattle from the Northeastern region of Pará, Brazil. Sera samples from 246 female adult cattle from municipalities of Castanhal and São Miguel do Guamá were used. Crude antigens ELISAs were used to detect antibodies to all agents, except to A. marginale, to which an indirect ELISA with recombinant major surface 1a protein (MSP1a) antigen was used. Overall frequencies of seropositive animals were: B. bigemina--99.2%; B. bovis--98.8%; A. marginale--68.3%; T. vivax--93.1% and B. burgdorferi--54.9%. The frequencies of seropositive cattle to B. bovis and B. bigemina suggest a high rate of transmission of these organisms by tick in the studied region, which can be classified as enzootically stable to these hemoprotozoans. The low frequency of seropositive cattle to A. marginale may be attributed to a lower sensitivity of the recombinant antigen ELISA utilized or a distinct rate of inoculation of this rickettsia by ticks, as compared with Babesia sp. transmission. The high frequency of seropositive cattle to T. vivax indicates that this hemoprotozoan is prevalent in herds from the Northeastern region of Pará. The rate of animal that showed homologues antibodies to B. burgdorferi indicates the presence of the tickborne spirochaetal agent in the cattle population in the studied region.

Epidemiologia das helmintoses pulmonares e gastrintestinais de bezerros em região de baixada do Estado do Rio de Janeiro

The epidemiology of pulmonary and gastrintestinal helmintoses of calves has been studied in cross-bred Zebu-Friesian calves, 6 to 9 month old, with natural infections. The calves were maintained in permanent pasturing in the lowland, with climate Aw, in the state of Rio de Janeiro. The experiments had the duration of 24 months. At the end the animals were bearer of natural infection of several helminth species. The research was based on fecal analysis and necropsies of at least four calves every 28 days. The animals maintained in permanent pasturing showed tendency to have larger helminth populations in autumn and spring. The graphical representation of an ellipse, obtained through the relationship of total rainfall and average minimum temperature per month, was effective in demonstrating the potential for clinical parasitism in autumn and spring.

Borrelioses, agentes e vetores

Borrelioses are infectous diseases caused by spirochaetes of the genus Borrelia. They are born mainly through ticks at animals and/or human beings. In this review are shown and discussed five groups of diseases determined by borrelia, general characteristics of the spirochaetes, aspects related to transmission by arthropods, biological and pathological aspects of the diseases in domestic and wild animals, Lyme disease as an important zoonosis, the association of borrelia with other hematozoa agents, the diagnostic methods and the comparative epidemiology with data obtained from Brazil and other countries. The borrelioses have pathological, clinical and epidemiological characteristics which vary according to physiographic regions due to the existence of different species, genospecies and strains of borrelia, of arthropod vectors, vector-agent relationship and of different ecocystems.

Experimental infection with Rickettsia rickettsii in an Amblyomma dubitatum tick colony, naturally infected by Rickettsia bellii

Amblyomma dubitatum engorged females, naturally infected by Rickettsia bellii, were used to establish a laboratory colony. Larvae, nymphs, and adults were exposed to two strains of Rickettsia rickettsii by feeding on needle-inoculated guinea pigs, and thereafter reared on uninfected guinea pigs. After acquisition feeding, engorged larvae and nymphs molted to nymphs and adults, respectively, which were shown to be infected (confirming transstadial perpetuation), and were able to transmit both strains of R. rickettsii to uninfected animals, as demonstrated by clinical, serological, and molecular analyses. However, the larval, nymphal, and adult stages of A. dubitatum showed to be only partially susceptible to R. rickettsii infection, since in all cases, only part of the ticks became infected by this agent, after being exposed to rickettsemic animals. While transovarial transmission of R. rickettsii was inefficient in the A. dubitatum engorged females of the present study, 100% of these females passed R. bellii transovarially. Because it has been reported that a primary infection by a Rickettsia species would preclude transovarial transmission of a second Rickettsia species, it is likely that the ineffectiveness of A. dubitatum to perpetuate R. rickettsii by transovarial transmission was related to its primary infection by R. bellii; however, it could also be related to unknown factors inherent to A. dubitatum. The relevance of A. dubitatum as a natural vector of R. rickettsii to humans or animals is discussed.

Desenvolvimento de Rhodnius pictipes Stal, 1872 alimentado atraves de membrana de silicone e em camundongos (Hemiptera, Reduviidae, Triatominae)

Rhodnius pictipes (Hemiptera, Reduviidae) from Serra Norte, State of Para, Brazil, acclimatized in an insectary at the Laboratorio Nacional e Internacional de Referencia em Taxonomia de Triatomineos, Departamento de Entomologia, Instituto Oswaldo Cruz, were fed through a silicone membrane. In order to know the viability and the efficiency of this membrane compared with insects fed on mice, the number of bloodmeals taken, period of development of the five nymphal instars, longevity of adults, average amount of blood intake in each meal and percent of mortality were observed. A total of 310 insects, were used, comprising 50 nymphs of each instar, as well as 30 male and 30 female adults. Insects fed artificially had reduced minimal and maximal periods of development than the group fed on mice. The largest relative increase of body weight was observed in the 2nd instar followed by the 1st, and the amount of blood ingested increased during the development, to the 5th instar for both groups. There were no significant differences between the groups fed artificially and in vivo according to Tukeys test for p > 0.05. The percent of mortality in the 1st instar was 18% for artificially fed and 16% for the group fed on mice; these percentages decreased as insects developed until the 4th instar, without mortality, returning to increase in the 5th instar. R. pictipes was shown to be easily adaptable to artificial feeding, and could be considered as an important and viable experimental model.

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale.

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.