Evaluation of an ELISA for detection of antibodies to Babesia bigemina in cattle and it's application in an epidemiological survey in Brazil

Um ensaio de imunoadsorção enzimática (ELISA) baseado em antígeno bruto foi avaliado na detecção de anticorpos contra Babesia bigemina. A sensibilidade e a especificidade do teste foram de 98,0% e 99,0%, respectivamente. Concordando com a alta especificidade do teste, não foram verificadas reações cruzadas com soros de bezerros inoculados três vezes com 10(7) merozoítos de Babesia bovis. Com relação à comparação do ELISA com a imunofluorescência indireta (IFAT) na detecção de anticorpos contra B. bigemina em bezerros experimentalmente infectados com cinco isolados brasileiros geograficamente distintos deste hemoparasito, o IFAT foi capaz de detectar anticorpos um dia antes do ELISA na maioria dos soros dos animais. Houve uma boa concordância entre os resultados encontrados no ELISA e no IFAT com soros de bovinos de região de estabilidade enzoótica (k=0.61). No entanto, não houve concordância entre os testes sorológicos com soros de animais de área de instabilidade enzoótica (k=0.33). O ELISA foi empregado em um inquérito epidemiológico com 1.367 soros de quatro municípios do Pantanal de Mato Grosso do Sul e caracterizou esta região como uma área de estabilidade enzoótica, uma vez que as prevalências variaram de 87,7 a 98,9%. Dessa forma, este ELISA, que apresentou alta sensibilidade, especificidade e desempenho similar ao IFAT, pode ser utilizado no diagnóstico sorológico de B. bigemina.

Isolation of Mycobacterium spp. in milk from cows suspected or positive to tuberculosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bacteriocin-like substances production by Streptococcus-salivarius against oral bacteria

Samples of tongue and bucal mucosa surfaces were obtained from six healthy subjects with the purpose of isolating S. salivarius. It was verified that 47 out of 48 S. salivarius strains produced bacteriocin-like substances against at least one of the indicator species: Actinomyces viscosus, Rothia dentocariosa, Streptococcus pyogenes, Staphylococcus aureus, Streptococcus mutans and Streptococcus sanguis. The method employed to test for bacteriocin production was that of deferred antagonism. The results showed that there was a high antagonism against R. dentocariosa, S. pyogenes and A. viscosus; extremely low against S. mutans and S. sanguis and no inhibition for S. aureus.

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to pyrazinamide

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular characterization of Cryptosporidium spp. in dairy calves from the state of So Paulo, Brazil

Cryptosporidiosis is a common protozoan disease observed in a wide range of vertebrate hosts, including ruminants. Cattle can be a potential reservoir of Cryptosporidium spp., leading to environmental contamination with oocysts of zoonotic species. The molecular characterization of Cryptosporidium spp. isolated from cattle from the state of So Paulo, Brazil, was accomplished using nested polymerase chain reaction for amplification of fragments of the 18S rRNA gene and the glycoprotein GP60 gene, following sequencing of amplified fragments. Positivity for Cryptosporidium was found in 10.7% (21/196) of the samples. Four species of Cryptosporidium were identified: C. andersoni, C. bovis, C. parvum subtype IIaA15G2R1, and C. ryanae. To the best of our knowledge, this is the first report of infection by C. ryanae and C. parvum IIaA15G2R1 in cattle from Brazil.

Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus)

Anaplasmataceae organisms comprise a group of obligate intracellular gram-negative, tick-borne bacteria that can infect both animals and humans. In the present work we investigate the presence of Ehrlichia, Anaplasrna, and Neorickettsia species in blood samples from Brazilian marsh deer (Blastocerus dichotomus), using both molecular and serologic techniques. Blood was collected from 143 deer captured along floodplains of the Parana River, near the Porto Primavera hydroelectric power plant. Before and after flooding, marsh deer were captured for a wide range research program under the financial support of São Paulo State Energy Company (CESP), between 1998 and 2001. Samples were divided into four groups according to time and location of capture and named MS01 (n = 99), MS02 (n = 18) (Mato Grosso do Sul, before and after flooding, respectively), PX (n = 9; Peixe River, after flooding), and AGUA (n = 17; Aguapei River, after flooding). The seroprevalences for Ehrlichia chaffeensis and Anaplasma phagocytophilum were 76.76% and 20.2% in MS01, 88.88% and 5.55% in MS02, 88.88% and 22.22% in PX, and 94.12% and 5.88% in AGUA, respectively. Sixty-one animals (42.65% of the total population) were PCR-positive for E. chaffeensis PCR (100.0% identity based on 16S rRNA, dsb, and groESL genes). Seventy deer (48.95% of the total population) were PCR-positive for Anaplasma spp. (99.0% of identity with A. platys, and in the same clade as A. phagocytophilum, A. bovis, and A. platys based on 16S rRNA phylogenetic analysis). Our results demonstrate that Brazilian marsh deer are exposed to E. chaffeensis and Anaplasma spp. and may act as reservoirs for these rickettsial agents, playing a role in disease transmission to humans and other animals. (c) 2012 Elsevier Ltd. All rights reserved.

Different inflammatory stimuli in the footpad of mice influence the kinetics of resident peritoneal cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anaerobic microorganisms in root canals of human teeth with chronic apical periodontitis detected by indirect immunofluorescence

Aiming to assess the presence of selected anaerobic microorganisms in root canals of human teeth with chronic apical periodontitis, 25 central and lateral upper incisors presenting with radiographic evidence of chronic apical periodontitis were studied. The pulp chamber was opened under aseptic conditions and samples of the root canal content were collected with sterile absorbent paper points, which were placed and dispersed in test tubes containing reduced transport medium (RTF). Aliquots were dried on glass slides and stained by indirect immunofluorcscencc for detection of Actinomyces viscosus, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia. The results showed a positive indirect immunofluorescence reaction in 24 of the 25 samples. Fourteen were positive for the specie Actinomyces viscosus, 12 for Prevotella intermedia, 10 for Fusobacterium nucleatum and 4 for Porphyromonas gingivalis. A semiquantitative assay was easily implemented for assessment of degree of infection by the organisms in individual cases. © Munksgaard, 1996.

Estreptomicina aumenta a aderência em células epiteliais de Bacteroides melaninogenicus associado às lesões peridentárias da cara inchada dos bovinos

Cara inchada of cattle (CI) is a periodontitis which occurs in calves in several regions of Brazil. Its progressive lesions, caused mainly by Bacteroides melaninogenicus and Actinomyces pyogenes, could lead to loss of teeth, malnutrition and possibly death of affected animals. For further analysis of the pathogenesis of this disease the influence of streptomycin on the adherence of the causative bacteria to oral epithelial cells of cattle was determined. Production of streptomycin by soil bacteria (Actinomycetes) in Brazil had been suggested by several authors. In our studies, streptomycin in subinibitory concentrations increased tenfold the rate of adherence with B. melaninogenicus (from 0.4 to 4.0 bacteria per bovine epithelial cell). On the contrary, streptomycin did not influence significantly the adherence of A. pyogenes nor streptococci of the viridans group. Thus, streptomycin could be a contributing factor to the development of the periodontal infections with B. melaninogenicus.

Adherence and experimental infection of bacteria associated with periodontal infections of young cattle in Brazil (Cara inchada)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

An enzyme-linked immunosorbent assay (elisa) for the detection of antibodies against babesia boris in cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Murine alpha-2-macroglobulin increase during inflammatory responses and tumor growth

Objective and Design: To determine the alpha-2-macroglobulin (alpha2M) levels in mice during acute and chronic inflammatory responses. Materials and Methods: Inflammation was induced by one of the following stimuli: carrageenin, zymosan, lipopolysacharide, thioglycollate, bacilli Calmette Guerin, PPD (in pre-immunized and non-immunized animals) and tumor cells. The concentration of alpha2M was determined in plasma or peritoneal liquid by electroimmunoassay. Results: In all the treatments employed, the plasma levels of alpha2M were higher than in untreated animals. This increase varied from 9%, 24 h after injection up a maximum of 66% 72 h post-injection. When compared to animals injected only with saline, the increases were significant 48 h after treatment with either zymosan or LPS, and 72 h after treatment with either thioglycollate or carrageenin. Treatment with BCG triggers an increase in alpha2M levels after 24 h (18.60%) and 48 h (27.90%). Immunized mice presented higher levels of this protein than non-immunized animals after challenge with PPD. The growth of Ehrlich tumor cells in the peritoneal cavity was directly correlated with the local levels of alpha2M which increased 3.5 fold, 10 days after injection. Conclusions: These results strongly indicate that in mice, the concentration of alpha2M can increase during acute and chronic inflammatory reactions with kinetics dependent on the particular kind of inflammatory agent.

Prevalência de reagentes ao teste de tuberculinização no rebanho bovino leiteiro da região de São Carlos - SP

Tuberculosis (TB), an infectious disease readily transmitted between animals and humans, is an great threat to public and animal health a like and has been declared an international emergency. According to the WHO, animal TB is a source of much concern, especially in the poorer countries where there is little awareness of the problem. Thus, improvements in veterinary health are essential, with regard to infection by Mycobacterium bovis, particularly in areas where the public is at risk. The prevalence of infected animals in Brazil is about 1%, whereas, in the dairy cattle region of São Carlos, the average proportion of positive tests was found to be 3. 1% from 1987 to 1996. The current research was carried out to investigate the prevalence, in the region, of dairy cattle reacting positively to the tuberculin test, from 1997 to the first half of 2001. The results were obtained by searching through the data collected by the Federal Inspection Authority, and they indicated an average prevalence of 1.3% over the period, similar to the official national average. It was concluded that bovine TB continues to be present in the region, posing a potential threat of infection to the human population.

Tuberculosis survey of free-ranging marsh deer (Blastocerus dichotomus) in Brazil

Esophageal-pharyngeal fluids from 53 free-ranging marsh deer (Blastocerus dichotomus) captured for a research program in the state of Mato Grosso do Sul, Brazil, were assayed for tuberculosis. Total DNA was extracted, amplified by polymerase chain reaction using specific primers for Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. microti, and M. africanum), and observed by agarose gel electrophoresis stained with ethidium bromide. All samples were negative. This, along with necropsy and histopathology data, suggests that these animals are not shedding and probably do not have active disease.