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)

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)

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.

Demodicose bovina no estado da Paraíba

Cases of bovine demodicosis caused by Demodex bovis were reported in a Sindhi herd from December 1989 to January 1992. Both localized and generalized forms were diagnosed. This is the first report of the generalized form in Brazil. In the first two years, demodicosis was diagnosed only in cattle < 2 years old, whereas animals of all ages were positive in the last two years. Prevalence varied from 20.4% (11/54) to 53.1% (26/49) and 13.2% (12/91) to 14.8% (9/61) for cattle < 2 years old and > 2 years old, respectively. Clinical signs varied from a few small nodules to a thickened skin with soft large nodules in the localized and generalized forms, respectively. Main microscopic features of the nodules in the generalized form consisted of acanthosis with hyperqueratosis, chronic sebaceous adenitis, subcutaneous muscular necrosis, focal cellular degeneration of the epidermis basal layer and presence of large number of mites inside the lumen of dilated hair follicles. In addition, a chronic perifoliculitis was observed, characterized by lymphoplasmocytic infiltrate which also contained macrophages and neutrophils. It is suggested that poor nutrition and stress due to prolonged drought probably contributed to the increase of susceptibility of the herd to mite infestation.

Effect of environmental mycobacteria (Mycobacterium avium) on immunity induced by a DNA vaccine (DNAhsp65) against tuberculosis

The efficacy of BCG vaccine (attenuated Mycobacterium bovis) against pulmonary tuberculosis varies enormously among different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment. Studies have revealed that most protective antigens expressed by the antituberculous vaccine are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates a cross-reactive immune response that interferes with BCG efficacy. In this study we investigated the effect of a prior exposure to heat-killed M. avium on the immune response and the protective efficacy induced by a genetic vaccine pVAXhsp65 (hsp65 gene from M. leprae inserted in pVAX vector) against experimental tuberculosis. To evaluate the effect on the immune response, female BALB/c mice were initially injected with distinct doses (0.08×106, 4×106, and 200×10 6) of heat-killed M. avium by subcutaneous route. Three weeks later, the animals were immunized with 3 doses of DNAhsp65 by intramuscular route (100μg/15 days apart). Control groups received only M. avium, vaccine (pVAXhsp65), vector (pVAX) or saline solution. Cytokine production and antibody levels were determined by ELISA. To evaluate the effect on the protective efficacy, animals were initially sensitized with 200×106 heat-killed CFU of M. avium by subcutaneous route and then immunized with 3 doses of pVAXhsp65 (100μg/15 days apart) by intramuscular route. Control groups were injected with saline, pVAX (4 doses), pVAXhsp65 (4 doses), M. avium or M. avium plus pVAX (3 doses). Fifteen days after last DNA dose, the animals were infected with 1×104 viable CFU of H37Rv M. tuberculosis by intratracheal route. Thirty days after challenge, the animals were sacrificed and the bacterial burden was determined by counting the number of CFU in the lungs. Lung histological sections were also analyzed. Splenic cells from primed animals produced more IL-5 but less IFN-gamma than non-primed ones. Also, prior contact with M. avium determined higher production of IgG1 and IgG2a anti-hsp65 antibodies in comparison to control groups. However, this higher immune response did not decrease the bacterial burden in the lungs. In addition, prior sensitization with M. avium decreased the parenchyma preservation observed in the group immunized only with pVaxhsp65. These results indicate that environmental mycobacteria can interfere with immunity and protective efficacy induced by DNAhsp65.

Soroepidemiologia da babesiose em rebanho de bovinos da raça Curraleiro

Bovine babesiosis is a blood parasitic disease. In Brazil it is caused by B. bovis and B. bigemina protozoa, both of which reveal the Boophilus microplus tick as the only biological vector. Animal samples were collected at Experimental Study Farm of Curraleiro Cattle (ESFC) in 2001 (n=117) and 2003 (n=113). The detection of antibodies against B. bovis and B. bigemina was carried out by ELISA-indirect method. This research was aimed at studing seroepidemiological aspects of bovine babesiosis in a Curraleiro herd, as well as obtain information about babesiosis stability in this population and relate the results with available climactic and management information. The occurrence rate of positive animals was 92.3% for B. bovis and 83.8% for B. bigemina in 2001; in 2003 it was 92.9% and 66.4%, respectively. There was a significant difference between seropositive frequency and age in 2003; such a frequency decreased with ageing. It was possible to conclude that despite environmental conditions and chemical controls against endo and ectoparasites, these animals were exposed to Babesia spp and they found themselves in a situation of enzootic stability for babesiosis.

Neurocysticercosis: The enigmatic disease

Neurocysticercosis (NCC) is an infection of the central nervous system (CNS) caused by the metacestode larval form of the parasite Taenia sp. Many factors can contribute to the endemic nature of cysticercosis. The inflammatory process that occurs in the tissue surrounding the parasite and/or distal from it can result from several associated mechanisms and may be disproportionate with the number of cysts. This discrepancy may lead to difficulty with the proper diagnosis in people from low endemic regions or regions that lack laboratory resources. In the CNS, the cysticerci have two basic forms, isolated cysts (Cysticercus cellulosae = CC) and racemose cysts (Cysticercus racemosus = CR), and may be meningeal, parenchymal, or ventricular or have a mixed location. The clinical manifestations are based on two fundamental syndromes that may occur in isolation or be associated: epilepsy and intracranial hypertension. They may be asymptomatic, symptomatic or fatal; have an acute, sub-acute or chronic picture; or may be in remission or exacerbated. The cerebrospinal fluid (CSF) may be normal, even in patients with viable cysticerci, until the patients begin to exhibit the classical syndrome of NCC in the CSF, or show changes in one or more routine analysed parameters. Computed tomography (CT) and magnetic resonance imaging (MRI) have allowed non-invasive diagnoses, but can lead to false negatives. Treatment is a highly controversial issue and is characterised by individualised therapy sessions. Two drugs are commonly used, praziquantel (PZQ) and albendazole (ABZ). The choice of anti-inflammatory drugs includes steroids and dextrochlorpheniramine (DCP). Hydrocephalus is a common secondary effect of NCC. Surgical cases of hydrocephalus must be submitted to ventricle-peritoneal shunt (VPS) immediately before cysticidal treatment, and surgical extirpation of the cyst may lead to an absence of the surrounding inflammatory process. The progression of NCC may be simple or complicated, have remission with or without treatment and may exhibit symptoms that can disappear for long periods of time or persist until death. Unknown, neglected and controversial aspects of NCC, such as the impaired fourth ventricle syndrome, the presence of chronic brain oedema and psychic complaints, in addition to the lack of detectable glucose in the CSF and re-infection are discussed. © 2011 Bentham Science Publishers.