In vivo and in vitro expression of five genes involved in Corynebacterium pseudotuberculosis virulence.

Caseous lymphadenitis (LC) is a chronic contagious disease caused by Corynebacterium pseudotuberculosis, which mainly affects goats and sheep. Vaccination is an effective but not yet well-established method, partly due to a lack of knowledge surrounding the most effective immunoprotective components. The present study aimed to quantify and compare the in vivo expression of genes pld (phospholipase D), cpp (CP40), nanH (neuraminidase H), sodC (superoxide dismutase C) and spaC (adhesin) using qRT-PCR, with the respective expression in vitro. Caseous material of abscesses removed from five animals was cultured, with colonies suggestive of C. pseudotuberculosis identified. RNA extraction was performed on these samples, as well as on the respective pellets derived from liquid cultures brain heart infusion. After evaluating RNA integrity, complementary DNA was synthesized, followed by the relative quantification each of the genes of interest. Mean mRNA expression of the five genes found in abscesses and in cultures differed significantly, with respective values of: nanH 811.50 ± 198.27 and 359.35 ± 75.45 (p = 0.009); cpp 856.31 ± 385.11 and 154.54 ± 94.34 (p = 0.0039); plD 922.70 ± 450.73 and 212.41 ± 153.10 (p = 0.016); sodC 1,293.53 ± 564.75 and 223.63 ± 145.58 (p = 0.016); spaC 1,157.10 ± 525.13 and 214.26 ± 125.70 (p = 0,016). Expression was observed to be 6-8 times higher in abscesses than in cultures, Indicative that is a genetic expression of the in vitro bacterium exists, yet in vivo has a greater magnitude corroborating to one of these virulence factors in the pathogenesis of LC.

Humoral and cellular immune responses in mice against secreted and somatic antigens from a Corynebacterium pseudotuberculosis attenuated strain: Immune response against a C. pseudotuberculosis strain.

BACKGROUND: Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CL), a chronic disease that affects goats and sheep. CL is characterized by the formation of granulomas in lymph nodes and other organs, such as the lungs and liver. Current knowledge of CL pathogenesis indicates that the induction of humoral and cellular immune responses are fundamental to disease control. The aim of this study was to evaluate the humoral and cellular immune responses in BALB/c mice inoculated with a C. pseudotuberculosis strain isolated in the state of Bahia, Brazil. RESULTS: The lymphocyte proliferation and in vitro production of IFN-γ, IL-4, IL-10, IL-12 and nitric oxide by spleen cells stimulated with secreted and somatic antigens from the studied strain were evaluated. IgG subclasses were also analyzed. Results showed a significant increase of Th1-profile cytokines after 60 days post-inoculation, as well as an important humoral response, represented by high levels of IgG2a and IgG1 against C. pseudotuberculosis. CONCLUSION: The T1 strain of C. pseudotuberculosis was shown to induce humoral and cellular immune responses in BALB/c mice, but, even at a dosage of 1x10(7) CFU, no signs of the disease were observed.

Spatio-temporal correlation networks of dengue in the state of Bahia.

BACKGROUND: Dengue is a public health problem that presents complexity in its dissemination. The physical means of spreading and the dynamics of the spread between municipalities need to be analyzed to guide effective public policies to combat this problem. METHODS: This study uses timing varying graph methods (TVG) to construct a correlation network between occurrences of reported cases of dengue between cities in the state of Bahia-Brazil. The topological network indices of all cities were correlated with dengue incidence using Spearman correlation. A randomization test was used to estimate the significance value of the correlation. RESULTS: The correlation network presented a complex behavior with a heavy-tail distribution of the network edges weight. The randomization test exhibit a significant correlation (P < 0.0001) between the degree of each municipality in the network and the incidence of dengue in each municipality. CONCLUSIONS: The hypothesis of the existence of a correlation between the occurrences of reported cases of dengue between different municipalities in the state of Bahia was validated. The significant correlation between the node degree and incidence, indicates that municipalities with high incidence are also responsible for the spread of the disease in the state. The method proposed suggests a new tool in epidemiological control strategy.

MAPK involvement in cytokine production in response to Corynebacterium pseudotuberculosis infection.

BACKGROUND: Caseous lymphadenitis (CL) is a contagious infectious disease of small ruminants caused by Corynebacterium pseudotuberculosis. Is characterized by the formation of abscesses in the lymph nodes and intestines of infected animals, induced by inflammatory cytokines. The production of cytokines, such as IL-10, TNF-α, IL-4 and IFN-γ, is regulated by mitogen-activated protein kinase (MAPK) pathway activation. The present study investigated the involvement of MAPK pathways (MAPK p38, ERK 1 and ERK 2) with respect to the production of cytokines induced by antigens secreted by C. pseudotuberculosis over a 60-day course of infection. CBA mice (n = 25) were divided into three groups and infected with 102 colony forming units (CFU) of attenuated strain T1, 102 CFU of virulent strain VD57 or sterile saline solution and euthanized after 30 or 60 days. Murine splenocytes were treated with specific inhibitors (MAPK p38 inhibitor, ERK 1/2 inhibitor or ERK 2 inhibitor) and cultured with secreted antigens obtained from pathogenic bacteria (SeT1 or SeVD57). RESULTS: The MAPK pathways evaluated were observed to be involved in the production of IL-10, under stimulation by secreted antigens, while the MAPK p38 and ERK 1 pathways were shown to be primarily involved in TNF-α production. By contrast, no involvement of the MAPK p38 and ERK 1 and 2 pathways was observed in IFN-γ production, while the ERK 2 pathway demonstrated involvement in IL-4 production only in the mouse splenocytes infected with VD57 under stimulation by SeT1. CONCLUSION: The authors hypothesize that MAPK p38 and ERK 1 pathways with respect to TNF-α production, as well as the MAPK p38 and ERK 1 and 2 pathways in relation to IL-10 production under infection by C. pseudotuberculosis are important regulators of cellular response. Additionally, the lack of the MAPK p38 and ERK 1/2 pathways in IFN-γ production in infected CBA murine cells stimulated with the two secreted/excreted antigens, in IL-4 production showing involvement only via the ERK 2 pathway under stimulation by SeT1 antigen during 60-day infection period with the virulent strain, suggests that these pathways regulated the production of pro-inflammatory and regulatory cytokines in the splenic cells of CBA mice.

Immunolocalization of FGF-2 and VEGF in rat periodontal ligament during experimental tooth movement.

OBJECTIVE: This article aimed at identifying the expression of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) in the tension and pressure areas of rat periodontal ligament, in different periods of experimental orthodontic tooth movement. METHODS: An orthodontic force of 0.5 N was applied to the upper right first molar of 18 male Wistar rats for periods of 3 (group I), 7 (group II) and 14 days (group III). The counter-side first molar was used as a control. The animals were euthanized at the aforementioned time periods, and their maxillary bone was removed and fixed. After demineralization, the specimens were histologically processed and embedded in paraffin. FGF-2 and VEGF expressions were studied through immunohistochemistry and morphological analysis. RESULTS: The experimental side showed a higher expression of both FGF-2 and VEGF in all groups, when compared with the control side (P < 0.05). Statistically significant differences were also found between the tension and pressure areas in the experimental side. CONCLUSION: Both FGF-2 and VEGF are expressed in rat periodontal tissue. Additionally, these growth factors are upregulated when orthodontic forces are applied, thereby suggesting that they play an important role in changes that occur in periodontal tissue during orthodontic movement.

Immunolocalization of FGF-2 and VEGF in rat periodontal ligament during experimental tooth movement

OBJECTIVE: This article aimed at identifying the expression of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) in the tension and pressure areas of rat periodontal ligament, in different periods of experimental orthodontic tooth movement. METHODS: An orthodontic force of 0.5 N was applied to the upper right first molar of 18 male Wistar rats for periods of 3 (group I), 7 (group II) and 14 days (group III). The counter-side first molar was used as a control. The animals were euthanized at the aforementioned time periods, and their maxillary bone was removed and fixed. After demineralization, the specimens were histologically processed and embedded in paraffin. FGF-2 and VEGF expressions were studied through immunohistochemistry and morphological analysis. RESULTS: The experimental side showed a higher expression of both FGF-2 and VEGF in all groups, when compared with the control side (P < 0.05). Statistically significant differences were also found between the tension and pressure areas in the experimental side. CONCLUSION: Both FGF-2 and VEGF are expressed in rat periodontal tissue. Additionally, these growth factors are upregulated when orthodontic forces are applied, thereby suggesting that they play an important role in changes that occur in periodontal tissue during orthodontic movement. .

OBJETIVO: o objetivo desse estudo foi identificar a expressão do fator de crescimento de fibroblastos 2 (FGF-2) e do fator de crescimento vascular endotelial (VEGF) nos lados de tensão e pressão do ligamento periodontal de ratos, durante movimento ortodôntico experimental, em diferentes períodos de tempo. MÉTODOS: uma força ortodôntica de 0,5N foi aplicada no primeiro molar superior direito de 18 ratos Wistar machos, por períodos de 3 (grupo I), 7 (grupo II) e 14 dias (grupo III). O primeiro molar do lado oposto foi utilizado como controle. Os animais foram sacrificados nos períodos de tempo mencionados, sendo a arcada superior removida e fixada. Após a desmineralização, os espécimes foram processados histologicamente e embebidos em parafina. A expressão do FGF-2 e do VEGF foram estudadas por meio de análise imuno-histoquímica. RESULTADOS: o ligamento periodontal dos dentes submetidos à movimentação ortodôntica mostraram maior expressão tanto de FGF-2 quanto de VEGF, em todos os grupos experimentais, quando comparados com os dentes do lado controle (p < 0,05). Diferenças estatisticamente significativas entre os lados de tensão e pressão também foram encontradas nos dentes submetidos à movimentação ortodôntica. CONCLUSÕES: tanto o FGF-2 quanto o VEGF são expressos no tecido periodontal de ratos, e esses fatores de crescimento são aumentados quando forças ortodônticas são aplicadas, sugerindo que esses desempenham um papel importante na reorganização do periodonto durante o movimento ortodôntico. .

Association between haptoglobin and IgM levels and the clinical progression of caseous lymphadenitis in sheep.

BACKGROUND: Sheep caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis (Cp), is associated with direct economic losses and presents significant zoonotic potential. Despite the importance of the disease, a satisfactory vaccine model has not been developed. Thus, this study aimed to investigate the association between haptoglobin (Hp) and IgM levels and the clinical progression of CLA in primarily infected sheep and in sheep immunized with Cp- secreted antigens adjuvanted with Quillaja saponaria saponins. These animals were kept with CLA-positive sheep to simulate natural exposure that occurs in field conditions. During the experiment, the Hp and IgM levels were monitored for 21 days, and the development of internal CLA lesions was investigated through necropsies on day182 post-immunization. RESULTS: Primarily infected sheep in Group 2 (inoculated with 2x105 Cp virulent strain) had higher Hp values between the first and ninth days post inoculation (PI) than sheep in Group 1 (control; P < 0.05). Immunized animals in Group 3 had significantly higher Hp values between the third and seventh days PI, compared with the control group (P < 0.01). Binary logistic regression (BLR) analysis of primarily infected sheep indicated an association between Hp concentration and CLA clinical progression: animals with high Hp values had 99.9% less risk of having CLA abscesses than animals with low Hp levels (Odds ratio = 0.001, P < 0.05). Both experimental groups had significantly higher IgM titers than the control group around the ninth and eleventh days PI (P < 0.05). The BLR analysis for immunized sheep indicated an association between IgM levels and clinical progression: sheep with high IgM titers had 100.0% less risk of having CLA abscesses than animals with low IgM levels (Odds ratio = 0.000, P < 0.05). CONCLUSIONS: Resistance to C. pseudotuberculosis infection is supported by the early acute phase response, in which up-regulation of Hp and IgM were predictive of a lower risk of CLA lesion development. Because the immunogen used in this study induced a high production of both Hp and IgM, Q. saponaria saponin should be considered a promising candidate in vaccine formulations against sheep CLA.

Porphyromonas gingivalis HmuY-induced production of interleukin-6 and IL-6 polymorphism in chronic periodontitis.

BACKGROUND: In chronic periodontitis (CP), the gene polymorphism of interleukin-6 (IL-6) to 174C/G has been associated with the altered production of this cytokine. The aim of this pilot study is to compare the allelic and genotypic frequencies in patients with CP with control individuals without periodontitis (NP) and to measure the production of IL-6 by whole blood cells stimulated with Porphyromonas gingivalis HmuY protein. METHODS: DNA was isolated from peripheral blood cells of 49 patients with CP and 60 control individuals classified as NP, and genotyping was performed by polymerase chain reaction using sequence-specific primers. Whole blood cells from 29 patients with CP and 30 control individuals were stimulated for 48 hours with HmuY, and IL-6 levels were measured using enzyme-linked immunosorbent assay. RESULTS: The proportion of individuals carrying the G allele at position -174 of the IL-6 gene was higher in the group with CP (85.7%) than in the normal control group (73.3%; P <0.03). P. gingivalis HmuY-induced production of IL-6 was higher in the group with CP (P <0.05). CONCLUSIONS: Our findings suggest that P. gingivalis HmuY may be associated with increased IL-6 production during CP. Furthermore, patients with periodontitis and individuals with higher HmuY-induced production of IL-6 show a high frequency of the G allele at position -174.

Estudo das condições microbiológicas de farinhas de mandioca (Manihot esculenta Crantz) comercializadas no centro de abastecimento de Alagoinhas, Bahia / Study of the microbiological conditions of marketing of cassava (Manihot esculenta Crantz) flour in supply center of Alagoinhas, Bahia

No município de Alagoinhas, Bahia, e na maioria dos estados do Nordeste, a farinha de mandioca (Manihot esculenta Crantz) é produzida de forma artesanal em casas-de-farinha, na maioria das vezes localizadas no próprio local de plantio da mandioca. Este trabalho teve como objetivo avaliar as condições microbiológicas de farinhas de mandioca comercializadas no Centro de Abastecimento em Alagoinhas-Bahia. Foram coletadas 100 gramas de 30 amostras de farinha de mandioca, as quais foram acondicionadas em frascos estéreis, em recipiente isotérmico. As amostras foram submetidas à pesquisa de bactérias heterotróficas empregando-se o método de contagem padrão em placas, seguido da coloração de Gram, e a pesquisa para coliformes totais e termotolerantes foi realizada pela técnica do Número Mais Provável (NMP). Os resultados indicam que as amostras estão em conformidade com a legislação vigente em relação à análise de coliformes totais e termotolerantes (<3NMPg-1), na contagem de bactérias heterotróficas algumas amostras mostraram-se fora dos padrões aceitáveis. Na coloração de Gram foram observados bacilos, diplobacilos e estreptobacilos Gram positivos e Gram negativos e cocos, diplococos e estafilococos Gram positivos. A avaliação sanitário-ambiental revelou más condições de comercialização da farinha de mandioca no Centro de Abastecimento de Alagoinhas.

In the city of Alagoinhas, Bahia, and in most Northeastern states, the cassava (Manihot esculenta Crantz) flour is produced in a craft house, most often located in the place of production. This study aimed to evaluate the microbiological conditions of cassava flours marketed Supply Centre in Alagoinhas, Bahia. We collected 30 samples of cassava flour, 100 grams, which were placed in sterile, insulated container. The samples were tested for heterotrophic bacteria using the method of standard plate count, followed by Gram staining, the search for total and fecal coliforms was performed by the Most Probable Number (MPN). The results indicate that the samples that the samples are in accordance with current legislation regarding the analysis of total coliforms and thermotolerant (<3NMPg-1), the count of heterotrophic bacteria some samples were outside the acceptable standards. In Gram staining bacilli were observed, and diplobacillus estreptobacilos Gram positive and Gram negative cocci, diplococci and staphylococci Gram posotivos. The assessment of health and environmental conditions revealed but the marketing of cassava flour in the Central Supply Alagoinhas.

Porphyromonas gingivalis antigens differently participate in the proliferation and cell death of human PBMC.

OBJECTIVE: Modulation of cell-mediated immunity by microorganisms in periodontal diseases has been widely studied; however, the proliferative activity and/or programmed death of mononuclear cells under periodontopathogenic stimuli are not yet well understood. The aim of this study was to investigate in vitro proliferation and death of peripheral blood mononuclear cells (PBMC) upon stimulation with Porphyromonas gingivalis (Pg) antigens. DESIGN: In 19 patients with chronic periodontitis (CP) and 16 controls without periodontitis (NP) the following clinical parameters were evaluated: bleeding on probing, probing depth, and clinical attachment level. PBMC were cultured under Pg stimuli and apoptosis/necrosis and proliferation assays were carried out for 18 and 48 h, respectively. Fluorescence of labelled cells was determined using flow cytometry. RESULTS: PBMC of CP and NP subjects exhibited a lower proliferative response to Pg LPS (p<0.05) and HmuY protein (p<0.001) compared with non-stimulated cells. Early apoptosis was induced by Pg LPS (p<0.01) and Pg extract (p<0.05), whilst all antigens induced late apoptosis (Pg LPS: p<0.001; Pg extract: p<0.001; HmuY: p<0.01) and necrosis (Pg LPS: p<0.01; Pg extract: p<0.001; HmuY: p<0.001). Pg LPS induced higher late apoptosis than HmuY (p<0.05). Only Pg LPS-induced necrosis tended to be higher in CP compared with NP. CONCLUSIONS: The inhibitory effect of cell proliferation caused by Pg LPS and HmuY protein is not observed when these antigens comprise Pg extract. Despite induced apoptosis, some still unknown mechanism determines the inflammatory outcome in cell death stimulated by HmuY.

Corynebacterium pseudotuberculosis secreted antigen-induced specific gamma-interferon production by peripheral blood leukocytes: potential diagnostic marker for caseous lymphadenitis in sheep and goats.

In the current study, the applicability of the quantification of gamma interferon (IFN-γ) levels for the detection of animals infected with Corynebacterium pseudotuberculosis and for determining caseous lymphadenitis (CLA) clinical status was evaluated. Peripheral blood leukocytes were collected from CLA nonendemic areas animals, from CLA seropositive animals without clinical signs of the disease, and from seropositive animals presenting CLA clinical signs. The leukocytes were stimulated with C. pseudotuberculosis-secreted antigens that were concentrated by the three-phase partitioning technique. An ovine IFN-γ enzyme-linked immunosorbent assay was used to quantify IFN-γ production. Goats and sheep with CLA had higher IFN-γ levels than uninfected seronegative animals. Leukocytes from sheep with CLA chronic abscesses produced higher IFN-γ levels when compared with seropositive sheep without CLA clinical signs, but this difference was not significant in goats. The sensitivity of the assay was 55.8% and 56%, whereas the specificity was 100% and 93%, for goats and sheep, respectively. In conclusion, IFN-γ is a potential marker for the determination of CLA infection status in small ruminants; however, further research is needed to improve assay sensitivity.

Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis.

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar-gel immunodiffusion (AGID) test for canine brucellosis, were used as the control panel for B. canis infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies.

The flavonoid rutin but not the alkaloid arborinine induces apoptosis in a B-cell hybridoma cell line.

The effects of arborinine, an alkaloid extracted from Erthela bahiensis and of rutin, a flavonoid obtained from Dimorphandra mollis (Benth.), Brazilian medicinal plants, on the viability and function of a murine B-cell hybridoma as a tumor model were investigated. The flavonoid rutin at 50 microM induced an increase in the number of apoptotic cells of one- to fivefold and reductions in cellular proliferation and monoclonal antibody production. Less but still significant necrosis was also induced by rutin under the same experimental conditions. On the other hand, the alkaloid arborinine exerted no significant effects on the studied parameters.

Serum antibody levels against Porphyromonas gingivalis extract and its chromatographic fraction in chronic and aggressive periodontitis.

UNLABELLED: Periodontitis is an inflammatory oral disease caused by multifactorial intrinsic and extrinsic agents, including Gram-negative bacteria such as Porphyromonas gingivalis. OBJECTIVE: To evaluate if there is difference in the serum levels of IgA, IgG and IgG subclasses reactive to Porphyromonas gingivalis in subjects with different periodontal conditions. METHODS: The study included 89 patients divided into four groups: 29 subjects with moderate or severe chronic periodontitis (CP), 12 with aggressive periodontitis (AP), 22 with gingivitis or mild periodontitis (GP), and 26 healthy controls (HC). Humoral response was assayed by enzyme-linked immunosorbent assay (ELISA) to verify serum levels of IgG, IgG1, IgG2, IgG3, IgG4 and IgA serum levels reacting to crude P. gingivalis ATCC 33277 sonicate extract and fraction IV, an enrichment of the immunoreactive bands of the crude extract obtained by chromatography. RESULTS: IgA, IgG (p < 0.01), IgG2, IgG3 and IgG4 serum level reactions to fraction IV were higher in the CP group compared with the healthy control. The CP group had higher levels of IgG and IgG4 to both antigens than the GP group, and higher levels of IgG and IgG4 to sonicate extract than the AP group. There were statistically significant differences in serum levels of IgG to both antigens (p < 0.01), IgG2 to fraction IV (p < 0.01), IgG3 to fraction IV (p < 0.05) and IgG4 to both antigens (p < 0.05) between AP and HC groups. IgG1 titers to sonicate extract were significantly higher (p < 0.05) in the GP group in comparison to the AP group. CONCLUSIONS: The findings of this study suggest that there are differences in the serum levels of IgA, IgG and IgG subclasses in patients with different periodontal conditions.

Existe correlação entre níveis séricos de anticorpos reativos a porphyromonas gingivalis e a condição periodontal clínica? / Is there any correlation between serum antibody levels reactive to porphyromonas gingivalis and clinical periodontal status?

Porphyromonasgingivalis has been shown to be shown to be one of the most important pathogen in chronic periodontitis development. The aim of this study was to evaluate the correlation among IgA, IgG and IgC and IgG isotype serum levels reactive Porphyromonas gingivalis ATTCC33277 and the clinical periodontal status. Twenty nine periodontitis patients were evaluated according to clinical parameters: probing depth, bleeding on probing, and clinical attachment loss measurements. Humoral immune response was assayed by enzyme linked immunosorbent assay (ELISA). There was no correlation between bleeding on probing (criterion 1) and the evaluated antibodies. Significant positive correlations were found between IgA levels and percentual of sites with clinical attachment loss (CAL) >3mm (criterion 2), percentual of sites with CAL > 5 mm (criterion 3), and percentual of sites with CAL > 3mm associated to probing depth > 4mm and bleeding on probing at the same site (criterion 4) (r=0,311; r=0,336; r=0,358, respectively; p<0,05). There were significant positive correlations between IgG serum levels and criteria 3 and 4 (r=0,400 and r=0,372, respectively; p<0,05), and between IgG1 and criteria 2, 3 and 4 (r=0,333, r=0,323 and r=0,340, respectively; p<0,05). The findings indicate that the antibody response to Porphyromonas gingivalis depends on the periodontal status, determined by clinical parameters

Antibody response to a chromatographic fraction of Porphyromonas gingivalis and its correlation with periodontal status

Porphyromonas gingivalis tem sido fortemente associado à gravidade das doenças periodontais e estimula respostas celulares e humorais no hospedeiro. Objetivo: Correlacionar os níveis de IgA, IgG e subclasses de IgG contra uma fração cromatogrßfica do extrato de Porphyromonas gingivalis ATCC33277 extract e descritores clínicos periodontais. Material e Métodos: Indivíduos com periodontite (29) e com saúde periodontal (26) foram avaliados de acordo com as medidas de profundidade de sondagem, sangramento à sondagem e nível de inserção clínica. O extrato de Porphyromonas gingivalis foi fracionado por cromatografia de troca iônica e a resposta humoral contra a fração IV foi avaliada por ôenzyme linked immunosorbent assayõ. Resultados e Conclusão: O percentual de sítios com sangramento à sondagem (critério 1) estava correlacionado significativamente com os níveis séricos de IgG2 (r = 0,385; p < 0,05). Os níveis de IgG total e IgG2 correlacionaram-se significativamente com o percentual de sítios com nível de inserção clínica (NIC) ≥ 3 mm (critério 2) (r = 0,428; p < 0,05 e r = 0,510; p < 0,01, respectivamente), NIC ≥ 5 mm (critérion 3) (r = 0,499 e r = 0,518, respectivamente; p < 0,01) e percentual de sítios com NIC ≥ 3 mm associado a profundidade de sondagem ≥ 4 mm e sangramento à sondagem no mesmo sítio (criterion 4) (r = 0,607; p < 0,001 e r = 0,487; p < 0,01, respectivamente). Foi observada uma correlação positiva estatisticamente significante entre os níveis de IgGA e IgG1 e o critério 4 (r = 0,339 e r = 0,345, respectivamente; p < 0,05), e entre os níveis de IgG3 e o critério 3 (p < 0,05; r = 0,370). Estes resultados indicam que quanto mais acurado for o critério de diagnóstico empregado para a determinação da doença periodontal, maiores os níveis séricos de imunoglobulinas.

SDS-PAGE and Western blot analysis of somatic and extracellular antigens of Corynebacterium pseudotuberculosis

Diferentes técnicas para isolar e descrever antígenos das frações secretadas de superfície e somáticas deCorynebacterium pseudotuberculosis foram estudadas por SDS-PAGE e imunoblot, utilizando-se pool de soros de cabras naturalmente infectadas. Os antígenos secretados foram obtidos do sobrenadante de cultura da bactéria cultivada em meio quimicamente definido ou em meio BHI (Brain Heart Infusion). A fração de superfície foi obtida por tratamento com NaCl 1M, e as frações somßticas foram obtidas por vários procedimentos (detergentes e ultra-som). Pela coloração por Coomassie blue, foram detectadas 20 bandas na fração secretada, 35 bandas no extrato de superfície e entre 40 e 50 bandas, a depender do processo de extração, na fração somßtica. Entre todas as frações estudadas, foram detectadas 16 proteínas imunorreativas. As bandas com pesos moleculares de 125, 108, 75, 68, 41, 40, 31 e 24 kDa foram reconhecidas commaior intensidade e todas elas foram encontradas na fração secretada. A utilização do meio quimicamente definido permitiu revelar, na fração secretada de C. pseudotuberculosis, a presença de proteínas de alto pesomolecular que não tinham sido previamente descritas.

Reconhecimento de antígenos por anticorpos de caprinos naturalmente infectados ou imunizados contra Corynebacterium pseudotuberculosis / Antigen recognition by antibodies against Corynebacterium pseudotuberculosisfrom naturally infected or immunized goats

Corynebacterium pseudotuberculosis é um cocobacilo gram-positivo, patógeno intracelular facultativo de macrófagos, filogeneticamente relacionado com o Mycobacterium tuberculosis. É amplamente distribuído em algumas espécies de animais, causando a linfadenite caseosa em ovinos e caprinos. A linfadenite caseosa, de ocorrência mundial, é uma doença infecciosa crônica cuja transmissão se dá principalmente através da pele. No presente experimento, foram estudados aspectos do reconhecimento antigênico, pela resposta humoral, em caprinos criados em regime extensivo, naturalmente infectados ou imunizados com uma vacina viva (cepa 1002), atenuada, desenvolvida pela Empresa Baiana de Desenvolvimento Agrícola (EBDA). As amostras de soros dos animais imunizados e de controle, coletadas mensalmente por um período de doze meses, foram analisadas pelo ensaio imunoenzimático ELISA e pelo Western blotting, utilizando-se como antígenos o extrato bacteriano contendo exotoxina e sonicado bacteriano de uma cepa selvagem e da cepa 1002. A análise através do Western blotting revelou que o padrão de antígenos reconhecidos pelos anticorpos séricos de animais imunizados, sem raça definida (SRD) ou de duas raças puras, bem como animais SRD naturalmente infectados, é semelhante, verificando-se bandas protéicas com pesos moleculares entre 20 e 94 kDa. Esta análise possibilitou observar-se que diferentes proteínas são reconhecidas ao longo da imunização.

Avaliação da resposta imune humoral em caprinos inoculados com uma vacina viva atenuada liofilizada contra Corynebacterium pseudotuberculosis / Evaluation of the goats humoral immune response induced by the Corynebacterium psudotuberculosis lyophilized live vaccine

A linfadenite caseosa (LC), comumente chamada de mal-do-caroço, é uma doença que acomete ovinos e caprinos e tem como agente etiológico Corynebacterium pseudotuberculosis. É um patógeno intracelular facultativo de macrófagos e é filogeneticamente relacionado com Mycobacterium tuberculosis. É uma doença de ocorrência mundial, caracterizada por formação de lesões necróticas encapsuladas caseosas, localizada principalmente nos linfonodos externos e pulmões. A transmissão se dá provavelmente através da pele, pelo contato de animais sadios com animais portadores de abscessos que evoluem a supuração. Eventualmente, tem sido encontrada em bovinos, porém é em ovinos e em caprinos que assume importância econômica e sanitária. Machos e fêmeas podem apresentar a doença em qualquer estação, porém encontram-se mais doentes com o desenvolver da idade, principalmente após um a dois anos de vida. A enfermidade causa sérios prejuízos a essas espécies, constituindo uma limitação zoopatológica às suas explorações. Com cerca de 80 por cento do rebanho nacional, a região Nordeste é a maior detentora de caprinos do Brasil, onde esses animais são essenciais ao sustento do pequeno agricultor. Corynebacterium pseudotuberculosis causa consideráveis prejuízos econômicos à região, que vão desde a condenação de pele e carcaças em razão de abscessos, até perdas em eficiência na reprodução ou na produção de carne e leite. O presente trabalho teve como objetivo avaliar em caprinos a resposta imune humoral induzida pela vacina viva liofilizada, elaborada com a cepa 1002, da Empresa Baiana de Desenvolvimento Agrícola (EBDA). Os resultados evidenciaram que o referido preparado liofilizado promove a produção de anticorpos específicos em doses iguais ou maiores do que 109 CFU e que o tratamento ministrado não provocou lesões nos animais vacinados.