Cyclosporin A treatment and decreased fungal load/antigenemia in experimental murine paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb). The cyclosporin A (CsA) is an immunosuppressant drug that inhibits calcineurin and has been described as a potential antifungal drug. The present study investigated the effect of CsA on the immune response, fungal load/antigenemia in experimental murine PCM. It was used four groups of BALB/c mice: (a) infected with 1 x 105 Pb18 yeast cells (Pb), (b) infected and treated with CsA every other day 10 mg/kg of CsA (s.c.) during 30 days (Pb/CsA), (c) treated with CsA (CsA) and (d) no infected/treated (PBS). The immune response was evaluated by lymphocyte proliferation, DTH assays to exoAgs, ELISA for IgG anti-gp43 (specific immune responses) and cytokine serum levels (IFN-γ, TNF-α, IL-4 and IL-10). Fungal load was determined by lung colony-forming units (CFU) counts, lung and liver histopathology analysis and antigenemia determined by inhibition-ELISA. As expected, CsA was able to inhibit the specific cellular and humoral immune response (P < 0.05), with decrease in serum IFN-γ, TNF-α and IL-4 levels (P < 0.05). Cyclosporin A treatment also resulted in significantly decreased lung Pb CFU (P < 0.05) as well as a lower number of yeasts in the lung and liver (P < 0.05) by histopathology. In concordance, the decreased antigenemia was observed in Pb/CsA group (P < 0.05). In conclusion, even with immunosuppressive action, treatment with CsA results in decreased lung fungal load/antigenemia in experimental PCM in BALB/c mice. Further study is required to determine whether this represents less severe disease or protection by CsA.

The effects of propolis on antibody production by laying hens.

Propolis is a honeybee product showing several biological properties that enhance the immune response, depending on the concentration and intake period. Because propolis possesses an immunomodulatory action on mammals, the objective of our study was to investigate the effects of propolis on the humoral immune response of laying hens by evaluating antibody production. Laying hens (ISA Brown) were divided into 5 groups with 7 birds each. Group 1 was a nonimmunized control, whereas birds in group 2 were immunized intravenously with SRBC, and those in groups 3, 4, and 5 were treated intraperitoneally with propolis (2, 10, and 50 mg/kg, respectively) on 3 consecutive days and then inoculated intravenously with SRBC. Hematological and serological analyses were carried out on d 0, 3, and 38. Natural and specific antibody levels were determined by hemagglutination with rabbit red blood cells and SRBC, respectively. Propolis-treated birds (50 mg/kg) showed a significant decline in heterophils and in the heterophil:lymphocyte ratio. After SRBC immunization, significant increases in levels of IgG were observed in groups 4 and 5. Furthermore, higher levels of natural antibodies were observed in propolis-treated laying hens. The administration of propolis to laying hens increased the production of IgG specific to SRBC and natural antibodies, and could be used to increase antigen-specific antibody responses to vaccines.

Activation of the alternative complement pathway in canine normal serum by Paracoccidioides brasiliensis.

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human granulomatous disease. Recently the first case of natural disease in dogs was reported. The complement system is an important effector component of humoral immunity against infectious agents. Therefore, the aim of this study was to evaluate the activation of the dog alternative complement pathway by P. brasiliensis. Initially, the ability of erythrocytes of guinea pig, rabbit, sheep, chicken and swine to activate the dog alternative pathway was evaluated. The guinea pig erythrocytes showed the greatest capacity to activate dog alternative pathway. The alternative (AH50) hemolytic activity was evaluated in 27 serum samples from healthy dogs and the mean values were 87.2 AH50/ml. No significant differences were observed in relation to sex and age. The alternative pathway activation by P. brasiliensis was higher in serum samples from adult dogs when compared to puppies and aged dogs (p ≤ 0.05). This is the first report of dog alternative complement pathway activation by P. brasiliensis and suggests that it may play a protective role in canine paracoccidioidomycosis.

Cloning, sequencing and antigenic characterization of rVirB9 of Anaplasma marginale isolated from Paraná State, Brazil.

Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.

Carcass characteristics and meat quality of broilers fed with different levels of Saccharomyces cerevisiae fermentation product.

Fermented products and components of Saccharomyces cerevisiae have been widely used in animal nutrition to promote the development and quality of broilers. This study aims to evaluate different levels of inclusion (0, 250, 750, 1,500 g/t) of S. cerevisiae fermentation product (SCFP) in broiler feed to gauge its effect on carcass characteristics and cuts beyond the quality of breast meat. For analyses of carcass yield, cuts, and meat quality, 16 broilers per treatment were slaughtered. The meat quality analyses were performed 24 h after slaughter and evaluated color, pH, water holding capacity, cooking loss, and shear force. Lipid oxidation was determined in frozen breast samples stored at -20°C for 45 d. The results indicate that different levels of inclusion of SCFP provided no changes in carcass yield, color, water holding capacity, cooking loss, and shear force; however, inclusion of 1,500 g/t of SCFP increased leg yield and reduced pH. The inclusion of 750 g/t of SCFP decreased the lipid oxidation of breast meat (P < 0.05). This study concluded that inclusion of SCFP may improve leg yield and the lipid oxidation of breast meat.

Características produtivas, carcaça, cortes e resposta imune humoral de frangos de corte alimentados com diferentes fontes de óleo e vitamina E / Productive and humoral immune response of broilers fed with different sources of oil and vitamin E

A adição de óleos na dieta de frangos de corte proporciona muitas vantagens, visto que, dependendo do perfil de ácidos graxos, pode melhorar o desempenho e atuar como estimulante do sistema imune. Assim, este estudo teve como objetivo avaliar as características produtivas, o rendimento de carcaça, os cortes e a resposta imune humoral de frangos de corte alimentados com diferentes fontes de óleos e vitamina E. Foram utilizados 312 pintainhos de corte machos da linhagem Cobb com um dia de idade, distribuídos em delineamento inteiramente ao acaso, com oito repetições compostas de 13 aves por parcela experimental. Os tratamentos experimentais consistiram em óleo de soja, óleo de canola e óleo de canola mais adição de vitamina E. As variáveis analisadas foram ganho de peso, consumo de ração, conversão alimentar, rendimento de carcaça, cortes comerciais e resposta imune humoral. Os resultados obtidos mostram que houve diferença significativa no desempenho somente na fase pré-inicial, quando as aves que receberam o tratamento com óleo de canola e vitamina E apresentaram piores ganhos de peso. Não foram observadas diferenças significativas para as outras variáveis analisadas. Conclui-se que a utilização de diferentes fontes lipídicas associadas ou não à vitamina E não afeta as características produtivas de carcaça, cortes e resposta imune humoral em frangos de corte em relação ao uso de óleo de soja.(AU)

Oil inclusion in poultry diets provides many advantages and according to the fatty acid profile it is possible to achieve performance improvement as well as immune system stimulation. Thus, the study aimed to evaluate productive performance, carcass and cuts yields and also the humoral immune response of broilers consuming diets formulated with different oil sources and vitamin E. A total of 312 one-day old male Cobb was distributed, in a completely randomized design, in three treatments with eight replications of 13 birds. The experimental treatments were the diets that had different oil source as follows: soybean oil, canola oil and canola oils with vitamin E. The analyzed parameters were weight gain, feed intake, feed conversion ratio, carcass and cut yields and humoral immune response. For the treatment with canola oil and vitamin E a reduction on weight gain during the pre-starter stage was observed. For the other evaluated parameters, no significant differences were observed. In conclusion, the use of canola oil or canola oil with added vitamin E does not affect the productive performance, carcass and cut yields and humoral immune response in broiler chicken in relation soybean oil use.(AU)

Simvastatin improves morphological and functional recovery of sciatic nerve injury in Wistar rats.

AIM: The purpose of this work is to investigate the effects of simvastatin on sciatic nerve regeneration in male Wistar Rats. MATERIALS AND METHODS: Forty animals were allocated into four groups: (1) control (C); (2) control+simvastatin (CS); (3) lesioned animals+sterile PBS (LC) and (4) lesioned animals+simvastatin (LS). Lesioned animals were submitted to crushing lesion of right sciatic nerve. Simvastatin (20mg/kg/day, i.p.) was administered for five days. Footprints were obtained weekly for evaluation of functional locomotor recovery by means of the Sciatic Function Index (SFI). Blood samples were obtained weekly for quantifying circulating leukocytes. Animals were sacrificed after 21 days for histological analyses of sciatic nerve and spleen. RESULTS: LS Animals presented increased SFI scores, decreased areas of oedema and mononuclear cell infiltration during Wallerian degeneration and nerve regeneration (7,14 and 21 days; P<0.05). Spleen weight and white pulp areas was increased in LC animals after 21 days. Increased numbers of circulating neutrophils were observed in simvastatin treated animals (CS e LS) at seven, 14 and 21 days, compared to non-treated groups (C and LC). CONCLUSION: The study suggests that simvastatin accelerates the morphological and functional recovery process of the peripheral nervous system interfering with innate and acquired immunity.

Efeito de dietas contendo plasma sanguíneo desidratado sobre características microbiológicas, imunológicas e histológicas de leitões leves ao desmame / Effect of spray-dried plasma diets on microbiological, immunological and histological parameters of light weight weaned pigs

Foram avaliados os efeitos do plasma sanguíneo desidratado (PSD) sobre desempenho, perfil imunológico, histológico, microbiológico e peso de órgãos de leitões leves, desmamados aos 21 dias de idade. Foram utilizados 24 leitões, com idade média inicial de 21 dias, em delineamento experimental completamente ao acaso. Os tratamentos foram: T1 - animais pesados ao desmame, sem suplementação com PSD; T2 - animais leves ao desmame, suplementados com 10g/animal/dia de PSD; T3 - animais leves ao desmame, suplementados com 20g/animal/dia de PSD; T4 - animais leves ao desmame, sem suplementação com PSD. A adição de 20g de PSD na dieta melhorou o ganho diário de peso, aumentou o peso (g/kg) do baço e o título de IgA no soro entre 21 e 31 dias de idade. A inclusão de 10g de PSD aumentou o comprimento e a largura do linfonodo ileocólico. A inclusão de PSD traz benefícios aos leitões nos primeiros 10 dias pós-desmame, atuando principalmente nos órgãos linfoides e na mucosa intestinal.

The aim of this experiment was to evaluate the effects of spray-dried plasma (SDP) on the growth performance, immunological, histological and microbiological profile and weight of organs of light weight weaned pigs. The trial was done using 24 pigs with an initial mean age of 21 days in a completely randomized experimental design. The treatments were: T1 - heavy weight weaned pigs, without SDP supplementation; T2 - light weight weaned pigs, supplemented with 10g/animal/day of SDP; T3 - light weight weaned pigs, supplemented with 20g/animal/day of SDP; T4 - light weight weaned pigs, without SDP supplementation. The inclusion of 20g of SDP in the diet improved the weight gain, spleen weight (g/kg) and serum IgA title between 21 and 31 days of age. The inclusion of 10g of SDP in the diet improved the length and width of the ileocolic lymph node. In the first 10 days after weaning, SDP improved the development of lymphoid organs and the protection of the intestinal mucosa.

Molecular identification of Paracoccidioides brasiliensis by PCR amplification of ribosomal DNA.

We have amplified and sequenced the 5.8S and 28S ribosomal DNA genes and intergenic regions of Paracoccidioides brasiliensis, strain Pb01. Using primers specifically designed for both ribosomal DNA regions, we were able to discriminate between P. brasiliensis and other human pathogenic fungi by PCR. The use of this molecular marker could be important for paracoccidiodomycosis diagnosis and ecological and molecular epidemiological studies of P. brasiliensis in Latin America.

Identification of differentially expressed transcripts in the human pathogenic fungus Paracoccidioides brasiliensis by differential display.

Paracoccidioides brasiliensis is a dimorphic human pathogenic fungus that is the causal agent of paracoccidioidomycosis, a systemic disease that predominantly affects rural communities in South and Central America. Dimorphism is a common characteristic of systemic human pathogenic fungi. Here we describe the use of differential display (DD) to isolate and identify differentially expressed genes of P. brasiliensis, in the two cell types, yeast (Y) and mycelium (M), as well as at different time intervals during temperature-induced M to Y transition. Using two oligo-deoxythymidine-anchored primers combined with 10 arbitrary ones, we were able to detect the presence of at least 20 differentially transcribed cDNA fragments. Some of these fragments were further analysed by reverse-northern blot and northern blot in order to confirm their differential expression. The M32, M51 and M73 cDNA fragments were specific for the mycelial form of P. brasiliensis. Furthermore, we found two cDNA fragments (M-Y1 and M-Y2) that were upregulated during M-Y transition. This method was efficient and useful in the detection of differentially expressed genes in P. brasiliensis.

Activation of the alternative complement pathway in canine normal serum by Paracoccidioides brasiliensis / Ativação da via alternativa do complemento em soro de cão normal por Paracoccidioides brasiliensis

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human granulomatous disease. Recently the first case of natural disease in dogs was reported. The complement system is an important effector component of humoral immunity against infectious agents. Therefore, the aim of this study was to evaluate the activation of the dog alternative complement pathway by P. brasiliensis. Initially, the ability of erythrocytes of guinea pig, rabbit, sheep, chicken and swine to activate the dog alternative pathway was evaluated. The guinea pig erythrocytes showed the greatest capacity to activate dog alternative pathway. The alternative (AH50) hemolytic activity was evaluated in 27 serum samples from healthy dogs and the mean values were 87.2 AH50/ml. No significant differences were observed in relation to sex and age. The alternative pathway activation by P. brasiliensis was higher in serum samples from adult dogs when compared to puppies and aged dogs (p < 0.05). This is the first report of dog alternative complement pathway activation by P. brasiliensis and suggests that it may play a protective role in canine paracoccidioidomycosis.

O fungo dimórfico Paracoccidioides brasiliensis é o agente etiológico da paracoccidioidomicose, uma doença granulomatosa humana. Recentemente, foi relatado o primeiro caso da doença natural em cães. O sistema complemento é um importante componente efetor da imunidade humoral contra agentes infecciosos. Portanto, o objetivo deste trabalho foi avaliar a ativação da via alternativa do complemento canina pelo P. brasiliensis. Inicialmente, foi avaliada a capacidade de eritrócitos de cobaia, coelho, carneiro, galinha e suíno ativarem a via alternativa do complemento canino. Os eritrócitos de cobaia apresentaram maior capacidade de ativar a via alternative do complemento canino. A atividade hemolítica da via alternativa (AH50) foi avaliada em 27 amostras de soro de cães saldáveis e os valores médios observados foram de 87,2 AH50/ml. Não foi observada diferença significativa ao sexo e idade. A ativação da via alternativa pelo P. brasiliensis foi maior nas amostras de soro de cães adultos quando comparada aos cães filhotes e idosos (p < 0.05). Este é o primeiro relato da ativação da via alternative do complemento canino pelo fungo P. brasiliensis e sugere que pode ter um papel protetor na paracoccidioidomicose canina.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis.

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.

Cloning, sequencing and antigenic characterization of rVirB9 of Anaplasma marginale isolated from Paraná State, Brazil

Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.