Período de absorção intestinal de macromoléculas em cabritos recém-nascidos após a ingestão de colostro bovino

Após o nascimento, os cabritos são dependentes das imunoglobulinas colostrais devido às características placentárias que não permitem a passagem de macromoléculas da circulação materna. De acordo com a literatura, os cabritos possuem capacidade absortiva por até quatro dias. Muitos aspectos fisiológicos de outras espécies são aceitos e utilizados para caprinos, mas aqueles relacionados à transferência de imunidade passiva precisam de investigação. Os objetivos do presente estudo foram determinar o período de passagem de macromoléculas da mucosa intestinal para a circulação e a duração da proteção humoral transferida passivamente pela ingestão de colostro bovino e caprino. Sessenta cabritos recém-nascidos foram distribuídos em seis tratamentos: T 0 (n=25), ingestão natural de colostro caprino à vontade; T 1 (n=7), colostro bovino entre o nascimento e duas horas pós-parto; T 2 (n=7), ingestão de colostro bovino entre quatro e seis horas pós-nascimento; T 3 (n=7), leite nas primeiras oito horas e colostro bovino entre 10 e 12 horas pós-parto; T 4 (n=7), ingestão de leite até 18 horas e colostro bovino entre 22 e 24 horas pós-nascimento; T 5 (n=7), leite até 30 horas e ingestão de colostro bovino entre 34 e 36 horas pós-parto. Determinaram-se as concentrações séricas de proteína total (PT), gamaglobulina, imunoglobulina G (IgG) e a atividade sérica de gama glutamiltransferase (GGT). Ao nascimento, todos os neonatos tiveram valores mais baixos das variáveis, com aumento significativo da PT e gamaglobulina, após dois dias, nos grupos T 0, T 1 e T 2; a IgG e GGT aumentaram em todos os grupos. Os tratamentos T 3, T 4 e T 5 foram considerados como indutores de falha de transferência de imunidade passiva. A absorção de macromoléculas pelo trato intestinal dos cabritos ocorreu até 36 horas pós-parto, sendo mais efetiva até 12 horas. Os níveis de anticorpos persistiram até 75 dias após a ingestão de colostro bovino, porém, com concentrações inadequadas.

Oxidized LDL and physical fitness in healthy young men: associations with body composition, smoking, metabolic syndrome and androgen status

Background: In the past, oxidized low density lipoprotein (ox-LDL) has been associated with an unbeneficial lipid profile. This atherogenic lipid profile increases the risk of atherosclerotic cardiovascular diseases. Physical fitness has substantial effect on serum lipoprotein concentration as well as body composition and humoral responses, however interrelationships between ox-LDL and physical fitness have not been widely examined in a nationally representative sample. Aims: This thesis evaluates how cardiorespiratory and muscular fitness associate with ox-LDL lipids and how the other known risk factors of atherosclerosis might alter these associations. Subjects and Methods: The study cohort consisted of 846 healthy young males (mean age 25.1, SD 4.6) who were gathered by voluntary nationwide recruitment. Each participant conducted a series of physical fitness tests (cardiorespiratory and muscular fitness) and answered a detailed questionnaire that included lifestyle habits (i.e. smoking and leisuretime physical activity). Venous blood samples including ox-LDL and serum lipids were also collected. Results: Higher levels of ox-LDL were found in overweight and obese men, however, high cardiorespiratory fitness seemed to protect the overweight from high levels of ox-LDL. Young men who smoked and had poor cardiorespiratory or muscular fitness possessed a higher concentration of ox-LDL lipids when compared to comparable levels of cardiorespiratory or muscular fitness non-smoking young men. Metabolic syndrome was associated with increased levels of ox-LDL and high levels of ox-LDL combined with poor cardiorespiratory and abdominal muscle fitness seems to predict metabolic syndrome in young men. Also, participants with poor cardiorespiratory fitness and low levels of testosterone had higher levels of ox-LDL when compared to participants with high cardiorespiratory fitness / low testosterone as well as those with poor cardiorespiratory fitness / high testosterone. Conclusions: Good cardiorespiratory and muscular fitness protects young men from increased levels of ox-LDL lipids. This association was discovered in young men who were categorized as being overweight, smokers, metabolic syndrome or with low levels of testosterone. Being fit seems to prevent higher levels of ox-LDL, even in young healthy

Effect of oral administration of Propionibacterium acnes on growth performance, DTH response and anti-OVA titers in goat kids

Immunostimulants are susbstances that stimuli the response of effector cells to activate the immune response such as antigen uptake, cytokine release or antibody response. These substances can increase resistence to infection by different types of microorganisms, reducing dependence of antibiotics used in livestock animals. Recent reports have demonstrated the positive effect of Propionibacterium acnes (P. acnes) to control animal diseases. In this study, we evaluated the effect of the non-specific immunostimulant P. acnes on immunological functions and growth performance in goat kids. Twenty five goat kids served as control group (A) and another 25 animals received P. acnes being the experimental group (B). Kids were challenged with ovalbumin (OVA) to assess humoral immunity. To assess in vivo cell immunity, delayed type hypersensitivity (DTH) test with phytohemagglutinin (PHA) was used, clinical signs and body weight were recorded each week until 9 weeks of age when the experiment ended. Blood samples were obtained to analyze serum proteins fractions and anti-OVA specific antibodies. No clinical signs of disease and no differences (p>0.05) on body weight between groups were recorded (7.32±0.81 kg in group A, 7.13±0.65 kg in group B). Goat kids from group B had more total protein (59.8±5g/l) and albumin levels (32.8±3.3g/l) than goat kids from group A (56.6±5.7 g/l, 29.6±3.9 g/l respectively) (p<0.05). DTH response in goat kids from group B on day 42 was higher (p<0.05) than group A. At day 63, goat kids from group receiving P. acnes had higher percentage (85.4) of anti-OVA IgM titers (p<0.05) than control group (57.7). In conclusion, the results showed that oral administration of P. acnes to goat kids improved some aspects of the immune system of the animals and it could be used to control goat diseases.

A multidisciplinary approach to diagnose naturally occurring bovine tuberculosis in Brazil

A herd infected naturally with tuberculosis was investigated by different diagnostic methods. Ninety days after a screening test that identified 21 cows as skin test positive, a Comparative Intradermal Tuberculin Test (CITT) was performed in those 21 cows and in 29 other randomly selected skin test negative cows. Milk samples and nasal swabs were collected prior to the CITT for bacteriological culture and PCR, while blood samples were collected for IFN release and antibody responses to MPB70 and MPB83, at three time points post tuberculin injection. Animals positive by CITT were slaughtered and disease confirmation undertaken. Based on the Kappa test, IFN was comparable to the standard tests (culture, PCR and CITT) at all three sampling points. Results from both antibody ELISAs were similar but were not comparable to the standard tests. T-test analysis of the CITT, IFN and ELISAs demonstrated that their performances were not correlated. There is increasing recognition that individually, available diagnostic tests do not detect all infected cattle. Therefore, a comprehensive strategy for the diagnosis of bovine TB should include test results for the detection of both cellular and humoral immune responses where there may be animals at different stages of infection.

Teste de ELISA indireto para diagnóstico sorológico de leishmaniose visceral em canídeos silvestres

Na América do Sul, alguns canídeos silvestres são considerados reservatórios naturais da Leishmania chagasi. A resposta imunológica desses animais à Leishmania é pouco conhecida, havendo a necessidade de métodos diagnósticos adequados para esse fim. No presente estudo, é descrita a padronização do ensaio imunoenzimático indireto (ELISA) para o diagnóstico sorológico de leishmaniose visceral em canídeos silvestres brasileiros. Foram estudadas amostras de soro e plasma de 12 canídeos cativos: sete lobos-guará (Chrysocyon brachyurus), três raposinhas (Lycalopex vetulus) e dois cachorros-do-mato (Cerdocyon thous). As amostras de um C. brachyurus e uma L. vetulus, cativos em área endêmica para LV, que apresentavam doença clínica e positividade em testes de Imunofluorescência Indireta e Reação em Cadeia de Polimerase, foram utilizadas como controles positivos. Foram comparados os conjugados anti-IgG de cão e proteína A, ambos ligados a peroxidase, cujos testes detectaram quatro (04/12) e três (03/12) C. brachyurus soropositivos para anticorpos anti-Leishmania sp., respectivamente. As médias das densidades ópticas (DOs) das amostras negativas foram nitidamente mais baixas do que as médias das DOs dos positivos tanto no ELISA com anti-IgG de cão (4,8 vezes) como com proteína A (15,5 vezes). Os soros de três C. brachyurus positivos no ELISA indireto foram avaliados por Western blotting e identificaram 22 bandas, sendo imunodominantes as de peso molecular de 19, 22, 24, 45 e 66 kDa. Os testes ELISA com a proteína A e o conjugado anti-IgG de cão apresentaram respectivamente concordância excelente (Kappa = 1; p<0,001) e moderada (Kappa = 0,8; p<0,0015), com o Western blotting. Ambos foram, portanto, considerados adequados a avaliações de triagem de animais cuja resposta humoral de anticorpos indica contato com o parasito, úteis para subsidiar estudos para adequação de metodologias específicas para os canídeos silvestres.

Kinetics of epsilon antitoxin antibodies in different strategies for active immunization of lambs against enterotoxaemia

Enterotoxaemia, a common disease that affects domestic small ruminants, is mainly caused by the epsilon toxin of Clostridium perfringens type D. The present study tested four distinct immunization protocols to evaluate humoral response in lambs, a progeny of non-vaccinated sheep during gestation. Twenty-four lambs were randomly allocated into four groups according to age (7, 15, 30 and 45 days), receiving the first dose of epsilon toxoid commercial vaccine against clostridiosis with booster after 30 days post vaccination. Indirect ELISA was performed after the first vaccine dose and booster to evaluate the immune response of the lambs. Results showed that for the four protocols tested all lambs presented serum title considered protective (≥0.2UI/ml epsilon antitoxin antibodies) and also showed that the anticipation of primovaccination of lambs against enterotoxaemia conferred serum title considered protective allowing the optimization of mass vaccination of lambs.

Induction of immune response in broiler chickens immunized with recombinant FliC and challenged by Salmonella Typhimurium

The study examined (1) the immune response in broiler chickens after oral immunization with recombinant flagellin (rFliC) from Salmonella Typhimurium conjugated with sodium alginate microparticles, and the immune response enhancement in association with recombinant cholera toxin B subunit protein (rCTB) and pool of Lactobacillus spp. (PL). The immune responses were evaluated by dosage of IgY serum and IgA from intestinal fluid and immunostaining of CD8+ T lymphocytes in the cecum. The immunized animals were challenged with Salmonella Typhimurium (ST) 21 days after treatment. In all immunized groups, a significant increase (p<0.05) was observed in IgA levels (μg/mL), especially three weeks after immunization. The serum IgY levels (μg/mL) were little affected by the treatments and differed significantly among groups only in the second post-immunization week (p<0.05). After the challenge, the number of CD8+ T cells differed significantly between the treatments and negative control. Retrieval of Salmonella Typhimurium was not detected at 48 hours after the challenge in T2 (rFliC+rCTb), T3 (rFliC+PL) and T4 (rFliC+rCTB PL). The rFliC administered orally with or without rCTB and Lactobacillus spp. produces significant induction of humoral immune response, and the immunized chickens were more effective in eliminating Salmonella after challenge.

Fontes de óleo na dieta e sua influência no desempenho e na imunidade de frangos de corte

A composição de ácidos graxos da dieta pode influenciar o desempenho produtivo e o sistema imune de frangos de corte. O objetivo deste estudo foi avaliar o efeito do consumo de óleos ricos em ácidos graxos poli-insaturados ômega-6 (PUFAs n-6) e ômega-3 (PUFAs n-3) sobre o desempenho e a resposta imunológica de frangos de corte frente a um desafio antigênico. Foram comparadas dietas formuladas com 7% de óleo de soja (OS), linhaça (OL) ou sardinha (OP), fornecidas a 240 frangos da linhagem Cobb, divididos em 24 grupos de 10 aves cada, num arranjo experimental 3x2 (3 tipos de óleo e aves vacinadas ou não vacinadas) e 4 repetições. O óleo de soja é rico em ácido linoleico, um PUFA n-6, o óleo de linhaça é fonte de ácido alfa-linolênico, um PUFA n-3, e o óleo de sardinha, de outros PUFAs n-3, como os ácidos eicosapentaenoico e docosahexaenoico. O consumo de ração, o ganho de peso e a conversão alimentar foram avaliados aos 21, 35 e 42 dias. Aos 7 e aos 21 dias de idade, metade das aves recebeu vacina contra doença de Newcastle. Quinze dias após a imunização, avaliou-se a produção de anticorpos pelo método de ELISA, expressa pela densidade óptica a 450 nm (D.O. 450nm). Apenas as aves alimentadas com ração contendo OS apresentaram maior imunidade humoral (P<0,05) após a vacinação. A resposta linfoproliferativa das aves, que expressa a imunidade celular, foi maior entre as aves vacinadas, em comparação às aves não vacinadas (P<0,05), independentemente do óleo utilizado. A fonte de óleo da ração ou a vacinação não influenciaram o ganho de peso das aves (P>0,05). Entre as aves que receberam dieta com OS, as aves vacinadas apresentaram pior conversão alimentar (P<0,05). Nos grupos que consumiram ração com OL ou OP, a vacinação não influenciou a conversão alimentar (P>0,05), considerando todo o período experimental. A utilização de óleo rico em PUFA n-6 na dieta de frangos de corte aumentou a resposta humoral, mas não influenciou a resposta celular frente a um desafio antigênico.

Immunization by subcutaneous implants of polyester-polyurethane sponges coupled with antigen

A new protocol is described for immunization of outbred Swiss mice. The procedure is based on subcutaneous implantation of antigen-coupled polyester-polyurethane sponges cut into disks of 10 mm in diameter vs 2 mm in thickness. Antigen coupling was performed by overnight incubation of the sponge with a solution of ovalbumin (Ova) (2 mg/ml) diluted in sodium carbonate buffer, pH 9.6. The amount of ovalbumin that was taken up by the sponge was between 71.4 to 82.5 µg. This was estimated by comparing the Ova absorbance at 280 nm in coating buffer solutions before and after incubation. To compare the efficiency of the proposed method, experimental groups immunized with the antigen in the presence of adjuvants (10 µg in Al(OH)3 or 100 µg in complete Freund's adjuvant (CFA)) were run in parallel. The data obtained after the 3rd week of immunization indicate that both cellular and humoral immune responses were achieved. These were assayed by antigen-induced footpad swelling and ELISA (specific antibodies), respectively. The levels of both immune responses elicited were similar to the responses observed in mice immunized with ovalbumin in the presence of Al(OH)3. The method might represent an advantage when immunizing with pathogenic antigens. Preliminary experiments have suggested that the antigen remains immobilized or bound to the sponge for a long period of time, since there is an increment on the cell population inside the sponges after boosting the animals. If so, the undesirable effects of immunization would be reduced.

A liquid phase blocking ELISA for the detection of antibodies against infectious bronchitis virus

A liquid phase blocking ELISA (LPB-ELISA) was developed for the detection and measurement of antibodies against infectious bronchitis virus (IBV). The purified and nonpurified virus used as antigen, the capture and detector antibodies, and the chicken hyperimmune sera were prepared and standardized for this purpose. A total of 156 sera from vaccinated and 100 from specific pathogen-free chickens with no recorded contact with the virus were tested. The respective serum titers obtained in the serum neutralization test (SNT) were compared with those obtained in the LPB-ELISA. There was a high correlation (r2 = 0.8926) between the two tests. The LPB-ELISA represents a single test suitable for the rapid detection of antibodies against bronchitis virus in chicken sera, with good sensitivity (88%), specificity (100%) and agreement (95.31%).

Effect of the injection of an extract of Ascaris suum on macrophage activation during the early phase of Mycobacterium bovis BCG infection in C57Bl/6 mice

Injection of an Ascaris suum extract (Asc) affects both the humoral and cellular immune responses to unrelated antigens when it is co-administered with these antigens. In the present study we evaluated the effect of Asc on macrophage activation in the early phase of Mycobacterium bovis BCG (Pasteur strain TMCC 1173) infection in C57Bl/6 mice. C57Bl/6 mice were injected intraperitoneally (ip) with 0.1 mg BCG (BCG group) or BCG plus 1 mg Asc (BCG + Asc group). The peritoneal exudates were obtained at 2, 7 and 14 days after infection. The numbers of IFN-g-secreting cells were assessed by the ELISPOT assay. Nitric oxide (NO) production was measured by the Griess method and by the evaluation of NADPH diaphorase activity in the peritoneal exudates. The administration of Asc extract increased NADPH diaphorase activity (2 days: control = 0, BCG = 7%, BCG + Asc = 13%, and Asc = 4%; 7 days: control = 4, BCG = 13%, BCG + Asc = 21%, and Asc = 4.5%) and TNF-a levels (mean ± SD; 2 days: control = 0, BCG = 169 ± 13, BCG + Asc = 202 ± 37, and Asc = 0; 7 days: control = 0, BCG = 545 ± 15.5, BCG + Asc = 2206 ± 160.6, and Asc = 126 ± 26; 14 days: control = 10 ± 1.45, BCG = 9 ± 1.15, BCG + Asc = 126 ± 18, and Asc = 880 ± 47.67 pg/ml) in the early phase of BCG infection. Low levels of NO production were detected at 2 and 7 days after BCG infection, increasing at 14 days (mean ± SD; 2 days: control = 0, BCG = 3.7 ± 1.59, BCG + Asc = 0.82 ± 0.005, Asc = 0.48 ± 0.33; 7 days: control = 0, BCG = 2.78 ± 1.54, BCG + Asc = 3.07 ± 1.05, Asc = 0; 14 days: control = 0, BCG = 9.05 ± 0.53, BCG + Asc = 9.61 ± 0.81, Asc = 10.5 ± 0.2 (2 x 106) cells/ml). Furthermore, we also observed that Asc co-injection induced a decrease of BCG-colony-forming units (CFU) in the spleens of BCG-infected mice during the first week of infection (mean ± SD; 2 days: BCG = 1.13 ± 0.07 and BCG + Asc = 0.798 ± 0.305; 7 days: BCG = 1.375 ± 0.194 and BCG + Asc = 0.548 ± 0.0226; 14 days: BCG = 0.473 ± 0.184 and BCG + Asc = 0.675 ± 0.065 (x 102) CFU). The present data suggest that Asc induces the enhancement of the immune response in the early phase of BCG infection.

Fever induction pathways: evidence from responses to systemic or local cytokine formation

The immune and central nervous systems are functionally connected and interacting. The concept that the immune signaling to the brain which induces fever during infection and inflammation is mediated by circulating cytokines has been traditionally accepted. Administration of bacterial lipopolysaccharide (LPS) induces the appearance of a so-termed "cytokine cascade" in the circulation more or less concomitantly to the developing febrile response. Also, LPS-like fever can be induced by systemic administration of key cytokines (IL-1ß, TNF-alpha, and others). However, anti-cytokine strategies against IL-1ß or TNF-alpha along with systemic injections of LPS frequently lead to attenuation of the later stages of the febrile response but not of the initial phase of fever, indicating that cytokines are rather involved in the maintenance than in the early induction of fever. Within the last years experimental evidence has accumulated indicating the existence of neural transport pathways of immune signals to the brain. Because subdiaphragmatic vagotomy prevents or attenuates fever in response to intraperitoneal or intravenous injections of LPS, a role for vagal afferent nerve fibers in fever induction has been proposed. Also other sensory nerves may participate in the manifestation of febrile responses under certain experimental conditions. Thus, injection of a small dose of LPS into an artificial subcutaneous chamber results in fever and formation of cytokines within the inflamed tissue around the site of injection. This febrile response can be blocked in part by injection of a local anesthetic into the subcutaneous chamber, indicating a participation of cutaneous afferent nerve signals in the manifestation of fever in this model. In conclusion, humoral signals and an inflammatory stimulation of afferent sensory nerves can participate in the generation and maintenance of a febrile response.

Immune response induced in mice oral immunization with cowpea severe mosaic virus

There is increasing interest in the immune response induced by plant viruses since these could be used as antigen-expressing systems in vaccination procedures. Cowpea severe mosaic virus (CPSMV), as a purified preparation (300 g of leaves, 2 weeks post-inoculation), or crude extract from cowpea (Vigna unguiculata) leaves infected with CPSMV both administered by gavage to Swiss mice induced a humoral immune response. Groups of 10 Swiss mice (2-month-old females) were immunized orally with 10 daily doses of either 50 µg viral capsid protein (boosters of 50 µg at days 21 and 35 after immunization) or 0.6 mg protein of the crude extract (boosters of 0.6 mg at days 21 and 35 after immunization). Anti-CPSMV antibodies were quantified by ELISA in pooled sera diluted at least 1:400 at days 7, 14, 21, 28, 35 and 42 after the 10th dose. IgG and IgA against CPSMV were produced systemically, but IgE was not detected. No synthesis of specific antibodies against the proteins of leaf extracts from V. unguiculata, infected or not with CPSMV, was detected. The use of CPSMV, a plant-infecting virus that apparently does not induce a pathogenic response in animals, induced a humoral and persistent (at least 6 months) immune response through the administration of low antigen doses by gavage. These results raise the possibility of using CPSMV either as a vector for the production of vaccines against animal pathogens or in quick and easy methods to produce specific antisera for viral diagnosis.

Immune response to respiratory syncytial virus in young Brazilian children

We have evaluated the cellular and humoral immune response to primary respiratory syncytial virus (RSV) infection in young infants. Serum specimens from 65 patients <=12 months of age (39 males and 26 females, 28 cases <3 months and 37 cases > or = 3 months; median 3 ± 3.9 months) were tested for anti-RSV IgG and IgG subclass antibodies by EIA. Flow cytometry was used to characterize cell surface markers expressed on peripheral blood mononuclear cells (PBMC) from 29 RSV-infected children. There was a low rate of seroconversion in children <3 months of age, whose acute-phase PBMC were mostly T lymphocytes (63.0 ± 9.0%). In contrast, a higher rate of seroconversion was observed in children >3 months of age, with predominance of B lymphocytes (71.0 ± 17.7%). Stimulation of PBMC with RSV (2 x 10(5) TCID50) for 48 h did not induce a detectable increase in intracellular cytokines and only a few showed a detectable increase in RSV-specific secreted cytokines. These data suggest that age is an important factor affecting the infants' ability to develop an immune response to RSV.

Effect of acute nitric oxide synthase inhibition in the modulation of heart rate in rats

Acute nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME) on chronotropic and pressor responses was studied in anesthetized intact rats and rats submitted to partial and complete autonomic blockade. Blood pressure and heart rate were monitored intra-arterially. Intravenous L-NAME injection (7.5 mg/kg) elicited the same hypertensive response in intact rats and in rats with partial (ganglionic and parasympathetic blockade) and complete autonomic blockade (38 ± 3, 55 ± 6, 54 ± 5, 45 ± 5 mmHg, respectively; N = 9, P = NS). L-NAME-induced bradycardia at the time when blood pressure reached the peak plateau was similar in intact rats and in rats with partial autonomic blockade (43 ± 8, 38 ± 5, 46 ± 6 bpm, respectively; N = 9, P = NS). Rats with combined autonomic blockade showed a tachycardic response to L-NAME (10 ± 3 bpm, P<0.05 vs intact animals, N = 9). Increasing doses of L-NAME (5.0, 7.5 and 10 mg/kg, N = 9) caused a similar increase in blood pressure (45 ± 5, 38 ± 3, 44 ± 9 mmHg, respectively; P = NS) and heart rate (31 ± 4, 34 ± 3, 35 ± 4 bpm, respectively; P = NS). Addition of L-NAME (500 µM) to isolated atria from rats killed by cervical dislocation and rats previously subjected to complete autonomic blockade did not affect spontaneous beating or contractile strength (N = 9). In vivo results showed that L-NAME promoted a tachycardic response in rats with complete autonomic blockade, whereas the in vitro experiments showed no effect on intrinsic heart rate, suggesting that humoral mechanisms may be involved in the L-NAME-induced cardiac response.