Use of anaerobic cecal microflora, lactose and acetic acid for the protection of broiler chicks against experimental infection with Salmonella Typhimurium and Salmonella enteritidis

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

Use of cecal microflora cultured under aerobic or anaerobic conditions in the control of experimental infection of chicks with Salmonella Enteritidis

One-day-old broiler chicks received cecal microflora (CM) cultured under aerobic, anaerobic conditions, or both (mixed) and were then infected with Salmonella Enteritidis, in order to compare the efficacy of these different types of culture in terms of the number of chicks infected, cecal colonization and faecal excretion of the challenging bacteria. Regardless of culture type, CM always led to a smaller number of S. Enteritidis for any of the parameters studied compared to untreated chicks. Aerobic CM demonstrated better efficacy in reducing the number of infected chicks and cecal colonization by S. Enteritidis, followed by mixed CM. No difference was observed in faecal excretion of S. Enteritidis between the chicks that received different types of CM culture. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Efeito protetor da microbiota cecal congelada e liofilizada sobre a infecção experimental de frangos de corte por Salmonella enterica sorovar Enteritidis

Estudaram-se os efeitos do tratamento de frangos de corte com microbiota cecal anaeróbia liofilizada (MCL) e congelada (MCC) sobre a infecção do trato digestivo das aves por Salmonella enterica sorovar Enteritidis. Foi usada microbiota intestinal sem prévia identificação bacteriana. A infecção foi persistente, em ordem, no ceco, inglúvio e duodeno. A infecção também foi autolimitante nos grupos tratados e no controle. Não ocorreu diferença entre o grupo-controle positivo e os tratados com MCL ou MCC. Houve redução da colonização do ceco no período de 12 dias após o desafio nos grupos tratados com MCL e MCC, o que não ocorreu no grupo-controle positivo. Não houve variação entre os tratamentos com MCL e MCC quanto às características pesquisadas. A S. Enteritidis reduziu o ganho de peso médio nas aves inoculadas. Os tratamentos com MCL e MCC minimizaram a redução de peso nos grupos infectados.

Efeito da vacina contra coccidiose sobre a colonização de Salmonella enteritidis em frangos de corte tratados com microbiota cecal anaeróbia

Avaliou-se o efeito da vacina contra coccidiose sobre a habilidade da microbiota cecal anaeróbia (MCA), administrada por diferentes vias (aves tratadas), em proteger pintos de corte da colonização por Salmonella enteritidis (Se). Utilizaram-se 120 aves assim divididas: grupo A, pulverização de MCA em aves vacinadas contra coccidiose e desafiadas com Se, grupo B, inoculação endoesofágica de MCA em aves vacinadas e desafiadas, grupo C, MCA na água de bebida de aves vacinadas e desafiadas, grupo D, aves não tratadas, vacinadas e desafiadas, grupo E, aves não tratadas, não vacinadas e desafiadas e, grupo F, aves não tratadas, não vacinadas e não desafiadas (controle negativo). Utilizaram-se como parâmetros a colonização do trato digestivo por Se e sua presença nas fezes, bem como o peso corporal das aves, avaliados aos dois, sete e 12 dias após o desafio. Nas aves tratadas com MCA, especialmente por meio de pulverização e inoculação endoesofágica, a colonização do ceco por Se e sua presença nas fezes foram menores, mostrando que a ação da MCA contra a colonização de ceco e excreção fecal não foi afetada pelo uso da vacina contra coccidiose em pintos de corte e que a associação MCA/vacina contra coccidiose pode ser utilizada sem que haja comprometimento na eficácia da MCA. Nos grupos que não receberam MCA, vacinados ou não contra coccidiose, houve aumento da colonização cecal, bem como excreção fecal da amostra desafio. O ceco foi o local de maior presença e persistência da Se. O resultado de administração de MCA pela água de bebida não foi tão eficiente, mas somente este tratamento resultou em peso corporal das aves significativamente superior ao das aves do grupo não tratado, não vacinado e desafiado, indicando que a presença de salmonelas paratifóides não interfere na produtividade de frangos de corte. Não se observou diferença no peso das aves dos grupos D (vacinados contra coccidiose) e E (não vacinadas) desafiadas com Se, demonstrando que a vacina não influenciou negativamente o ganho de peso das aves desafiadas com Salmonela enteriditis.

Efeito da microbiota cecal e do Lactobacillus salivarius inoculados in ovo em aves desafiadas com Salmonella enterica sorovar Enteritidis

Ovos embrionados provenientes de matrizes pesadas foram inoculados na câmara de ar com microbiota cecal total, microbiota cecal diluída e cultura de Lactobacillus salivarius, no 18º dia de incubação. Dois dias após o nascimento, as aves foram desafiadas com Salmonella enterica sorovar Enteritidis (SE) e, cinco dias após o desafio, avaliou-se a presença da bactéria no fígado e ceco. O efeito de exclusão competitiva, após o desafio com SE, somente foi observado pela ausência da bactéria no fígado das aves tratadas in ovo com L. salivarius. A inoculação in ovo de microbiota cecal indefinida ou diluída não reduziu a colonização de SE no fígado e no ceco das aves, incluindo, neste último, também o tratamento com L. salivarius. Nenhum dos tratamentos in ovo determinou índice de eclodibilidade superior a 65%.

Ácidos orgânicos e microbiota cecal anaeróbia no controle da infecção experimental de frangos por Salmonella typhimurium e Salmonella enteritidis

The aim of this investigation was to study the effect of organic acids and/or anaerobic cecal microflora (ACM) on systemic and digestive infection of broilers by Salmonella typhimurium and S. enteritidis. ACM was used without previous bacterial identification. The treatment with ACM increased the resistance to Salmonella spp infection. Infection was more evident in caeca, followed by rectum and crop and did not interfere on body weight of broilers. Treated and control groups showed the same degree of infection at the end of the experiment. The use of ACM isolated or combined with acetic acid, reduced the colonization of the chick's digestive system by S. typhimurium and S. enteritidis. Acetic acid added to ACM did not potentiate the reduction of digestive system colonization. Except for the crop, the isolated use of acetic, propionic or formic acids did not reduce S. typhimurium and S. enteritidis, in caeca and rectum. The use of organic acids and ACM had little effect on reduction of caecum pH. The treatment with ACM reduced the quantity of S. enteritidis in the faeces. The reduction of caecum pH did not reduce the quantity of S. enteritidis in faeces. S. enteritidis was much more invasive than S. typhimurium and use of organic acids and ACM had little effect on reduction of systemic infection.

Detection of T lymphocytes in intestine of broiler chicks treated with Lactobacillus spp. and challenged with Salmonella enterica serovar enteritidis

The expression of immune response in the form of leukocytic infiltrate by CD3+, CD4+, and CD8+ cells in the epithelium and in the intestinal lamina propria of chicks was studied in the present work by means of immunohistochemical reaction. The chicks were treated with Lactobacillus spp. or cecal microflora (CM) and experimentally challenged or not with Salmonella enterica serovar Enteritidis. The 320 birds utilized were divided into 4 groups containing 80 chicks each and submitted to treatments with Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus acidophilus, and CM. Each group was subdivided into 4 subgroups of 20 birds each and classified into a subgroup that did not receive treatment (negative control), subgroup treated, subgroup treated and challenged with Salmonella Enteritidis, and subgroup only challenged with Salmonella Enteritidis (positive control). The results obtained show that the treatment with L. reuteri, L. salivarius, L. acidophilus, or CM and challenged or not with Salmonella Enteritidis determine immune response in the form of leukocytic infiltrate by CD3+ and CD8+ lymphocytes followed by CD4+ in the epithelium and in the lamina propria of the duodenum, jejunum, and cecum of chicks up to 12 d of age. The quantity of CD3+ lymphocytes was significantly higher (P < 0.05) in the intestine of chicks treated with L. acidophilus or CM and challenged or not with Salmonella Enteritidis; however, the higher quantity of CD8+ lymphocytes was in the intestine of chicks treated with CM and challenged with Salmonella Enteritidis. The duodenum was the segment in which the immune response by T cells (CD3+, CD4+, and CD8+) was stimulated with the greatest intensity, followed by, respectively, the jejunum and cecum. The quantity of CD3+ lymphocytes present in the duodenum, jejunum, and cecum increases with the age of chicks, independent of the stimulus determined by treatments or challenge.

Detection of CD4+ and CD8+ lymphocytes in the intestine of broiler chicks treated with lactobacillus spp. and challenged with Salmonella enterica serovar enteritidis

The expression of immune response as a leukocytic infiltrate by CD4+ and CD8+ cells in the epithelium and in the intestinal lamina propria of chicks fed Lactobacillus spp or cecal microflora (CM) and experimentally challenged or not with Salmonella enterica serovar Enteritidis (SE) was studied using immunohistochemistry. Three hundred and twenty day-of-hatch broiler chicks were divided into four groups of 80 birds each and orally received L. reuteri, L. salivarius, L. acidophilus, or CM. Each group was subdivided into four subgroups of 20 birds each, classified as follows: a subgroup did not receive any oral treatment (negative control), subgroup treated with L. spp or CM, subgroup treated with L. spp or CM and challenged with SE, and subgroup only challenged with SE (positive control). The results show that the oral treatment with L. reuteri, L. salivarius, L. acidophilus, or CM and challenge or not with SE stimulated bird immune response as determined by the leukocytic infiltrate by CD8+ lymphocytes followed by CD4+ in the epithelium and in the lamina propria of the duodenum, jejunum, and cecum of chicks up to 12 days of age. CD8+ lymphocyte number was significantly higher in the intestine of chicks receiving CM and challenged with SE. The duodenum, followed by the jejunum, were the segments in which the immune response, as shown by T, CD4+ and CD8+ cells, was stimulated with the greatest intensity.

Enrichment and hydrogen production by marine anaerobic hydrogen-producing microflora

Acid, alkali, heat-shock, KNO3 and control pretreatment methods applied to anaerobic sludge were evaluated for their ability to selectively enrich the marine hydrogen-producing mixed microflora. Seawater culture medium was used as the substrate. The hydrogen yield of pretreated microflora was higher than that of the un-pretreated control (P < 0.05). Among the pretreatment methods studied, heat-shock pretreatment yielded the greatest hydrogen production, which was 14.6 times that of the control. When the effect of initial pH on hydrogen production of heat-shock pretreated samples was studied, hydrogen was produced over the entire pH range (pH 4-10). The hydrogen yield peaked at initial pH 8 (79 mL/g sucrose) and then steadily decreased as the initial pH increased. Sucrose consumption was high at neutral initial pH. During the process of hydrogen production, pH decreased gradually, which indicated that the acquired microflora consisted of acidogenic bacteria.

Anaerobic digestion process for biogas and biomolecules production: microflora identification and characterization

The anaerobic process was efficient in organic matter removal. During the process, an interesting compound as quercetin was produced inside of reactor. Phylogenetic analysis showed the presence of phylotypes affiliated with gamma-Proteobacteria, Choroflexi, and Bacteroidetes. Archaea were represented by phylotypes belonging to the genus Methanosarcina and Methanosaeta.

Multi-Study Assessment of Vaginal Cytokines and Microflora in the Safety Evaluation of Intravaginal Rings in Pig-Tailed Macaques

BACKGROUND: HIV microbicide trials have emphasized the need to evaluate the safety of topical microbicides and delivery platforms in an animal model prior to conducting clinical efficacy trials. An ideal delivery device should provide sustainable and sufficient concentrations of effective products to prevent HIV transmission while not increasing transmission risk by either local mucosal inflammation and/or disruption of the normal vaginal microflora.

METHODS: Safety analyses of macaque-sized elastomeric silicone and polyurethane intravaginal rings (IVRs) loaded with candidate antiretroviral (ARV) drugs were tested in four studies ranging in duration from 49 to 73 days with retention of the IVR being 28 days in each study. Macaques were assigned to 3 groups; blank IVR, ARV-loaded IVR, and naïve. In sequential studies, the same macaques were used but rotated into different groups. Mucosal and systemic levels of cytokines were measured from vaginal fluids and plasma, respectively, using multiplex technology. Changes in vaginal microflora were also monitored. Statistical analysis (Mann-Whitney test) was used to compare data between two groups of unpaired samples (with and without IVR, and IVR with and without ARV) for the groups collectively, and also for individual macaques.

RESULTS: There were few statistically significant differences in mucosal and systemic cytokine levels measured longitudinally when the ring was present or absent, with or without ARVs. Of the 8 proinflammatory cytokines assayed a significant increase (p = 0.015) was only observed for IL8 in plasma with the blank and ARV loaded IVR (median of 9.2 vs. 5.7 pg/ml in the absence of IVR). There were no significant differences in the prevalence of H2O2-producing lactobacilli or viridans streptococci, or other microorganisms indicative of healthy vaginal microflora. However, there was an increase in the number of anaerobic gram negative rods in the presence of the IVR (p= < 0.0001).

CONCLUSIONS: IVRs with or without ARVs neither significantly induce the majority of potentially harmful proinflammatory cytokines locally or systemically, nor alter the lactobacillus or G. vaginalis levels. The increase in anaerobic gram negative rods alone suggests minimal disruption of normal vaginal microflora. The use of IVRs as a long-term sustained delivery device for ARVs is promising and preclinical studies to demonstrate the prevention of transmission in the HIV/SHIV nonhuman primate model should continue.

Molecular Identification and Characterization of Ileal and Cecal Fungus Communities in Broilers Given Probiotics, Specific Essential Oil Blends, and Under Mixed Eimeria Infection

Broiler digestive tract fungal communities have gained far less scrutiny than that given corresponding bacterial communities. Attention given poultry-associated fungi have focused primarily on feed-associated toxin-producers, yeast, and yeast products. The current project focused on the use of pyrosequencing and denaturing gradient gel electrophoresis (DGGE) to identify and monitor broiler digestive fungal communities. Eight different treatments were included. Four controls were an Uninfected-Unmedicated Control, an Unmedicated-Infected Control, the antibiotic bacitracin methylene disalicylate plus the ionophore monensin as Positive Control, and the ionophore monensin alone as a Negative Control. Four treatments were two probiotics (BC-30 and Calsporin) and two specific essential oil blends (Crina Poultry Plus and Crina Poultry AF). All chickens except the Unmedicated-Uninfected Control were given, at 15 days of age, a standard oral Eimeria inoculum of sporulated oocysts. Ileal and cecal digesta were collected at pre-Eimeria infection at 14 days of age and at 7 days post-Eimeria infection at 22 days of age. Extracted cecal DNA was analyzed by pyrosequencing to examine the impact of diet supplements and Eimeria infection on individual constituents in the fungal community, while DGGE was used to compare more qualitative changes in ileal and cecal communities. Pyrosequencing identified three phyla, seven classes, eight orders, 13 families, 17 genera, and 23 fungal species. Ileal and cecal DGGE patterns showed fungal communities were clustered mainly into pre- and post-infection patterns. Post-infection Unmedicated-Uninfected patterns were clustered with pre-infection groups demonstrating a strong effect of Eimeria infection on digestive fungal populations. These combined techniques offered added versatility towards unraveling the effects of enteropathogen infection and performance enhancing feed additives on broiler digestive microflora.

Use of Pyrosequencing and Denaturing Gradient Gel Electrophoresis to Examine the Effects of Probiotics and Essential Oil Blends on Digestive Microflora in Broilers Under Mixed Eimeria Infection

A protective digestive microflora helps prevent and reduce broiler infection and colonization by enteropathogens. In the current experiment, broilers fed diets supplemented with probiotics and essential oil (EO) blends were infected with a standard mixed Eimeria spp. to determine effects of performance enhancers on ileal and cecal microbial communities (MCs). Eight treatment groups included four controls (uninfected-unmedicated [UU], unmedicated-infected, the antibiotic BMD plus the ionophore Coban as positive control, and the ionophore as negative control), and four treatments (probiotics BC-30 and Calsporin; and EO, Crina Poultry Plus, and Crina PoultryAF). Day-old broilers were raised to 14 days in floor pens on used litter and then were moved to Petersime batteries and inoculated at 15 days with mixed Eimeria spp. Ileal and cecal samples were collected at 14 days and 7 days postinfection. Digesta DNA was subjected to pyrosequencing for sequencing of individual cecal bacteria and denaturing gradient gel electrophoresis (DGGE) for determination of changes in ileal and cecal MC according to percentage similarity coefficient (%SC). Pyrosequencing is very sensitive detecting shifts in individual bacterial sequences, whereas DGGE is able to detect gross shifts in entire MC. These combined techniques offer versatility toward identifying feed additive and mild Eimeria infection modulation of broiler MC. Pyrosequencing detected 147 bacterial species sequences. Additionally, pyrosequencing revealed the presence of relatively low levels of the potential human enteropathogens Campylobacter sp. and four Shigella spp. as well as the potential poultry pathogen Clostridiun perfringens. Pre- and postinfection changes in ileal (56%SC) and cecal (78.5%SC) DGGE profiles resulted from the coccidia infection and with increased broiler age. Probiotics and EO changed MC from those seen in UU ilea and ceca. Results potentially reflect the performance enhancement above expectations in comparison to broilers not given the probiotics or the specific EO blends as feed supplements.

Assessment of anaerobic sewage sludge quality for agricultural application after metal bioleaching

The effects of metal bioleaching on nutrient solubilization, especially nitrogen and phosphorous, from anaerobically-digested sewage sludge were investigated in this work. The assessment of the sanitary quality of the anaerobic sludge after bioleaching was also carried out by enumerating indicator (total coliforms, fecal coliforms, and fecal streptococci) and total heterotrophic bacteria. The experiments of bioleaching were performed using indigenous sulphur-oxidizing bacteria (Thiobacillus spp.) as inoculum and samples of anaerobically-digested sludge. Nitrogen and phosphorous solubilization from sewage sludge was assessed by measuring, respectively, the concentration of Total Kjeldahl Nitrogen, ammonia, nitrate/nitrite, and soluble and total phosphorous before and after the bioleaching assays. At the end of the experiment, after 4 days of incubation (final pH of 1.4), the following metal solubilization yields were obtained: zinc, 91%; nickel, 87%; copper, 79%; lead, 52%; and chromium, 42%. As a result of sludge acidification, the viable counts of selected indicator bacteria were decreased to below the detection limit (4 × 103 cfu 100 ml-1), followed by an increase in the mineral fraction of nitrogen (from 6 to 10%) and in the soluble fraction of phosphorous (from 15 to 30%). Although some loss of sludge nutrients can occur during solid-liquid separation following bioleaching, its beneficial effects as metal removal and reduction of pathogenic bacteria are sufficient to consider the potential of this treatment before sludge disposal onto agricultural fields.

Tamponamento cecal: aspectos clínico, fisiopatológico e terapêutico na laminite experimental em eqüinos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)