Studies on the phagocytic activity of the reticuloendothelial system (RES) to rough and smoothEscherichia coli in young conventional and germfree guinea pigs

The functional (phagocytic) capacity of the reticuloendothelial system (RES) of young conventional and germfree guinea pigs was studied using thein vivo blood clearance test of living bacteria (rough and smoothEscherichia coli). It was found that as previously shown in newborn germfree piglets, the smooth strain was taken up from the blood stream of germfree guinea pigs very slowly whereas roughEscherichia coli was phagocytosed effectively. The inability of the RES of germfree guinea pigs to phagocytose the smooth strain is not due to a functional incapability of phagocytic cells, but it reflects rather the lack of serum opsonins to this strain. This was demonstrated in experiments in which smooth bacteria, sensitized prior to injection into the blood circulation with specific antiserum, were phagocytosed as effectively as the rough strain. It is assumed that effective phagocytosis of rough strain is due to the presence of non-specific opsonins (e.g. components of the complement system). In young conventional guinea pigs both strains,i.e. smooth and rough, were taken up from the blood stream very effectively thus indicating that sufficient levels of serum opsonins for both strains were present. This fact could be correlated with the finding that in sera of conventional guinea pigs haemagglutinating antibodies to both strains ofEscherichia coli could be detected, whereas in sera of young germfree guinea pigs, no antibodies to usedEscherichia coli strain were found. The importance of serum opsonins for effective phagocytosis of bacteria by RE cellsin vivo is discussed.     

Selective decontamination,induced colonization resistance and connected immunological changes in piglets

14-d-old conventional piglets were picked from normal piggery, washed with disinfectants, placed into isolators suitable for germfree work, fed a sterile diet and treated with peroral antibiotics (nalidixic acid, kanamycin, and nystatin). Beginning with day 5 or 7, Enterobacteriaceae were not found in feces. The absence of these bacteria was proved by inoculation of germfree newborn piglets with caecal content. In selectively decontaminated piglets, the white blood cell count in blood had fallen to 6 X 10(9)/L; this decrease was due to an extremely low number of granulocytes (to 0.8 X 10(9)/L). On day 35, IgG-positive cells almost disappeared from the spleen, whereas IgA cells were found in an unusually great amount. Corresponding changes in serum levels were established. The colonization resistance effect in Enterobacteriaceae-deprived piglets was confirmed; settling of introduced various E. coli strains did not occur or was delayed.     

Influencing the immune parameters in germ‐free piglets by administration of seal oil with increased content of ω‐3 PUFA

Oral administration of oil with an increased content of ω‐3 polyunsarurated fatty acids to germfree piglets resulted in a significant increase in the total values of CD4, CD8 lymphocytes, B lymphocytes, and monocytes, in peripheral blood in comparison with the controls. The metabolic activities of phagocytes as well as the polyclonal activation of lymphocytes were not significantly influenced. The level of growth factor was significantly higher, as determined on the basis of somatomedin in the blood serum. Biochemical indices showed a significant increase in the level of eicosapentaenoic and docosahexaenoic acids in blood serum and the decrease in the level of arachidonic acid at the same time.     

Effect of the EM Bokashi® Multimicrobial Probiotic Preparation on the Non-specific Immune Response in Pigs

The aim of the study was to determine the effect of EM Bokashi® on the phagocytic activity of monocytes and granulocytes, oxidative burst, SWC3, and CD11b + CD18+ expression on monocytes and granulocytes, and the serum concentration of cytokine and lysozyme in pig. 60 Sixty female piglets were divided into two groups: I – control and II – experimental. For the experimental group, a probiotic in the form of the preparation EM Bokashi® was added to the basal feed. Flow cytometry was used to determine selected non-specific immune response parameters, intracellular production of hydrogen peroxide by peripheral granulocytes and monocytes, and surface particles in peripheral blood. The EM Bokashi® preparation used in the study was found to increase phagocytic activity mainly in monocytes, with an increased percentage of phagocytic cells in the experimental group. The highest serum lysozyme concentration in the piglets in the experimental group (2.89 mg/dl), was noted on day 42 of the study. In the group of pigs receiving EM Bokashi®, the percentage of phagocytic cells with SWC3 (monocyte/granulocyte) expression was statistically significantly higher than in the control. The increase in the number of cells with SWC3 (monocyte/granulocyte) expression in the peripheral circulation in combination with the greater capacity of the cells for phagocytosis and respiratory burst confirms that the non-specific immune response was modulated in the pigs supplemented with EM Bokashi®.

     

Lactobacillus gasseri LF221 and K7 — from isolation to application

The article presents research findings on two human strains with probiotic activity. On the basis of API 50 CHL fermentation pattern, PCR by species-specific primers and sequencing of the V2–V3 region of 16S rRNA both strains designated as LF221 and K7 were identified as members of the Lactobacillus gasseri species. Two LF221 bacteriocins, acidocin LF221 A and B were purified and sequenced. They were classified as members of the two-component class II bacteriocins. Among basic probiotic properties, the survival under conditions in gastro-intestinal tract, ability to adhere to cultured intestinal enterocytes and pig’s mucosa and stimulation of the immune response were demonstrated. In in vivo study of 24 weaned piglets, the survival rate of K7 Rif^r and LF221 Rif^r was quantified by selective enumeration on MRS agar with rifampicin. The survival of both strains was good (2.9 × 10⁵ cfu of K7 Rif^r /g faeces; 4.8 × 10⁵ cfu of LF221 Rif^r /g) and the LF221 Rif^r /K7 Rif^r viable cells were found either in the mucosa of duodenum, jejunum or in the ileum. The possible effect of K7 to inhibit adhesion of E. coli O8:K88 to enterocytes was studied on Caco-2 cultured cells, on tissue obtained from small intestines of pigs and in vivo on gnotobiotic piglets. Lactobacilli were found to be effective in reducing E. coli adhesion to enterocytes in Caco-2 model, but not on mucosa of pig’s jejunum under ex vivo conditions. Competitive exclusion, production of organic acids and stimulation of immune response, were involved in inhibition of E. coli by K7 strain in gnotobiotic piglets. Any inflammatory change in intestines of piglets treated with K7 was observed, which confirmed its safe use. Among the technological parameters the survival and activity of the strains during cheese-making are presented. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Distribution and effects of a defined six-member murine-derived microflora in gnotobiotic gerbils.

The gnotobiotic gerbil was selected as a model with which to study the effects of colonization with a defined microflora on organ morphology, histology, and selected blood biochemical parameters. Gerbils were maintained germfree for 13 months but failed to reproduce, presumably because of the enlarged cecum. A colony of gnotobiotic gerbils that was associated with a bacterial flora consisting of Lactobacillus brevis, Streptococcus faecalis, Staphylococcus epidermidis, Bacteroides vulgatus, Enterobacter aerogenes, and a Fusobacterium sp. was established. These gnotobiotic gerbils had smaller ceca than germfree gerbils and proved capable of reproduction. Except for the presence of large numbers of Bacteroides organisms in the stomach and greater numbers of S. epidermidis in gnotobiotic gerbils, the number and location of gastrointestinal bacteria were similar in conventional and gnotobiotic gerbils. Bacteroides sp. was the second most predominant microorganism present in gnotobiotic gerbils, whereas clostridia were reported to be the second most predominant microorganism in conventional gerbils. Microscopic examination of direct-impression smears indicated that fusobacteria were present on mucosal surfaces. Intestines of gnotobiotic gerbils weighed twice as much as the intestines of conventional gerbils. Intestinal tissue water weight values from conventional and gnotobiotic gerbils were similar. Histological examination of gerbil intestinal tissue revealed no cellular hypertrophy and no evidence of inflammation in gnotobiotic gerbil intestines. Spleens of gnotobiotic gerbils showed no germinal center stimulation. Statistical differences in total serum glucose, serum protein, and hematocrit levels were found between conventional and gnotobiotic gerbils.     

Effects of age, gender and time on receptor expression and anti-Aspergillus functions of human phagocytes

Phagocytes play a central role in immune defense. Their dysfunction predisposes to infections. This study determined the expression level of nine receptors involved in Aspergillus immune response as well as the values of phagocytosis and production of radical oxygen species after Aspergillus stimulation, in a healthy adult population.The expression values of the CD11b, CD11c, CD14, CD18, CD35, CD181, CD182, CD282 and CD284 receptors on peripheral human monocytes and granulocytes was established. A heterogenous expression of the CD282 on granulocytes was observed as CD181, CD182 and CD284 on monocytes. Similarly, we observed considerable variation in the expression of these receptors over time. Only CD282 on granulocytes varied with sex. No variation with age was observed. Adherence of Aspergillus conidia to phagocytes was dependent of individual, sex, age and time. A better characterization of these innate immunity parameters is necessary to develop in the future an immunologic surveillance strategy for transplant recipients.     

Nonspecific resistance in germ-free and E. coli monocontaminated miniature piglets]

Phagocytic activity of leukocytes, as well as the complement, properdin, and lysozyme levels in the blood serum of miniature piglets, germfree and monocontaminated with E. coli 055 and E. coli 083, were studied. E. coli 055 phagocytosis was decreased in the presence of autologous serum and complement and increased under the effect of specific opsonins (antibodies to E. coli 055). Complement, properdin, and lysozyme levels were decreased in the germfree, in comparison with conventional animals. In the E. coli contaminated piglets properdin and complement production was stimulated most, and lysozyme formation--less. No antibodies to E. coli 055 were revealed in monocontaminated piglets. The highest lysozyme levels were found in the ex-germfree animals, this indicating the participation of factors other than E. coli contamination in lysozyme stimulation. It is concluded that microbial contamination played an important role in the development of cellular and humoral factors of the organism resistance.     

Immunological state of adult germfree miniature minnesota pigs

Fundamental hematological and immunological data were obtained on sexually mature germfree miniature pigs fed, after the milk diet period, with cereal-type diet sterilized by γ-radiation, and were compared with data of control conventional animals. Germfree adult pigs had a lower count of peripheral blood leukocytes with a lower percentage of neutrophil granulocytes and without any younger forms, a lower total serum protein level with a negligible amount of γ and α₂ globulin fractions and a higher serum albumin and β globulin level. In the mesenteric lymph nodes and in spleen, surface IgA-bearing cells predominated over surface IgG-bearing cells. Also a large amount of IgA-containing cells was found in the intestinal lamina propria, where the IgG cells were present in a negligible amount. IgM cells were the most frequent surface isotype in peripheral blood. The count of blood T lymphocytes was more than doubled.     

Effects of Lactobacillus casei and Enterococcus faecalis on growth performance,immune function and gut microbiota of suckling piglets

This study was carried out to investigate the effects of orally administrated Lactobacillus casei and Enterococcus faecalis on performance, immune function and gut microbiota of suckling piglets. Neonatal piglets (n = 120) were randomly assigned to 4 groups, with 30 suckling piglets in each group. The piglets were from 15 litters, one male and one female piglet were selected for each group in each litter. The Control group was administrated with normal saline, the other groups with L. casei or E. faecalis or a combination of L. casei and E. faecalis at a ratio of 3:1. Each piglet was orally administrated with 1, 2, 3 and 4 ml probiotics or normal saline at the age of 1, 7, 14 and 21 d, respectively. The piglets were weaned at the age of 21 d. The results showed that compared with the Control group, the average daily gain of piglets administrated with probiotics was significantly increased, and the diarrhoea rate and mortality were significantly decreased (p < 0.05). After supplementation of the combined probiotics, the protease activity in stomach, duodenum and colon was increased and in all supplemented groups, the immunoglobulin A concentration in plasma was significantly higher (p < 0.05). The combined probiotics significantly increased villus length and the expression level of transforming growth factor-β in the jejunum (p < 0.05) but decreased the expression level of the jejunal tumour necrosis factor-α (p < 0.05). In addition, probiotics could regulate gut microbiota and increase microbial similarity coefficients for keeping piglet gut microbiota stable.     

Endogenous ileal losses of nitrogen and amino acids in pigs and piglets fed graded levels of casein

Abstract

In order to determine ileal losses of nitrogen (N) and amino acids (AA) and the coefficients of apparent and true ileal digestibility (AID, TID) of N and AA from casein in piglets and pigs, two experiments were conducted. In Experiment 1, 24 piglets were used. The piglets were weaned at 17 days of age, weighing 6.4 kg and cannulated at terminal ileum. Ileal digesta was collected at 28 – 29 and 35 – 36 days of age in period 1 and 2, respectively. Feed intake was 150 and 300 g · d^?1 during the first and second period. In Experiment 2, 16 castrates weighing 52.5 kg and cannulated at terminal ileum were used. The intake level of digestible energy was 2.5 times their maintenance requirement. The experiment lasted 7 days and ileal digesta was collected on day 6 – 7. Treatments consisted of four levels of N from casein: 8, 16, 24 and 32 g N · kg^?1 feed, respectively. Results showed that N level did not increase N or AA ileal losses. In piglets, N and AA ileal losses were similar between periods, except for period 2, where losses per kg DMI were about 47 and 64% higher for glycine and proline, respectively (p < 0.05). When ileal losses from pigs and piglets were compared, piglets had higher (p < 0.05) ileal losses of N and AA (excepted glutamic acid and alanine). A lower (p < 0.05) AID was observed in piglets in period 2 for N, methionine, glutamic acid, glycine and proline. With exception of glycine in pigs, all values for TID of N and AA of casein were superior to 0.90. Piglets had higher TID of N, leucine, isoleucine, valine and phenylalanine. These results showed that piglets have higher ileal losses than pigs.     

Oxygen Affects Gut Bacterial Colonization and Metabolic Activities in a Gnotobiotic Cockroach Model

The gut microbiota of termites and cockroaches represents complex metabolic networks of many diverse microbial populations. The distinct microenvironmental conditions within the gut and possible interactions among the microorganisms make it essential to investigate how far the metabolic properties of pure cultures reflect their activities in their natural environment. We established the cockroach Shelfordella lateralis as a gnotobiotic model and inoculated germfree nymphs with two bacterial strains isolated from the guts of conventional cockroaches. Fluorescence microscopy revealed that both strains specifically colonized the germfree hindgut. In diassociated cockroaches, the facultatively anaerobic strain EbSL (a new species of Enterobacteriaceae) always outnumbered the obligately anaerobic strain FuSL (a close relative of Fusobacterium varium), irrespective of the sequence of inoculation, which showed that precolonization by facultatively anaerobic bacteria does not necessarily favor colonization by obligate anaerobes. Comparison of the fermentation products of the cultures formed in vitro with those accumulated in situ indicated that the gut environment strongly affected the metabolic activities of both strains. The pure cultures formed the typical products of mixed-acid or butyrate fermentation, whereas the guts of gnotobiotic cockroaches accumulated mostly lactate and acetate. Similar shifts toward more-oxidized products were observed when the pure cultures were exposed to oxygen, which corroborated the strong effects of oxygen on the metabolic fluxes previously observed in termite guts. Oxygen microsensor profiles of the guts of germfree, gnotobiotic, and conventional cockroaches indicated that both gut tissue and microbiota contribute to oxygen consumption and suggest that the oxygen status influences the colonization success.     

Effects of weaning age and formic acid-based feed additives on pigs from weaning to slaughter

Abstract

Two hundred and forty piglets were used in a 2×6 factorial experiment to study the effects of weaning age (26 or 36 d) and four formic acid-based feed additives on the performance of pigs from weaning to slaughter. Either formic acid (F) or a mixture of formic acid, propionic acid, and potassium sorbate (FPS) or a mixture of formic acid, propionic acid, and sodium benzoate (FPB) or formic acid in a diatomaceous earth carrier (FD) were added to the diets of weaned piglets (from weaning to 60 d of age) and growing (18 – 46 kg) and finishing pigs (46 – 107 kg) to provide 8, 6, and 6 g acid per kg feed, respectively. The negative control treatment's (C) diets contained no growth promoters, whereas the positive control treatment's weaner and grower diets were supplemented with 40 mg/kg of avilamycin (A). The piglets weaned at the age of 26 and 36 d weighed 7.6 and 10.7 kg at weaning (p < 0.001), and 18.5 and 17.9 kg at the age of 60 d (p > 0.05), respectively. There was a weaning age×feed additive interaction in the weight gain of piglets after weaning (p < 0.05). The weight gain of piglets weaned on day 26 was enhanced by A, FPS, and FD (p < 0.05), and that of piglets weaned on day 36 by A and FPB (p < 0.05). The feed conversion ratio was not affected by weaning ages but was decreased in groups A, F, FBS, and FPB (p < 0.05). The severity of post-weaning diarrhoea was less in groups A, F, FPS, and FD than in C (p < 0.05). In piglets weaned on day 26, faecal water content and the total Escherichia coli count were highest 9 d after weaning. The total E. coli count was reduced only by FD (p < 0.05). Increased faecal water content was characterized by increased faecal Na⁺ and decreased K⁺ concentrations. Weaning age did not influence performance or carcass quality in the growing-finishing pigs. Feed additives did not affect weight gain in the growing pigs, but FPS and FPB enhanced weight gain during finishing period and total fattening (p < 0.05). In summary, the pigs' growth performance from weaning to slaughter was not affected by weaning age but it was enhanced by mixtures of formic and propionic acids with small amounts of sorbate or benzoate.     

The phagocytic activity of reticuloendothelial system of newborn precolostral pigs to smooth and roughEscherichia coli

The fate of smooth and rough strains ofEscherichia coli in the blood stream of newborn, germ-free, colostrum deprived piglets was studied using blood clearance test with live and radioisotope-labelled (P³²) bacteria. Smooth strains are eliminated at an extremly slow rate (hours) whereas rough strains are taken up within 30 minutes. Specific immune serum accelerates rapidly the clearance rate of smooth strains after bothin vitro andin vivo opsonisation of bacteria. Clearance of smooth liveEscherichia coli from the blood stream cannot be stimulated by an endotoxin treatment of newborn piglets. The possible role of complement system as a nonspecific opsonin in phagocytosis of rough strains is discussed. The results demonstrate that RES cells of newborn precolostral piglets are functionally competent as to the capacity to remove gramnegative bacteria from the blood stream provided specific and/or nonspecific opsonins are available.     

Indigenous bacteria influence the number of Salmonella typhimurium in the ileum of gnotobiotic mice.

Gnotobiotic BALB/c mice associated with indigenous Lactobacillus, Bacteroides, and two fusiform-shaped Clostridium strains had fewer S. typhimurium present in the ileum 3 days after intragastric challenge with the pathogen than did similarly challenged germfree mice. Acetic and butyric acids were detected in the caecal contents of the gnotobiotic mice, but in smaller concentration than was present in conventionalized mice. No difference in the motility of the small intestine was detected between germfree, gnotobiotic, and conventionalized mice.     

Liquid preservation of highly purified human granulocytes

Highly purified human granulocytes isolated from continuous flow centrifugation leukapheresis concentrates by counterflow centrifugation-elutriation were stored at 4 °C in concentrations of 6 × 10⁶ to 1 × 10⁷ granulocytes per milliliter for up to 14 days. The in vitro physiological function assays of phagocytosis, oxygen consumption associated with phagocytosis, bacterial growth inhibition, chemotaxis, and five enzyme analyses indicated good storage survival for up to 4 days. Stored granulocytes separated from other blood cells have greater storage stability than granulocytes stored as leukapheresis concentrates. After 14 days of storage a small percentage of granulocytes still maintained all physiological functions, with the exception of chemotaxis. Of the five enzymes assayed, only the enzyme activity of leucine aminopeptidase decreased significantly by the 14th day of storage. The storage stability of each physiological function assayed decreased as follows: bacterial growth inhibition (most stable), phagocytosis, oxygen consumption, and chemotaxis (least stable).     

Local Shwartzman phenomenon in germ-free guinea pig]

Eleven germfree and two monoassociated with the Citrobacter guinea pigs, and 25 conventional animals were injected with the heat-inactivated Citrobacter or E. coli for the induction of the local Schwartzman phenomenon. All the gnotobiotic pigs gave a positive reaction. Infiltration at the site of intracutaneous injection was found in all the conventionals, but in none of the gnotobiotics. The data are discussed from the aspect of primary and secondary sequelae of the absence of host microflora.     

Influence of <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> I5007 on the intestinal and systemic immune responses of healthy and <Emphasis Type="Italic">E. coli</Emphasis> challenged piglets

The effect of feeding Lactobacillus fermentum I5007 on the immune system of weaned pigs with or without E. coli challenge was determined. Twenty-four weaned barrows (6.07 ± 0.63 kg BW) were randomly assigned to one of four treatments (N = 6) in a factorial design experiment. The first two treatments consisted of healthy piglets with half of the pigs receiving no treatment while the other half was orally administered with L. fermentum I5007 (10⁸ CFU/ml) at a daily dose of 20 ml. Pigs in the second two treatments were challenged on the first day with 20 ml of E. coli K88ac (10⁸ CFU/ml). Half of these pigs were not treated while the remaining pigs were treated with 20 ml of L. fermentum I5007 (10⁸ CFU/ml). Peripheral blood lymphocytes subsets were determined using flow cytometry. The intestinal mucosal immunity of the pigs was monitored by real time polymerase chain reaction. The cytokine content of the pig’s serum was also analyzed. Oral administration of L. fermentum I5007 increased blood CD4⁺ lymphocyte subset percentage as well as tumor necrosis factor-α and interferon-γ expression in the ileum. Pigs challenged with E. coli had elevated jejunal tumor necrosis factor-α while interferon-γ expression was increased throughout the small intestine. There was no difference in the concentration of the cytokines interleukin-2, interleukin-6, tumor necrosis factor-α and interferon-γ in the serum. CD8⁺ and CD4⁺/CD8⁺ in peripheral blood were not affected by treatment. In conclusion, L. fermentum I5007 can enhance T cell differentiation and induce ileum cytokine expression suggesting that this probiotic strain could modulate immune function in piglets.     

Dietary Enterococcus faecalis LAB31 Improves Growth Performance,Reduces Diarrhea,and Increases Fecal Lactobacillus Number of Weaned Piglets

Lactic acid bacteria (LAB) have been shown to enhance performance of weaned piglets. However, few studies have reported the addition of LAB Enterococcus faecalis as alternatives to growth promoting antibiotics for weaned piglets. This study evaluated the effects of dietary E. faecalis LAB31 on the growth performance, diarrhea incidence, blood parameters, fecal bacterial and Lactobacillus communities in weaned piglets. A total of 360 piglets weaned at 26 ± 2 days of age were randomly allotted to 5 groups (20 pens, with 4 pens for each group) for a trial of 28 days: group N (negative control, without antibiotics or probiotics); group P (Neomycin sulfate, 100 mg/kg feed); groups L, M and H (supplemented with E. faecalis LAB31 0.5×10⁹, 1.0×10⁹, and 2.5×10⁹ CFU/kg feed, respectively). Average daily gain and feed conversion efficiency were found to be higher in group H than in group N, and showed significant differences between group H and group P (P₀ < 0.05). Furthermore, groups H and P had a lower diarrhea index than the other three groups (P₀ < 0.05). Denaturing gradient gel electrophoresis (DGGE) showed that the application of probiotics to the diet changed the bacterial community, with a higher bacterial diversity in group M than in the other four groups. Real-time PCR revealed that the relative number of Lactobacillus increased by addition of probiotics, and was higher in group H than in group N (P₀ < 0.05). However, group-specific PCR-DGGE showed no obvious difference among the five groups in Lactobacillus composition and diversity. Therefore, the dietary addition of E. faecalis LAB31 can improve growth performance, reduce diarrhea, and increase the relative number of Lactobacillus in feces of weaned piglets.