Colonization of preterm gnotobiotic piglets with probiotic Lactobacillus rhamnosus GG and its interference with Salmonella Typhimurium.

A balanced microbiota of the gastrointestinal tract (GIT) is a prerequisite for a healthy host. The GIT microbiota in preterm infants is determined by the method of delivery and nutrition. Probiotics can improve the GIT microbiota balance and suitable animal models are required to verify their harmlessness. Preterm gnotobiotic piglets were colonized with Lactobacillus rhamnosus GG (LGG) to evaluate its safety and possible protective action against infection with an enteric pathogen, Salmonella Typhimurium (ST). Clinical signs (anorexia, somnolence, fever and diarrhea), bacterial interference and translocation, intestinal histopathology, transcriptions of claudin-1, occludin and interferon (IFN)-γ, intestinal and systemic protein levels of interleukin (IL)-8, IL-12/23 p40 and IFN-γ were compared among (i) germ-free, (ii) LGG-colonized, (iii) ST-infected and (iv) LGG-colonized and subsequently ST-infected piglets for 24 h. Both LGG and ST-colonized the GIT; LGG translocated in some cases into mesenteric lymph nodes and the spleen but did not cause bacteremia and clinical changes. ST caused clinical signs of gastroenteritis, translocated into mesenteric lymph nodes, the spleen, liver and blood, increased claudin-1 and IFN-γ transcriptions, but decreased occludin transcription and increased local and systemic levels of IL-8 and IL-12/23 p40. Previous colonization with LGG reduced ST colonization in the jejunum and translocation into the liver, spleen and blood. It partially ameliorated histopathological changes in the intestine, reduced IL-8 levels in the jejunum and plasma and IL-12/23 p40 in the jejunum. The preterm gnotobiotic piglet model of the vulnerable preterm immunocompromised infant is useful to verify the safety of probiotics and evaluate their protective effect.

Interference of Bifidobacterium choerinum or Escherichia coli Nissle 1917 with Salmonella Typhimurium in gnotobiotic piglets correlates with cytokine patterns in blood and intestine.

The colonization, translocation and protective effect of two intestinal bacteria - PR4 (pig commensal strain of Bifidobacterium choerinum) or EcN (probiotic Escherichia coli strain Nissle 1917) - against subsequent infection with a virulent LT2 strain of Salmonella enterica serovar Typhimurium were studied in gnotobiotic pigs after oral association. The clinical state of experimental animals correlated with bacterial translocation and levels of inflammatory cytokines [a chemokine, interleukin (IL)-8, a proinflammatory cytokine, tumour necrosis factor (TNF)-α and an anti-inflammatory cytokine, IL-10] in plasma and intestinal lavages. Gnotobiotic pigs orally mono-associated with either PR4 or EcN thrived, and bacteria were not found in their blood. No significant inflammatory cytokine response was observed. Mono-association with Salmonella caused devastating septicaemia characterized by high levels of IL-10 and TNF-α in plasma and TNF-α in the intestine. Di-associated gnotobiotic pigs were given PR4 or EcN for 24 h. Subsequently, they were infected orally with Salmonella and euthanized 24 h later. Pigs associated with bifidobacteria before Salmonella infection suffered from severe systemic infection and mounted similar cytokine responses as pigs infected with Salmonella alone. In contrast, EcN interfered with translocation of Salmonella into mesenteric lymph nodes and systemic circulation. Pigs pre-associated with EcN thrived and their clinical condition correlated with the absence of IL-10 in their plasma and a decrease of TNF-α in plasma and ileum.

Susceptibility of bifidobacteria to lysozyme as a possible selection criterion for probiotic bifidobacterial strains.

Resistance or susceptibility of bifidobacteria to lysozyme and growth of bifidobacteria in human milk were tested. Susceptible bifidobacterial strains stopped their growth almost immediately after the addition of lysozyme (400 microg/ml), moderately susceptible strains exhibited reduced growth rate, and growth curves of resistant strains were not affected. Strains of human origin were more resistant to lysozyme than animal strains. While strains of B. bifidum grew well in human milk samples, the growth B. animalis strains was inhibited after inoculation to human milk. The resistance to lysozyme seems to be a promising criterion for the selection of new probiotic bifidobacterial strains.

The piglet as a model for B cell and immune system development.

The ability to identify factors responsible for disease in all species depends on the ability to separate those factors which are environmental from those that are intrinsic. This is particularly important for studies on the development of the adaptive immune response of neonates. Studies on laboratory rodents or primates have been ambiguous because neither the effect of environmental nor maternal factors on the newborn can be controlled in mammals that: (i) transmit potential maternal immunoregulatory factors in utero and (ii) are altricial and cannot be reared after birth without their mothers. Employing the newborn piglet model can address each of these concerns. However, it comes at the price of having first to characterize the immune system of swine and its development. This review focuses on the porcine B cell system, especially on the methods used for its characterization in fetal studies and neonatal piglets. Understanding these procedures is important in the interpretation of the data obtained. Studies on neonatal piglets have (a) provided valuable information on the development of the adaptive immune system, (b) lead to important advances in evolutionary biology, (c) aided our understanding of passive immunity and (d) provided opportunities to use swine to address specific issues in veterinary and biomedical research and immunotherapy. This review summarizes the history of the development of the piglet as a model for antibody repertoire development, thus providing a framework to guide future investigators.

Cytokine response to Escherichia coli in gnotobiotic pigs.

One-week-old-germ-free pigs were inoculated with 10(8) CFU of E.coli bacteria-either commensal 086 strain or virulent 055 strain for 1 d. Bacteria were counted in the small intestine, mesenteric lymph nodes, blood and lungs. The O55 strain reached higher levels in circulation and lungs. IL-8, IL-10 and TNF-alpha concentrations were determined by ELISA in plasma and intestinal washes . No difference in cytokine levels was found between control germ-free pigs and their counterparts associated with commensal O86 strain in spite of its high concentration in the gut and circulation.

Probiotics manipulate host cytokine response and induce antimicrobial peptides.

Probiotics modulate production of both cytokine and antimicrobial peptides. This effect can be regarded as a part of complex interplay between them and the host.

Ontogeny of interferon alpha secreting cells in the porcine fetal hematopoietic organs.

We examined the ontogeny of IFN-alpha Secreting Cells (IFN-alpha SC) in different hematopoietic organs and blood of porcine fetuses at different stages of gestation. Cells were induced to produce IFN-alpha by incubation with the coronavirus TGEV and IFN-alpha SC were detected by ELISPOT. A striking finding was that IFN-alpha SC could be detected in the fetal liver as early as at 26 days of gestation, i.e., during the first quarter of gestation, a period at which T-cell markers could not be detected by flow cytometry. In addition, IFN-alpha SC could be detected in the cord blood, the spleen and the bone marrow of fetuses at later stages of gestation. These data indicate that IFN-alpha SC appear very early during the ontogeny of the immune system, long before the development of the specific immune system, and may therefore represent an early antiviral defence mechanism. IFN-alpha SC were found to be associated with hematopoietic organs, which argues for their hematopoietic lineage.

Early ontogeny of immune cells and their functions in the fetal pig.

The origin of immune cells and their products have been studied in the prenatal period in miniature pigs. Macrophages were first detected on day 25, and myelocytes and lymphoid cells by day 28. Membrane antigens SLA-DR and CD45 were found by day 22, membrane molecules MG-7, 8/1, CD1, CD2 and 74-22 by day 28, Gamma/delta T cells were found initially in extrathymic sites (in the liver). The first gamma/delta T cells were detected as early as 40 days of gestation. The expression of fibronectin, Thy-1 and its message, Ig isotypes and the first induction of IFN alpha were described.

Escherichia coli administered into pig amniotic cavity appear in fetal airways and attract macrophages into fetal lungs.

Escherichia coli (2 x 10(4) bacteria) of the non-pathogenic O86 strain or enteropathogenic O55 strain were administered into the pig amniotic cavity at 79 to 86 days of gestation for six or ten hours. Translocation of bacteria into fetal lungs was confirmed by cultivation as well as by light and electron microscopy. Infection caused an influx of macrophages that were immunostained in cryostat sections by monoclonal antibody recognizing calprotectin.

Early ontogeny of monocytes and macrophages in the pig.

Prenatal development of cord blood monocytes and tissue macrophages was studied in pig foetuses by immunophenotyping and functional assays. The function of peripheral blood monocytes was compared in germ-free and conventional piglets. First macrophages were identified by electron microscopy in foetal liver on the 25th day of gestation. Monoclonal antibodies against porcine CD45 and SWC3 antigens were used for flow cytometric identification of myelomonocytic cells in cell suspensions prepared from the yolk sac, foetal liver, spleen and cord blood. Leukocytes expressing the common myelomonocytic antigen SWC3 were found in all organs studied since the earliest stages of development. Opsonized zymosan ingestion assay was used to determine the phagocytic capacity of foetal mononuclear phagocytes isolated from cord blood, liver and spleen. In the foetal liver, avid phagocytosis of apoptic cells had been found to occur before cells were able to ingest zymosan in vitro. The first cells capable of ingesting zymosan particles were found on the 40th day of gestation in umbilical blood and 17 days later in foetal spleen and liver. Their relative proportion increased with age. Cord blood monocytes and peripheral blood monocytes in germ-free piglets had low oxidatory burst activity as shown by iodonitrophenyl tetrazolium reduction assay. A remarkable increase of oxidatory burst activity was observed in conventional piglets, probably due to activation of immune mechanisms by the microflora colonizing gastrointestinal tract.

Induction of inflammatory cytokines by Nocardia fractions.

NDCM and NLD fractions of Nocardia opaca cell walls were used for in vitro stimulation of human and porcine peripheral blood mononuclear cells. Inflammatory cytokines IL-1, IL-6, tumour necrosis factor (TNF) alpha and interferon (IFN) gamma were detected at the single-cell level using paraformaldehyde-fixed and saponin-permeabilized mononuclears stained with cytokine-specific antibodies and indirect immunofluorescence method. IL-1, IL-6 and TNF were produced by human monocytes stimulated for 2 h, IFN gamma-positive lymphocytes were detected later. IFN gamma was produced also by activated porcine lymphocytes. Cells expressing apoptotic features were found among blood mononuclears treated with Nocardia fractions.

Distribution of gamma delta T cells in the pig foetus.

The distribution of gamma delta T cells during different developmental stages of pig foetuses was studied using monoclonal antibodies directed against a ruminant WC1 antigen, a T-cell receptor (TCR) subset epitope, and pig Null T-cell antigen. Binding was revealed by the avidin/biotin/peroxidase technique on cryostat sections and flow cytometry analysis. The extrathymic origin of gamma delta T cells was confirmed: gamma delta T cells appeared in the liver, spleen and peripheral blood sooner than in the thymus. In the course of the second half of gestation, these T cells were found in the dermis and intestinal mucosal compartments which represented the predominant loci of gamma delta T cells after birth.

Cytokines and other important inflammatory mediators in gestation and bacterial intraamniotic infections.

Intraamniotic infections caused by viruses, bacteria or mycoplasmas are frequently followed by damage of fetus or increased perinatal morbidity and mortality. Cytokines are key substances regulating a number of biological processes including reproductive and inflammatory processes. An association between intraamniotic infections, rising concentrations of inflammatory cytokines in amniotic fluid and preterm labor is suggested. A great effort is made to find reliable markers typical for intraamniotic infections with high predictive value that make possible prompt identification of patients with intraamniotic infection. This review concerns inflammatory mediators, especially IL-1, IL-6, IL-8, TNF-alpha, and other important biologically active substances as prostaglandins and NO metabolites and their roles in intraamniotic infections. Finally, we discuss their relevance for diagnosis of intraamniotic infections.

Expression of inflammatory markers in pig amnion after intraamniotic infection with nonpathogenic or enteropathogenic Escherichia coli.

The pig amnion was in vivo intraamniotically infected with E. coli for 10 h at 80-85 d of gestation either with the nonpathogenic O86 strain or enteropathogenic O55 strain. TNF-alpha, IL-10, IL-1beta and IFN-gamma were determined in amniotic fluids by ELISA, the expression of cytokines and some other inflammatory markers was determined by immunohistochemistry. Intraamniotic infection induced high levels of TNF-alpha in amniotic fluids which correlated with bacterial virulence whereas IL-10 was induced only by O86. The IL-1beta level did not increase significantly and was expressed in all infected membranes. IFN-gamma was negligible or absent. TNF-alpha, IL-12p40, calprotectin, HSP65 and gp91phox were found by immunohistochemistry only in amnion membranes infected with the enteropathogenic strain 055.

Prenatal expression of the 65-kDa heat-shock protein homologue in pig tissues.

A monoclonal antibody (ML30) recognizing the 65-kDa heat-shock protein of mycobacteria and reacting with homologous human protein was found to stain various porcine tissues. Development of this reactivity was studied. The first ML30-positive cells were embryonic hepatocytes. The protein reacting with the ML30 antibody was localized predominantly in the Golgi area and mitochondria of hepatocytes. Cell membranes of some peripheral blood lymphocytes were also found to bind ML30.

Expression of TNF-alpha in pig fetal cells stimulated in vitro.

Macrophages and lymphocytes of pig fetuses stimulated in vitro with bacterial mitogens such as lipopolysaccharide and Nocardia opaca delipidated cell mitogen showed a high TNF-alpha cytoplasmic expression. TNF-alpha was detected by immunofluorescence in peripheral blood lymphocytes and lymphocytes from the thymic region as early as at 34 d of gestation. Macrophages were the main producers of TNF-alpha at later developmental stages.

Nitric oxide metabolites in gnotobiotic piglets orally infected with Salmonella enterica serovar typhimurium.

Reactive NO metabolites play a distinct role in the control of Salmonella enterica serovar Typhimurium (ST; a facultative intracellular pathogen) in susceptible host. A significant increase of nitrite and/or nitrate plasma levels, 3-nitro-tyrosine expression and pathological changes in mesenteric lymph nodes have been observed in gnotobiotic piglets orally infected for 1 d with a virulent strain of ST but not in piglets infected with a rough mutant of ST.

Immunomodulatory effects of Bacillus firmus.

3-day-old miniature piglets were stimulated in vivo with Bacillus firmus by the intraperitoneal or intragastric route for 1 d. Cells containing IgA and IgG2 were detected in the ileum in all stimulated but not in control animals. The frequency of blood CD3+ cells increased after intraperitoneal administration of B. firmus, the ratio of polymorphonuclears to lymphocytes increased in all stimulated piglets. B. firmus induced antitumor immunity in rats with transplanted Yoshida sarcoma cells. Granular lymphocytes and dead tumor cells were found in peritoneal exudate of stimulated animals. B. firmus induced IFN-gamma synthesis in human blood lymphocytes stimulated in vitro for 1 d. The amount of TNF-alpha produced by these stimulated human peripheral blood mononuclears (PBMC) was lower than that of PBMC stimulated with some other bacterial immunomodulators. Cells containing TGF-beta or IL-8 were not found in human PBMC stimulated with B. firmus.

Systemic and local cytokine response of young piglets to oral infection with Salmonella enterica serotype Typhimurium.

One-week-old breast-fed miniature piglets were orally infected either with virulent LT2 strain or with a non-virulent SF1591 rough mutant of Salmonella Typhimurium for 1 d. Both microorganisms were cultivated from mesenteric lymph nodes but not from the blood of infected piglets. Interleukins (IL) 1 beta, 8, 18, tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) were quantified by ELISA in plasma and washes of a terminal part of the small bowel. In plasma, cytokines were mostly missing in non-infected piglets and either missing or low in infected piglets. In the gut of non-infected piglets, IL-1 beta, IL-8 and IL-18 were detected whereas TNF-alpha and IFN-gamma were mostly missing. IFN-gamma levels highly increased (p < 0.05) after infection with nonvirulent salmonellae. The variability of cytokine levels in the gut of suckling piglets is discussed.

Effect of controlled antigenic stimulation on lymphocyte subsets in pigs and pig fetuses.

The effect of controlled antigenic stimulation in immunologically virgin organisms, i.e. pig fetuses treated with NDCM (Nocardia delipidated cell mitogen) and germ-free (GF) piglets associated with a non-pathogenic E. coli 086, on peripheral blood lymphocyte subsets defined by the expression of CD5 and CD8 was studied by double color flow cytometry. Stimulation of both fetuses and GF piglets increased the frequency of CD8low+ lymphocytes. A prominent subset of CD5-CD8low+NK cells was present in GF and E. coli associated piglets and their frequency was slightly higher in E. coli associated animals. The most pronounced difference between stimulated and non-stimulated animals was in a relative proportion of an ill-defined lymphocyte subset with an unusual CD5low+CD8low+ expression. Both NDCM injection into fetal blood circulation and association of GF piglets with E. coli resulted in a marked increase of frequency of CD5low+CD8low+ lymphocytes in peripheral blood.