Tacrolimus-FK506 induced immunosuppression in gnotobiotic piglets

Tacrolimus (FK506), an inhibitor of calcineurin, is an immunosuppressive agent used in clinical trials of transplant patients. Although FK506 targets Ca(2+)-mediated T-cell signaling, phenotype(s) of the specific target cells and the corresponding cytokine pathways are not well known. In this study, the impact of FK506 on number and characteristic of T-cells in selected lymphoid tissues of gnotobiotic (GB) piglets was determined. FK506-treated GB piglets were compared with untreated GB and conventional piglets. The T-helper, cytotoxic, natural killer, double-positive, and activated T-cell populations were analyzed in suspensions of mononuclear cells isolated from thymus, mesenteric lymph nodes and peripheral blood. In vitro secretion of interleukin-8 and interferon-gamma in concanavalin A-stimulated lymphoid cell-cultures was measured by ELISA. Daily intramuscular treatment of GB piglets with 1mg/kg of FK506 from birth for 4 weeks resulted in lowered (P<0.05) in vitro secretion of interferon-gamma and interleukin-8. Moreover, depletions of MNC in systemic and mucosa-associated lymphoid tissues were observed in piglets treated with FK506. The depletions of mononuclear cells and low levels of interferon-gamma and interleukin-8 in piglets treated with FK506 were accompanied by lower proportion of CD3+, CD2+CD4+ and CD2+CD8+ T-cell phenotypes in peripheral blood but not in thymus and mesenteric lymph nodes. These results indicate that FK506-treatment causes immunosuppression in GB piglet, and this effect could be exploited further to study opportunistic pathogens in pig model.     

Pathogenicity of Isospora suis in gnotobiotic and conventionalised piglets

Isospora suis is unequivocally a primary pathogen of swine. Inoculation of I suis in conventionalised and germ-free piglets caused a biphasic disease course with marked diarrhoea, villous atrophy and necrosis of the intestinal epithelium at four to six and eight to 10 days after inoculation. The presence of a normal bacterial flora markedly (P less than 0.05) influenced the survival rate of piglets but did not appear to affect the histopathological changes observed. Mild limited focal necrosis and bile stasis were present in the liver at eight to 10 days after inoculation. In this period there was also ectasia of lymph vessels in the intestinal lymph nodes.     

Dynamics of Campylobacter jejuni invasion in gnotobiotic piglets

The study deals with the results of the investigation of C. jejuni populations in the intestines of gnotobiotic piglets after oral infection with C. jejuni in the span of one to five days after infection (DAI). The infection of the whole large intestine was revealed, with the onset of pathological lesions 3rd DAI. C. jejuni was isolated from liver, gallbladder, ileum, rectum, colon, mesenterial lymphatic node, rectal smears and blood. From 1st to 5th DAI C. jejuni was demonstrated in liver, gallbladder and posterior part of ileum. In the cecum and rectum C. jejuni was detected as late as on 2nd-3rd DAI to 5th DAI and in mesenterial lymphatic nodes from 2nd to 4th DAI. In rectal smears C. jejuni was found out regularly during the whole period of experiment. C. jejuni was also isolated from inflammatory infiltrate. Histological examination revealed C. jejuni between the villi and in the contents of cup-shaped cells from 2nd to 5th DAI.     

Pathogenesis of intestinal cryptosporidiosis in conventional and gnotobiotic piglets.

The pathogenesis of intestinal cryptosporidiosis was studied in 52 conventionally reared and 20 gnotobiotically reared piglets by inoculation with different doses of Cryptosporidium parvum oocysts. The prepatent period of C. parvum in both groups of animals were variable, depending on the number of oocysts administered. The patent period of C. parvum in conventionally reared piglets was 8 or 9 days; in gnotobiotic piglets cryptosporidia were found in feces until Day post infection (DPI) 16, when the last piglet was necropsied. Cryptosporidiosis in conventionally reared piglets is a self-limited diarrheal disease associated with morphological changes within the intestine. The most severe lesion was seen in the posterior jejunum and ileum from DPI 3 to DPI 7, and consisted of villous atrophy, crypt hyperplasia and inflammatory infiltration in the lamina propria. In gnotobiotic piglets cryptosporidia induced severe enterocolitis which occurred at least until DPI 16. The characteristics of enteric lesions were similar to those found in conventionally reared piglets. Intestinal cryptosporidiosis in both groups of animals shifted in the course of infection in the caudal direction and terminated in the large intestine. Examination by scanning electron microscope showed that infected absorptive cells had thicker and longer microvilli than those on non-infected cells; neighboring non-infected cells were hypertrophic, bulbously protuberant with minute microvilli with no distinct intercellular borders. Numerous cryptosporidia in the heterotopic glandular epithelium in the submucosa of cecum and colon on DPI 9 and 10 were found. No differences in the location and degree of cryptosporidial infection between colostrum-fed and colostrum-deprived conventionally reared piglets were found. Sow's colostrum does not appear to protect piglets from C. parvum infection. The role of intestinal microflora in the pathogenesis of cryptosporidiosis in piglets is discussed.     

Immune response of gnotobiotic piglets against Mycoplasma hyopneumoniae

In this study, several cytokine responses were investigated during Mycoplasma hyopneumoniae (Mhp) infection using a gnotobiotic infection model. We found that several inflammatory cytokines (IL-1beta, IL-8, IL-18, and TNF-alpha) and an anti-inflammatory cytokine IL-10 were induced from peripheral blood mononuclear cells (PBMC) of germ-free (GF) piglets stimulated with heat killed Mhp whole antigens, but no IFN-gamma and IL-4 were induced by Mhp. After the intranasal infection of Mhp, IL-1beta, IL-8, IL-18, and IFN-gamma were also detected in the broncho-alveolar lavage fluids (BALF). The antigen-specific IFN-gamma and IL-10 responses after infection of Mhp were gradually suppressed during Mhp infection as well as non-specific immune response to concanavalin A (ConA) and lipopolysacchalide (LPS) at early stage of infection. These results suggested that Mhp infection modulates the immune response of pigs by inducing several cytokines, and causes immuno-suppression of pigs in a gnotobiotic condition.     

Intranasal infection with Bordetella bronchiseptica in gnotobiotic piglets.

Nineteen gnotobiotic piglets, four to six days old, from three different litters, were inoculated intranasally on three consecutive days with a pure culture of Bordetella bronchiseptica. Necropsy 10 to 30 days after inoculation revealed atrophic rhinitis lesions in all and pneumonia in 13 (73 per cent)infected piglets. One died of septicaemia two days after inoculation. Agglutinating antibodies were detected in the sera of all piglets necropsied from 17 to 30 days after inoculation. Five control piglets showed no lesions or serological changes.     

Experimental colibacillosis in gnotobiotic piglets exposed to 3 enterotoxigenic serotypes

Three strains of enterotoxigenic Escherichia coli (ETEC) (064:KSNT, K88ac; 020:KSNT, K88ac and 08:K85ab, K99) originally cultured from outbreaks of diarrhoea in piglets a few hours old, were administered orally to gnotobiotic piglets. There was a marked age-related difference in the clinical response to infection between the 3 strains although they all produced heat-stable toxin. All 3 strains produced severe clinical signs of depression, anorexia, vomiting, diarrhoea, followed by dehydration and death in one-day-old piglets. In piglets infected at 3 days of age the two K88+ ETEC caused diarrhoea and death but the K99+ ETEC induced moderate diarrhoea only. In piglets infected at 7 days of age, the 064 strain produced severe diarrhoea and death, and 020 strain caused mild diarrhoea in 3 of 6 piglets with one death while the 08 strain caused no illness. Pathological changes in the intestinal tract associated with these infections were minimal, or absent. Immunofluorescent staining with homologous hyperimmune sera demonstrated adherence of the 3 ETEC strains to the brush border of small intestinal epithelial cells. Fluorescing organisms were observed in all infected piglets irrespective of the severity of clinical signs but the degree and extent of colonisation varied with the age of the piglets and the infecting strain. This may explain the difference in clinical response between the 3 strains.     

Intestinal and serum antibody response in gnotobiotic piglets to oral immunization with Escherichia coli

The local and systemic immune response to a formolized E. coli oral vaccine was investigated in 13 gnotobiotic piglets. Beginning at ten days of age animals received a daily dose of 10¹⁰ or 10¹¹ bacteria, on ten consecutive days. Intestinal loop tests with one animal of each group on day 26 showed protection which was more pronounced in the animal dosed 10¹⁰ bacteria compared with the other immunized piglet. Immunoglobulin class-specific antibodies to O and K antigens were determined by ELISA technique. In serum no IgG or IgA antibodies were found, whereas IgM-anti O149 antibodies in both immunized groups reached their highest level at day 4 of dosing and decreased thereafter. IgM-anti K88 antibodies were first detected at day 10 of dosing. Both immunized groups had comparable serum levels at days 20 and 30. Also in gut secretion the IgM antibody response was predominant, and higher levels were found in the 10¹⁰ group than in the 10¹¹ group. IgG and IgA antibody response were also detected in secretion.     

Seroprevalence and risk factors of Mycoplasma suis infection in pig farms in central China

Mycoplasma suis, the causative agent of porcine infectious anemia, causes large economic losses to the swine industry worldwide. A questionnaire-based survey was conducted in 69 pig farms in Hubei Province, China, from November 2011 to August 2013 to ascertain the prevalence and associated risk factors of M. suis. Four thousand and four blood samples from pigs of all the age groups were tested for M. suis antibodies using the established rMSG1–ELISA assay. Among these 4004 samples, 1615 blood samples from multiparous sows were examined to identify the association between seroprevalence and different seasons. Information on risk factors collected from farmers or attending veterinarians was recorded on a pre-designed questionnaire. The overall test seroprevalence of M. suis infection at the animal level was 31.9% (1277/4004; 95% CI: 30.5%, 33.4%), whereas at the farm level, this value was 95.65% (66/69; 95% CI: 87.8%, 99.1%). The seroprevalence of M. suis was higher in replacement gilts (40.6%; 95% CI: 35.1%, 46.3%), multiparous sows (48.2%; 95% CI: 45.8%, 50.7%) and boars (44.4%; 95% CI: 34.5%, 54.8%), as compared to piglets (13.0%; 95% CI: 9.4%, 17.3%), weaned-piglets (10.8%; 95% CI: 8.9%, 13.0%), and growing-finishing pigs (25.0%; 95% CI: 22.0%, 28.3%). In terms of seasons, the prevalence of M. suis in pigs was significantly higher in summer (65.3%; 95% CI: 61.0%, 69.5%) and autumn (65.0%; 95% CI: 59.0%, 70.6%) compared to spring (30.1%; 95% CI: 26.0%, 34.4%) and winter (36.4%; 95% CI: 31.4%, 41.5%). Farm-level risk factors were identified by multivariable logistic regression analysis. The associated factors retained in the final multivariable logistic regression model were drug treatment, presence of mosquitoes and flies, and frequency of disinfection. Drug treatment (OR = 0.24; 95% CI: 0.07, 0.88; P = 0.031) and frequency of disinfection (OR = 0.23; 95% CI: 0.06, 0.90; P = 0.035) were protective factors, and the presence of mosquitoes and flies (OR = 5.994; 95% CI: 1.56, 23.00; P = 0.009) was a risk factor for M. suis infection on farms. The results of the present study provide the first insight into the impact of associated determinants on M. suis infection in central China.     

Immunisation of pregnant sows with a live Salmonella cholerae suis vaccine

Sows were immunised subcutaneously with a live Salmonella cholerae suis (SCS) vaccine prior to farrowing. Serological tests demonstrated a high level of SCS colostral antibodies at parturition. Piglet sera negative for antibodies at birth contained a high level of SCS antibodies 24 h after the ingestion of vaccinated sow colostrum. Experiments were carried out to establish if these maternally derived antibodies could protect the piglets against an intranasal (IN) challenge with a field isolate of SCS. It was concluded that pregnant sows could be safely immunised with a live SCS vaccine thus providing their piglets with a passive immunity which protected them against an intranasal challenge.     

Effects of age and diet on small intestinal structure and function in gnotobiotic piglets

The effects of age and dietary change on the structure and function of piglet small intestine were investigated in gnotobiotic pigs. The small intestinal mucosa was damaged when gnotobiotic pigs were weaned on to a pelleted meal diet; villus height, crypt cell production rate, and activities of brush border enzymes were reduced. Small intestinal damage was associated with reduced weight gain over a three week period; diarrhoea was not observed. The continuous consumption of meal appeared to perpetuate the intestinal damage for three weeks, although evidence of intestinal repair, as indicated by an increased crypt cell production rate, was present three weeks after weaning. Antibodies to soya antigen were detected in serum after weaning and the intestinal damage could have been caused by antibody-mediated immune reactions to soya proteins. Villi of milk fed gnotobiotic pigs shortened significantly with age.     

Early cytokine response of gnotobiotic piglets to Salmonella enterica serotype typhimurium

Cytokine response against Salmonella Typhimurium is traditionally studied in conventional animals. Germ-free animals, however, enable to study response against infection without background effect of other microorganisms. Plasma and ileal inflammatory cytokines in germ-free piglets orally infected with virulent LT2 strain or, with a non-virulent SF1591 rough mutant were quantified by ELISA. In plasma and ileal washes, IFN-gamma levels significantly increased in both infected groups. TNF-alpha and IL-18 were mostly missing in plasma 24 h after infection. In the ileum, IFN-gamma, TNF-alpha, and IL-1beta were induced mainly by the virulent strain, whereas IL-18 was induced in highest quantity by non-virulent Salmonella. These data confirmed an important role of IFN-gamma, as well as other inflammatory cytokines in early stage of salmonellosis.     

Relationship of porcine cytomegalovirus and B bronchiseptica to atrophic rhinitis in gnotobiotic piglets.

Both porcine cytomegalovirus (PCMV) and Bordetella bronchiseptica produced rhinitis and pneumonia when inoculated intranasally into young gnotobiotic pigs. With PCMV the nasal lesions were confirmed to the lamina propria, while Bordetella produced atrophy of the turbinate bones and hyperplasia and degeneration of the nasal epithelium. Some exacerbation of the lesions was observed in the nasal mucosa of pigs given both agents, but the degree of bone atrophy was not increased.     

Differences in the development of the small intestine between gnotobiotic and conventionally bred piglets

The effects of age, weaning and breeding conditions on the small intestinal morphology and the distribution of immunocompetent cells were investigated. The villus height and numbers of CD3+ T-lymphocytes, measured in the duodenum, jejunum and ileum, were determined in both the gnotobiotic and conventionally bred piglets. The diet of gnotobiotic piglets was composed of milk-replacement and feed mixtures. The application of milk replacement was finished on day 28. Conventional piglets were fed on sow's milk and feed mixtures. The animals were weaned on day 28. Small intestines were collected from 12 conventional and 12 gnotobiotic piglets slaughtered at the age of 2, 7, 14, 21, 28 and 35 days. The morphology results demonstrated that duodenal and ileal villi were significantly higher (p < 0.001) in gnotobiotic piglets during the entire period of the experiment. However, the weight of conventional piglets was higher during the experiment, in some cases significantly (p < 0.05). A marked reduction, in some cases significant (p < 0.001, p < 0.01 respectively), of the villi height on day 7 after the diet change for both groups of animals was recorded. The results demonstrate the differences in the gut development between both groups and their relationship to the breeding conditions. Nevertheless, the effect of crucial diet changes was observed independently of them. The immunohistochemistry results showed significantly (p < 0.001, p < 0.01 respectively) higher numbers of CD3+ T-lymphocytes in the jejunal villi of conventionally bred piglets. Similar results, in some cases significant (p < 0.001, p < 0.01 respectively), were also obtained from the other parts of the small intestine. These observations confirm reduced microorganism exposure under the gnotobiotic conditions.