Effect on the immune system of germ-free piglets of probiotics potentiated with polyunsaturated fatty acids

The present study investigated the influence of polyunsaturated fatty acids (PUFA) on the immune system of germ-free piglets. Oil with increased content of omega-3 PUFA was administered to piglets from the experimental group (EG) for four weeks. Piglets from the control group (CG) received identical volumes of saline solution. At the age of 21 days both groups of germ-free piglets were inoculated perorally with Lactobacillus casei subsp. casei at a dose of 2 ml (1x10(8) mli). At the age of 28 days, i.e. after one-week colonisation of germ-free piglets with Lactobacillus casei subsp. casei, significant differences were recorded in phagocytic activity of neutrophils (PANe) and phagocytic activity of potentially phagocytizing cells (PA) (P < 0.05). Between EG and CG there have been observed no significant differences in absolute numbers of CD4+ and CD8+ T lymphocytes and numbers of IgM cells and in additional investigated parameters - number of CD2+ T lymphocytes, index of phagocytic activity of neutrophils (IPANe) and index of phagocytic activity (IPA).The total number of leukocytes (Le) in EG was also higher. Of the parameters determined in blood serum we observed a significant increase in concentration of alpha linolenic, eicosapentaenoic and docosahexaenoic acids and a parallel decrease in the level of arachidonic acid.     

Gene-conversion in rabbit B-cell ontogeny and during immune responses in splenic germinal centers

Combinatorial diversity is limited in rabbits because only a few V(H) genes rearrange. Most diversification of the primary repertoire is generated by somatic hypermutation and gene conversion-like changes of rearranged V(H) in B cells that migrate to appendix and other gut associated lymphoid tissues (GALT) of young rabbits. The changes are referred to as gene conversion-like because the non-reciprocal nature of the alterations introduced has not yet been demonstrated. There are many similarities between rabbits and chickens in how their B cells develop and diversify their repertoires. However, although the majority of rabbit B cells may have rearranged and diversified their V genes early in life, some B cells in adult rabbits have rearranged VH sequences that are identical or nearly identical to germline sequences. We found these cells in splenic germinal centers (GC) on days 7 and 10 after immunization of normal adult rabbits with DNP-BGG. By day 15, all rearranged V(H) sequences were diversified. We find an overall pattern of splenic precursor cells whose germline or near germline sequences change both by gene conversion and point mutations during early divisions and mainly by point mutations during later divisions. These events, in parallel with diversification of light chain sequences, may produce the diverse combining sites that serve as substrates for further affinity maturation by selection either within GC or later among emigrant cells in sites such as bone marrow. Some of the sequences altered by gene conversion in splenic germinal centers may also produce new members of the B-cell repertoire in adult rabbits comparable to those produced in GALT of neonatal rabbits.     

Somatic hypermutation leads to diversification of the heavy chain immunoglobulin repertoire in cattle

The availability of unique variable (VH), diversity (D), and joining (JH) gene segments in the vertebrate germline determines the extent to which a primary immunoglobulin (Ig) repertoire can be generated through combinatorial rearrangement. Although bovine D segments possess unusual properties, the diversity of the primary Ig heavy chain (IgH) repertoire in cattle is restricted by the dominance of a single family of germline VH genes of limited number and diversity. Cattle therefore must employ other diversification strategies in order to generate a functional IgH repertoire, the main candidates being gene conversion and somatic hypermutation. In considering these possibilities, we predicted that if somatic hypermutation was active during B lymphocyte development, the process would introduce nucleotide substitutions to the VDJ exon and also non-coding region lying downstream of the rearranged JH segment. In contrast, our expectation was that gene conversion would show a greater tendency to confine modification to the IgH coding sequence, leaving intron regions substantially unmodified. An analysis of rearranged IgH sequences from cattle of different ages revealed that the diversification of germline sequences could be observed in very young calves and that substitution frequency increased with age. The age-dependent accumulation of mutations was particularly apparent in the second IgH complementarity-determining region (CDR2). Single base substitutions were found to predominate, with purines targeted more frequently than pyrimidines and transitions favoured over transversions. In non-coding regions, mutations were detected at a normalised frequency that was indistinguishable from that observed in CDR2. These data are consistent with a process of IgH diversification driven predominantly by somatic hypermutation.     

B-1 and B-2 B-cells in the pig cannot be differentiated by expression of CD5

In a number of species, such as mice, humans and cattle, B-cells can be differentiated into two populations based on the surface expression of CD5, a marker normally found on T-cells. These B-cell subsets have been found to differ with regard to location, development and phenotypic characteristics. The B-1 (CD5(+)) B-cells have also been shown to have a more restricted immunoglobulin isotype expression profile, limited combinatorial diversity in immunoglobulin heavy chains and lower somatic hyper-mutation. They are potent producers of IL-10. In the pig, CD5(+) and CD5(-) B-cell populations have previously been described in this laboratory. Here, we show that B-cells isolated and separated into CD5(+) and CD5(-) populations do not differ with regard to immunoglobulin isotype or IL-10 RNA expression, nor do the immunoglobulin heavy chain V(D)J re-arrangements differ in terms of gene usage, CDR3 length and composition or the frequency of hyper-mutations. In conclusion, expression of CD5 cannot be used to differentiate between pig blood B-1 and B-2 B-cells.     

Intermittent Escherichia coli O157:H7 colonisation at the terminal rectum mucosa of conventionally-reared lambs

In cattle, the lymphoid rich regions of the rectal-anal mucosa at the terminal rectum are the preferred site for Escherichia coli O157:H7 colonisation. All cattle infected by rectal swab administration demonstrate long-term E. coli O157:H7 colonisation, whereas orally challenged cattle do not demonstrate long-term E. coli O157:H7 colonisation in all animals. Oral, but not rectal challenge of sheep with E. coli O157:H7 has been reported, but an exact site for colonisation in sheep is unknown. To determine if E. coli O157:H7 can effectively colonise the ovine terminal rectum, in vitro organ culture (IVOC) was initiated. Albeit sparsely, large, densely packed E. coli O157:H7 micro-colonies were observed on the mucosa of ovine and control bovine terminal rectum explants. After necropsy of orally inoculated lambs, bacterial enumeration of the proximal and distal gastrointestinal tract did suggest a preference for E. coli O157:H7 colonisation at the ovine terminal rectum, albeit for both lymphoid rich and non-lymphoid sites. As reported for cattle, rectal inoculation studies were then conducted to determine if all lambs would demonstrate persistent colonisation at the terminal rectum. After necropsy of E. coli O157:H7 rectally inoculated lambs, most animals were not colonised at gastrointestinal sites proximal to the rectum, however, large densely packed micro-colonies of E. coli O157:H7 were observed on the ovine terminal rectum mucosa. Nevertheless, at the end point of the study (day 14), only one lamb had E. coli O157:H7 micro-colonies associated with the terminal rectum mucosa. A comparison of E. coli O157:H7 shedding yielded a similar pattern of persistence between rectally and orally inoculated lambs. The inability of E. coli O157:H7 to effectively colonise the terminal rectum mucosa of all rectally inoculated sheep in the long term, suggests that E. coli O157:H7 may colonise this site, but less effectively than reported previously for cattle.     

The experimental infection of piglets with a porcine reovirus.

A strain (Quebec) of reovirus isolated from the faeces of a pig with dysentery was neutralised by reovirus type 1 antiserum. Four of eight hysterectomy-produced, colostrum-deprived (HPCD) piglets dosed orally with the third cell culture passage of the virus developed diarrhoea and showed focal areas of villous atrophy in the small intestine. The virus was isolated from the intestinal tract of all eight specific pathogen free piglets, but not from three control animals. Nine germ-free piglets dosed orally with the eight cell culture passage of the virus showed neither clinical signs nor lesions, but virus was recovered from their intestinal tracts for 14 days after infection. No virus was isolated from four control germ-free piglets.     

Immune development in jejunal mucosa after colonization with selected commensal gut bacteria: a study in germ-free pigs

The immunological structure of the porcine jejunal lamina propria in germ-free piglets was compared with that of their counterparts associated with two strains of commensal Escherichia coli, A0 34/86 serotype O83:K24:H31 and the O86 E. coli strain, up to 20 days post-colonization. In the antigen-presenting compartment, both dendritic cells (DC) and cells expressing CD163, probably macrophages were investigated. In addition we also assessed the number of CD2+/CD3+ (T) cells. In contrast to some previous reports, we show a total lack of both DC and T cells for germ-free animals in the diffuse lymphoid tissue of villi and crypts of the jejunum. Association with either strain of commensal E. coli had a profound effect on the immune structure and resulted in extensive recruitment of DC to the lamina propria and of T cells to epithelium and lamina propria. The data suggest that the earliest immigrant cells were monocytes, which soon acquired the phenotype of mucosal DC. T cells migrated in at a slightly slower rate. Nevertheless, the response could be extremely rapid: within 3 days of colonization with O83, the magnitude of this response was comparable to that observed 20 days post-colonization.     

Phage display selection and characterization of single-chain recombinant antibodies against Eimeria tenella sporozoites

A single-chain antibody library against Eimeria tenella sporozoites was constructed by phage display. Antibody-displaying phage was selected in five panning rounds against cryopreserved E. tenella sporozoites. A 1000-fold increase in phage output and a 3000-fold enrichment were obtained after three rounds of panning, as the binding clones became the dominant population in the library. Ten clones were randomly selected from the last selection round, and their nucleotide sequences were aligned and compared to chicken germ-line sequences. Analysis of the light chain variable regions revealed possible donor pseudogenes which act as donors in gene conversion events, and contribute to the diversification of the V(L) immune repertoire. Possible somatic hypermutation events, a consequence of affinity maturation, were also identified. Soluble antibody was produced in a non-suppressor E. coli strain, purified by nickel affinity chromatography, and characterized by immunoblotting. In an immunofluorescence assay, this recombinant antibody showed specific binding to E. tenella sporozoites.     

The effect of intramuscular administration of colistin on the development and course of experimentally induced oedema disease in weaned piglets

Shiga-toxigenic E. coli (STEC) strains that produce Shiga toxin Stx2e cause oedema disease in weaned piglets. The purpose of the present study was to investigate the impact of Stx2e released in mesenteric lymph nodes on disease pathogenesis. Colistin and ampicillin were intramuscularly administered to piglets of the experimental group simultaneously challenged with STEC strain, type O139:F18ab, Stx2e+. Piglets of the control group were challenged with STEC only. The strain was naturally resistant to ampicillin and susceptible to colistin. After the challenge, colonisation of the intestines was observed in both antibiotic-treated piglets and control piglets without antibiotic treatment. Histochemistry and scanning electron microscopy revealed sporadic colonisation of the small intestine in the piglets. STEC was detected in the mesenteric lymph nodes of untreated piglets. The clinical manifestations of oedema disease were observed in both groups. In the antibiotic-treated group (11 piglets), oedema disease developed in 10 piglets, eight of which died or were euthanized ante finem. In the untreated group (11 piglets), oedema disease developed in five piglets, four of which died or were euthanized ante finem. We therefore propose that the STEC lysed by colistin suddenly released the toxin from bacterial cells immediately after their passage through the intestinal wall. That could explain a more severe course of oedema disease in the treated piglets. Even though high amounts of STEC were present in the lymph nodes of untreated piglets, the toxin was not released abruptly because the bacterial cells were not damaged.     

Biochemical intestinal parameters in pigs reared outdoors and indoors,and in germ-free pigs

Intestinal microbial functions reflect cross-talk between a host and its flora, and external factors may influence these functions. The aim of this investigation was to follow the development of six biochemical microbial-related functions of piglets, raised outdoors (OPs) or indoors (IPs), from birth to slaughter age. The following parameters (microflora-associated characteristic; MAC) were consecutively measured at five different ages: production of short-chain fatty acids (SCFA), conversion of cholesterol to coprostanol and of bilirubin to urobilinogens, inactivation of trypsin, degradation of beta-aspartylglycine and of mucin. Additionally, four parameters (production of SCFA. conversion of cholesterol to coprostanol, inactivation of trypsin, degradation of beta-aspartylglycine) were investigated in faecal samples from germ-free minipigs. The differences in MAC patterns between OPs and IPs were most pronounced at 20 days of age. Differences were found in the total amount of SCFAs, proportions of the acetic, propionic and butyric acids, conversion of bilirubin to urobilinogens, degradation of faecal tryptic activity and degradation of mucin. The values found in the minipigs were within the range of a germ-free animal characteristic (GAC) pattern. Our results show that environmental factors influence the development of some intestinal microbial functions in pigs.     

Attempted eradication of methicillin-resistant staphylococcus aureus colonisation in horses on two farms

REASONS FOR PERFORMING STUDY: Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging equine and zoonotic pathogen. Infection control protocols can be used to control MRSA in human hospitals, but measures to eradicate MRSA on horse farms have not been evaluated. OBJECTIVES: To describe an MRSA eradication programme that was used to attempt to eliminate MRSA colonisation among horses and horse personnel on 2 equine farms. METHODS: Active surveillance cultures and infection control protocols were implemented on 2 farms with endemic MRSA. RESULTS: Active screening and strict implementation of infection control protocols resulted in a rapid decrease in number of colonised horses on both farms. The majority of horses eliminated MRSA without antimicrobial treatment. On one farm colonisation was eradicated, while only 2 (3%) colonised horses remained on the other farm at the end of the study. CONCLUSIONS: Although at this stage the benefit of eradication of MRSA from populations of horses and cost-benefit studies have not been established, this study illustrates that short-term eradication can be achieved with a policy of segregation, enhanced infection control precautions and repeated testing of groups of animals. POTENTIAL RELEVANCE: Infection control practices should form the basis of MRSA control. Antimicrobial therapy does not appear to be required for eradication of MRSA colonisation in horses and control of MRSA on farms. In appropriate circumstances, these methods may be useful for controlling the spread of this potentially serious pathogen.     

Identification of epitopes recognised by mucosal CD4+ T-cell populations from cattle experimentally colonised with Escherichia coli O157:H7

Vaccines targeting enterohaemorrhagic Escherichia coli (EHEC) O157:H7 shedding in cattle are only partially protective. The correlates of protection of these vaccines are unknown, but it is probable that they reduce bacterial adherence at the mucosal surface via the induction of blocking antibodies. Recent studies have indicated a role for cellular immunity in cattle during colonisation, providing an impetus to understand the bacterial epitopes recognised during this response. This study mapped the epitopes of 16 EHEC O157:H7 proteins recognised by rectal lymph node CD4⁺ T-cells from calves colonised with Shiga toxin producing EHEC O157:H7 strains. 20 CD4⁺ T-cell epitopes specific to E. coli from 7 of the proteins were identified. The highly conserved N-terminal region of Intimin, including the signal peptide, was consistently recognised by mucosal CD4⁺ T-cell populations from multiple animals of different major histocompatibility complex class II haplotypes. These T-cell epitopes are missing from many Intimin constructs used in published vaccine trials, but are relatively conserved across a range of EHEC serotypes, offering the potential to develop cross protective vaccines. Antibodies recognising H7 flagellin have been consistently identified in colonised calves; however CD4⁺ T-cell epitopes from H7 flagellin were not identified in this study, suggesting that H7 flagellin may act as a T-cell independent antigen. This is the first time that the epitopes recognised by CD4⁺ T-cells following colonisation with an attaching and effacing pathogen have been characterised in any species. The findings have implications for the design of antigens used in the next generation of EHEC O157:H7 vaccines.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-016-0374-5) contains supplementary material, which is available to authorized users.     

黄芪茎叶对断奶仔猪盲肠微生物区系的影响

本试验旨在探讨日粮中添加不同比例黄芪茎叶对断奶仔猪盲肠微生物区系的影响。选用24头健康的长×大二元杂交仔猪,按体重随机分成对照组、2.5%黄芪茎叶组、5%黄芪茎叶组和7.5%黄芪茎叶组4组,每组3个重复,每个重复2头仔猪,试验期为28 d。试验结束后,采用高通量测序方法对仔猪盲肠内容物进行分析。结果表明:断奶仔猪盲肠微生物的多样性随黄芪茎叶的添加量不同发生改变,其中2.5%组的微生物多样性最高,盲肠内容物中微生物菌群以Firmicutes(厚壁菌门)和Bacteroidetes(拟杆菌门)为主,占总含量92%以上;在属水平上,2.5%黄芪茎叶组与7.5%黄芪茎叶组的优势菌属数量最高为11种,其中Prevotella菌属的丰度最高。综上所述,在仔猪日粮中添加适量的黄芪茎叶,可降低盲肠p H及提高微生物多样性,有利于断奶仔猪的健康生长。     

Protection of gnotobiotic pigs against Salmonella enterica serotype Typhimurium by rough mutant of the same serotype is accompanied by the change of local and systemic cytokine response

We have demonstrated that severe systemic disease caused by virulent LT2 strain Salmonella enterica serotype Typhimurium in gnotobiotic piglets can be alleviated by oral inoculation with an avirulent rough (R) mutant of the same serotype 24 h before challenge with the virulent strain. Protected piglets had no signs of enteritis. The concentrations of TNF-alpha, IL-1beta, IL-8 and IL-10 were measured by ELISA in ileal washings and plasma of uninfected and infected pigs. The cytokines were not detected in plasma of germ-free piglets, and low concentrations of IL-1beta and IL-8 were found in their ileal washings. The pre-inoculation of the rough mutant induced an increase in IL-8 and decrease in IL-1beta and IL-10 in plasma. The virulent LT2 strain induced very high TNF-alpha concentrations in the ileum which were reduced in the pigs pre-inoculated with the R mutant.     

Salmonella enterica serovar Typhimurium and Enteritidis infection of pigs and cytokine signalling in palatine tonsils

Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1β and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.     

The effect of in vitro antibacterial properties of bovine milk diets on the natural colonisation of newborn piglets with coliform bacteria.

The diets suitable for the hand rearing of piglets in incubators were examined. Diet A was based on cows' milk heated to 56 degrees C and had bacteriostatic and antiadhesive properties against Escherichia coli. Diet B was based on evaporated cows' milk and did not have these properties. The numbers of coliform bacteria naturally colonising the small intestines of newborn piglets fed entirely on these diets for one week did not differ significantly, however both were significantly higher than in control piglets suckled from birth. Faecal counts of coliforms were similar in all three groups. Examination of the bacteriostatic sensitivity of the isolated strains to sows' milk indicated a predominance of milk-sensitive strains colonising the suckled piglets and a predominance of milk-resistant strains in the piglets fed diet B. This diet-dependent colonisation could not be explained by the in vitro bacteriostatic properties of the diets alone.     

The redox potential of the large intestine in swine in relation to swine dysentery

The redox potential of (Eh) +111 +/- 25 mV was measured in the large intestine of newly born piglets. In the post-weaning period the Eh values decreased significantly to -173 +/- 27 mV and remained at this level also in the healthy sows (-214 +/- 55 mV). The Eh value recorded in dysenteric pigs was -188 +/- 5 mV, and this was not statistically significant in relation to the healthy weaned piglets. The Eh level measured in the blood agar prepared with cysteine and covered by a thick growth of the strain Treponema hyodysenteriae, which had been incubated in an anaerobic medium for five days, was -218 +/- 18 mV. The Eh of piglets after weaning was not the decisive condition for the development of dysentery. However, it can be assumed that the impossibility of eliciting dysentery in microbe-free and gnotobiotic pigs is associated with a relatively high redox potential of a microbially unpopulated or insufficiently populated intestine.