Antagonistic effect of indigenous skin bacteria of brook charr (Salvelinus fontinalis) against Flavobacterium columnare and F. psychrophilum

Industrial fish production exposes fish to potentially stressful conditions, which in turn may induce infections by opportunistic pathogens. Probiotics appear to be a promising way to prevent opportunistic infections in aquaculture. In this study, we tested the inhibitory potential of endogenous bacterial communities found in the mucus of brook charr (Salvelinus fontinalis) against two major pathogens Flavobacterium columnare and Flavobacterium psychrophilum. Nine bacterial strains were isolated from brook charr skin mucus and tested for potential antagonistic activity. Results from both agar diffusion assays and broth co-culture assays showed the presence of antagonism. We identified seven bacterial strains, collected from unstressed fish, which exerted strong antagonism against F. psychrophilum and/or F. columnare. These strains were mixed and used to treat columnaris disease in an in vivo experiment in which four distinct fish families were tested. This treatment resulted in a decrease of mortality (54-86%) across fish families indicating that candidates from the host microbiota are potentially suitable for probiotic development. This would allow for the efficient (ability to adhere and colonize the host mucus) and durable management (antagonistic effect against pathogens which would be harmless for the host and safe for its environment) of opportunistic diseases in aquaculture.     

Adhesion Properties of Enterococci to Intestinal Mucus of Different Hosts

The adhesive capacity of selected enterococci to human, canine and porcine intestinal mucus was investigated in order to select for potential probiotic strains with good adhesive properties for human or animal use. The adhesion to the human intestinal mucus of the tested strains was found to range from log10 3.8 to log10 8.6 cfu per microtitre plate well. The highest adhesion to the human intestinal mucus was found among strains from horse faeces, dog faeces and dog feed. The adhesion to canine mucus was observed to range from log10 3.8 to log10 8.3 cfu/well, with the highest adhesive capacity among strains from dog faeces, horse faeces and dog's feed; on average log10 7.9, 7.3 and 7.0 cfu/well, respectively. Isolates from dogs did not bind at higher levels to canine mucus than to human mucus. A strong correlation was observed for the adhesion to human and canine intestinal mucus (p < 0.0001) and also between porcine and canine or human mucus (p < 0.05 for both). When comparing the adhesion of Enterococcus faecium and E. faecalis, no statistical significant differences were observed for any of the tested mucus types. The tested Enterococcus strains were found to exhibit a strain dependent on in vitro adhesion to human, canine and porcine intestinal mucus and did not exhibit host specificity in their adhesion, though some sources appeared to contain more adhesive strains than others. To our knowledge, this is the first report on the in vivo adhesion to intestinal mucus of a large number of enterococci from different sources.     

1株鲤鱼源植物乳杆菌的分离鉴定及其生物学特性分析

【目的】筛选具有优良益生特性的鲤源乳酸菌作为水产养殖用微生态制剂候选菌株。【方法】以鲤鱼肠道内容物及黏膜为菌株的分离来源，采用MRS培养基进行菌株分离纯化，经16S rRNA测序鉴定后，对分离菌株生长特性、产酸能力、耐酸、耐胆盐、抑菌特性、药物敏感性及安全性等生物学特性进行分析。【结果】试验成功分离到1株植物乳杆菌，将其命名为YY001，该分离株在MRS培养基上菌落形态为圆形、表面光滑、边缘整齐、呈乳白色，革兰氏染色为阳性、无芽孢、两端钝圆的短小杆菌；4 h后进入对数期生长，12 h后生长达到稳定期；具有较强的产酸能力，产酸曲线显示，0~12 h pH迅速下降，培养16 h的菌液pH达3.8；在pH 3.0和0.3%胆盐的条件下具有一定耐受性；该分离株对水产常见致病菌柱状黄杆菌、维气氏单胞菌和嗜水气单胞菌均具抑菌效果，且对柱状黄杆菌的抑菌效果最显著，抑菌圈直径达34.19 mm；药物敏感性试验结果显示，分离株对卡那霉素、链霉素和万古霉素耐药；每天一次连续1周灌胃10⁸CFU分离菌株对小鼠无毒害作用。【结论】本研究分离到的菌株YY001具有优良的生物学特性，可作为水产养殖用微生态制剂候选菌株，为后续制备鲤鱼用微生态制剂奠定基础。     

Interaction of Campylobacter spp. and Human Probiotics in Chicken Intestinal Mucus

Campylobacter is the most common cause of bacterial food‐borne diarrhoeal disease throughout the world. The principal risk of human contamination is handling and consumption of contaminated poultry meat. To colonize poultry, Campylobacter adheres to and persists in the mucus layer that covers the intestinal epithelium. Inhibiting adhesion to the mucus could prevent colonization of the intestine. The aim of this study was to investigate in vitro the protective effect of defined commercial human probiotic strains on the adhesion of Campylobacter spp. to chicken intestinal mucus, in a search for alternatives to antibiotics to control this food‐borne pathogen. The probiotic strains Lactobacillus rhamnosus GG and Propionibacterium freudenreichii ssp. shermanii JS and a starter culture strain Lactococcus lactis ssp. lactis adhered well to chicken intestinal mucus and were able to reduce the binding of Campylobacter spp. when the mucus was colonized with the probiotic strain before contacting the pathogen. Human‐intended probiotics could be useful as prophylactics in poultry feeding for controlling Campylobacter spp. colonization.     

Selection of enterococci for potential canine probiotic additives

Enterococci are important inhabitants of animal intestine and are widely used in probiotic products. A potentially successful probiotic strain is expected to have several desirable properties in order to be able to exert its beneficial effects. Forty enterococcal isolates from dog faeces were tested for characters believed to be important for probiotic strains; bacteriocin production, resistance or tolerance to antibiotics, low pH, bile tolerance and adhesive activity. The total count of enterococci was found to be 3.3-7.3log(10)CFU/g of faeces. Most identified strains were Enterococcus faecium. All strains were sensitive to vancomycin, ampicillin, penicillin and chloramphenicol. Thirty-three percentage of strains were resistant to erythromycin and 28% to tetracycline. Among 40 isolates, 75% showed a broad inhibitory spectrum only against Gram-positive indicator bacteria. Seven strains with broad bacteriocin activity were selected for further assays. In the presence of 1% bile, the survival rate of selected strains ranged between 72 and 98%. Survival of strains at pH 3.0 was found in the range between 76 and 87% after 3h. The adhesion of the tested strains to intestinal mucus ranged from 4 to 11% for canine mucus and from 5 to 8% for human mucus. E. faecalis EE4 and E. faecium EF01 showed the best probiotic properties. It indicates that they could be used as new candidate probiotic strains after in vivo testing.     

Requirement for capsular antigen KX105 and fimbrial antigen CS1541 in the pathogenicity of porcine enterotoxigenic Escherichia coli O8:KX105 strains.

The requirement for capsular antigen KX105 and fimbrial antigen CS1541 in the pathogenicity of porcine enterotoxigenic Escherichia coli O8:KX105 strains lacking the colonization factor antigens K88, K99, 987P and F41 was investigated using two encapsulated strains and their acapsular variants, one of which produced the fimbrial antigen CS1541 in vitro. None of the strains adhered in vitro to enterocytes isolated from newborn colostrum-deprived piglets. All of the strains caused diarrhea in orally infected, hysterotomy-derived, colostrum-deprived piglets although a great variability in the clinical response of the piglets was observed. Colonization of the small intestine of infected piglets by these strains was only moderate and no differences in the ability to colonize the small intestine was noted between the strains. All of the strains reacted in the indirect fluorescent antibody test with both CS1541 and 987P antisera when applied to organisms in the intestines of infected piglets. A control strain expressing the 987P fimbrial adhesin also reacted with the CS1541 antiserum applied to organisms in the intestines of an infected piglet. It was concluded that capsular antigen KX105 was not essential for intestinal colonization and production of diarrhea in hysterotomy-derived colostrum-deprived pigs, and that fimbrial antigen CS1541 does not promote in vitro adherence to enterocyte brush borders but could be important in bacterial colonization in vivo.     

饲粮纤维对家禽肠道健康的调控及作用机制

近年来由于抗生素在临床上的滥用,使得抗菌药物的抗菌效果大大降低,抗生素滥用还会导致有害菌定殖增加,影响人和动物的健康。因此,很多国家开始禁止用抗生素作为饲料添加剂,且在饲料中添加低剂量的抗生素作为肠道微生态调节剂已不再受欢迎,而探寻新的抗生素替代品来调节畜禽肠道微生物区系和维持肠道健康显得非常重要。人们一直误认为饲粮纤维是一种抗营养因子,指出纤维能稀释饲粮能量水平,影响家禽的生产性能。但最新研究表明,在家禽饲粮中添加适量纤维是改善家禽肠道健康、减少家禽肠道紊乱的一种有效的营养调控措施。本文从家禽肠道发育、肠道黏膜形态及上皮组织健康、肠道消化生理、肠道微生物区系等方面综述饲粮纤维对家禽肠道健康的影响,为进一步研究纤维对家禽肠道健康的调控机制提供参考,也为今后在家禽饲粮中添加纤维性物质提供一定的科学依据。     

Probiotic alternatives to reduce gastrointestinal infections: the poultry experience

The intestinal mucosa represents the most active defense barrier against the continuous challenge of food antigens and pathogenic microorganisms present in the intestinal lumen. Protection against harmful agents is conferred by factors such as gastric acid, peristalsis, mucus, intestinal proteolysis, and the intestinal biota. The establishment of beneficial bacterial communities and metabolites from these complex ecosystems has varying consequences for host health. This hypothesis has led to the introduction of novel therapeutic interventions based on the consumption of beneficial bacterial cultures. Mechanisms by which probiotic bacteria affect the microecology of the gastrointestinal tract are not well understood, but at least three mechanisms of action have been proposed: production/presence of antibacterial substances (e.g., bacteriocins or colicins), modulation of immune responses and specific competition for adhesion receptors to intestinal epithelium. The rapid establishment of bacterial communities has been thought to be essential for the prevention of colonization by pathogenic bacteria. Some animal models suggest that the reduction in bacterial translocation in neonatal animals could be associated with an increase in intestinal bacterial communities and bacteriocin-like inhibitory substances produced by these species. This review emphasizes the role of the intestinal microbiota in the reduction of the gastrointestinal infections and draws heavily on studies in poultry.     

一株母猪产道益生性粪肠球菌的分离鉴定

产道作为母猪生殖系统的重要器官组织,与繁殖性能密切相关。本试验无菌采集江西省某养殖场窝产14头母猪产道中段阴道分泌物进行传统细菌分离培养,初步筛选出4株细菌,挑选其中一株通过生化反应和16S rDNA序列分析方法进行鉴定,同时进行抗生素抵抗能力试验和牛津杯法体外抑制试验。生化鉴定结果表明该菌可降解果糖、乳糖、半乳糖等,该菌代谢产物与葡萄糖酸盐无反应,葡萄糖产气试验说明该菌产酸不产气。分离菌株通过NCBI同源性比对为粪肠球菌（登录号EU794735.1）。分离菌对青霉素、头孢哌酮等高度敏感,对红霉素、庆大霉素等耐药。体外抑制试验中分离菌对大肠杆菌k88和变形杆菌具有良好的抑制效果,可以作为候选益生菌株进行深入研究。这为今后益生菌代替抗生素及产道菌群移植防治母猪产道炎症提供理论基础。     

猪源乳酸杆菌的分离鉴定及生物学特性分析

乳酸杆菌制剂可以替代抗生素,用于预防和治疗细菌性腹泻。为获取可作为微生态制剂的候选菌株,从健康仔猪肠道分离到5株乳酸杆菌,经抑菌试验、生长曲线测定、耐酸、耐胆盐、耐胰酶试验等,筛选到1株对大肠杆菌、沙门氏菌、金黄色葡萄球菌等致病菌有较好的抑制作用,对低pH值、高胆盐、胰酶均有一定耐受能力的乳酸杆菌,命名为LY7。经PCR鉴定,该菌株为约氏乳杆菌。     

Bile tolerant <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> isolated from pig feces inhibits enteric bacterial pathogens and porcine rotavirus

Lactic acid producing bacterial strain Probio-16 was isolated from the swine excrements under anaerobic conditions and characterized by morphology and biochemical characteristics. The strain was further identified by 16S rRNA gene sequencing and phylogeneitc analysis. The antimicrobial activity of the strain was assayed by testing for growth inhibition of thirteen pathogenic microorganisms. The strain was tested for antiviral activity against porcine rotavirus in vitro in African green monkey epithelial cell line TF-104. Antibiotic susceptibility of the strain against 13 antibiotics was tested using disk diffusion method. Phenotypically and through 16S rRNA gene sequences, Probio-16 was identified and named as Lactobacillus reuteri Probio-16. This strain was resistant to pH 2.0, 5% porcine bile and exhibited antimicrobial activity against all the thirteen enteric bacterial pathogens tested. Probio-16 supernatant inhibited porcine rotavirus in vitro in TF-104 cell lines. Except for erythromycin and penicillin G at a concentration of 4 μg/ml, Probio-16 showed resistance to all other thirteen antibiotics tested. This study indicates L. reuteri Probio-16 as a novel strain with its tolerance to low pH and bile, antimicrobial activity, antibiotic resistance and antiviral activity against rotavirus, and an ideal probiotic candidate for animal and human application after the proper in vivo experiments.     

猪源芽孢杆菌的分离鉴定及生物学特性研究

从自然生长的健康猪肠道中分离到13株芽孢杆菌,通过生理生化试验初步鉴定其中9株为枯草芽孢杆菌,2株为巨大芽孢杆菌,1株为地衣芽孢杆菌,1株为凝结芽孢杆菌;对这13株芽孢杆菌分别进行了耐酸性试验、耐胆盐试验、体外抑菌试验和动物安全性试验。结果表明,其中1株地衣芽孢杆菌B7株和1株枯草芽孢杆菌B9株均有较强的耐酸、耐胆盐能力,对致病性大肠杆菌具有较好的体外抑制能力,且均安全无毒,可作为动物用益生菌制剂的候选菌种。     

Expression of type 1 pili by Escherichia coli strains of high and low virulence in the intestinal tract of gnotobiotic turkeys

Highly virulent (strain 1) and weakly virulent (strain 3) Escherichia coli were examined using immunofluorescent and electron microscopic techniques to determine their ability to express type 1 pili in the intestinal tract of 3-week-old gnotobiotic turkeys. Turkeys were necropsied on postinoculation day (PID) 1, 2, 5, 8, and 12. Nonpiliated forms of strains 1 and 3 were more numerous than piliated forms in cecal and colonic contents examined by negative staining electron microscopy. A piliated form of strain 1 was seen in intestinal contents on each PID and was more numerous in cecal contents than in colonic contents. The mucus blanket of the cecum and colon contained large numbers of bacteria, although organisms were rarely intimately associated with the intestinal epithelium. Immunofluorescent staining indicated large numbers of piliated forms of strains 1 and 3 within the mucus blanket of the cecum and colon on PID 2, 5, 8, and 12. Piliated bacteria were infrequently seen in the ileal mucus blanket. Serum antibody titers to type 1 pili increased markedly by PID 5 and persisted in turkeys inoculated with strain 1. In contrast, antibody titers in turkeys exposed to strain 3 increased gradually and varied markedly among birds at each PID. Type 1 pili may not be important for adherence of pathogenic E coli to intestinal epithelium of turkeys.     

Putative probiotic Lactobacillus spp. from porcine gastrointestinal tract inhibit transmissible gastroenteritis coronavirus and enteric bacterial pathogens

A total of 310 bacterial strains isolated from the porcine gastrointestinal tract were tested for their activity against transmissible gastroenteritis (TGE) coronavirus and other enteric pathogens. Based on activity, the strains Probio-38 and Probio-37 were selected as potential probiotics and identified as Lactobacillus plantarum Probio-38 and Lactobacillus salivarius Probio-37 respectively by 16S rRNA gene sequencing. Supernatants of these strains inhibited TGE coronavirus in vitro in ST cells, without any cytopathic effect even after 72 h of incubation. Both the strains exhibited high survival in synthetic gastric juice. The strains were resistant to 5% porcine bile and exhibited antimicrobial activity against all the 13 enteric bacterial pathogens tested. These strains also exhibited resistance to most of the antibiotics analyzed. The inhibition of transmissible gastroenteritis coronavirus and enteric bacterial pathogens as well as the bile tolerance, high survival in gastric juice, and the antibiotic resistance indicate that the two isolated bacterial strains are ideal probiotic candidates for animal application after proper in vivo experiments.     

Potential use of characterised hyper-colonising strain(s) of Campylobacter jejuni to reduce circulation of environmental strains in commercial poultry

Sixty-two persistently colonising Campylobacter jejuni strains were tested for their ability to dominate colonisation of the chicken gastrointestinal tract in competition with each other leading to selection of dominant or "hyper-colonising"Campylobacter strains, which are able to displace others in the chicken intestinal tract. One such strain was shown to be a hyper-efficient coloniser of chickens, as it was able to displace other colonising strains, as well as maintain itself in the chicken intestinal tract for the duration of the 56-day broiler production cycle. Once colonisation was established, this hyper-colonising C. jejuni strain, 331, could not be displaced by other colonising or hyper-colonising strains. We proposed that a defined, hyper-colonising strain, or a cocktail of defined strains with a similar phenotype, could form the basis for biological control of unknown/uncharacterised Campylobacter strains from the environment that continuously colonise chicken flocks. To validate this approach, three different chicken infection trials were carried out. These trials demonstrated that the dominant strain of C. jejuni was able to colonise broiler chickens consistently and for the entire life of the birds irrespective of the day of inoculation and antimicrobial agents used in the feed to control other pathogenic micro-organisms. In addition, we have shown that the bio-control strain was able to replace other colonising strains at various points of a 56-day broiler production cycle irrespective of time and type of inoculation. This strain was also capable of re-establishing itself following the challenge with other strains, with and without re-challenge. This work represents a "proof of principle" that a defined C. jejuni strain could be used to biologically control circulation of uncharacterised environmental strains in commercial poultry flocks.     

Association of Streptococcus gallolyticus strains of high and low virulence with the intestinal tract of pigeons

We investigated the ability of a high virulence (STR 357) and a low virulence (STR 598) strain of Streptococcus gallolyticus to attach to the intestinal tract of pigeons. For that purpose, first of all, two groups of six pigeons were anesthetized and ligatures were placed at the beginning of duodenum, jejunum, ileum, and colon. The obtained intestinal loops of the birds of the first and second group were injected with S. gallolyticus strains STR 357 and STR 598, respectively. At 15, 30, and 60 min postinoculation, two pigeons of each group were euthanatized and the various intestinal loops were sampled for histologic, immunohistochemical, and electron microscopic examination. Both the high and low virulence strains were able to adhere to the intestinal mucosa. Indeed, all samples dearly showed numerous coccal-shaped bacteria that stained positively with S. gallolyticus antiserum and were lining up against the intestinal epithelium. Likewise, on electron microscopic examination, cocci were seen in the mucus covering the intestinal epithelium. Second, the association of S. gallyticus strains of differing virulence with the intestinal tissue was determined quantitatively. Experiments were performed as described above. The number of S. gallolyticus bacteria that adhered to the intestinal epithelium was determined by plating out 10-fold serial dilutions of the segments. No significant differences in the number of adhered bacteria were found between the strains of high and low virulence.     

Intestinal colonisation-inhibition and virulence of Salmonella phoP, rpoS and ompC deletion mutants in chickens

Administration of live Salmonella strains to day-old chicks provides profound protection against superinfection with a related strain within a matter of hours by a colonisation-inhibition mechanism, which is primarily a bacterial physiological process. Although currently available, commercial, live attenuated Salmonella vaccines induce protection by adaptive immunity, none of them is able to induce protection against Salmonella organisms by colonisation-inhibition and, therefore, they are unable to protect newly-hatched birds immediately after oral vaccination. In this study, mutants of Salmonella Typhimurium and Enteritidis with deletions in phoP and rpoS, either alone or in combination with ompC, were characterised and tested for their level of attenuation and their ability to inhibit the intestinal colonisation of the isogenic parent strains in chickens. Mutants with deletions only in rpoS demonstrated an unaffected potential to inhibit the intestinal colonisation of the challenge strain but were still fully virulent for the chickens. Mutants with deletions in phoP, either alone or in combination with rpoS, resulted in a high level of attenuation, unimpaired ability to colonise the gut and a nearly unaffected potential to inhibit the challenge strain from caecal colonisation. Mutants with an additional deletion in ompC revealed a reduced capacity of intestinal colonisation-inhibition when compared to the control strains and both the single rpoS and the phoP deletion mutants. Mutations in phoP- or phoP-regulated genes may therefore be used for the development of live attenuated Salmonella vaccines possessing these novel characteristics.     

Immunogenicity of a Salmonella Enteritidis mutant as vaccine candidate and its protective efficacy against salmonellosis in chickens

A novel Salmonella Enteritidis (SE) vaccine candidate strain, JOL919 was constructed by deleting the lon and cpxR genes from a wild-type SE using an allelic exchange method. The study was carried out to evaluate the strain as a vaccine candidate against salmonellosis. The strain showed the enhanced macrophage invasion, early bacterial clearance and higher immune responses as compared to the other mutants, JOL917 (Δlon) and JOL918 (ΔcpxR), and the wild type. In further analysis, the chickens immunized with JOL919 showed a significant increase in plasma IgG and intestinal secretory IgA levels, which was an indication of robust humoral and mucosal immune responses induced by the candidate. The lymphocyte proliferation response and CD45(+)CD3(+) T cells, associated with an activation of T helper and cytotoxic cells, were also significantly increased in the immunized group, which indicated that the candidate also induced cellular immune responses. The immune cell influx into caecal tissues analyzed by immunohistochemistry showed that CD8(+) T cells were predominated in the immunized group, suggesting that the candidate can clear the invaded pathogen in the intestines by a more direct way involving cytotoxic activity. By the examination of the protection efficacy measured by observations of gross lesions in the organs and bacterial recovery, the candidate can provide an efficient protection upon virulent challenge.     

Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo

Glässer’s disease is a re-emerging swine disease characterized by a severe septicaemia. Vaccination has been widely used to control the disease, although there is a lack of extended cross-protection. Trimeric autotransporters, a family of surface exposed proteins implicated in host-pathogen interactions, are good vaccine candidates. Members of this family have been described in Haemophilus parasuis and designated as virulence-associated trimeric autotransporters (VtaA). In this work, we produced 15 recombinant VtaA passenger domains and looked for the presence of antibodies directed against them in immune sera by immunoblotting. After infection with a subclinical dose of H. parasuis Nagasaki, an IgG mediated antibody response against 6 (VtaA1, 5, 6, 8, 9 and 10) of the 13 VtaA of the Nagasaki strain was detected, indicating that they are expressed in vivo. IgA production against VtaA was detected in only one animal. VtaA were more likely to be late antigens when compared to early (Omp P5 and Omp P6) and late (YaeT) defined antigens. Antibody cross-reaction with two orthologs of Nagasaki’s VtaA5 and 6, VtaA15 and 16 of strain HP1319, was also detected. No antibodies against VtaA were detected in the sera of animals immunized with a bacterin of the Nagasaki strain, suggesting poor expression in the in vitro conditions used. Taken together, these results indicate that VtaA are good candidate immunogens that could be used to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be further studied.     

Colonization strategy of Campylobacter jejuni results in persistent infection of the chicken gut

Although poultry meat is now recognized as the main source of Campylobacter jejuni gastroenteritis, little is known about the strategy used by the bacterium to colonize the chicken intestinal tract. In this study, the mechanism of C. jejuni colonization in chickens was studied using four human and four poultry isolates of C. jejuni. The C. jejuni strains were able to invade chicken primary cecal epithelial crypt cells in a predominantly microtubule-dependent way (five out of eight strains). Invasion of cecal epithelial cells was not accompanied by necrosis or apoptosis in the cell cultures, nor by intestinal inflammation in a cecal loop model. C. jejuni from human origin displayed a similar invasive profile compared to the poultry isolates. Invasiveness of the strains in vitro correlated with the magnitude of spleen colonization in C. jejuni inoculated chicks. The C. jejuni bacteria that invaded the epithelial cells were not able to proliferate intracellularly, but quickly evaded from the cells. In contrast, the C. jejuni strains were capable of replication in chicken intestinal mucus. These findings suggest a novel colonization mechanism by escaping rapid mucosal clearance through short-term epithelial invasion and evasion, combined with fast replication in the mucus.