Characterization of the bacterial gut microbiota of piglets suffering from new neonatal porcine diarrhoea

BMC Veterinary Research

Mette Boye²  Lars Mølbak³  Hans-Christian Ingerslev²  Hanne Kongsted¹  Niels Larsen⁴  Mikael Lenz Strube²  Anders Stockmarr⁵  Kerstin Skovgaard²  Marie Louise Hermann-Bank² 

[1] Danish Pig Research Centre, Danish Agriculture and Food Council, Vinkelvej 13, Kjellerup, 8620, DK, Denmark;National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, Frederiksberg C, 1870, DK, Denmark;Present address: Chr. Hansen, Bøge Allé 10-12, Hørsholm, 2970, DK, Denmark;Danish Genome Institute, Skt. Lucas Kirkeplads 8, Århus, 8000, DK, Denmark;Department of Applied Mathematics and Computer Science, Technical University of Denmark, Matematiktorvet, Building 324, Lyngby, 2800, DK, Denmark

关键词: 454 sequencing;    Gut Microbiotassay;    Microbiota;    qPCR;    Diarrhoea;    Piglet;    Neonatal;    NNPD;   

Others  :  1219110
DOI  :  10.1186/s12917-015-0419-4

 received in 2014-11-12, accepted in 2015-04-27,  发布年份 2015

【 摘 要 】

Background

In recent years, new neonatal porcine diarrhoea (NNPD) of unknown aetiology has emerged in Denmark. NNPD affects piglets during the first week of life and results in impaired welfare, decreased weight gain, and in the worst-case scenario death. Commonly used preventative interventions such as vaccination or treatment with antibiotics, have a limited effect on NNPD. Previous studies have investigated the clinical manifestations, histopathology, and to some extent, microbiological findings; however, these studies were either inconclusive or suggested that Enterococci, possibly in interaction with Escherichia coli, contribute to the aetiology of NNPD. This study examined ileal and colonic luminal contents of 50 control piglets and 52 NNPD piglets by means of the qPCR-based Gut Microbiotassay and 16 samples by 454 sequencing to study the composition of the bacterial gut microbiota in relation to NNPD.

Results

NNPD was associated with a diminished quantity of bacteria from the phyla Actinobacteria and Firmicutes while genus Enterococcus was more than 24 times more abundant in diarrhoeic piglets. The number of bacteria from the phylum Fusobacteria was also doubled in piglets suffering from diarrhoea. With increasing age, the gut microbiota of NNPD affected piglet and control piglets became more diverse. Independent of diarrhoeic status, piglets from first parity sows (gilts) possessed significantly more bacteria from family Enterobacteriaceae and species E. coli, and fewer bacteria from phylum Firmicutes. Piglets born to gilts had 25 times higher odds of having NNPD compared with piglets born to multiparous sows. Finally, the co-occurrence of genus Enterococcus and species E. coli contributed to the risk of having NNPD.

Conclusion

The results of this study support previous findings that points towards genus Enterococcus and species E. coli to be involved in the pathogenesis of NNPD. Moreover, the results indicate that NNPD is associated with a disturbed bacterial composition and larger variation between the diarrhoeic piglets.

【 授权许可】

   
2015 Hermann-Bank et al.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Wittum TE, Dewey CE, Hurd S, Dargatz DA, Hill GW. Herd- and litter-level factors associated with the incidence of diarrhea morbidity and mortality in piglets 1 to 3 days of age. J Swine Health Prod. 1995; 3:99-104.
• [2]Kongsted H, Stege H, Toft N, Nielsen JP. The effect of New Neonatal Porcine Diarrhoea Syndrome (NNPDS) on average daily gain and mortality in 4 Danish pig herds. BMC Vet Res. 2014; 10:90. BioMed Central Full Text
• [3]Koenig JE, Spor A, Scalfone N, Fricker AD, Stombaugh J, Knight R et al.. Succession of microbial consortia in the developing infant gut microbiome. Proc Natl Acad Sci U S A. 2011; 108:4578-85.
• [4]Kenworthy R, Crabb WE. The intestinal flora of young pigs, with reference to early weaning, Escherichia coli and scours. J Comp Pathol Therap. 1963; 73:215-28.
• [5]Ley RE, Hamady M, Lozupone C, Turnbaugh PJ, Ramey RR, Bircher JS et al.. Evolution of mammals and their gut microbes. Science. 2008; 320:1647-51.
• [6]Bezirtzoglou E. The intestinal microflora during the first weeks of life. Anaerobe. 1997; 3:173-7.
• [7]Bauer E, Williams BA, Smidt H, Verstegen MWA, Mosenthin R. Influence of the gastrointestinal microbiota on development of the immune system in young animals. Curr Issues Intest Microbiol. 2006; 7:35-51.
• [8]Guarner F, Malagelada JR. Gut flora in health and disease. Lancet. 2003; 361:512-9.
• [9]Willing BP, Van Kessel AG. Enterocyte proliferation and apoptosis in the caudal small intestine is influenced by the composition of colonizing commensal bacteria in the neonatal gnotobiotic pig. J Anim Sci. 2007; 85:3256-66.
• [10]Pabst R, Geist M, Rothkötter HJ, Fritz FJ. Postnatal development and lymphocyte production of jejunal and ileal Peyer’s patches in normal and gnobiotic pigs. Immunology. 1988; 64:539-44.
• [11]Svensmark S. New neonatal diarrhoea syndrome in Denmark. In: Proceedings of the 1st ESPHM. Stege H, Kristensen CS, editors. Faculty of Life Sciences, University of Copenhagen, Copenhagen, Denmark; 2009: p.27.
• [12]Melin L, Wallgren P, Mattsson S, Stampe M, Löfstedt M. Neonatal diarrhoea in piglets from E.coli vaccinated sows in Sweden. In: D’Allaire S, Friendship R, editors. Proceedings of the 21st IPVS Congres. Vancouver, British Columbia, Canada; 2010. p. 290.
• [13]Gin T. Diagnostic des nouveaux cas de diarrhées néonatales anzootiques du porcelet: évaluation de la prise colostrale. PhD Thesis. Ecole Nationale Vétérinaire de Toulouse - ENVT; 2008.
• [14]Astrup P, Larsen KV, Jorsal SE, Larsen LE. Neonatal Diarrhea in Danish pig farms - a questionnaire among veterinarians. In: D’Allaire S, Friendship R, editors. Proceedings of the 21st IPVS Congres. Vancouver, British Columbia, Canada; 2010. p. 751.
• [15]Kongsted H, Jonach B, Haugegaard S, Angen O, Jorsal SE, Kokotovic B et al.. Microbiological, pathological and histological findings in four Danish pig herds affected by a new neonatal diarrhoea syndrome. BMC Vet Res. 2013; 9:206. BioMed Central Full Text
• [16]Wallgren P, Mattson S, Merza M. New neonatal porcine diarrhoea. II. aspects on etiology. In Proceedings of the 22nd IPVS Congres. Jeju, Korea; 2012. p. 76.
• [17]Sialelli JN, Lautrou Y, Oswald I, Quiniou N. Peut-on établir une relation entre les caractéristiques de la truie et de sa portée et l’apparition de diarrhées néonatales? Proposition de réponse à partir de mesures réalisées en élevage de production. Journées Recherche Porcine. 2009; 41:167-72.
• [18]Jonach B, Boye M, Stockmarr A, Jensen TK. Fluorescence in situ hybridization investigation of potentially pathogenic bacteria involved in neonatal porcine diarrhea. BMC Vet Res. 2014; 10:68. BioMed Central Full Text
• [19]Hermann-Bank ML, Skovgaard K, Stockmarr A, Larsen N, Mølbak L. The Gut Microbiotassay: a high-throughput qPCR approach combinable with next generation sequencing to study gut microbial diversity. BMC Genomics. 2013; 14:1-14. BioMed Central Full Text
• [20]Promega: Maxwell® 16 Cell LEV DNA Purification Kit. Instructions for Use of Product AS1140. Promega Corporation, WI, USA; 2010.
• [21]Access array system™ user guide v3. 2010.
• [22]R: A language and environment for statistical computing. Version 3.1.0. 2014.
• [23]Johnson DD. Enteric Streptococcus durans--An adhering Streptococcus as a cause of diarrhea in suckling piglets? In: Twenty Fifth Annual George A. Young Conference. DigitalCommons@University of Nebraska-Lincoln, USA Lincoln, Nebraska; 1984. p. 1-8.
• [24]Tzipori S, Hayes J, Sims L, Withers M. Streptococcus durans: an unexpected enteropathogen of foals. J Infect Dis. 1984; 150:589-93.
• [25]Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P et al.. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 2013; 41:D590-6.
• [26]Oksanen J, Blanchet G, Kindt R, Legendre P, Minchin P, O’Hara RBS, et al. vegan: Community Ecology Package. Version 2.0-2. [http://CRAN.R-project.org/package=vegan], 2011.
• [27]Ahmad A, Ghosh A, Zurek L, Schal C. Insects in confined swine operations carry a large antibiotic resistant and potentially virulent enterococcal community. BMC Microbiol. 2011;11:23.
• [28]Ramos S, Igrejas G, Capelo-Martinez JL, Poeta P. Antibiotic resistance and mechanisms implicated in fecal enterococci recovered from pigs, cattle and sheep in a Portuguese slaughterhouse. Ann Microbiol. 2012; 62:1485-94.
• [29]Larsson J, Lindberg R, Aspán A, Grandon R, Westergren E, Jacobson M. Neonatal Piglet Diarrhoea Associated with Enteroadherent Enterococcus hirae. J Comp Pathol. 2014; 151:137-47.
• [30]Cheon DS, Chae CH. Outbreak of diarrhea associated with Enterococcus durans in piglets. J Vet Diagn Invest. 1996; 8:123-4.
• [31]Suchodolski JS, Markel ME, Garcia-Mazcorro JF, Unterer S, Heilmann RM, Dowd SE, et al. The fecal microbiome in dogs with acute diarrhea and idiopathic inflammatory bowel disease. PLoS One. 2012;7:12.
• [32]Rajilic-Stojanovic M, Shanahan F, Guarner F, de Vos WM. Phylogenetic analysis of dysbiosis in ulcerative colitis during remission. Inflamm Bowel Dis. 2013; 19:481-8.
• [33]Portrait V, Cottenceau G, Pons AM. A Fusobacterium mortiferum strain produces a bacteriocin-like substance(s) inhibiting Salmonella enteritidis. Lett Appl Microbiol. 2000; 31:115-7.
• [34]Rendon MA, Saldana Z, Erdem AL, Monteiro-Neto V, Vazquez A, Kaper JB et al.. Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization. Proc Natl Acad Sci U S A. 2007; 104:10637-42.
• [35]Taras D, Vahjen W, Macha M, Simon O. Performance, diarrhea incidence, and occurrence of Escherichia coli virulence genes during long-term administration of a probiotic Enterococcus faecium strain to sows and piglets. J Anim Sci. 2006; 84:608-17.
• [36]STEC Center at Michigan State University. http://www. shigatox.net/ webcite
• [37]Hazen TH, Sahl JW, Redman JC, Morris CR, Daugherty SC, Chibucos MC et al.. Draft genome sequences of the diarrheagenic Escherichia coli collection. J Bacteriol. 2012; 194:3026-7.
• [38]Petri D, Hill JE, Van Kessel AG. Microbial succession in the Gastrointestinal Tract (GIT) of the preweaned pig. Livest Sci. 2010; 133:107-9.
• [39]Osawa R, Fujisawa T, SLY LI. Streptococcus gallolyticus sp. nov.; Gallate degrading organisms formerly assigned to Streptococcus bovis. Syst Appl Microbiol. 1995; 18:74-8.
• [40]Leser TD, Amenuvor JZ, Jensen TK, Lindecrona RH, Boye M, Møller K. Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited. Appl Environ Microbiol. 2002; 68:673-90.
• [41]Vaissaire J, Marcon C, Kobisch M, Menec M, Carnero R, Laroche M et al.. Importance of streptococcal disease (Streptococcus suis, group R) in France. Annales De Zootechnie. 1985; 34:372-3.
• [42]Bramley AJ. Beecham Mastitis Series- Streptococcus uberis udder infection - a major barrier to reducing mastitis incidence. Br Vet J. 1984; 140:328-35.
• [43]Lippke RT, Borowski SM, Marques SMT, Paesi SO, Almeida LL, Moreno AM et al.. Matched case–control study evaluating the frequency of the main agents associated with neonatal diarrhea in piglets. Pesq Vet Bras. 2011; 31:505-10.
• [44]Pryde SE, Duncan SH, Hold GL, Stewart CS, Flint HJ. The microbiology of butyrate formation in the human colon. FEMS Microbiol Lett. 2002; 217:133-9.
• [45]Frank DN, Amand ALS, Feldman RA, Boedeker EC, Harpaz N, Pace NR. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proc Natl Acad Sci U S A. 2007; 104:13780-5.
• [46]Sarbini SR, Rastall RA. Prebiotics: metabolism, structure, and function. Funct Food Rev. 2011; 3:93-106.
• [47]Robinson IM, Whipp SC, Bucklin JA, Allison MJ. Characterization of predominant bacteria from the colons of normal and dysenteric pigs. Appl Environ Microbiol. 1984; 48:964-9.
• [48]Krogius-Kurikka L, Lyra A, Malinen E, Aarnikunnas J, Tuimala J, Paulin L, et al. Microbial community analysis reveals high level phylogenetic alterations in the overall gastrointestinal microbiota of diarrhoea-predominant irritable bowel syndrome sufferers. BMC Gastroenterol. 2009;9:95.
• [49]Cho JH, Zhao PY, Kim IH. Probiotics as a dietary additive for pigs: a review. J Anim Vet Adv. 2011; 10:2127-34.
• [50]Fuller R. Probiotics in man and animals. J Appl Bacteriol. 1989; 66:365-78.
• [51]Carey CM, Kostrzynska M, Ojha S, Thompson S. The effect of probiotics and organic acids on Shiga-toxin 2 gene expression in enterohemorrhagic Escherichia coli O157: H7. J Microbiol Methods. 2008; 73:125-32.
• [52]Gill HS, Shu Q, Qu F. Probiotic treatment using Bifidobacterium lactis HN019 reduces weanling diarrhea associated with rotavirus and Escherichia coli infection in a piglet model. J Pediatr Gastroenterol Nutr. 2001; 33:171-7.
• [53]Amao H, Akimoto T, Komukai Y, Sawada T, Saito M, Takahashi KW. Detection of Corynebacterium kutscheri from the oral cavity of rats. Exp Anim. 2002; 51:99-102.
• [54]Murphy J, Devane ML, Robson B, Gilpin BJ. Genotypic characterization of bacteria cultured from duck faeces. J Appl Microbiol. 2005; 99:301-9.
• [55]Loubinoux J, Bronowicki JP, Pereira IAC, Mougenel JL, Le Faou AE. Sulfate-reducing bacteria in human feces and their association with inflammatory bowel diseases. Fems Microbiol Ecol. 2002; 40:107-12.
• [56]Thompson CL, Wang B, Holmes AJ. The immediate environment during postnatal development has long-term impact on gut community structure in pigs. ISME J. 2008; 2:739-48.
• [57]Inglis G, Thomas MC, Thomas DK, Kalmokoff ML, Brooks SP, Selinger L. Molecular methods to measure intestinal bacteria: a review. J AOAC Int. 2012; 95:5-23.