Safety of antibiotic and probiotic feed additives for Gallus gallus domesticus

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Stellenbosch : Stellenbosch University
ENGLISH ABSTRACT: The inclusion of antibiotics in broiler feed is of great concern, as many resistant pathogenic bacteria may spread to other farming animals and humans. Alternative methods are thus required to improve broiler health and performance without detrimental consequences. The objective of this study was to evaluate the effect of a multi-species probiotic on the health and growth performance of Gallus gallus domesticus. Bacteria from different segments of the gastrointestinal tract (GIT) of healthy free-range broilers were isolated, identified to species level by amplifying the genes encoding 16S rDNA, recA and gyrB, and comparing the sequences with those listed in GenBank. A select few isolates were screened for probiotic characteristics. Among the 609 isolates sampled from the GIT, Lactobacillus johnsonii DPN184, Lactobacillus salivarius DPN164, Lactobacillus crispatus DPN167, Lactobacillus gallinarum DPN164, Enterococcus faecalis DPN94 and Bacillus amyloliquefaciens DPN123 tolerated acidic conditions (pH 2 to 3), were resistant to bile salts (0.2 to 2.0 % w/v) and produced exopolysaccharides. Bacillus amyloliquefaciens DPN123, isolated from the duodenum, produced extracellular amylase, phytase and antimicrobial lipopeptides (surfactin and iturinA1). Enterococcus faecalis DPN94, isolated from the jejunum and ileum, produced phytase and bile salt hydrolase. The genome of E. faecalis DPN94 contained several genes that may encode virulence, but not the production of cytolysin. Differences in opinion exist regarding the role virulence genes may play in the colonisation of epithelial cells. Lactobacillus johnsonii DPN184, isolated from the cecum, produced hydrogen peroxide. Lactobacillus salivarius DPN181, isolated from the colon, produced hydrogen peroxide and high levels of lactic acid. Lactobacillus crispatus DPN167 was isolated from the crop, proventriculus and ventriculus, and produced hydrogen peroxide and bile salt hydrolase. Lactobacillus gallinarum DPN164 was isolated from the jejunum and ileum. A multi-species probiotic, consisting of L. johnsonii DPN184, L. salivarius DPN164, L. crispatus DPN167, L. gallinarum DPN164, E. faecalis DPN94 and B. amyloliquefaciens DPN123, was added to the feed of broilers and its effect on growth performance, size of the lymphoid organs, gizzard mass, mineral content of the tibia bones and red blood cell parameters determined. A separate group of broilers was administered a combination of sulphadiazine, colistin and trimethoprim through their feed and subjected to the same tests. A third group of birds received standard feed without additives and served as control. On day 19, the villi of broilers on antibiotics had larger surface areas, and higher lymphocyte and basophil counts compared to broilers from probiotic and control treatment groups. On day 29, the cecal microbiome of broilers from the control and probiotic treatment groups were similar but differed significantly from broilers that received antibiotics. Probiotic administration did not alter homeostasis of the normal GIT microbiome, suggesting that probiotics rather modulate the microbiome by preventing dysbiosis induced by pathogenic microorganisms. Birds on antibiotics had lower levels of Enterobacteriaceae and higher levels of unidentified Clostridiales, Brucellaceae, Synergistaceae, Erysipelotrichaceae and Coriobacteriaceae in their ceca. The multi-species probiotic repressed the growth of Listeria monocytogenes EDGE in vivo, most probably by lowering the cell’s metabolic activity, by competing with Listeria for receptor sites on the gut wall or mucosa, or by production of antimicrobial compounds such as short-chain fatty acids, hydrogen peroxide and lipopeptides. Salmonella Enteritidis 147 invaded Caco-2 cells and altered claudin-3 tight junctions between the cells, leading to monolayer disruption. Salmonella decreased tight junctions by invading eukaryotic cells which led to cell death. Interaction of S. Enteritidis with broiler epithelial cells led to the up-regulation of lysozyme C and G, cathelicidin 2 and 3, myeloid protein 1, trypsin inhibitor CITI-1, gallinacin-2 and ubiquitin-fold modifier 1, and the down-regulation of glutaredoxin-1, gallicin-7 and vigilin. Up-regulated proteins acted as chemotactic compounds, inhibitors of microbial enzymes, and played critical roles during stress. Down-regulated proteins activated natural killer cells, and regulated apoptosis and antimicrobial defence systems. The multi-species probiotic was not cytotoxic, but the metabolic end products were. The probiotic bacteria adhered to Caco-2 cells but did not invade them, and decreased claudin-3 tight junctions but did not disrupt the monolayer. Probiotics decreased claudin-3 tight junctions by producing short-chain fatty acids, hydrogen peroxide and antimicrobial lipopeptides. In broilers administered with the multi-species probiotic, transgelin 2/3, elongation factor-1 beta and anterior gradient 2 were up-regulated, but carnitine O-acetyltransferase, adenylate kinase 2, superoxide dismutase Cu-Zn and protein SET down-regulated. Upregulated proteins were involved in the proliferation, migration and healing of cells and regulation of the cytoskeleton, whereas down-regulated proteins were important in fatty acid transport, energy homeostasis, nucleotide metabolisms, free-radical elimination and signal transduction. Concluded from these studies, the multi-species probiotic was non-toxic and interacted with epithelial cells in a symbiotic manner. Feeding of Salmonella enterica serovar Enteritidis A9 to broilers had no effect on body mass, and no significant differences were observed with respect to immune organ weights, haematological parameters and serum interferon gamma levels. Colonisation of Salmonella in the cecum of broilers that received oxytetracycline was, however, lower on days 11 (one day post infection, dpi 1) and 14 (dpi 4), but then increased to levels corresponding to those of birds in the control and probiotic groups. At first, the antibiotics decreased the cell numbers of Salmonella in the cecum, but higher levels were recorded with continuous administration. The increase in cell numbers may be due to antibiotics disturbing the microbiome in the GIT, indirectly favouring the colonisation of Salmonella. On day 29 (dpi 19), the cell numbers of Salmonella in the cecum of broilers administered with the multi-species probiotic were similar to those of infected and uninfected birds. Broilers that received oxytetracycline displayed higher serum bactericidal activity against Salmonella on day 11 (dpi 1) compared to birds from the probiotic and control groups. In addition, on day 29 (dpi 19) birds on probiotics had higher serum bactericidal activity against Salmonella than birds in the control group. Broilers receiving the multi-species probiotic had higher levels of lysozyme in their serum on day 11 (dpi 1) compared to uninfected broilers. Broilers receiving the antibiotic and probiotic had higher T lymphocyte responses compared to broilers from the control treatment groups on day 17 (dpi 7). These results suggested that antibiotic and probiotic feed additives stimulated the immune response of broilers infected with Salmonella. The designed multi-species probiotic possessed numerous beneficial characteristics and its daily use as a feed additive was deemed safe, as probiotic use did not negatively affect the performance of healthy birds. The probiotic strains adhered to intestinal epithelial cells and crosstalk between these cells did not induce negative proteomic changes. The multi-species probiotic also increased broiler immune responses during Salmonella infection, which suggests that the strains may be used as an alternative feed additive to improve broiler health and performance.
AFRIKAANSE OPSOMMING: Toevoeging van braaikuiken voer met antibiotika is 'n groot bedreiging vir die mensdom, omdat weerstandbiedende patogeniese bakterieë na ander plaasdiere of mense kan versprei. Alternatiewe metodes word benodig om braaikuiken gesondheid en groei prestasie te verbeter, sonder enige nadelige uitwerking. Die doelstelling van hierdie studie was om die effek van 'n multi-stam probiotikum op die gesondheid en groei prestasie van Gallus gallus domesticus te bestudeer. Bakterieë is vanuit verskillende segmente in die spysveteringskanaal (SVK) van gesonde vrylopende braaikuikens geïsoleer, en is tot op spesievlak geïdentifiseer deur die gene wat vir 16S rDNA, recA en gyrB kodeer, te amplifiseer en met DNA volgordes in GenBank te vergelyk. Die multi-stam probiotikum verteenwoordig isolate uit elke segment in die SVK. Van die 609 bakterieë geïsoleer uit die SVK, het Lactobacillus johnsonii DPN184, Lactobacillus salivarius DPN164, Lactobacillus crispatus DPN167, Lactobacillus gallinarum DPN164, Enterococcus faecalis DPN94 en Bacillus amyloliquefaciens DPN123 hoë verdraagsaamheid getoon vir suurtoestande (pH 2 to 3) en gal soute (0.2 tot 2.0 % m/v), en het so ook hetero-eksopolisakkariede geproduseer. Bacillus amyloliquefaciens DPN123, wat uit die duodenum geïsoleer is, het ekstrasellulêre amilase, fitase en antimikrobiese lipopeptiede (surfactin en iturin A1) geproduseer. Enterococcus faecalis DPN94, geïsoleer uit die jejunum en ileum, het fitase en galsout hidrolase geproduseer. Die genoom van E. faecalis DPN94 het vir verskeie virulensie gene gekodeer, maar nie vir die produksie van sitolisien nie. ‘n Mengingsverskil betaan oor die rol wat virulensie gene in kolonisasie van epiteelselle speel. Lactobacillus johnsonii DPN184, geïsoleer uit die sekum, het waterstofperoksied geproduseer. Lactobacillus salivarius DPN181, geïsoleer uit die kolon, het waterstofperoksied en hoë vlakke melksuur geproduseer. Lactobacillus crispatus DPN167 is uit die krop en maag geïsoleer, en het waterstofperoksied en galsout hidrolase geproduseer. Lactobacillus gallinarum DPN164 is uit die jejunum en ileum geïsoleer. Die multi-stam probiotikum, bestaande uit L. johnsonii DPN184, L. salivarius DPN164, L. crispatus DPN167, L. gallinarum DPN164, E. faecalis DPN94 and B. amyloliquefaciens DPN123, is by die voer van braaikuikens gevoeg en die uitwerking daarvan op groeiprestasie, grootte van limfoïede organe, spiermaag massa, mineraal inhoud van die tibia bene en rooibloedsel parameters bepaal. Braaikuikens van 'n aparte groep het ‘n kombinasie van sulfadiasien, kolistien en trimetoprim deur middel van hul voer ontvang en is aan dieselfde toetse onderwerp. 'n Derde groep voëls het normale voer, sonder bymiddels, ontvang en het as kontrole gedien. Op dag 19 het braaikuikens wat antibiotika ontvang het 'n groter villi oppervlak getoon, asook hoër limfosiet- en basofiel getalle in vergelyking met braaikuikens uit die probiotika en kontrole groepe. Op dag 29 was die sekum mikrobioom van braaikuikens in die kontrole- en probiotika groepe soortgelyk, maar aansienlik verskillend van die mikrobioom in die antibiotika groep. Die sekum mikrobioom van braaikuikens wat antibiotika ontvang het, het laer vlakke Enterobacteriaceae en hoër vlakke onbekende Clostridiales, Brucellaceae, Synergistaceae, Erysipelotrichaceae en Coriobacteriaceae bevat. Die multi-stam probiotikum het die groei van Listeria monocytogenes EDGE in vivo onderdruk, waarskynlik deur die metaboliese aktiwiteit van die selle te verlaag, met Listeria mee te ding vir vashegting aan reseptore op die epiteelselle of mukosa, deur antimikrobiese komponente soos kortketting vetsure, waterstofperoksied en lipopeptiede te produseer. Salmonella Enteritidis 147 het Caco-2-selle binnegedring en die claudin-3-digte kruisings tussen die selle, asook die monolaag, ontwrig. Die interaksie van S. Enteritidis met braaikuiken epiteel selle het tot die verhoging in lisosiem C en G, katelicidin 2 en 3, myeloïde proteïen 1, trypsien inhibeerder CITI-1, gallisien -2 en ubiquitin-vou modifikator 1 vlakke aanleiding gegee, maar ‘n verlaaging in gluteredoksien-1, gallisien-7 en vigilien teweeg gebring. Die proteïene wat verhoog is speel ‘n rol in chemotaktiese verbindings, inhibeer ensieme en beheer stres. Die proteïene wat verlaag is speel ‘n belangrike rol in aktivering van natuurlike moordenaarselle, regulering van apoptose en antimikrobiese verdedigingstelsels. Die multi-stam probiotika was nie sitotoksies nie, maar hul metaboliese eindprodukte was wel. Probiotiese bakterieë het aan Caco-2 selle gebind, maar het hulle nie binnegedring nie, en het claudin-3 stywe kruisings veminder, maar het nie die monolaag ontwrig nie. Epiteel selle van braaikuikens wat blootgestel is aan die multi-stam probiotikum het tot die verhoging van transgelin 2/3, verlengings faktor-1 beta en anterior gradiënt 2, en verlaaging van karnitien O-asetieltransferase, adenylaatkinase 2, superoksied dismutase [Cu-Zn] en proteïen SET aanleiding gegee. Die proteïene wat verhoog is, is betrokke by sel proliferasie, sel migrasie, genesing en sitoskelet regulering. Die proteïene wat verlaag is speel ‘n belangrike rol in die vervoer van vetsure, handhawing van energievlakke, nukleotied metabolisme, die eliminasie van vryradikale en seintransduksie. Hierdie resultate het getoon dat die probiotiese bakterieë nie toksies is nie en in ‘n simbiotiese verhouding met epiteelselle is. Salmonella enterica serovar Enteritidis A9 toediening het geen effek op liggaamsmassa gehad nie en geen beduidende verskille is waargeneem met betrekking tot immuun orgaangewigte, hematologiese parameters en serum interferon gamma vlakke nie. Kolonisasie van Salmonella in die sekum van braaikuikens wat oksitetrasiklien ontvang het, was egter laer op dag 11 (1 dag na infeksie, dni 1) en dag 14 (dni 4), maar het daarna toegeneem tot vlakke wat ooreenstem met dié van voëls in die beheer- en probiotikum groepe. Aanvanklik het antibiotika die selgetalle van Salmonella in die sekum verlaag, maar hoër vlakke is aangeteken met aaneenlopende toediening. Die toename in selgetalle mag die gevolg wees van mikrobioom versteuring in die SVK, wat indirek die kolonisasie van Salmonella bevoordeel. Op dag 29 (dni 19) was die selgetalle van Salmonella in die sekum van braaikuikens wat die multi-stam probiotikum toegedien is, soortgelyk aan dié van besmette en onbesmette voëls. Braaikuikens wat oksitetrasiklien ontvang het, het op dag 11 (dni 1) hoër serum bakteriedodende aktiwiteit teen Salmonella getoon as voëls van die probiotikum- en kontrole groepe. Daarbenewens het voëls op probiotika teen dag 29 (dni 19) hoër serum bakteriedodende aktiwiteit teen Salmonella getoon as voëls in die kontrole groep. Braaikuikens op probiotika het op dag 11 (dni 1) hoër vlakke lisosiem in hul serum gehad, in vergelyking met onbesmette braaikuikens. Braaikuikens wat antibiotika en probiotika ontvang het, het hoër T-limfosiet reaksies gehad in vergelyking met braaikuikens van die kontrole behandelingsgroepe op dag 17 (dni 7). Hierdie resultate dui daarop dat die toediening van antibiotika en probiotika die immuunrespons van braaikuikens wat met Salmonella besmet is, gestimuleer het. Die multi-stam probiotikum het talle voordelige eienskappe getoon en die insluiting daarvan in daaglikse voer is as veilig beskou, aangesien dit nie die ontwikkeling van gesonde voëls negatief beïnvloed het nie. Die aanhegting van probiotiese bakterieë aan epiteelselle het nie negatiewe proteomiese veranderinge tot gevolg gehad nie. Die multi-stam probiotikum het ook die immuunrespons van braaikuikens tydens Salmonella infeksie verhoog, wat daarop dui dat die multi-stam probiotikum gebruik kan word as 'n alternatiewe toevoeging tot die voer van braaikuikens.
Thesis (PhD)--Stellenbosch University, 2018.
Probiotics, Broilers, Lactic acid bacteria, Broiler feed and feeding, UCTD