Sponsor Day on animal feeding: Studies of feed additives in experimental conditions

Studies of feed additives in experimental conditions

Dr. Ignacio BadiolaHead of the BACPAR SubProgram, IRTA-CReSA

SPONSOR DAYSEMINAR ON

ANIMAL FEEDING

IntroductionAgence Française de Sécurité Sanitaire des Aliments (AFSSA), Maisons-AlfortDepartamento de Ciencia Animal, ETSI Agrónomos, UPVDepartamento de Producción Animal, ETSI Agrónomos, UPMDepartment of Agronomy, Food, Natural Resources, Animal and Environment, Università de PadovaDepartment of Comparative Biomedicine and Food Science, Università de PadovaDepartment of Genetics and Microbiology, UABFaculty of Animal Sciences of the Agricultural University of AthensGut and Immunology Group, Rowett Institute of Nutrition and Health, University of AberdeenInstitut National de la Recherche Agronomique, ToulouseInstitut National de la Recherche Agronomique, ToursInstituto de Estudios Biofuncionales. Facultad de Farmacia. UCMInstituto de Investigación en Recursos Cinegéticos, IREC (CSIC-UCLM-JCCM)IRTA, Centre de Sant Carles de la Ràpita, Unitat de Cultius AqüícolesIRTA, Centre Mas de Bover, Nutrició de MonogàstricsNational Veterinary School, ToulouseResearch Institute for Animal Breeding and Nutrition, Godollo UniversityRoyal Veterinary and Agricultural University (KVL), FrederiksbergStatens Veterinärmedicinska Anstalt, Uppsala University

ADISSEO France S.A.S.DSM Nutritional ProductsElanco Animal Science ResearchIndukern, S.A.Laboratorios Andrés Pintaluba, S.A.Laboratorios Calier, S.A.Laboratorios CENAVISALaboratorios Esteve Veterinaria, S.A.Laboratorios Intervet, S.A.Laboratorios Lamons, S.A.Laboratorios Maymo S.A.Laboratorios S.P. Veterinaria, S.A.NANTA, S.A.Novartis Animal HealthNovozymes Biologicals FR S.A.ONDAX ScientificRubinum Animal Health

• Public projects with in vivo feed additive trials (9) EU: 5 Nationals: 4

• Private company contracts with in vivo feed additive trials (26) International

Antimicrobials: 3 Prebiotics and probiotics: 10

Nationals Antimicrobials: 12 Prebiotics and probiotics: 1

Introduction

• Feed additives are used to supplement essential components in the diet of farm animals (essential aminoacids, vitamins).

• Another important role of feed additives is to increase the digestibility of raw materials (enzymes).

• After the ban of antimicrobials growth promoters, the interest for feed additives with antimicrobial activity has been increased (plant extracts, organic acids...)

• To reduce pathogenic and to increase saprophytic microorganisms are fundamental aims for feed additives (probiotics, prebiotics).

• To stimulate the immunologic system is another point of interest for feed additives.

• To improve the knowledge of intestinal physiology, and specially changes in the intestinal microbiota (components and metabolism), is essential for the development of new feed additives.

Experimental infection models

Salmonella Enteritidis in chickens. Salmonella Enteritidis in hens. Salmonella Typhimurium in pigs. Escherichia coli in pigs. Escherichia coli in chickens. Clostridium perfringens in chickens.

The gut microbiota contributions to host physiology

Sekirov et al., Physiol. Rev. 2010;90:859-904

DC tolerization

Cellular immunityLymphoid organogenesis

Mucosal Immunity

↑BacteroidetesB. fragilis (PSA)SCFAs

AMPs

Gm-PGGM-LPS

↑IgA

↑Iapactivation

NFKBinactivation

Lactobacillus spp. E.coli

Barriermaintenance

B. thetaiotaomicron

Bifidobacterium spp.Clostridium spp.

SCFAmetabolism

Conjugation oflinoleic acid

Nutrition Xenobiotics metabolismDrug disposition

↓Oxalateexcretion

Lipidmetabolism Behavior O.formigenes

Normalization ofHPA stress response

B.infantis

AngiogenesisPresistalsisGlycosylation

GIT surface maturationGIT functional maturation

ImmunocompetenceTolerance

The intestinal microbiota of productive animals is composed by:

16-23% cultured bacteria.

77-84% uncultured bacteria.

To analyse the cultured and the uncultured intestinal bacteria we initially used the Restriction Fragment

Length Polymorphism (RFLP) method

RFLP profiles

AGL2008-00627-GAN

0 100 200 300 400 500 600 700 800

0

500

1000

1500

2000

2500

T1-D27-A1403-CeT3-D27-A1401-Ce

Lactobacillus helveticusUncultured alpha proteobacteriumUncultured gamma proteobacteriumParabacteroides sp.Prevotella nigrescensNo sequence homology

No sequence homology

Uncultured bacteriumUncultured DesulfovibrioUncultured SuccinivibrioUncultured VeillonellaNo sequence homology

Lactobacillus spp.Rodobacter spp.Uncultured DesulfovibrioNo sequence homology

Lactobacillus salivariusNo sequence homologyParabacteroides sp.

No sequence homology

Identification of Bacteria Species by Cloning and Sequencing

Effect of strains L-92 and L-94 (Bacteroides spp.) on mortality rate by ERE

0

10

20

30

40

50

L-94 Control -

Trial 1

23.33

46.66

Mortality by ERE

At weaning At the beginning of ERE

0

10

20

30

40

50

60

L-92 L-94 Control -

Trial 2

29.17 31.25

58.33

Mortality by ERE

OT00-040-C2-2

Effect of fiber source and enzyme supplementation on digesta’s and mucosa’s biodiversity of ileum and caecum

C = CornWBR = Wheat+Barley+Rye- = Without enzyme+ = With enzyme

New molecular biology methods

RNA-Later

Etanol Etanol

0

1

2

3

4

5

C-C-

C/L

C-C-

C/H

C-C-

L/L

C-C-

L/H

L-C-

C/L

L-C-

C/H

L-C-

L/L

L-C-

L/H

C-C-

C/L

C-C-

C/H

C-C-

L/L

C-C-

L/H

L-C-

C/L

L-C-

C/H

L-C-

L/L

L-C-

L/H

1st period 2nd period

02468

101214161820

Q-PCR and 2-ΔΔCт22d 30d

RNA-Later

Ion Torrent 316™ Chip Kit

Summary of Ion-Torrent analysis of Ileal Microbiota

First Period Second Period

C-C/GFC C-C/BFC L-C/GFC L-C/BFC C-C-C/GFC C-C-C/BFC C-C-L/GFC C-C-L/BFC L-C-C/GFC L-C-C/BFC L-C-L/GFC L-C-L/BFC

Number of sequences 345221 353196 382219 311926 125949 181309 173262 201476 197613 200657 166206 155863

% Lactobacillus 91.74 86.38 83.36 93.99 91.19 83.62 91.07 90.61 86.99 84.98 90.82 93.75

% Other bacteria 8.26 13.62 16.64 6.01 8.81 16.38 8.93 9.39 13.01 15.02 9.18 6.25

Biodiversity 78 60 89 57 84 93 71 50 86 135 101 68

Kruskal-Wallis rank sum test & Period P-value Kruskal-Wallis rank sum test & FCR P-value Kruskal-Wallis rank sum test & SD P-value Kruskal-Wallis rank sum test & FD P-value

Bacillus 0.001 Anaerostipes 0.027 Jeotgalicoccus 0.027 Butyricicoccus 0.006unclas_Bacillales 0.001 Aerococcus 0.035 unclas_Bacteroidetes 0.044 unclas_Betaproteobacteria 0.011unclas_Porphyromonadaceae 0.003 unclas_Burkholderiales 0.044 Catenibacterium 0.064 unclas_Clostridiales 0.016Prevotella 0.003 unclas_Lactobacillaceae 0.059 Comamonas 0.064 unclas_Clostridia 0.019unclas_Prevotellaceae 0.003 Brachybacterium 0.064 Deinococcus 0.064 Bacteroides 0.020unclas_Bacillaceae.1 0.004 Curvibacter 0.064 Finegoldia 0.064 Clostridium.XI 0.021unclas_Chitinophagaceae 0.004 Delftia 0.064 Peptoniphilus 0.064 unclas_Bacteria 0.021Paracoccus 0.006 Parabacteroides 0.064 unclas_Bradyrhizobiaceae 0.064 unclas_Lachnospiraceae 0.021Enterococcus 0.006 Erysipelotrichaceae_incerta_sedis 0.074 Methylobacterium 0.025Dorea 0.008 Neisseria 0.097 Streptophyta 0.027Lactococcus 0.011 unclas_Rhodobacteraceae 0.097 Cloacibacterium 0.027Roseburia 0.011 Fusobacterium 0.027Sarcina 0.011 Ruminococcus 0.027unclas_Acidaminococcaceae 0.011 unclas_Peptostreptococcaceae 0.027Selenomonas 0.011 Clostridium.IV 0.037unclas_Bacilli 0.012 Sphingomonas 0.043unclas_Erysipelotrichaceae 0.020 unclas_Firmicutes 0.046unclas_Alphaproteobacteria 0.025 unclas_Ruminococcaceae 0.046Aquabacterium 0.027 Barnesiella 0.046Parasutterella 0.027 Alistipes 0.061unclas_Planococcaceae 0.027 Brachybacterium 0.064unclas_Clostridiaceae.1 0.033 Coprobacillus 0.064Chryseobacterium 0.033 Coprococcus 0.064Barnesiella 0.035 Gemella 0.064unclas_Lactobacillales 0.036 Granulicatella 0.064Flavonifractor 0.037 Megasphaera 0.064Phenylobacterium 0.037 Peptoniphilus 0.064unclas_Enterococcaceae 0.045 Clostridium.XlVb 0.073Clostridium.XVIII 0.061 unclas_Bacteroidales 0.073Comamonas 0.065 Sarcina 0.074Coprobacillus 0.065 Pelomonas 0.074Deinococcus 0.065 unclas_Comamonadaceae 0.093Granulicatella 0.065 Actinomyces 0.097Lysinibacillus 0.065 Megasphaera 0.065 Sphingobacterium 0.065 unclas_Cytophagaceae 0.065 unclas_Leptotrichiaceae 0.065 unclas_Veillonellaceae 0.065 Clostridium.XlVb 0.073 Sphingomonas 0.073 unclas_Ruminococcaceae 0.074 unclas_Bacteroidetes 0.081 Anaerococcus 0.083 Veillonella 0.083 Clostridium.sensu.stricto 0.091 Oscillibacter 0.092 unclas_Neisseriaceae 0.093 Clostridium.XI 0.093 Staphylococcus 0.093 Neisseria 0.097

Significant differences for Ileal Microbiota vs Trial Factors

Effect of feed supplemented with marine algae extract on some immunological parameters of intestinal mucosa by RT-PCR

CENIT VIDA (CEN-20101026)

Importance of variance homogeneity as productive parameter

Clinical efficacy evaluation of the use of tylosin for the treatment and control of porcine proliferative enteropathy (ileitis) associated with Lawsonia intracellularis

Mean Variance

  Control TylosinP-

value1 Control Tylosin P-value2

Animal weight (kg/pig) Day 0 28.42 29.20 0.77 25.40 15.10 0.16

Day 21 42.72 45.66 0.36 51.80 21.70 0.02Day 31 50.90 53.98 0.35 76.10 23.00 0.002

Daily growth rate (kg/pig/day)Day 0 to 21 0.681 0.773 0.036 0.022 0.009 0.02

Day 21 to 31 0.818 0.833 0.76 0.064 0.018 0.001

Daily feed consumption (kg/pig/day) Days 0 to 21 1.471 1.569 0.21 0.005 0.008 0.65

Days 21 to 31 1.862 1.922 0.5 0.006 0.014 0.53

Feed conversion ratioDays 0 to 21 2.160 2.030 0.050 0.535 0.073 <0.001

Days 21 to 31 2.280 2.300 0.770 0.550 0.137 <0.0011Anova test2Bartlett test

Importance of variance homogeneity as productive parameter

This batch will be punished if all animals are sent

together to slaughterhouse

Stay o

n farm

Troubles for the correct cleaning and disinfection of farm facilities. Increasing the risk of maintenance of infectious agents.

Higher prevalence of respiratory and digestive disorders in the next batch.

Conclusions

• It is important to normalise experimental infection models.• The intestinal microbiota is essential for animal health

and welfare.• The gut microbiota plays an essential role on digestive

physiology.• Some microbial components have direct effect on

intestinal mucosa (barrier effect and gut associated lymphoid tissue).• The intestinal microbiome can be modified by feed

composition (raw materials and feed additives).• Feed additives can reduce the variance of productive

parameters, representing a significant improvement on the economical balance of farm.

Edifici CReSA. Campus UAB. 08193 Bellaterra (Barcelona) Spain.Tel. (+34) 93 581 32 84 Fax. (+34) 93 581 44 90e-mail: [email protected] - www.cresa.cat

THANKS FOR YOUR INTEREST

SPONSOR DAYSEMINAR ON

ANIMAL FEEDING