7. Friday Monogastric Sessions dr peter selle university of sydney - phytase changes the hepatic flow of amino acids

1. Scientifically proven methods to reduce
the reliance on antibiotics
D.J. CADOGAN

2. The Aim
To offer “cost effective alternatives” to antibiotics
to limit resistance so they are highly effective
when required
… and keep the supermarkets happy

3. Aims to promote good gut health
• To create a strong and stable beneficial microflora in the GI tract
• Reduce rapid diet changes
• Raw ingredients and additives that increase Butyrate production
• Additives that reduce gut inflammation
• And dietary means to improve but not stimulate the immune
response

4. So what are the “alternatives”?
• Acids (short and medium chain fatty acids)
• Probiotics (single to multi-strained bacillus)
• Prebiotics (yeast cell/metabolites, inulin, β glucan)
• Essential oils
• Phytobiotics
• Algae extracts (Fucoidan, Laminarin)
• Betaine
• Spray Dried Plasma (weaer pigs and broiler starters)
• Zinc Oxide/HiZox
• Toxin binders/deactivators

9. Conclusions to Professor Lyte’s studies
• Gut bugs aren’t Dumb!
Google: Mark Lyte… You Tube

10. Gut microflora colonies and biofilms

11. Acids – the good and the bad
• Short chain fatty acids mostly effective against gram negative
bacteria (eg Salmonella and E. coli)
• Medium chain acids (C6-C12), particularly Lauric Acid, against
all bacteria and some viruses, but slightly better on gram
positive.

12. Which is the best acid?
• In vitro work showed short chain (fatty) acids could be ranked
as follows: formic > propionic > lactic. Diebold and Eidesburger 2006
• Medium chain acids carry the most anti bacterial effect against
salmonella than short chain acids. Van Immerseel et al (2006)
• Short chain acids such as butyric and formic have the capacity
to reduce the efficacy of salmonella to invade epithelial cells. Van
Immerseel et al (2006) Ricke, 2002

13. Acids ain’t Acids
• Remember it’s the R-COO- NOT
the H+ ion for longer term effects
• Dietary and Gut Bicarbonate nullify
most H+

14. Antibacterial mode of action of organic acids
0
200
400
600
800
1000
E. coli Staphylococcus
aureus
Bacillus cereus
MIC(micromolar)
Undissociated
Dissociated
Minimum Inhibitory Concentration
(MIC) of propionic acid against
different bacteria depending on
the chemical form (adapted from
Eklund, 1989)
Undissociated
Disassociated

15. Fumaric
Citric
Tartaric
Malic
Phenylacetic
Benzoic
Sorbic
Lactic
Butyric
Propionic
Acetic
Formic
2.00
2.50
3.00
3.50
4.00
4.50
5.00
0 50 100 150 200 250
Molecular Weight
pKavalue
Relationship between molecular weight and pKa value of organic
acids

16. Antibacterial mode of action of organic acids
Gizzard
pH= 2 to 4
R-COOH
R-COO-
+
H+
R-COOH
R-COO-
+
H+
Bacteria cytoplasm
pH= 7
diffusion
(depending on
environmental pH,
concentration and pKa
of acids)

17. R-COOH
R-COO- + H+
H+-ATPase pump
H+
ATP
Loss of energy
Antibacterial mode of action of organic acids
accumulation
toxicity

18. Acids ain’t Acids
• Remember it’s the R-COO- NOT
the H+ ion for longer term effects
• Dietary and Gut Bicarbonate nullify
most H+

19. Bacterial colonies, biofilms and acid tolerance

21. Campylobacter challenge day 14 Broilers
Acids
Probiotics
Prebiotic like
Plant extracts

22. Campylobacter challenge day 42
Acids
Probiotics
Prebiotic like
Plant extracts

23. Is there long term tolerance to acids by pathogens?
• Yes there is reasonable evidence (S. Ricke, 2002, Foster, 1995; Leyer
and Johnson, 1992; Arnold et al, 2001 and Kwon et al 2002)
• At least use regular changes/shuttles in the acid
program.
• Best acids are Protected Butyrate and medium chain
fatty acids/Lauric acid…. But
………..…. We need an alternative to acids too!!

24. Formaldehyde has a high level of broad antibacterial activity. It acts directly on forming
irreversible cross-linking of functional proteins within the bacterial cell.
Most cost efficient effective chemical treatment against salmonella for stockfeed (Davis
and Wray, 1995)
In commercial settings, formaldehyde treatment is generally combined with technologies
that offer cross protection such as organic acids either straight or embedded in slow-
release carriers.
An extensive study by JJ Carrique-Mas et al, 2006, compared four commercial
technologies for the reduction of artificially contaminated meals and finished feeds with
Salmonella. Treatment with formaldehyde appeared to be the only effective method,
Formaldehyde

25. Prebiotics
• Functional carbohydrate that promote beneficial
bacteria…. Competitive Exclusion
• Beneficial fibre/NSP
– Insoluble arabinoxylan (wheat, wheat bran/millmix), beta-
glucan (barley and oats)
– Enhanced by Xylanase and beta glucanase enzymes
• Prebiotic like additives (Yeast metabolites, inulin, mixed linkage beta
glucan from cereal, yeast or algae, xylo-oligosaccarides etc.)

26. The amount and type of functional
carbohydrate/prebiotic and will depend on type
GUT and size and development

27. Dried Yeast
metabolites
Inulin
(chicory)
FOS
(Fructo-
oligosacchirides)
Acetate (%) 48 57 64
Propionate (%) 30 30 26
Butyrate (%) 21 14 9
High butyrate concentrations in the large intestine support a healthy
intestinal epithelium, and help control inflammatory and malignant
diseases of the intestine
Possemiers et al (2013) J Ag Food Chem
Butyrate Production

28. Butyrate (not Butyric acid):
The Wonder Molecule!?
• Small chain volatile fatty acid (pH 4.82) BUT!
• Primary source of energy (ATP) in the colon
• Increases growth of “normal” mucosa cells
• Reduces inflammation
• Reduces leaky gut
• Provides innate immunity
• Inhibits growth of pathogens
• Reduces cancer

29. Butyrate production is crucial for gut health

30. The beneficial bugs
• Butyrate producers
– Clostridium (now Blautia) coccoides and Clostridium leptum
– Faecalibactrium pranusnitzii
• Acetate producers – the ↑ bacteria convert acetate to Butyrate
– Bifidobacteria and Akkermansia (mucin feeders)

31. Minimise fermentable protein !!
• Increased levels of ammonia, hydrogen sulphide and phenols, polyamines (eg
cadaverine)
• These are toxic and damage gut lining → leaky gut
• Undigested protein or excessive amounts produce pathogens (E coli, pathogenic Clostridia
and salmonella)
• Decreases Butyrate!
• Produces inflammation of the gut
• Increase cytokine production which reduce feed intake

32. Manno Oligosacharides (MOS) or Refined
Fraccionated Carbohydrates (RFCs)
MOS or RFCs is a relatively new technology available to the feed industry. These molecules contain certain
physicochemical structures that allow for the agglutination/binding of type-1 fimbrie (hairs) of Salmonella and other
gram negative species.
A study by Borowsky et al 2009, demonstrated that out of 108 salmonella strains, 74% demonstrated strong
agglutination to MOS in vitro.
In vivo studies have also demonstrated the efficacy of these molecules to reduce Salmonella caecal counts in chickens
when added as a feed supplement in the diet (Fernandez et al 2000; Spring et al 2000; Spencer 1997)

33. Observation of agglutination with
Salmonella (x400):
Salmonella agglutination
No agglutination

34. Xylanase decreases mortalities in pigs
• Xylanase creates prebiotics from fibre/NSP breakdown
• Boyd et al 2015;
– High levels of Xylanase activity (6000 units/kg) reduced mortalities by 40% in a
typical USA diet
• Beckers, 2016
– Lower levels of Xylanase 1500 units/kg decreased mortalities by 27.9% in
wheat/soy/DDGS
– 1500 to 3000 units/kg xylanase decreased morbidities/removals by 20.1 and
50.1% respectively.

35. Probiotics
• The best probiotic is lactobacillus based, BUT this are unstable and
have poor shelf life (Stanley et al 2016)
• The majority of Probiotics are Bacillus
• Bacillus spores that are poor colonisers so must be offered all the
time
• The Bacillus spores are heat stable and long shelf life… so it’s a
compromise!

36. Early colonisation
Dana Stanley
• Life time microflora population strongly influenced by exposure in
the first 2 days
• Feeding a live lactobacillus probiotic in the first day significant long
term benefit
• Potential for hatchery gels

37. Bespoke Probiotics: Bacillus strains selected for following extensive in-field
investigations
In vitro studies
Identify basic &
functional
properties of
Bacillus strains
In vitro studies
Test Bacillus
strains through
in-vitro model
systems
In vivo studies
Test Bacillus
strains in birds
Proven benefits
Bacillus:
Supports a
balanced gut
microbiota
More
profitable bird
performance
Thousands potential Bacillus strains 3 strains of
Bacillus
Identify optimal Bacillus strainsIdentify the problem
Screening
studies
Identify specific
bacteria
How was Enviva® PRO developed?

38. Gastrointestinal Analysis: Enumeration of intestinal microbes: CSI
Enumeration of specific bacterial species
Information on the micro-
organisms populating the
GIT may identify underlying
problems.
Selective culture of specific microorganisms
GITs dissected and
processed in a local
laboratory
C. perfringens Lactic acid
bacteria
E. coli

39. In vitro growth inhibition assays to identify the most efficacious
Bacillus blend: Dupont Enviva CSI
Inoculate wells with
bacteria isolated from the GITs
Add Bacillus products to
the bacteria in the wells
Incubate togetherSummary highlighting the overall level of inhibition
provided by the 8 Bacillus strains
Determine level of
growth inhibition
provided by each
Bacillus strain
Note: Inhibition levels over 50% are considered efficacious.
Strains D, G & H showed the highest levels of inhibition. A
blend of these 3 strains would provide the greatest
challenge coverage.
Inhibition level: A B C D E F G H
75%- 100% 3 10 13 15 3 5 15 17
50%- 75% 3 6 8 4 15 12 4 2
25%- 50% 5 5 1 2 4 4 3 2
0%-25% 11 1 0 1 0 1 0 1

40. Epidemiology of APEC in Australian broilers
Investigating the epidemiology of APEC in Australian broilers
2015:
3 broiler farms
(A, B, C)
30 birds collected for
analysis at 14 & 28
days of age
Microbial
enumeration
E. coli genetic
material shipped to
U.S.
APEC prevalence and
predictive inhibition
of 3 Bacillus probiotic
strains
Wealleans et al., 2017 (DUPONT)

41. Enumeration of E. coli and Avian pathogenic E.
coli
5 log threshold
Epidemiology of APEC in Australia broilers

42. Epidemiology of APEC in Australian broilers
51% of total E. coli population sampled identified as potential avian
pathogenic E. coli
10% high
virulence

43. Predictive inhibition of 254 potential APEC isolates with 3 strains of Bacillus Direct-
fed microbials
Epidemiology of APEC in Australian broilers
80% of
Australian
isolates had a
U.S. “match”
Wealleans et al., 2017

44. Probiotics in combination with enzymes reduced faecal shedding and
eradicated extra-intestinal spread of Campylobacter jejuni
Red bar represents the median value for each group. Significant difference
was evaluated using a Wilcoxon test in JMP v 11.0.
Could a reduction in early shedding of
C. jejuni reduce spread within a flock?
5.06 logs
3.43 logs
98% ↓
C. jejuni counts in Liver 7 dpi (log10 CFU/g)
Livers positive for Campylobacter (%) after
enrichment86%
0%
Awad et al, 2016

47. Bespoke or CSI Probiotics from Dupont
• Significantly reduces pathogenic Clostridia and Ecoli etc
• Gut samples taken and pathogen type and load assessed
• Unique blend of 3-4 from a library of over 8 Bacillus Spores
• Cost effective at around $2/tonne inclusion

48. Essential oils
• Extracted concentrated from particular plant materials (flowers,
roots, bark, leaves, seeds)
• Aromatic and volatile liquids called essential oils
• Effective against pathogenic bacteria, coccidia, some viruses
and nematodes.
• Best review EVER = O’Bryan et al 2017 Frontiers in Veterinary Science. 2:35. doi:
10.3389/fvets.2015.00035

49. Target of antibacterial action
Target Essential oil or active
component
Reference/s
Cell wall or membrane Carvacrol (active) Helander et al, Utee et al, Fitzgerald et al, Xu
et al
Thymol (active) Walsh et al Helander et al, , Fitzgerald et al,
Xu et al
Cinnamaldhyde (active) Gill and Holley; Boudhid et al
Eugenol (active) Walsh et al
Vanillin Fitzgerald et al
Oregano Bouhdid et al De Souza et al
Tea tree oil Cox et al
Respiration Vanillin Fitzgerald et al
Cinnamaldhyde (active) Bouhdid et al
Tea tree oil Cox et al
Quorum Sensing Oregano Alvarez et al
Clove Khan et al
Rosemary Szabo et al

50. Are essential oils the “most” effective natural anti-
microbial?
MIC’s = Minimum Inhibitory Concentrations for common food safety pathogens
Range ug/ml
E coli 0157:H7 0.225 to 5.0
Salmonella typhimurium 0.225 to 0.25
Staphylococcus aureus 0.175 to 0.45
Listeria monocytogenes 0.375 to 5.0
Bacillus cereus 0.188 to 0.90
Also active against Streptococci and Campylobacter
Beneficial Lactobacillus spp. are spared – MICs are 10-fold higher than other
bacteria
Michiels J et al. 2007. Livestock Science 109:157-160
Lambert RJW et al. 2001. J Appl Microbiol 91:453-463
Burt SA et al 2005. J Food Prot 68:919-926.
Ben Arfa A. 2006. Lett Appl Microbiol 43:149-154

51. Mechanism of Action
 Carvacrol and thymol are primary antimicrobials (O’Byran et al. 2017)
 Carvacrol and thymol damage to the bacterial cell wall
 Unlike antibiotics, resistance is unlikely
Lambert RJW et al. 2001. J Appl Microbiol 91:453-463
Oussalah M et al. 2006. J Food Prot 69:1046-1055.
Aiello AE et al. 2004. Antimicrob Agents Chemother 48:2973-2979
Leaky
membrane
Essential ions move
out of the cell

52. Essential Oils in Oregano
• Contains 34 components
1. -Pinene
2. Camphene
3. -Pinene
4. Sabinene
5. Myrcene
6. -Phelandrene
7. -Terpinene
8. Limonene
9. 1,8-Cyneole
10. -Phelandrene
11. -Terpinene
12. -Ocimene
13. -Cimene
14. -Terpinolene
15. l-Octen-3-ol
16. trans-Sabinene
hydrate
17.cis-Sabine hydrate
18. Linalool
19. -Caryophyllene
20. Methyl carvacrol
21. trans-Dihydrocarvone
22. cis-Dihydrocarvone
23. Isoborneol
24. -Terpineol
25. -Alemene
26. -Bisatolene
27. -Cadinene
28. trans-Carvacrol
29. Calemene
30. -Cimene-8-ol
31. Carvacrol acetate
32. Sparcholerol
33. Thymol
34. Carvacrol
Ratio 42:1

53. Common Anti-Oxidant Levels
Oregano oil
2,520,600
Total ORAC: TE/100g
Red Wine: 5,034
Cranberries: 9,584
Dark Chocolate: 20,823
Natural Vitamin E: 48,200
BHT: 72,000

54. Lactobacillus are less susceptible to Essential oils
• Lactic acid bacilli (LAB) are part of the microflora of
vegetables
• Adapted to plant environment
• Phenolic compounds are part of innate defense of
plants
• LAB’s produce enzymes to utilize phenols:
 Tannase
 Phenolic acid decarboxylase
 Benzyl alcohol dehydrogenase
Rodriguez H et al. 2009. Int J Food Microbiol. 132:79-90. Phenol Carvacrol

55. Oregano oil….The treatment!?
• Best non chemical to reduce coccidiosis infection. Evidence that in its very effective in
combination with Cocci Vaccine and coccidostats (Alp et al 2012)
• Very effective at controlling Black head in turkey and broiler breeders (Andres 2012; Gray AVPA 2015)
• Reduction in salmonella in poultry (O’Bryan et al, 2017)
• Reduction in the severity of spotty liver in free range layers (Stanely unpublished)
• Effective at reducing oedema caused by E. coli in pigs
• Evidence of reducing ileitis and Brachyspira in grower pigs** (Vande Maela et al, 2016; Omonijo et al, 2018
• Effective treatment against Strep Suis in pigs (de Agiar et al 2018)
• Reduces Salmonella and Ecoli counts in lamb feedlots and in beef processing (Omonijo et al,
2018)

56. Regano can potentially replace chemical coccidiostats
Adj to 2.7kg and for mortality and culls
1.610
1.620
1.630
1.640
1.650
1.660
1.670
1.680
Negative Control:
Salinomycin + BMD
Positive Control:
Salinomycin + 3-Nitro
+ BMD
Test#1: Salinomycin +
Regano (low level) +
BMD
Test#2: Regano (high
level) + NutriFibe
Complex
Adj 1.675 1.668 1.635 1.657
Ralco Nutrition Regano Trial October 2011
Adjusted Feed Conversion MCA
by Treatment

57. Half life 1.5 to 2 hours in the GIT
Plasma concentrations peaked about 1hr
Cinnamaldehyde showed the lowest levels
of degradation
Pharmacokinetics

58. • Natural blends and sources are more effective than synthetic
individual oils
• Essential oils can vary, so must be blended and analysed for
minimum active content
• Minimum and maximum dose required and be careful of low
dose recommendations
• Water treatment of emulsified essential oils most cost
effective
Caution !

59. Feedworks - Ralco Essential Oil
– Regano (In feed and water treatments)
• Oregano + Thymol + Prebiotic fiber
• Microencapsulated
– Prosper EO
• Oregano + Thymol + Cinnamaldehyde + Prebiotic fiber
• Microencapsulated

61. Intestinal Colonization
Feeding XPC prebiotic decreased intestinal
colonization.
a,bP < 0.05
Feye et al., 2016
29,023 CFU/g
CONa
3,875 CFU/g
XPCb

62. CON XPC
0
200000
400000
600000
800000
CFU/g
Dot Plot of Cloaca Swab Salmonella Numbers
Layer Field Samples; LC03
Super shedders

63. NARMS Resistance Testing
NARMS: National Antimicrobial Resistance Monitoring System
• Established by FDA (1996)
• Collaboration with the CDC and USDA
• Panel represents common antimicrobials used in both
human and veterinary medicine.
Source: www.ars.usda.gov
Goals of NARMS
1. Monitor AMR trends in foodborne bacteria from humans, retail
meats, and animals
2. Distribute information to promote interventions that reduce AMR
3. Conduct research to better understand the emergence,
persistence, and spread of AMR
4. Assist the FDA in making decisions related to the approval of safe
and effective antimicrobial drugs for animals

64. Feye et al., 2016
Salmonella DNA contains an integron
Integron contain genes for antibiotic resistance
Loss of the integron = Loss of Antibiotic Resistance
Presence or absence of integron is measurable
Salmonella Antibiotic Resistance

65. 64.6a
96.9a
0.3
97.4a
88.7a
37.8b
53.3b
0.0
48.9b
26.7b
0.0
20.0
40.0
60.0
80.0
100.0
120.0
Gentamicin Streptomycin Aztreonam Ampicillin Amox-cla
SalmonellaColoniesResistantto
anAntibiotic(%)
CON XPC
Antibiotic Resistance of Salmonella Recovered from
Ceca
a,bP < 0.0003
Broiler Field Samples; BC13
Colonies Tested: 4,999
Prebiotic

66. 87.1a
46.4a
39.7a
87.1a
45.8b
23.0b
12.4b
38.8b
0.0
20.0
40.0
60.0
80.0
100.0
Azithromycin Sulfisoxazole SMZ-TMP Tetracycline
SalmonellaColoniesResistantto
anAntibiotic(%)
CON XPC
Antibiotic Resistance of Salmonella Recovered from
Cloaca Swabs
Layer Field Samples; LC03
a,bP < 0.0001
Colonies Tested: 5,451
Macrolides
Sulfonamide
Tetracycline

67. Dried yeast
ferment
Antibiotic
Prebiotics Have No Direct Antibiotic Activity
S. Typhimurium; Ampicillin

69. Concluding remarks
• It is possible to limit the use of anti-biotics and achieve
similar health and growth
(in combination with vaccines, good hygiene and management)

70. Concluding remarks
• Acids aren’t the answer
• Functional fibre is essential
• Prebiotic/yeast/lactobacillus metabolites for competitive exclusion and
reduced inflammation
• Bespoke Probiotics (CSI) are very cost effective
• Particular essential oils are the best “natural” anti-microbial