Prevention of Salmonella with Calcium-butyrate Globamax Performant®
Introduction ● A common disease in chickens, pigs and cows ● Responsible for enormous economic burdens due to the costs of the production losses and the costs of prevention measures ● Problem for human health: Humans get infected by eating contaminated meat and eggs Prevention of Salmonella is very important, and has to start from the beginning of the production process
Etiology
● ● ● ●
Salmonella bacteria Gram negative bacteria Divided in species and serotypes Disease in humans and animals is caused by the species Salmonella Enterica
Pathogenesis: 2 phases 1/Oral uptake => intestinal phase: o Colonization of tonsils (mainly pig) o Colonization gut (mainly large intestine: caecum, colon, but also small intestine: ileum): 2 phases adhesion (attachment of the bacteria to the gut wall cell) and invasion (penetrating of the bacteria into the gut wall cell) Gut inflammation and diarrea Production of toxin General illness
2/ Systemic phase: colonization organs General illness
Invasion
Arrows: Invasion of Salmonella typhimurium into the epithelial cells of the small intestines.
Pathogenesis of Salmonella
Pathogenesis
Organic acids ● Added to feed and drinking water ● Most important: short chain fatty acids and medium chain fatty acids ● Resorption in small intestine => antibacterial effect in drinking water/feed and the upper part of the digestive system ● Coated acids: no resorption => ileum, colon, caecum (large intestine) ● An important SCFA is butyric acid (C4) => specific action on gut health
Calcium-butyrate •
= Calcium
+ 2 butyric acid molecules
• Coated of non-coated • mode of action: 3 pathways: Fysiological Microbiological Semi-therapeutic
Mode of action of encapsulated calcium-butyrate Biochemical – Physiological Effects VIA BUTYRATE IN THE STOMACH (absorption, crypt effect) AND SMALL INTESTINE (Villi effect)
-Villi development and protection - Fuel of the gutwallcell - Energy metabolism (Liver / fat metabolism) - Insuline production
B Semi Therapeutic effects
Microbiological
- (Chronic) gutwallcell inflammation (new theory on mode of action of ABGP’s)
- Normal Microflora : balancing
- Immune modulation : dilemma between health and performance
- Pathogenic flora :
Coli, Clostridium, Salmonella, Campylobacter…(Streptococcus S.)
BUTYRATE PRESENCE NEEDED IN THE SMALL INTESTINE !!
Health effects of Calciumbutyrate
Picture of Salmonella Genetics of Salmonella Hil A = Invasion, Fim A = Attachment
Encapsulated Calcium-butyrate and Salmonella: 5-way action 1. Genetics : Down regulation of the expression of several genes responsible for invasion and attachment 2. Gut health and intestinal lining : reinforcement of intestinal lining (tight junctions) leading to a decreased invasion through epithelial cells 3. Colonization : decrease of growth of various Salmonella strains in the different parts of the intestines 4. Excretion : decrease of excretion (pig) and accumulation in ceca (poultry) (coated form only) 5. Transmission : decrease of transfer of Salmonella to other animals (pigs)
1/Genetics: key words ● Gene expression: a highly specific process in which a gene is switched on at a certain time and "speaks out.“ ● Up regulation: the process to activate or “switch on” a gene. ● Down regulation: the process to inactivate or “swich off” a gene.
1/Genetics Adhesion For colonization Salmonella has to attach to the epithelial cells of the gut wall. Butyrate decreases the attachment of Salmonella by down regulating the gene responsible for adhesion: FimA gene, coding for the filaments (fimbrae) the bacteria need to attach to the gut wall cell. Reference: Coated fatty acids alter virulence properties of Salmonella Typhimurium and decrease intestinal colonization of pigs. F. Boyen, F. Haesebrouck, A. Vanparys, J. Volk, M. Mahu, F. Van Immerseel, I. Rychlik, J. Dewulf, R. Ducatelle, F. Pasmans. Veterinary Microbiology 132 (3-4), 2008, Pages 319-327
1/Genetics Invasion For colonization Salmonella has to penetrate the epithelial cells of the gut wall. Butyrate decreases the invasion of Salmonella by down regulating the genes responsible for invasion. The genes responsible for the invasion are located on SPI-1 (salmonella pathogenicity island 1). This island is down regulated by butyric acid. For example, the genes HilA and InvF, both stimulators of SPI-1, are inhibited by butyric acid. Reference: I. Gantois, R. Ducatelle, F. Pasmans, F. Haesebrouck, I. Hautefort, A. Thompson, J. C. Hinton, F. Van Immerseel (2006). Butyrate Specifically Down-Regulates Salmonella Pathogenicity Island 1 Gene Expression. Applied and environmental microbiology, p001 Reference: Van Immerseel, De buck, Meulemans, Pasmans, Velge, Bottreau, Fievez, Haesebrouck, Ducatelle (2004). Invasion of Salmonella Enteritidis in avian intestinal epithelial cells in vitro is influenced by SCFA. Journal of food microbiology, 85(3); 237-248.
2/Gut health and intestinal lining Salmonella is a gut associated pathogen. It causes damage and inflammation to the gut. Indeed, a healthy gut is important in the prevention of Salmonella! It is known that butyrate has beneficial effects on the gut wall: o It stimulates villi development and protection o It is the fuel of the gut wall cell o Enforcement of the tight junctions, this leads to a better intestinal lining and a stronger barrier against invasion o Stimulation and balancing of the ‘good’ intestinal flora Reference: R. Ducatelle*, F. Pasmans, F. Haesebrouck , and F. Van Immerseel Feeding for improved gut health in broilers. World Nutrition Forum (2008)
3/Colonization It has been proven in several trials that butyrate decreases the colonization of the caecum by Salmonella. Butyric acid significantly reduces Salmonella counts (amount of Salmonella bacteria) in the caecum and does not influence the counts in spleen and liver in pigs and chickens. Reference: Van Immerseel F., Fievez V., De Buck J., Pasmans F., Haesebrouck F., Ducatelle R. (2004). Micro-encapsulated short-chain fatty acids in feed modify colonization and invasion early after infection with salmonella enteritidis in young chickens. Poultry science 83, 69-74. Reference: Coated fatty acids alter virulence properties of Salmonella Typhimurium and decrease intestinal colonization of pigs. F. Boyen, F. Haesebrouck, A. Vanparys, J. Volk, M. Mahu, F. Van Immerseel, I. Rychlik, J. Dewulf, R. Ducatelle, F. Pasmans. Veterinary Microbiology 132 (3-4), 2008, Pages 319-327
4/Excretion As butyrate decreases the salmonella counts in caecum, a new hypotheses was born: Does a decreased colonization of the gut leads to a decreased excretion of Salmonella bacteria in the faeces? Indeed! The addition of calcium butyrate in the feed leads to an decreased amount of Salmonella in the faeces, the excretion. Reference: F. Boyen, F. Haesebrouck, A. Vanparys, J. Volk, M. Mahu, F. Van Immerseel, I. Rychlik, J. Dewulf, R. Ducatelle, F. Pasmans. Coated fatty acids alter virulence properties of Salmonella Typhimurium and decrease intestinal colonization of pigs. Veterinary Microbiology 132 (3-4), 2008, Pages 319-327.
5/ Transmission Butyrate decreases the amount of Salmonella in the feces: the excretion. Less excretion of Salmonella leads to a decreased contamination of the environment by Salmonella and to a lower transfer of the bacteria towards other animals. Reference: L. De Ridder (2010). CODA-CERVA. Control of Salmonella infections in pigs: Transmission study. Unpublished.
Salmonella and Calciumbutyrate Trials in poultry Globamax Performant®
1/ Invasion of Salmonella Enteritidis in avian intestinal epithelial cells in vitro is influenced by SCFA. Reference: Van Immerseel, De buck, Meulemans, Pasmans, Velge, Bottreau, Fievez, Haesebrouck, Ducatelle (2004). Journal of food microbiology, 85(3); 237-248.
Aim: Evaluate effect of SCFA on the invasion properties of S. Enteritidis in avian epithelial cell line in vitro.
1/ Invasion of Salmonella Enteritis in avian intestinal epithelial cells in vitro is influenced by SCFA.
● Results: Salmonella invasion through epithelial cells of broiler chickens was studied at two different pH levels 6 and 7. In the research 4 different organic acids were studied : formic, propionic, acetic and butyric acid. Butyrate decreased significantly the invasion at all levels. Two other organic acids did increase invasion (one significantly and one numerically), whereas a third had only a numerical effect.
Globamax performant and Salmonella control in broilers (Van Immerseel 2004 - University Ghent) Salmonella invasion through epithelial cells at pH 7 and 6 with graded concentrations of Globamax performant (Greencab 70 Coated ) pH = 6
pH = 7
7.0
7.0
6.0
6.0
5.0 4.0
0
25
50
5.0 0
25
50
100
4.0
3.0
3.0
2.0
2.0
1.0
1.0
0 Different concentrations of Greencab 70 Coated
0
Different concentrations of Greencab 70 Coated
100
2/Protection against Salmonella colonization in chickens by feed supplementation with short-chain fatty acids.
Reference: Van Immerseel, Fievez, De Buck, Pasmans, Haesebrouck, Ducatelle. (2004). Poultry science 83, 69-74. Aim: Evaluating the efficacy of microencapsulated SCFA in reducing the colonization of caeca and internal organs of chickens by Salmonella Enteritidis
2/Protection against Salmonella colonization in chickens by feed supplementation with short-chain fatty acids.
Results: The effect of the 4 acids in coated form was studied on the Salmonella counts in caecum, spleen and liver of broilers. Globamax Performant significantly reduced Salmonella counts in the caecum and didn’t influence the counts in spleen and liver, opposite to other SCFA’s that increased counts.
Salmonella in various organs after supplementation of coated butyric acid Van Immerseel-University of Ghent-2004 7.0 6.0
Negative control
5.0
Greencab 70 Coated
4.0 3.0
2.0 1.0 0
Caecum
Liver
Spleen
Coated butyric acid significantly reduced Salmonella counts in the caecum and didn’t influence the counts in spleen and liver, opposite to other SCFA’s that increased counts.
Salmonella in different organs after administration of encapsulated Short Chain Fatty Acids Van Immerseel –University of Ghent- 2004
Log cfu/gr 7.0
control Formic acid Acetic acid Propionic acid Butyric acid
6.0 5.0 4.0 3.0
2.0 1.0 0
a
a
b
ac c
Caecum
a bc
b ac Liver
a
a
b
c
a
a
Spleen
Coated butyric acid significantly reduced salmonella counts in the caecum and didn’t influence the counts in spleen and liver, opposite to other SCFA’s that increased counts.
MIC values Salmonella Java University Ghent, Belgium 2009
● Minimum inhibitory concentration : preventing growth at pH 6 ● 3 Different strains of S. Java: MIC : 80 mM butyric acid ● S. Typhimurium strains: MIC : 160 - 320 mM butyric acid
Salmonella and Calciumbutyrate Trials in pigs Globamax performant®
1/Globamax Performant and Salmonella in pigs Ghent University – Faculty of veterinairy medicine IWT project 2006-2007
a) Study of the influence of SCFA’s and MCFA’s on the gen expression for Salmonella, in vitro study : • Hil A : gene responsible for invasion • Fim A : gene responsible for adhesion o The influence of fatty acids on the expression of virulence genes of Salmonella Typhimurium and the colonization of pigs. I. Lardon, F. Pasmans, F. Boyen, A. Decostere, F. Haesebrouck (2007) Proceedings of the 7th International Safe Pork Symposium o Short- and medium-chain fatty acids influence Salmonella Typhimurium virulence gene expression. M. Mahu, F. Pasmans, F. Boyen, A. Decostere, F. Haesebrouck (2006) Proceedings of the International Symposium Salmonella and Salmonellosis
b) Study on the invasion through the epithelial cell wall, in vitro and evaluation of the excretion in the litter and colonization of Salmonella in the organs in pigs, in vivo study Coated fatty acids alter virulence properties of Salmonella Typhimurium and decrease intestinal colonization of pigs. F. Boyen, F. Haesebrouck, A. Vanparys, J. Volk, M. Mahu, F. Van Immerseel, I. Rychlik, J. Dewulf, R. Ducatelle, F. Pasmans. Veterinary Microbiology 132 (3-4), 2008, Pages 319-327.
1a/ Study of the influence of SCFA’s and MCFA’s on the gene expression of Salmonella in vitro ● Outcome: - Gen expression Fim A (Adhesion): Butyric acid decreased Fim A expression - Gen Expression Hil A (Invasion): Butyric, capronic and caprylic decreases HilA gen expression
1a/Study of the influence of SCFA’s and MCFA’s on the gene expression of Salmonella in vitro M. Mahu, F. Pasmans, F. Boyen, A. Decostere, F. Haesebrouck (2006) 1,00E+10
Control boterzuur mierenzuur
1,00E+08
caprinezuur capronzuur caprylzuur
1,00E+07
LB
1,2
relatieve lichtproductie
0,4 0,2
LB
30
bo te rz uu r m ie re nz uu r pr op io nz uu r ca pr yl zu ur ca pr in ez uu r ca pr on zu ur
20
Relative gen expression for hilA by Salmonella in LB-medium with and without different fatty acids (GPI-1,invasion)
6
Control 4
1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0
LB
bo te rz uu r m ie re nz uu r pr op io nz uu r ca pr in ez uu r ca pr on zu ur ca pr yl zu ur
0
az i
jn zu ur
2
Control Butyric acid
LB
Butyric acid
Relative invasion of IPI-21 cells by Salmonella in LBmedium with and without different fatty acids
relatieve lichtproductie
10
jn zu ur
0,6
jn zu ur
10 tijd (uren)
relatief aantal intracellulaire kiemen
0,8
az i
0
az i
1
0
1,00E+06
8
Butyric acid
bo te rz uu r m ie re nz uu r pr op io nz uu r ca pr yl zu ur ca pr in ez uu r ca pr on zu ur
log 10 kve/ml
azijnzuur 1,00E+09
Relative gene expression for fimA by Salmonella in LB-medium with and without different fatty acids (attachment type 1 fimbrae)
1b/Coated fatty acids alter virulence properties of Salmonella Typhimurium and decrease intestinal colonization of pigs. Reference: F. Boyen, F. Haesebrouck, A. Vanparys, J. Volk, M. Mahu, F. Van Immerseel, I. Rychlik, J. Dewulf, R. Ducatelle, F. Pasmans. Veterinary Microbiology 132 (3-4), 2008, Pages 319-327.
Aim: Evaluating the usefulness of SCFA and MCFA in controlling Salmonella infections in pigs. The influence of concentrations of these acids on virulence gene expression and invasive capacities of Salmonella Typhimurium was evaluated. The efficacy of coated and uncoated fatty acids in reducing the early colonization of piglets inoculated with Salmonella Typhimurium was assessed in two in vivo trials.
1b/ Coated fatty acids alter virulence properties of Salmonella Typhimurium and decrease intestinal colonization of pigs. Results ● Gene Expression Hil A (Invasion) in vitro: o
Butyric, proprionic, capronic and caprylic decrease HilA gen expression
● In vivo excretion of Salmonella (manure, excreta): -
Butyrate decreased significantly the excretion!!! (coated form, not uncoated form)
● Colonization : o
o
Globamax performant significantly reduced colonization of Salmonella in the intestines, but had no influence of colonization of tonsils, liver, spleen. Uncoated butyrate had no influence on colonization of internal organs and excretion.
Effect of butyric acid on invasion : in vitro 1.2
Relative invasion
1
0.8
0.6
0.4
0.2
0 Butyrate
LB * LB = control medium
Butyrate decreases the invasion in vitro!
Globamax performant and Salmonella in pigs: in vivo trial (University of Ghent – Faculty of Veterinary Sciences, 2007)
PROTOCOL : • Diets during 12 days : - control - 2 kg globamax performant - 3,1 kg coated caprylic (50%) • Infection with 5.10 exp7 cfu Salmonella Typhimurium • Samples of litter every day • Day 4 after infection : euthanasy and bacterial analysis of the organs
Butyrate decreases the excretion of Salmonella
(= Excretion in the litter) Butyric acid
Globamax performant and Salmonella in pigs: in vivo trial (University of Ghent – Faculty of Veterinary Sciences, 2007) Colonization of different organs 8 Controle
Control Butyric
LOG10(kve/gram staal)
Caprylic
7
Boterzuur
6
Caprylzuur
5 4 3 2 1
ile
lo n co
in h
co
lo
ln n
n
lo n co
ln n ale ec
oc a
ca ec um
um ec
in ho ud
ca
ile
um
um in ho ud
ile
nn tl
jej
m es en
inhoud = content lnn = lymphenods
jej
-1
in ho ud
un um
0
Globamax performant and Salmonella in pigs: in vivo trial (University of Ghent – Faculty of Veterinary Sciences, 2007) Both NON-COATED additives !!!!!!!!!!
Need for Coated Product
Excretion 6
LOG10(kve/gram mest)
5 4 Controle
3
Capronzuur 2
Boterzuur
1
Control Ca-Butyrate
0 Dag 1 pi
Dag 2 pi
-1 dagen pi
Dag 3 pi
Na-Caproate
2/Globamax performant and Salmonella Field case study - Belgium
Background info :
• Home mix farm with proper sows (source of Salmonella) • Farm ranked as “risk farm” (Regional Health Centre) • Veterinarian advised to acidify the drinking water, but result was not satisfactory. • Heard about Globamax performant: dosage at 2 kg/ton in sow feed
Salmonella field case: results S/P Ratio 2,5
2,16 2 1,5
Adding to piglet feed
1,46
Adding to sow feed
0,89
1
0,68 s/p = 0.6
0,5 0 8/05/2007
'22/09/2007
6/02/2008
9/06/2008
S/P ration decreases with Globamax : need to apply in all phases : sows, feed and pre-fattening.
3/Flanders Food Salmonella project
‘Innovative feed additives for reduction of Salmonella in pig meat” (University Ghent – Ilvo : 2007-2009) Two Feed suppliers – two farms – various consecutive sheds (fattening periods) – Evolution of S/P ratio and prevalence – 2 kg Globamax /ton in round 2 till 4 FARM 1
FARM 2 start
Control :
end Prevalence
0.12
5.0 - 40.0%
start
end Prevalence
Control : 2.08 0.78 1.1 – 6.8%
Round 2 : 0.28
0.20
5.0%
Round 2 :
0.08
3.3 %
Round 3 : 0.14
0.29
3.7%
Round 3 :
0.38
6.6 %
Round 4 : 0.14
0.22
1.3%
Round 4 :
0.31
4.9 %
Conclusions : “Remarkable improvement of the Salmonella status in trial period compared with control period”. Due to either the use of the product or other origin of the pigs “
Conclusions : “Only Typhimurium found. Still running. Prevalence remains the same, s/p titers decrease”
5/Control of Salmonella infections in pigs: Transmission study (part 1) L. De Ridder, CODA-CERVA, 2010
● Aim: Search for strategies to prevent transfer of Salmonella in a herd ● Experimental design: o 69 Salmonella-negative piglets (age: 3 weeks) o treatments Calcium-butyrate (Globamax performant®, Sanluc International) 3kg/ton (2x8 pigs) Acidified water pH 3,5-3,8 (formic acid (50%), proprionic acid, acetic acid and lactic acid) (2x8 pigs ) oral vaccination with salmonella vaccin (Salmoporc®, IDT) (2x8 pigs) positive control group, 2x8 pigs negative control group, 1x5 pigs
o 8 weeks of age: 2 piglets of each group, except the negative control piglets, were infected orally with 108 cfu of Salmonella Typhimurium.
Experimental design:
Results: Serology
Vaccination leads to an antibody response with a high level of protective antibodies. Due to infection with Salmonella there is an antibody response in the animal with an antibody level correlating to the severity of infection: treatment with acidified water leads to high antibody levels, this means there was a severe infection with Salmonella.
Results: Isolation of Salmonella in feces
Acidified water leads to a high excretion level of Salmonella. Treatment with calciumbutyrate or vaccination decreases shedding of Salmonella.
Isolation of Salmonella in organs
Treatment with calcium-butyrate leads to a decrease of Salmonella colonization of the organs!
Results: isolation of Salmonella of the digestive tract
Treatment with calcium-butyrate leads to an decrease of salmonella colonization of the intestinal tract!
Results: isolation from… Basis reproductie Ratio R0 = number of secondary cases caused by 1 infectious animal in an infectious period in a fully susceptible population Rate of disease spread in a population Calculations according SI-model (with Rn = Rnursery):
Weight gain from inoculation (8w) till euthanasia (14w)
Treatment with calcium-butyrate increased significantly the final weight comparing the positive control group. There was no weight loss due to the infection pressure of Salmonella, the final weight of the calcium-butyrate group and the negative control group was similar.
Isolation of Salmonella in organs
Treatment with calcium-butyrate leads to a decrease of Salmonella colonization of the organs!
Results: isolation of Salmonella of the digestive tract
Treatment with calcium-butyrate leads to an decrease of salmonella colonization of the intestinal tract!
Results: isolation from… Basis reproductie Ratio R0 = number of secondary cases caused by 1 infectious animal in an infectious period in a fully susceptible population Rate of disease spread in a population Calculations according SI-model (with Rn = Rnursery):
Weight gain from inoculation (8w) till euthanasia (14w)
Treatment with calcium-butyrate increased significantly the final weight comparing the positive control group. There was no weight loss due to the infection pressure of Salmonella, the final weight of the calcium-butyrate group and the negative control group was similar.
Results: Weight gain
The vaccine and the combination strategy do not have negative effects on Daily weight gain
Results: Serology
Lowest S/P ratio’s in combination strategy.
Results: Excretion in faeces
Lowest Salmonella excretion in control strategy group.
Results: isolation of Salmonella of the digestive tract
Lowest contamination of the digestive tract by salmonella
6/Controle van Salmonella infecties in varkens: Transmissie studie (part 2) - L. De Ridder, CODA-CERVA, 2010 ● ●
Aim: Search for strategies to prevent transfer of Salmonella in a herd Materials and methods: o o
72 Salmonella-negative, 20-21 day old piglets in 5 groups (2 replicates per treatment) Treatments (3-15 weeks of age): Vaccination with DIVA merker vaccine of University of Ghent (2x8 pigs) Vaccination and Globamax performant® supplemented feed 3kg/ton feed (2x8pigs) Feed premix with benzoic acid (Vevostart®) Positive control group (infected/untreated), 2x8 pigs Negative control group (uninfected/untreated), 1x5 pigs
●
●
9 weeks of age: two pigs of each group (=seeders), except from the negative control, were challenged orally with 107 cfu of Salmonella Typhimurium and placed back after 24h. Measurements: fecal samples for salmonella counts , body weight, blood for salmonella specific antibodies, gut samples (week 15)
Results: Weight gain
The vaccine and the combination strategy do not have negative effects on Daily weight gain
Results: Serology
Lowest S/P ratio’s in combination strategy.
Results: Excretion in faeces
Lowest Salmonella excretion in control strategy group.
Results: isolation of Salmonella of the digestive tract
Lowest contamination of the digestive tract by salmonella
Salmonella field trial with DGZ Belgium, 2012
● Set-up: o 15 fattening pigs (in 12 compartments) o Duration: whole fattening period o farm with salmonella problems in the past: s/p ratio >0,6: Barn with hygienic problems: only partly slatted floor o Treatment: Globamax Performant 2kg per ton of feed in starter, grower and finisher o Measurements: Blood at 20kg/40kg/70kg/slaughter for serology Rectal feces at 20kg/40kg/70kg/slaughter for bacterial examination Floor samples at 20kg/40kg/70kg/slaughter for bacterial examination
Salmonella field trial with DGZ Belgium, 2012
● Pretrial results: o Floor samples: POSITIVE for Salmonella Livingstone and Salmonella Derby. o S/p ratio: 0,84 →No cleaning of floor between rounds and start trial!
Salmonella field trial with DGZ Belgium, 2012
Overview of S/P ratio of farm
Globamax performant 2kg/ton
1,6 1,4 1,2 1 0,8 0,6 0,4
0,2 0 25/03/2011
26/07/2011
05/12/2011
05/03/2012
09/07/2012
Salmonella field trial with DGZ Belgium, 2012
S/p ratio during trial 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 20 kg
40kg
70 kg
slaughter
Salmonella field trial with DGZ Belgium, 2012
● Floor samples remain NEGATIVE during whole trial ● Rectal feces samples remain NEGATIVE during whole trial
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