Lactobacillus acidophilus D2/CSL (CECT 4529)

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2 Lactobacillus acidophilus D2/CSL (CECT 4529) HISTORY SAFETY EFFICACY 1985 to 2018

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4 4 SYNERGETIC COMPANIES Today, four different companies work in synergy within a single business system, validly maintaining their autonomy, characteristics and production specificity.

5 AGROZOOTECHNICAL CULTURES POULTRY AGRO ZOOTECHNICAL SILAGE

6 HOW CSL WORKS MATERIALS: - FOODS - FEEDS - EXCRETA... Isolation of LAB strains Production Pilot plant Characterization Selection Strain collection

7 Lactobacillus acidophilus D2/CSL CECT 4529 HISTORY

8 1985 CSL isolates 19 strains of faecal lactobacilli from a «rural» adult chicken

9 1985 Choice of L. acidophilus D2/CSL from the 19 LAB strains The choice of the strain L. acidophilus D2/CSL over the other 18 isolated strains was due to: better bile-resistance better adhesion to the gut epithelium better resistance to the jonophorous antibiotic better performance in the production plant (recovery of UFC after the freeze-drying process)

10 Lactobacillus acidophilus D2/CSL CECT 4529 SAFETY

11 SAFETY OF L. acidophilus (FEEDAP, 2014) The species L. acidophilus is considered by EFSA to be suitable for the Qualified Presumption of Safety (QPS) approach to safety assessment (EFSA, 2007; EFSA BIOHAZ Panel, 2013). This approach requires: - THE IDENTITY OF THE STRAIN to be conclusively established and - evidence that it does not show RESISTANCE TO ANTIBIOTICS of human and veterinary importance.

12 2012 Results of the last A-R test (FEEDAP, 2012) NOT TRASMISSIBLE

13 Lactobacillus acidophilus D2/CSL CECT 4529 EFFICACY

14 1993 Efficacy trial - D2/CSL laying hens - ISA WARREN HENS KEPT IN CAGE - TRIAL 1 = 7150 HENS - 3 GROUPS = 2 TR + 1 CO - TRIAL 2 = 672 HENS - 8 REPETITIONS = 4 TR + 4 CO - PROBIOTIC IN DRINKING WATER

15 1994 Efficacy trial D2/CSL broilers - SHAVER REDBRO BROILERS KEPT ON LITTER - TOT. 216 CHICKS - 12 GROUPS = 6 TR + 6 CO - PROBIOTIC IN DRINKING WATER

16 1995 Efficacy trial - D2/CSL broilers - SHAVER REDBRO BROILERS KEPT IN CAGE = 3 CHICKS/CAGE - TOT. 54 CHICKS - 18 CAGES = 9 CAGES TR (27 CHICKS ) + 9 CAGES CO (27 CHICKS) - PROBIOTIC IN DRINKING WATER

17 2007 No. 3 «in field» efficacy trials - broilers - ROSS 508 BROILES KEPT ON LITTER CHICKS 3 SEASONS CHICKS/SEASON - 6 POULTRY HOUSES 3 TR + 3 CO ( CHICKS/SHELTER) - PROBIOTIC IN DRINKING WATER

18 2008 n. 1 Efficacy trial laying hens - HI-LINE BROWN HENS KEPT IN CAGE HENS - 8 GROUPS = 4 TR + 4 CO - PROBIOTIC IN FEED

19 2008 n.2 Second efficacy trial laying hens - HI-LINE W 36 HENS KEPT IN (SINGLE) CAGE HENS 3 GROUPS = CO (D2) ; TR1 (D2 + SMP 3%); TR2 (D2 + SMP 4%) - PROBIOTIC IN FEED

20 2008 n. 3 Study of an analysis method for feed Ital.J.Anim.Sci. vol. 7, , 2008

21 2012 Efficacy trial of «MIX AVI» - laying hens - HI-LINE BROWN HENS KEPT ON LITTER - 2 POULTRY FARMS 480 HENS IN TOTAL - 2 GROUPS PER FARM = CO; TR (D2 + 14D) - 3 REPLICATES PER GROUP - PROBIOTIC VIA THE DRINKING WATER

22 2014 Efficacy trial- laying hens - HI-LINE W-36 HENS KEPT IN CAGE LAYERS - 3 GROUPS = CO (D2) ; TR1 (D2 + SMP 3%); TR2 (D2 + SMP 4%) - PROBIOTIC IN FEED

23 2015 Authorization for D2/CSL (CECT 4529) in the EU: laying hens (feed + water)

24 Identification number of the additive 2017 Authorization for D2/CSL (CECT 4529) in the EU: chickens for fattening Name of the holder of authorisation Additive Category of zootechnical additives. Functional group: gut flora stabilisers 4b1715 Centro Sperimentale del latte Lactobacillus acidophilus CECT 4529 ANNEX COMMISSION IMPLEMENTING REGULATION (EU) 2017/2275 Composition, chemical formula, description, analytical method Additive composition: Preparation of Lactobacillus acidophilus CECT 4529 containing a minimum of: 5x10 10 CFU/g of additive (solid form) Characterisation of the active substance: Viable cells of Lactobacillus acidophilus CECT Analytical method 1 Enumeration: spread plate method using MRS agar (EN 15787). Identification: Pulsed Field Gel Electrophoresis (PFGE). Species or category of animal Chickens for fattening Maximum age Minimum content Maximum content CFU /kg of complete feedingstuff with a moisture content of 12% Other provisions - 1 x In the directions for use of the additive and premixture, the storage conditions and stability to heat treatment shall be indicated. 2. For users of the additive and premixtures, feed business operators shall establish operational procedures and organisational measures to address potential risks resulting from their use. Where those risks cannot be eliminated or reduced to a minimum by such procedures and measures, the additive and premixtures shall be used with personal protective equipment, End of period of authorisation [10 years from the date of entry into force of this Regulation To be completed by the Service responsible for the publication] 1 Details of the analytical methods are available at the following address of the European Union Reference Laboratory for Feed Additives:

25 The 5 more recent experimental trials 1. UniPg-IZSUM & UniTe: D2/CSL in organic layers 2. UniPg & IZSUM: D2/CSL in organic layers 3. UniBo: D2/CSL on broiler performance 4. UniBo: D2/CSL on intestinal chicken microbioma 5. UniPg & IZSUM: D2/CSL in rural broilers

26 2015 Efficacy trial UniPg-IZSUM-UniTe organic hens

27 2016 Efficacy trial UniPg-IZSUM organic hens

28 2016 Efficacy trial UniBo intestinal microbioma and metaboloma of broilers

29 2017 Efficacy trial UniBo D2/CSL effects on performance and microbioma of broilers

30 2018 Efficacy trial IZSUM-UNIPG D2/CSL effects on rurally reared broilers

31 : summary of the efficacy results Laying hens The better GUT HEALTH (or EUBIOSIS) due to L. acidophilus D2/CSL has led to significant improvements in terms of: 1) Intestinal microbiota composition 2) FCR (kg feed/kg eggs) 3) Egg production 4) Egg quality (ESG, shell thickness, Haugh Units) 5) Saleable eggs (less wastage: dirty eggs, cracked eggs, etc. 6) Environment: reduction of volatile N compounds and faecal moisture within the poultry house.

32 : summary of the efficacy results Broilers The better GUT HEALTH (or EUBIOSIS) due to L. acidophilus D2/CSL has led to significant improvements in terms of: 1) Gut microbiota modifications 2) Gut morphology (villus height) 3) Morbidity and pasty vents 4) Weight increment 5) FCR (Kg feed/kg weight increment) 6) Environment: reduction of volatile N compounds and faecal moisture within the poultry house.

33 SUMMARY OF THE ACTIVITIES OF L. ACIDOPHILUS D2/CSL L. ACIDOPHILUS D2/CSL PRODUCES BY FERMENTATION LACTIC ACID REBALANCES BY COMPETITION WITH PATHOGENS REBALANCES THROUGH ACIDIFICATION DEVELOPS AND STIMULATES GALT GASTRO-INTESTINAL MICROBIOTA MORE GUT HEALTH AND ANIMAL PRODUCTIONS

34 Commercial chickens are subject to INTESTINAL DYSBIOSIS. The outcome can be a TRUE PATHOLOGY or, more frequently, a SUB- CLINICAL MALAISE that worsens the flock PERFORMANCE. The INTESTINAL DYSBIOSIS: CONCLUDING REMARKS produces INFLAMMATION and alters the DIGESTIVE FUNCTIONS; damages the EGG PRODUCTION in quanti-qualitative terms (dirty and thin egg shells, liquid albumen, etc.); damages the BROILER PERFORMANCES and increases BROILER MORBIDITY; reduces the FEED DIGESTIBILITY and the FAECAL CONSISTENCY harms the ENVIRONMENT in the shelter (more pathogens, humidity, ammonia, H2S, etc.).

35 CONCLUDING REMARKS Each flock is composed by a mixture of HEALTHY, SUB- CLINICALLY ILL, and CLINICALLY ILL birds. The primary aim of feeding birds with L. acidophilus D2/CSL is to prevent or resolve the gastro-intestinal dysbiosis, namely to protect the HEALTHY birds (PROPHYLAXIS) and/or "SUB-CLINICALLY ILL birds (METAPHYLAXIS). That is why L. acidophilus D2/CSL is the NATURAL WAY to improve the productive performance of the so called HEALTHY flocks.

36 Probiotics and pathogens: the DOSE make the difference! THE DOSE OF D2CSL MUST BE HIGH ENOUGH TO EFFECTIVELY INFECT THE HOST. On the contrary a cause-effect relationship is not evident probably because you have generated a ASYMPTOMATIC CARRIER! PATHOGENIC STRAIN PATHOLOGY ASYMPTOMATIC CARRIER (NO EFFECT) LOW (NON INFECTING) DOSE HIGH (INFECTING) DOSE HEALTHINESS PROBIOTIC STRAIN

37 THANK YOU FOR YOUR ATTENTION! takes care of the gut health since 1948