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EFSA Journal 2012;10(2):2574

EFSA Journal 2012;10(2):2574

SCIENTIFIC OPINION

Scientific Opinion on Lactiferm ® (Enterococcus faecium) as a feed additive for weaned piglets and calves 1

EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3

European Food Safety Authority (EFSA), Parma, Italy

ABSTRACT

Lactiferm ® is a feed additive containing viable cells of Enterococcus faecium. It is currently authorised for use in piglets and calves and provisionally authorised for its use in chickens for fattening in powder and coated forms. The applicant is requesting the authorisation of a new water-miscible form of the product, and the renewal of the

powder form and the withdrawal of the coated form. Lactiferm ® is intended for use at a range of 5 x 10 8 - 2 x 10 10 CFU/kg feedstuffs. It is also intended to be used in water for drinking for piglets in the range of 2 x 10 8 to 8

x 10 9 CFU/L water. E. faecium NCIMB 11181 does not contain marker genes typical of hospital associated

isolates responsible for clinical infections and does not harbour acquired genes coding for antibiotic resistance. Lactiferm ® is safe for weaned piglets and calves at the recommended dose range. Since neither the active agent nor the other components of the additive give rise to concerns, the FEEDAP Panel considers the use of the additive safe for consumers. Given the lack of specific information and its proteinaceous nature and the high dusting potential, in particular of the water soluble form, Lactiferm ® should be considered to have the potential

to be an irritant to eye and skin and a skin/respiratory sensitiser. E. faecium is a natural component of gut

microbiota and its use as Lactiferm ® in animal feeding would not be expected to pose any additional risk for the environment. Lactiferm ® is efficacious in improving zootechnical performance of piglets and calves. The minimum effective dose for piglets is in the order of 1 x 10 10 CFU/kg feed and for calves in the region of 2 x 10 9 CFU/kg milk replacer. The FEEDAP Panel considers evidence of efficacy to be independent of route of delivery provided that the same dose is given.

© European Food Safety Authority, 2012

KEY WORDS

Zootechnical additive, Lactiferm ® , Enterococcus faecium, piglets, calves, efficacy

1 On request from the European Commission, Question No EFSA-Q-2009-00679, adopted on 1 February 2012.

2 Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Albert Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Reinhard Kroker, Lubomir Leng, Secundino López Puente, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Giovanna Martelli, Miklós Mézes, Derek Renshaw, Maria Saarela, Kristen Sejrsen and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu

3 The Panel wishes to thank the members of the Working Group on Micro-organisms, including Guido Rychen, for the preparatory work on this scientific opinion.

For citation purposes: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); Scientific Opinion on Lactiferm ® (Enterococcus faecium) as a feed additive for weaned piglets and calves. EFSA Journal 2012;10(2):2574. [15 pp.] doi:10.2903/j.efsa.2012.2574. Available online: www.efsa.europa.eu/efsajournal

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

SUMMARY

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of Lactiferm ® (Enterococcus faecium) for piglets and calves.

This product is currently authorised for use in piglets and calves in two forms: a powder form with a guaranteed minimum concentration of 4 x 10 11 CFU of E. faecium per gram of product, and a coated form with a minimum guaranteed concentration of 5 x 10 10 CFU of E. faecium per gram of additive. With the current application, the applicant is requesting the authorisation of a new water-miscible form of the product and the renewal of the current authorisation with a modification in the formulation of the powder form and withdrawal of the coated form. The product is intended for use in feeds for weaned piglets and calves up to six months at the minimum recommended dose of 5 x 10 8 and at the maximum recommended dose of 2 x 10 10 CFU/kg feedstuffs. It is also intended to be used in water for drinking for piglets at concentrations in the range of 2 x 10 8 to 8 x 10 9 CFU/L water.

E. faecium NCIMB 11181 does not contain marker genes typical of hospital associated isolates responsible for clinical infections and does not harbour acquired genes coding for antibiotic resistance.

Lactiferm ® is safe for weaned piglets and calves at the recommended dose range.

Since neither the active agent nor the other components of the additive give rise to concerns, the FEEDAP Panel considers the use of the additive safe for consumers.

Given the lack of specific information and its proteinaceous nature and the high dusting potential, in particular of the water soluble form, Lactiferm ® should be considered to have the potential to be an irritant to eye and skin and a skin/respiratory sensitiser.

E. faecium is a natural component of gut microbiota and its use as Lactiferm ® in animal feeding would not be expected to pose any additional risk for the environment.

Lactiferm ® is efficacious in improving zootechnical performance of piglets and calves. The minimum effective dose for piglets is in the order of 1 x 10 10 CFU/kg feed and for calves in the region of 2 x 10 9 CFU/kg milk replacer. The FEEDAP Panel considers evidence of efficacy to be independent of route of delivery provided that the same dose is given.

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

TABLE OF CONTENTS

Abstract

 

1

Table of contents

3

Background

4

Terms of reference

 

4

Assessment

7

1. Introduction

7

2. Characterisation

7

2.1.

Characterisation of the active agent

7

2.1.1. Virulence

7

2.1.2. Antimicrobial resistance

7

2.2. Manufacturing process

8

2.3. Characterisation of the product

8

2.4. Stability and homogeneity

8

2.5. Proposed conditions of use

9

2.6. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL)9

3. Safety

9

3.1. Safety for the target species

9

3.2. Safety for the consumer

10

3.3. Safety for the user

10

3.4. Safety for the environment

10

4. Efficacy

10

4.1. Efficacy for weaned piglets

10

4.2. Efficacy for calves

12

5. Post-market monitoring

13

Conclusions

13

Documentation provided to EFSA

14

References

14

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

BACKGROUND

Regulation (EC) No 1831/2003 4 establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7. Article 10(2) of that Regulation also specifies that for existing products within the meaning of Article 10(1), an application shall be submitted in accordance with Article 7, at the latest one year before the expiry date of the authorisation given pursuant to Directive 70/524/EEC for additives with a limited authorisation period, and within a maximum of seven years after the entry into force of this Regulation for additives authorised without time limit or pursuant to Directive 82/471/EEC.

The European Commission received a request from the company Chr. Hansen A/S 5 for the re- evaluation and authorisation of the product Lactiferm ® , Enterococcus faecium, when used as a feed additive for piglets and calves (category: zootechnical additives; functional group: gut flora stabilisers) under the conditions mentioned in Table 1.

According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4(1) (authorisation of a feed additive or new use of a feed additive) and under Article 10(2) (re-evaluation of an existing authorisation). EFSA received directly from the applicant the technical dossier in support of this application. 6 According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 13 August 2010.

The applicant requested the application of Article 4(2) of Regulation (EC) No 429/2008.

The additive Lactiferm ® is a preparation of Enterococcus faecium NCIMB 11181. This product is currently authorised for use in diets for piglets and calves. 7

The Scientific Committee on Animal Nutrition (SCAN) issued one opinion on the use of Lactiferm ® as a feed additive (EC, 2003). EFSA issued also one opinion on the safety of the safety of Lactiferm ® for chickens for fattening (EFSA, 2005).

TERMS OF REFERENCE

According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the safety for the target animal(s), consumer, user and the environment and the efficacy of the product Lactiferm ® , Enterococcus faecium, when used under the conditions described in Table 1.

4 Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. OJ L 268, 18.10.2003, p.29

5 Chr. Hansen A/S, 10-12 Boege Allé, 2970 Hoersholm, Denmark

6 Dossier reference: FAD-2009-0022

7 Commission Regulation (EC) No 1333/2004 of 20 July 2004 concerning the permanent authorisation of certain additive in feedingstuffs. OJ L 247, 21.7.2004, p.11

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

Table 1:

Description and conditions of use of the additive as proposed by the applicant

Additive

Enterococcus faecium M74 ® , NCIMB 11181 (Lactiferm ® )

Registration number/EC No/No

E1708

Category of additive

Zootechnical additive

Functional group of additive

Gut flora stabiliser

 

Description

 

Composition, description

Chemical formula

 

Purity criteria

Method of analysis

 

Powder form/L Basic 50

     

5

x 10 10 CFU/g

 

Complies with EU law on undesirable substances

 

Validated

A preparation of Enterococcus faecium M74 ® , NCIMB 11181

 

microbiological

Water soluble/L WS 200

 

methods

2

x 10 11 CFU/g

   

Trade name

Lactiferm ® (other names may also be used)

 

Name of the holder of authorisation

Chr. Hansen A/S

 

Conditions of use

Species or

Maximum

Minimum content

 

Maximum content

 

Withdrawal

category of

Age

 

CFU/kg of complete feedingstuffs or CFU/L of water

 

period

animal

Calves

6 months

 

Powder:

   

Powder:

   

5

x 10 8

2

x 10 10

     

Powder:

   

Powder:

 

Not relevant

Piglets

5

x 10 8

2

x 10 10

-

Water soluble:

   

Water soluble:

   
   

2

x 10 8

8 x 10 9

Other provisions and additional requirements for the labelling

Other provisions and additional requirements for the labelling

Specific conditions or restrictions for use

Specific conditions or restrictions for handling

Post-market monitoring

Specific conditions for use in complementary feedingstuffs

Store refrigerated (4-5ºC) in original, closed packaging

For user safety: recommended to use a facemask, gloves and goggles to reduce contact with dust.

Chr. Hansen A/S will conduct post-marketing monitoring in compliance with EU law on feed hygiene, namely by use of HACCP and traceability systems, and formal monitoring of customer feedback through product or service complaints

Content of Enterococcus faecium M74 ® , NCIMB 11181 in final feeds should be 2.5 x10 8 to 2 x 10 10 CFU/kg

Maximum Residue Limit (MRL)

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

Marker residue

Species or category of animal

Target tissue(s) or food products

Maximum content in tissues

Not relevant

Not relevant

Not relevant

Not relevant

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

ASSESSMENT

1. Introduction

The additive Lactiferm ® is a microbiological feed additive containing viable cells of a strain of Enterococcus faecium. This product is currently authorised for use in piglets and calves in two forms:

a powder form with a guaranteed minimum concentration of 4 x 10 11 CFU of E. faecium per gram of product, and a coated form with a minimum guaranteed concentration of 5 x 10 10 CFU of E. faecium per gram of additive. With the current application the Company is requesting the authorisation of a new water-soluble form of the product, the re-evaluation of the product with a modification in the formulation of the powder form, and withdrawal of the coated form.

2.

Characterisation

2.1.

Characterisation of the active agent

The active agent of Lactiferm ® are viable cells of E. faecium. The strain is deposited at the National Collection of Industrial and Marine Bacteria collection (UK) with the accession number NCIMB 18111. 8 The strain has been identified as E. faecium by Multilocus Sequence Typing of atpA, rpoA and pheS genes. 9

2.1.1. Virulence

In a first set of experiments E. faecium NCIMB 11181 was demonstrated to be free of the following genes coding putative enterococcal virulence factors by Polymerase Chain Reaction: aggregation substance, esp, efaAfs, gelatinase, and pheromone production determinants. 10

Moreover, the applicant provided a new study based on the analysis of complete genome sequence, aimed to demonstrate the absence of the genetic determinants typical of hospital isolated strains, including MSCRAMM (microbial surface components recognising adhesive matrix molecules, Nallapareddy et al., 2003), predicted major pilus protein, (Sillanpää et al., 2009), collagen adhesin gene (acm), putative hyaluronidase (hyl) (Rice et al., 2003), esp pathogenicity island and IS16 (Werner et al. 2011).

None of these virulence determinants were present other than the gene coding for collagen binding protein Acm and the part of the Pilin cluster 3 (PilB), two intact genes and two truncated, were detected. 11 Pilin and acm genes were found to be widely distributed in both clinical isolated and commensal strains of E. faecium (Sillanpää et al., 2010). However the Acm protein is expressed almost exclusively by clinical isolates (Sillanpää et al., 2009). Sequence analysis of acm gene of E. faecium NCIMB 11181 revealed the presence of deletions which give rise to frame-shifts, which results in a truncated protein.

The FEEDAP Panel concludes that E. faecium NCIMB 11181 does not contain marker genes typical of hospital associated isolates responsible for clinical infections.

2.1.2. Antimicrobial resistance

The susceptibility of the production strain to the antibiotics recommended by the FEEDAP Panel in its Technical guidance on the updating the criteria used in the assessment of bacteria for resistance to antibiotics of human or veterinary importance (EFSA, 2008) was tested by a dilution method. 12 The

8 Technical dossier/Section II/Annex II.2.1.2a

9 Technical dossier/Supplementary info Sept 2011/ Annex II.2.1.2c

10 Technical dossier/Section II/Annex II.2.1.2a

11 Technical dossier/Supplementary info Sept 2011/ Annex II.2.1.2c

12 Technical dossier/Section II/Annexes II.2.2.2b and d

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

minimum inhibitory concentrations of E. faecium NCIMB 11181 were lower than the EFSA breakpoints for all but kanamycin and clindamycin, which exceeded the breakpoints in each case by a single dilution and is not considered of concern.

2.2. Manufacturing process

Cells were grown in a sterilised medium typical of those used for lactic acid bacteria and then separated from the growth medium by centrifugation. Cryoprotectants (maltodextrin, sorbitol) are added and the cell mix is freeze-dried and ground. The excipients used are all food-grade (material safety data sheet (MSDS) provided). 13

2.3. Characterisation of the product

The additive is prepared in two different forms:

Powder form (Basic 50): Concentrate of E. faecium M74 ® NCIMB 11181 ca. 12-16 %, maltodextrin as carrier ca. 84-88 % , guaranteeing a with a minimum concentration of active agent of 5 x 10 10 CFU/g. 14

Water soluble form (WS 200): Concentrate of E. faecium M74 ® NCIMB 11181 ca. 45-50 %, sorbitol as carrier ca. 50-55 %, guaranteeing a minimum concentration of active agent of 2 x 10 11 CFU/g. 15

Data on a number of production batches of both powder form (nine batches, average 9.31 x 10 10 CFU/g) and water soluble form (seven batches, average 3.4 x 10 11 CFU/g) showed that the minimum specification was exceeded in all cases. 16

The particle size distribution was measured by laser diffraction in one batch of both forms. 17 The powder form has 30 % of particles with a diameter of approximately 100 µm, and 3 % of particles with a diameter 10 µm. Water soluble form has 23 % of particles with a diameter of approximately 100 µm and 2 % of particles with a diameter <10 µm.

The dusting potential of the two Lactiferm forms (powder and water soluble) was assessed with the Stauber-Heubach test. 18 Lactiferm powder showed a moderate dusting potential (0.50 g/m 3 ). In contrast, the water-soluble form presents an extremely high dusting potential (13.62 g/m 3 ).

The additive is routinely monitored for microbial contamination at various points in the manufacturing process and in the final product. Limits are set for total aerobic count, Escherichia coli, yeasts, filamentous fungi and Salmonella. Given the nature of the fermentation medium and the food grade excipients, the probability of contamination with heavy metals or mycotoxins is considered to be low and, consequently, not included in routine monitoring.

2.4. Stability and homogeneity

The stability of Lactiferm powder form (three batches) during storage in hermetic sealed plastic bags was tested at different temperatures (-20 °C, 5-8 °C and 20 °C) for 12-15 months. 19 During this time, the bacterial counts slightly decreased (less than 0.3 log) at 20 °C, while remained at their original count when refrigerated or frozen, guaranteeing the minimum content of 5 x 10 10 CFU/g. The stability of the water soluble form was assessed on four batches in sealed plastic bags at 20 °C. No significant losses were observed over a 12 months period.

Data on stability in premixtures for piglets were not provided.

13 Technical dossier/Section II and Supplementary info Sept 2011/Annex II.1.1.LBasic50 and Annex II_3_2a

14 Technical dossier/Section II/Annex II.1.1.LBasic50

15 Technical dossier/Section II/Annex II.1.1.LWS200

16 Technical dossier/Section II/ Annex_II_1_3_Var_batch.pdf

17 Technical dossier/Section II/Annex II.1.5a

18 Technical dossier/Section II/Annex II.1.5b

19 Technical dossier/Section II/Annex II.4.1

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

Stability in feed for piglets was assessed in three studies with the powder form at 8 and 20-22 °C (based on three batches). 20 The product was stable over six months at the lower temperature, but showed significant losses in two of the three experiments after two months at 20 °C. No data were provided on stability during pelleting and the single experiment made with pelleted feed the additive was sprayed after the thermal treatment.

Data on the stability of Lactiferm water soluble form (one batch) in dry milk replacer indicate that the product is stable up to six months. When dissolved in water (without heating), data indicated that the product should be used on the day of mixing (based on three batches). 21 This limitation would also apply to use of the additive in water for drinking for use with piglets.

The homogeneous distribution of Lactiferm powder in feed was assessed in one study by analysing ten sub-samples. The product showed a good distribution (cv 7 %). 22

2.5. Proposed conditions of use

The product is intended for use in feeds for weaned piglets and calves up to six months at the minimum recommended dose of 5 x 10 8 and at the maximum recommended dose of 2 x 10 10 CFU/kg feedstuffs. It is also intended to be used in water for drinking for piglets at concentrations in the range of 2 x 10 8 to 8 x 10 9 CFU/L water.

2.6. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL)

EFSA has verified the EURL report as it relates to the methods used for the control of E. faecium in animal feed. The Executive Summary of the EURL report can be found in the Appendix.

3.

Safety

3.1.

Safety for the target species

Two studies were provided. The first study involved 16 piglets weaned at 21 days allocated to two groups (control and treatment with the powder form used at 2.6 x 10 11 CFU/kg feed (corresponding to approximately 13 times the maximum recommended dose, confirmed by analysis), four replicates per treatment of two piglets each. The administration of the treatment lasted 28 days. 23

The second study was made with 12 calves (between 3 and 6 days of age) allocated to two groups (control and treatment with the powder form used at 2.1 x 10 11 CFU/kg feed, confirmed by analysis). Animals were housed individually. The study lasted 24 days. 24

Only feed intake, performance parameters and general health status were monitored in both studies. No significant differences were found between groups.

Although these studies do not meet the requirements of a tolerance study, taking into consideration that:

E. faecium is not pathogenic for the target species,

the strain lacks the marker genes associated with human clinical isolates,

the strain is free from acquired antibiotic resistance determinants,

20 Technical dossier/Section II/Annex II.4.1a

21 Technical dossier/Section II/Annexes II.4.1c and d

22 Technical dossier/Section II/Annex II.4.2b

23 Technical dossier/Section III/Annex III.1.11

24 Technical dossier/Section III/Annex III.1.1.2

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

the end-products of the metabolism of the species are typical of lactic acid bacteria, and do not raise concerns,

the additive does not contain excipients of concern,

the FEEDAP Panel concludes that Lactiferm ® is safe for weaned piglets and calves at the recommended dose range.

3.2. Safety for the consumer

E. faecium NCIMB 11181 does not contain marker genes typical of hospital associated isolates responsible for clinical infections and does not harbour acquired genes coding for antibiotic resistance.

The metabolism of E. faecium is well known and when the potential for infection is excluded, no other harmful metabolites or substances are expected to be produced during fermentation. In addition, cells are washed prior to incorporation to the additive and therefore, any carry-over from the fermentation would be negligible. The additive does not contain excipients of concern. Consequently, the FEEDAP Panel does not see the need for toxicological studies. This view is supported by the history of use, both in animals and directly in humans.

3.3. Safety for the user

Data on safety for the users were not provided. Given the lack of specific information and its proteinaceous nature and the high dusting potential, in particular of the water soluble form, Lactiferm ® should be considered to have the potential to be an irritant to eye and skin and a skin/respiratory sensitiser. These potential hazards are identified in the MSDS as proposed by the applicant accompanying the product.

3.4. Safety for the environment

E. faecium is a natural component of gut microbiota and its use as Lactiferm ® in animal feeding would not be expected to pose any additional risk for the environment.

4.

Efficacy

4.1.

Efficacy for weaned piglets

Six studies performed in five Member States have been provided. They have similar experimental design with weaned piglets allocated to two treatments according to their weights (control and Lactiferm ® ), except for one case in which different dose-groups were included. The animals in the treatment group received the additive via feed in all cases except in trial one in which the water soluble form was administered via water for drinking. In all cases health and performance parameters of piglets were monitored and reported.

In one of the trials, the dose range used was about two orders of magnitude greater than the maximum recommended dose, and therefore, was not further considered. 25

The first study involved 40 weaned piglets (30 days of age) allocated to two treatments (see Table 2). Each treatment was replicated twice with ten piglets each. A one-way analysis of variance (ANOVA) was used in a randomised complete block design, group differences were evaluated by the Scheffe’s test. A group of ten pigs was used as an experimental unit when analysing for feed intake and feed conversion ratio (FCR), whereas individual pigs were used as experimental units when analysing for daily weight gain (ADG).

In the second study, 50 piglets weaned at 28 days were used (Table 2). A one-way generalised linear model (GLM) analysis was used in a randomised complete block design with the Lactiferm ® treatment

25 Technical dossier/Section IV/ Annex IV_3_1_1bReport

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

as the main factor. When feed intake and feed conversion rates were statistically analysed, the pen was used as the experimental unit. This was done because feed-intake was only determined for a group of piglets. For weight and weight gain respectively, each piglet within a pen was used as the experimental unit.

The third study involved two rounds of weaned piglets amounting to a total of 48 (21 days of age, see Table 2). Piglets were individually housed (12 per treatment). Four treatments were included in the trial, control and Lactiferm ® at three different doses: 0.5 x 10 10 , 1.0 x 10 10 , 2.0 x 10 10 CFU/kg feed.

Studies 4 and 5 were field trials. Study 4 involved 747 weaned piglets (21 days of age) of two successive farrowing batches divided in two rooms (one per treatment) and within a room in three pens (see Table 2). Faecal samples were collected (27 samples per treatment collected throughout the experiment) to monitor E. coli and Enterococcus spp counts. After comparison of the initial weights by group to verify their homogeneity, mean average daily gain were compared between groups using three factorial ANOVA (group, sex and farrowing batch) taking the initial weight as covariate. The farrowing batch effect and the treatment group were nested into the pen effect. Mortality and morbidity were compared by the Fisher’s exact test. Faeces samples parameters were compared between groups using the Kruskal-Wallis non parametric test at each date.

Study 5 was divided in two phases according to the accommodation of animals. Animals were housed in one establishment, for 24 days during phase one and then relocated during the second phase of 50 days. The study involved 511 weaned piglets (21 days of age) allocated to two treatments (control and Lactiferm ® at 1 x 10 9 CFU/kg feed in the first phase, 5 x 10 8 in the second phase). During the first phase piglets were penned in 27 groups of ten animals each, while in the second phase they were redistributed in 16 pens with up to 20 animals each (see Table 2).

Results of all the studies are summarised in Table 2.

Table 2:

Effect of Lactiferm ® on the performance of piglets

Total No of animals

Trial No

 

Initial

Final

Daily

 

(duration,

days)

(replication/treatment

×

animals/pen)

Dose

(CFU/kg feed)

weight

(kg)

weight

(kg)

weight gain

(g/day)

Feed:gain

(kg/kg)

1

26

40

0

7.7

27.9

a

482

a

1.96

(42)

(2 × 10)

3

x

10 9

7.7

29.6

b

522

b

1.88

2

27

50

0

6.9

24.0

a

348

a

2.03

(49)

(3 × 7-10)

2

x

10 10

6.9

25.6

b

381

b

1.96

 

0

23.3

426

1.70

a

3

28

48

0.5

x 10 10

24.3

451

1.61

b

(49)

(12 × 1)

1.0

x 10 10

-

22.9

418

1.70

a

 

2.0

x 10 10

23.7

438

1.70

a

4

29

747

0

5.7

476

1.51

(49)

(3 × 121-131)

2

x

10 10

5.8

-

479

1.36

5

30

511

0

5.7

21.9

305

1.86

a

(54)

(27 × 9/10-16 × 15)

1 x 10 9 /5 x 10 8

5.8

22.7

319

1.61

b

a,b : Treatment means differ significantly from control P < 0.05.

26 Technical dossier/Section IV/ Annex IV_3_1_2

27 Technical dossier/Section IV/ Annex IV_3_1_3

28 Technical dossier/Section IV/ Annex IV_3_1_6

29 Technical dossier/Section IV/ Annex IV_3_1_4

30 Technical dossier/Section IV/ Annex IV_3_1_5

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

Three studies showed an increase in final body weights and daily weight gain for the treated group(s) compared to those of the control reaching significance in two studies. There was a significant improved feed to gain ratio in another study. However, the FEEDAP Panel is unable to relate these results to the dose range proposed by the applicant. The minimum proposed dose was included in only one study and conclusions were further confounded by the use of different doses in different phases in one of the positive studies. The evidence presented only justifies a minimum dose in the order of 1 x 10 10 CFU/kg feed, close to the maximum dose proposed by the applicant.

4.2. Efficacy for calves

Five studies have been provided, performed in three Member States. All studies showed similar experimental design with calves homogeneously distributed to two groups according to the weights. All animals were fed milk/milk replacer twice a day plus a complementary feed ad libitum, except for study 5 in which only milk replacer was fed to animals. The administration of Lactiferm ® in the treatment groups was done via milk replacer. Performance and health parameters of animals were monitored and recorded through the whole experimental periods.

Study 1 was made with 30 Lithuanian Black-and-White calves (both sexes, 4 days of age) allocated to two treatments (control and Lactiferm ® at 1.5 x 10 10 CFU/kg complete feed in the first 3 weeks and 2.5 x 10 9 in the last six weeks, confirmed by analysis), three pens per treatment of five animals (see Table 3). One-way ANOVA was carried out with treatment as the main factor and live weight at turn-out as covariate. For weight data the experimental unit was the individual animal while for feed intake it was the pen.

Study 2 involved 20 Lithuanian Black-and-White calves (both sexes, 4-6 days of age) and followed the same design as study 1.

Study 3 involved 20 Holstein calves (both sexes, 5-8 days of age) distributed in two groups according to the treatment (control and Lactiferm ® , see Table 3). Calves were individually housed. A one-way ANOVA was used to analyse the results in a randomised complete block design.

Study 4 involved 24 Holstein calves (both sexes, 6 days of age) allocated to two treatments (control and Lactiferm ® , see Table 3). A one-way ANOVA was used to analyse the results in a randomised complete block design.

Study 5 involved 30 female German Fleckvieh calves (40±8 d) allocated to two treatments (control and Lactiferm ® , see Table 3). Animals were individually housed until slaughter. Results were analysed by ANOVA using initial body weight as a covariate.

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

Table 3:

Summary of results from the efficacy trials on calves with Lactiferm ®

Trial N°

 

Dose (CFU/kg milk or milk replacer)

Initial

Final

Daily

 

(Duration

days)

animals

per

treatment

weight

(kg)

weight

(kg)

weight gain

(kg/day)

Feed:gain 1

1

31

15

0

39.7

74.7

A

0.56

A

(63)

15

1.5 x 10 10 /2.5 x 10 9

39.8

78.4

B

0.61

B

-

2

32

10

0

41.0

81.1

A

0.72

A

(56)

10

2

x

10 10

40.8

88.7

B

0.86

B

-

3

33

10

0

42.2

63.7

0.51

2.18

a

(42)

10

2

x

10 9

42.4

67.0

0.58

2.01

b

4

34

12

0 10 9

x

48.4

77.3

0.58

1.71

a

(50)

12

3

47.7

78.3

0.61

1.53

b

5

35

15

0

65

166.1

a

1.16

a

1.70

a

(84-91)

15

5

x 10 8

65

172.7

b

1.23

b

1.61

b

Treatment means differ significantly from control a,b P < 0.05, A,B P<0.01

1 : Feed as the sum of milk replacer and concentrate

The results of the experiments support the efficacy of Lactiferm ® for calves, but only at a minimum effective dose in the region of 2 x 10 9 CFU/kg milk replacer.

5. Post-market monitoring

The FEEDAP Panel considers that there is no need for specific requirements for a post-market monitoring plan other than those established in the Feed Hygiene Regulation 36 and Good Manufacturing Practice.

CONCLUSIONS

E. faecium NCIMB 11181 does not contain marker genes typical of hospital associated isolates responsible for clinical infections and does not harbour acquired genes coding for antibiotic resistance.

Lactiferm ® is safe for weaned piglets and calves at the recommended dose range.

Since neither the active agent nor the other components of the additive give rise to concerns, the FEEDAP Panel considers the use of the additive safe for consumers.

Given the lack of specific information and its proteinaceous nature and the high dusting potential, in particular of the water soluble form, Lactiferm ® should be considered to have the potential to be an irritant to eye and skin and a skin/respiratory sensitiser.

E. faecium is a natural component of gut microbiota and its use as Lactiferm ® in animal feeding would not be expected to pose any additional risk for the environment.

Lactiferm ® is efficacious in improving zootechnical performance of piglets and calves. The minimum effective dose for piglets is in the order of 1 x 10 10 CFU/kg feed and for calves in the region of 2 x 10 9 CFU/kg milk replacer. The FEEDAP Panel considers evidence of efficacy to be independent of route of delivery provided that the same dose is given.

31 Technical dossier/Annex_IV_3_2_1b

32 Technical dossier/Annex_IV_3_2_4

33 Technical dossier/Annex_IV_3_2_2

34 Technical dossier/Annex_IV_3_2_3

35 Technical dossier/Annex_IV_3_2_5

36 Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements for feed hygiene. OJ L 35, 8.2.2005, p. 1.

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

DOCUMENTATION PROVIDED TO EFSA

1. Lactiferm ® (Enterococccus faecium M74 ® , NCIMB 11181). Zootechnical additive for piglets and calves. May 2009. Submitted by Christian Hansen A/S.

2. Lactiferm ® (Enterococccus faecium M74 ® , NCIMB 11181). Zootechnical additive for piglets and calves. Supplementary information. September 2011. Submitted by Christian Hansen A/S.

3. Lactiferm ® (Enterococccus faecium M74 ® , NCIMB 11181). Zootechnical additive for piglets and calves. Supplementary information. December 2011. Submitted by Christian Hansen A/S.

4. Evaluation report of the Community Reference Laboratory for Feed Additives on the methods(s) of analysis for Lactiferm for calves and piglets.

5. Comments from Member States received through the ScienceNet.

REFERENCES

EC (European Commission), 2003, online. Report of on the use of certain micro-organisms as additives in feedingstuffs. Available from: http://ec.europa.eu/food/fs/sc/scan/out93_en.pdf

EFSA (European Food Safety Authority), 2005. Opinion of the Scientific Panel on additives and products or substances used in animal feed on a request from the Commission on the safety and efficacy of the product Lactiferm for chickens for fattening for use as a feed additive. The EFSA Journal (2005), 207, 1-6.

http://www.efsa.europa.eu/en/scdocs/scdoc/207.htm

Nallapareddy SR, Weinstock GM, Murray BE (2003). Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family. Molecular Microbiology 2003 Mar;47(6):1733-47

Rice LB, Carias L, Rudin S, Vael C, Goossens H, Konstabel C, Klare I, Nallapareddy SR, Huang W, Murray BE (2003). A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin. The Journal of Infectious Diseases. 2003 Feb 1;187(3):508-12. Epub 2003 Jan 8.

Sillanpää J, Prakash VP, Nallapareddy SR, Singh KV, and Murray BE (2009). Distribution of Genes Encoding MSCRAMMs and Pili in Clinical and Natural Populations of Enterococcus faecium. Journal of Clinical Microbiology, Apr. 2009, p. 896–901

Sillanpää J, Nallapareddy S R, Singh K V, Prakash V P, Fothergill T, Ton-That H, and Murray B E (2010). Characterization of the ebpfm pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection. Virulence. 2010 July ; 1(4): 236–246.

Werner G, Fleige C, Geringer U, van Schaik W, Klare I, Witte W. (2011). IS element IS16 as a molecular screening tool to identify hospital-associated strains of Enterococcus faecium. BMC Infectious Diseases 2011 Mar 31;11:80.

Lactiferm ® for piglets and calves

Lactiferm ® for piglets and calves

APPENDIX

Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Lactiferm for piglets and calves

In the current application authorisation is sought under Articles 4(1) (new use) and 10 (2) (re- evaluation of an authorised additive) for feed additive Lactiferm ® under the category 'zootechnical additives', functional group 4(b), 'gut flora stabilisers' according to Annex I of Regulation (EC) No 1831/2003. The active component of Lactiferm ® is a pure culture of strain Enterococcus faecium M74 ® NCIMB 11181. The feed additive is intended to be marketed in three forms with different concentration of Enterococcus faecium M74 ® NCIMB 11181: - powder form Lactiferm ® Basic 50 (5 x 10 10 CFU/g), - coated form Lactiferm ® Caps 50 (5 x 10 10 CFU/g), and - water soluble form Lactiferm ® WS 200 (2 x 10 11 CFU/g).

It is intended to be mixed in powder and coated form at a dose ranging from 5 x 10 8 to 2 x 10 10 CFU/kg of complete feedingstuffs for calves and piglets and from 2.5 x 10 8 to 1.5 x 10 10 CFU/kg for chickens for fattening. In water the Applicant proposed a non classical value, but a minimum content of Lactiferm ® per 100 piglets and 1000 birds in drinking water is recommended in the registry entry.

For the enumeration of Enterococcus faecium M74 ® NCIMB 11181 in feed additives, premixtures, feedingstuffs and water the Applicant proposes ring trial validated spread plate method developed by CEN for the enumeration of Enterococcus spp (EN 15788). The EURL recommends the CEN spread plate method using Bile Esculin Azide Agar. The performance characteristics of the EN 15788 method reported after logarithmic transformation (CFU) are:

a repeatability standard deviation (s r ) ranging from 0.12 to 0.2 log 10 CFU/g,

a reproducibility standard deviation (s R ) ranging from 0.23 to 0.41 log 10 CFU/g, and

a limit of detection (LOD) of 1 x 10 5 CFU/kg, well below the minimum dose proposed by the

applicant (2.5 x 10 8 CFU/kg of feedingstuffs). Molecular methods were used by the Applicant for identification of the active agent. The EURL recommends for official control Pulsed Field Gel Electrophoresis (PFGE), a generally recognised standard methodology for microbial identification.

Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by Article 10 (Commission Regulation (EC) No 378/2005) is not considered necessary.