Вы находитесь на странице: 1из 15

EFSA Journal 2012;10(2):2574

SCIENTIFIC OPINION

Scientific Opinion on Lactiferm (Enterococcus faecium) as a feed additive


for weaned piglets and calves1
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 108 - 2 x
1010 CFU/kg feedstuffs. It is also intended to be used in water for drinking for piglets in the range of 2 x 108 to 8
x 109 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 1010 CFU/kg feed and for calves in the region of 2 x 109
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

On request from the European Commission, Question No EFSA-Q-2009-00679, adopted on 1 February 2012.
Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Nol
Albert Dierick, Mikolaj Antoni Gralak, Jrgen Gropp, Ingrid Halle, Christer Hogstrand, Reinhard Kroker, Lubomir Leng,
Secundino Lpez Puente, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Giovanna Martelli, Mikls Mzes,
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.
2

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

European Food Safety Authority, 2012

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 1011 CFU of E. faecium per gram of product, and a coated
form with a minimum guaranteed concentration of 5 x 1010 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 108 and at the
maximum recommended dose of 2 x 1010 CFU/kg feedstuffs. It is also intended to be used in water for
drinking for piglets at concentrations in the range of 2 x 108 to 8 x 109 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 1010 CFU/kg feed and for calves in the region of 2 x 109
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.

EFSA Journal 2012;10(2):2574

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

EFSA Journal 2012;10(2):2574

Lactiferm for piglets and calves

BACKGROUND
Regulation (EC) No 1831/20034 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/S5 for the reevaluation 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.

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 All, 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

EFSA Journal 2012;10(2):2574

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
Chemical formula

Composition, description
A preparation of Enterococcus
faecium M74, NCIMB 11181

Powder form/L Basic 50


5 x 1010 CFU/g
Water soluble/L WS 200
2 x 1011 CFU/g

Purity criteria

Method of analysis

Complies with
EU law on
undesirable
substances

Validated
microbiological
methods

Trade name

Lactiferm (other names may also be used)

Name of the holder of


authorisation

Chr. Hansen A/S

Conditions of use
Species or
category of
animal

Maximum
Age

Calves

6 months

Piglets

Minimum content

Maximum content

CFU/kg of complete feedingstuffs or


CFU/L of water
Powder:
5 x 108

Powder:
2 x 1010

Powder:
5 x 108

Powder:
2 x 1010

Water soluble:
2 x 108

Water soluble:
8 x 109

Withdrawal
period

Not relevant

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 (4-5C) 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 x108 to 2 x 1010 CFU/kg

Maximum Residue Limit (MRL)

EFSA Journal 2012;10(2):2574

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

EFSA Journal 2012;10(2):2574

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 1011 CFU of E. faecium per gram of
product, and a coated form with a minimum guaranteed concentration of 5 x 1010 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.
2.1.

Characterisation
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, (Sillanp 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 (Sillanp et al., 2010). However the Acm protein is expressed
almost exclusively by clinical isolates (Sillanp 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


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
9

EFSA Journal 2012;10(2):2574

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 1010
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 1011 CFU/g.15
Data on a number of production batches of both powder form (nine batches, average 9.31 x 1010
CFU/g) and water soluble form (seven batches, average 3.4 x 1011 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 m, and 3 % of particles
with a diameter 10 m. Water soluble form has 23 % of particles with a diameter of approximately
100 m and 2 % of particles with a diameter <10 m.
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/m3). In
contrast, the water-soluble form presents an extremely high dusting potential (13.62 g/m3).
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 1010 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
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
14

EFSA Journal 2012;10(2):2574

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 108 and at the maximum recommended dose of 2 x 1010 CFU/kg
feedstuffs. It is also intended to be used in water for drinking for piglets at concentrations in the range
of 2 x 108 to 8 x 109 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 1011 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 1011 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


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
21

EFSA Journal 2012;10(2):2574

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 Scheffes
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

EFSA Journal 2012;10(2):2574

10

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 1010, 1.0 x 1010, 2.0 x 1010 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 Fishers 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 109 CFU/kg feed in the first phase, 5 x 108 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
(replication/treatment

animals/pen)

Dose
(CFU/kg feed)

Initial
weight
(kg)

Final
weight
(kg)

Daily
weight gain
(g/day)

Feed:gain
(kg/kg)

126
(42)

40
(2 10)

0
3 x 109

7.7
7.7

27.9a
29.6b

482a
522b

1.96
1.88

227
(49)

50
(3 7-10)

6.9
6.9

328
(49)

48
(12 1)

24.0a
25.6b
23.3
24.3
22.9
23.7

429
(49)
530
(54)

747
(3 121-131)
511
(27 9/10-16 15)

0
2 x 1010
0
0.5 x 1010
1.0 x 1010
2.0 x 1010
0
2 x 1010
0
1 x 109/5 x 108

348a
381b
426
451
418
438
476
479
305
319

2.03
1.96
1.70a
1.61b
1.70a
1.70a
1.51
1.36
1.86a
1.61b

Trial No
(duration,
days)

5.7
5.8
5.7
5.8

21.9
22.7

a,b

: Treatment means differ significantly from control P < 0.05.

26

Technical dossier/Section IV/ Annex IV_3_1_2


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
27

EFSA Journal 2012;10(2):2574

11

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
1010 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 1010 CFU/kg complete feed in the first 3 weeks and 2.5
x 109 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 (408 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.

EFSA Journal 2012;10(2):2574

12

Lactiferm for piglets and calves

Summary of results from the efficacy trials on calves with Lactiferm

Table 3:
Trial N
(Duration
days)
131
(63)
232
(56)
333
(42)
434
(50)
535
(84-91)

N
animals
per
treatment
15
15
10
10
10
10
12
12
15
15

Dose
(CFU/kg milk or milk
replacer)

Initial
weight
(kg)

Final
weight
(kg)

Daily
weight gain
(kg/day)

0
1.5 x 10 /2.5 x 109
0
2 x 1010
0
2 x 109
0
3 x 109
0
5 x 108

39.7
39.8
41.0
40.8
42.2
42.4
48.4
47.7
65
65

74.7A
78.4B
81.1A
88.7B
63.7
67.0
77.3
78.3
166.1a
172.7b

0.56A
0.61B
0.72A
0.86B
0.51
0.58
0.58
0.61
1.16a
1.23b

10

Feed:gain1

2.18a
2.01b
1.71a
1.53b
1.70a
1.61b

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 109 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 Regulation36 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 1010 CFU/kg feed and for calves in the region of 2 x 109
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
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.
32

EFSA Journal 2012;10(2):2574

13

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.
Sillanp 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. 896901
Sillanp 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): 236246.
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.

EFSA Journal 2012;10(2):2574

14

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) (reevaluation 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
1010 CFU/g), - coated form Lactiferm Caps 50 (5 x 1010 CFU/g), and - water soluble form Lactiferm
WS 200 (2 x 1011 CFU/g).
It is intended to be mixed in powder and coated form at a dose ranging from 5 x 108 to 2 x 1010
CFU/kg of complete feedingstuffs for calves and piglets and from 2.5 x 108 to 1.5 x 1010 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 (sr) ranging from 0.12 to 0.2 log10 CFU/g,

a reproducibility standard deviation (sR) ranging from 0.23 to 0.41 log10 CFU/g, and

a limit of detection (LOD) of 1 x 105 CFU/kg, well below the minimum dose proposed by the
applicant (2.5 x 108 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.

EFSA Journal 2012;10(2):2574

15

Вам также может понравиться