U.S. patent application number 15/005590 was filed with the patent office on 2016-05-19 for gastrointestinal health, immunity and performance by dietary intervention.
The applicant listed for this patent is BIOATLANTIS LTD. Invention is credited to Michael GALLAGHER, John O'DOHERTY, John T. O'SULLIVAN, Torres SWEENEY.
Application Number | 20160136200 15/005590 |
Document ID | / |
Family ID | 42470703 |
Filed Date | 2016-05-19 |
United States Patent
Application |
20160136200 |
Kind Code |
A1 |
O'SULLIVAN; John T. ; et
al. |
May 19, 2016 |
GASTROINTESTINAL HEALTH, IMMUNITY AND PERFORMANCE BY DIETARY
INTERVENTION
Abstract
This invention relates to the improvement of gastrointestinal
health, immunity and performance by direct dietary intervention
with a composition comprising a glucan and/or a fucan, and relates
in particular to the transfer of associated health benefits to
offspring via glucan and/or a fucan supplementation of the maternal
diet. Accordingly the present invention provides a composition
comprising at least one glucan, at least one fucan, or at least one
glucan and at least one fucan for use in improving or maintaining
the gastrointestinal health or function of a progeny of a maternal
animal by administration to the maternal animal; and a method for
improving or maintaining the gastrointestinal health or function of
a progeny of a maternal animal, the method comprising administering
a composition comprising at least one glucan, at least one fucan,
or at least one glucan and at least one fucan to the maternal
animal.
Inventors: |
O'SULLIVAN; John T.;
(Tralee, IE) ; GALLAGHER; Michael; (Milstreet,
IE) ; O'DOHERTY; John; (Clane, IE) ; SWEENEY;
Torres; (Clane, IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOATLANTIS LTD |
Tralee |
|
IE |
|
|
Family ID: |
42470703 |
Appl. No.: |
15/005590 |
Filed: |
January 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13321412 |
Apr 30, 2012 |
9241951 |
|
|
PCT/EP2010/003088 |
May 21, 2010 |
|
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15005590 |
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Current U.S.
Class: |
514/54 |
Current CPC
Class: |
A61P 17/06 20180101;
A61K 31/716 20130101; A61P 43/00 20180101; A61P 17/00 20180101;
A61P 37/04 20180101; A61P 29/00 20180101; A61P 7/00 20180101; A61P
1/00 20180101; A61K 31/737 20130101; C08B 37/0024 20130101; A61K
2300/00 20130101; A61K 36/02 20130101; A61K 2300/00 20130101; A61P
19/02 20180101; A61K 31/737 20130101; A61K 45/06 20130101; A61K
31/716 20130101; A61P 37/00 20180101; A61P 31/04 20180101; A61P
1/04 20180101; A61K 36/02 20130101; A61P 31/12 20180101; A61K
2300/00 20130101; A61P 37/02 20180101; A61P 31/00 20180101 |
International
Class: |
A61K 31/716 20060101
A61K031/716 |
Foreign Application Data
Date |
Code |
Application Number |
May 21, 2009 |
IE |
2009/0398 |
Claims
1. A method for improving or maintaining the health, structure,
function, immunity or performance of the progeny of a maternal
animal or human; or preventing, treating or ameliorating disorders
in structure or function or immunity in the progeny of a maternal
animal or human, to levels equivalent to or greater than those
achieved by direct administration to each individual progeny at
weaning or later; the method comprising administering a composition
comprising at least one glucan, at least one fucan, or at least one
glucan and at least one fucan to the maternal animal or human.
2. The method according to claim 1, wherein the at least one glucan
is a beta glucan.
3. The method according to claim 1, wherein the at least one glucan
is beta (1.fwdarw.3, 1.fwdarw.4) glucan or beta (1.fwdarw.3,
1.fwdarw.6) glucan.
4. The method according to claim 1, wherein the at least one glucan
is laminarin.
5. The method according to claim 1, wherein the at least one fucan
is an alpha-fucan.
6. The method according to claim 1, wherein the at least one glucan
and/or the at least one fucan is isolated from a brown macroalga of
the class Phaeophycea.
7. The method according to claim 1, wherein the at least one glucan
and/or the at least one fucan is isolated from a red alga selected
from Florideophyceae.
8. The method according to claim 1, wherein the composition is
administered to the maternal animal or human perinatally and/or
prenatally, and/or postnatally.
9. The method according to claim 1, wherein the composition is
administered daily to the maternal animal or human.
10. The method according to claim 1, wherein the composition is
administered to the maternal animal or human in an amount such that
about 2-40 milligrams of fucan per kilogram of body weight is
administered to the maternal animal or human.
11. The method according to claim 1, wherein the maternal animal or
human is a monogastric animal selected from the group consisting of
pigs, poultry, fish, cats, dogs and humans; and/or the animal is a
hind-gut fermenter selected from the group consisting of horses and
rabbits; and/or the animal is a foregut fermenter selected from the
group consisting of sheep.
12. The method according to claim 1, wherein the disorder of
structure or function is prevented, treated or ameliorated by
altering immunological function and/or the immunological profile;
and/or altering immune gene expression and/or the expression or
secretion of signalling proteins; and/or altering the expression of
intra-cellular and/or extra-cellular receptors.
13. The method according to claim 12, wherein immunological
function and/or immunological profile is improved by altering
leukocyte numbers and/or leukocyte function and/or leukocyte
phenotype and/or leukocyte trafficking and/or leukocyte
distribution and/or cytokine gene expression and/or cytokine
secretion and/or cytokine receptor expression.
14. The method according to claim 13, wherein leukocyte numbers are
reduced and/or phagocytic activity is increased.
15. The method according to claim 13, wherein the cytokine is
selected from pro-inflammatory and/or anti-inflammatory
cytokines.
16. The method according to claim 1, wherein the disorder of
structure or function in the progeny is a chronic disease selected
from the group consisting of Crohn's disease, Irritable Bowel
syndrome, and Colitis.
17. The method according to claim 1, wherein the disorder of
structure or function in the progeny is selected from the group
consisting of an autoimmune, inflammatory, autoinflammatory, and
atopic disease.
18. The method according to claim 1, wherein the disorder of
structure or function in the progeny animal is prevented, treated
or ameliorated by increasing the concentration of immunoglobulin in
the colostrum or milk of a maternal animal or human.
19. The method according to claim 1, wherein the disorder of
structure or function in the animal progeny animal is prevented,
treated or ameliorated by decreasing bacterial infection and/or
decreasing viral infection.
20. The method according to claim 1, wherein the disorder of
structure or function in the progeny is prevented, treated or
ameliorated by increasing the expression of mucins.
21. The method according to claim 1, wherein the disorder of
structure or function in the progeny is one which gives rise to
wasting away of muscle and/or fat tissue.
22. The method according to claim 1, wherein the disorder of
structure or function in the progeny is prevented, treated or
ameliorated by decreasing infection or load of viral pathogen.
23. The method according to claim 24, wherein the viral pathogen is
a non-enveloped virus.
24. The method according to claim 1, wherein the disorder of
structure or function in the progeny is prevented, treated or
ameliorated by increasing the production of straight-chain volatile
fatty acids and/or reduction of branched-chain fatty acids.
25. The method according to claim 1, wherein the at least one fucan
is fucoidan.
26. The method according to claim 1, wherein the at least one
glucan and/or the at least one fucan is derived from at least one
family selected from the group consisting of Laminariaceae,
Fucaceae and Lessoniaceae.
27. The method according to claim 1, wherein the at least one
glucan and/or the at least one fucan is selected from at least one
species from the group consisting of Ascophyllum species; Laminaria
species and Sargassum species.
28. The method according to claim 1, wherein the at least one
glucan is derived from a species of fungi.
29. The method according to claim 1, wherein the at least one
glucan is derived from yeast.
30. The method according to claim 1, wherein the at least one
glucan is derived from Saccharomyces cerevisiae.
31. The method according to claim 1, wherein the disorder of
structure or function in the progeny is selected from the group
consisting of psoriasis, rheumatoid arthritis, psoriatic arthritis,
atopic dermatitis and juvenile idiopathic arthritis.
32. The method according to claim 1, wherein the disorder of
structure or function in the animal progeny is prevented, treated
or ameliorated by decreasing infection selected from the group
consisting of Escherichia coli infection, Campylobacter infection,
Salmonella, porcine circovirus infection and PCV-2 infection.
33. The method according to claim 1, wherein glucans are derived by
approaches which include synthetic chemistry and
biotechnology-related approaches.
34. The method according to claim 1, wherein fucans are derived by
approaches which include synthetic chemistry and
biotechnology-related approaches.
35. The method according to claim 1, wherein administration of the
composition to the maternal animal or human confers benefits to a
single progeny from the maternal animal or human.
36. The method according to claim 1, wherein administration of the
composition to the maternal animal or human confers benefits to
multiple birth progeny offspring from the maternal animal or
human.
37. The method according to claim 1, wherein administration of the
composition to the maternal animal or human confers benefits to
multiple birth progeny offspring such that 2 or more progeny
offspring from the maternal animal or human are affected.
38. The method according to claim 1, wherein the disorder of
structure or function gives rise to early life mortality in
progeny.
39. The method according to claim 1, wherein early life mortality
is reduced during perinatal, prenatal and postnatal periods, during
gestation, pregnancy, viparity, ovoviviparity or viviparity,
surrounding the time of birth, hatching or spawning or during post
birth, post hatch, neonatal or weaning periods, optionally with
reductions in morbidity, mortality, numbers of stillborn,
un-hatched, un-fertilized or mummified offspring or reductions in
pre- or post-birth or hatch deaths.
40. The method according to claim 1, wherein parity is
improved.
41. The method according to claim 1, wherein performance is
increase throughout the animals lifetime, during the post birth
period, weanling period, juvenile period, fattening period,
finisher period, slaughter age periods, adult stage and periods
during which animals grow to reach their genetic growth
potential.
42. The method according to claim 1, wherein immunoglobulins are
increased in blood serum of the progeny offspring of the maternal
animal.
43. The method according to claim 1, wherein the uniformity of
progeny offspring weaning weight is significantly improved.
44. The method according to claim 1, wherein the percentage of
offspring weaned per maternal animal on weaning day is increased.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a U.S. patent application
continued from U.S. Ser. No. 13/321,412 filed on Apr. 30, 2012,
which is a U.S. National Stage application under 35 U.S.C. 371 of
International Application Number PCT/EP2010/003088, filed in
English on May 21, 2010, which claims the benefit of patent
application IE 2009/0398 filed in English on May 21, 2009. The
entirety of each application is incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to the improvement of gastrointestinal
health, immunity and performance by direct dietary intervention
with laminarin and/or alpha-fucan and the transfer of associated
health benefits to offspring via laminarin and/or alpha-fucan
supplementation of the maternal diet.
[0003] In particular, the invention has the purpose of improving
the nutritional, immunological and microbiological status of
suckling and weaned offspring by supplementation of the maternal
diet with laminarin and/or alpha-fucans. In another aspect, this
invention relates to the utilisation of laminarin and/or
alpha-fucan-containing preparations or feedstuffs to improve the
immune status and immune response in pigs, poultry, sheep, horses,
rabbits, fish, cats, dogs, humans and other monogastric subjects.
Other aspects relate to the use of such compounds to increase
performance in livestock as manifested by increased weight gain and
feed conversion indices in weaned stock through maternal transfer
of beneficial compounds in utero or via colostrum and breast milk
during suckling, following supplementation of the maternal diet
with laminarin and/or alpha-fucan.
[0004] In another aspect, the invention relates to manipulating the
sterile conditions of the intestine of neonates by reducing total
microbiological populations or by selectively encourage beneficial
bacteria and inhibit growth of pathogens within the
gastrointestinal system. Another aspect relates to the increasing
the production of straight chain volatile fatty acids and reducing
the production of branched chain volatile fatty acids within the
gut by increasing fermentation from carbohydrate substrate and
reducing fermentation from protein substrate. Yet another aspect
relates to the synthesis of long chain polyunsaturated fatty acids
including conjugated linoleic acid and omega-3 fatty acids by
selectively stimulating Bifidobacteria in the intestinal tract.
[0005] In another aspect, the invention relates to the upregulation
of mucin and/or trefoil factor (TFF) production in-vivo, thereby
enhancing protection and stability of the gastrointestinal mucosa
against insult, infection or injury.
BACKGROUND OF THE INVENTION
[0006] Paediatric clinicians and veterinarians are well-informed on
the importance of achieving optimal nutrition during pregnancy to
achieve successful physical, cognitive and neural development.
Several trials have demonstrated the effects associated with
deficiencies or toxicities of nutrients and other compounds on
foetal development and its subsequent phenological
characterisation. This has highlighted the importance of prenatal
and perinatal dietary interventions to achieve optimal development
during the critical stages and a healthy growth rate post
partum.
[0007] To compound these issues, the publication of the Swann
report (1969) encouraged a more stringent control of antibiotic
usage in animal feeds due to the risks associated with antibiotic
resistance, specifically the imposing threat on public health. This
led to the EU prohibition of growth promoting antibiotics in animal
feeds in January 2006. The prohibition of these growth promoters
created a void in the market for intensive farming producers and
also presented an opportunity for sourcing of a natural, safe
alternative. The inclusion of laminarin and/or alpha-fucan in
lactation diets of pigs, poultry, horses, sheep, rabbits, fish,
humans and other monogastric subjects will have a major effect on
the critical immunological and microbiological status at and
immediately following parturition and will therefore have a major
effect on consequent welfare, development and growth rates.
[0008] Algal beta-glucans, called laminarin, consist of beta
(1.fwdarw.3)-D glucosyl subunits with occasional (1.fwdarw.6)
linked branches. Laminarin from Laminaria digitata occurs as two
homologous series of molecules, a minor G series containing 22-28
glucosyl residues and a more abundant M series consisting of 20-30
glucosyl residues linked to a mannitol residue. Laminarin from many
species of Laminaria (including Laminaria hyperborea) is relatively
insoluble and consists of predominantly beta (1.fwdarw.3) chains
while laminarin from Laminaria digitata is soluble and consists of
small but significant levels of beta (1.fwdarw.6) linked branches.
(Read et al, 1996).
[0009] Yeast beta glucans are found in long linear chains of up to
1300-1500 glucose residues linked by beta (1.fwdarw.3) bonds with a
minor incidence of beta (1.fwdarw.6) chains. Laminarin has much
smaller chain lengths (average=24 residues) with occasional beta
(1.fwdarw.6) branches, depending on the species. Laminaria digitata
has the beta (1.fwdarw.6) branching which make the glucans derived
from them water soluble. Other Laminaria species, like Laminaria
hyperborea, do not have this branching which makes the linear
chains aggregate and makes the glucans derived from it,
insoluble.
[0010] Natural polysaccharides built essentially of sulfated
alpha-L-fucose residues are known as fucoidan (or alpha-fucans).
These are present in brown algae, some echinoderms and are the
second most predominant polysaccharide in brown seaweed, like
Ascophyllum nodosum and species of Laminaria. alpha-Fucans have
been extensively studied due to their diverse biological
activities, since they are potent anticoagulant, antitumor, and
antiviral agents.
[0011] The present invention encompasses the use of alpha-fucans,
in particular the fucans present in sea plants, such as the sea
cucumber body wall; in particular the alpha-fucan present in the
cell walls of marine algae, and the egg jelly coat of sea urchin
eggs. Ideally the present invention utilises fucoidan, the
alpha-fucan present in macroalgae.
OBJECT OF THE INVENTION
[0012] It is an object of this invention to provide a novel method
of controlling microbiological, immunological and performance
related attributes of livestock such as pig, poultry, horse as well
as rabbits, fish, cats, dogs and human neonates through maternal
transfer mechanisms, by ensuring early delivery of beneficial
compounds at critical growth stages. Another object is to provide
prenatal dietary intervention with a laminarin and/or alpha-fucan
containing preparation in the maternal diet for delivery through
prenatal exchange in-utero or by postnatal transfer in colostrum or
breastmilk. Another object is to provide a dosing regimen for
laminarin and/or alpha-fucan containing preparations for
controlling microbiological, immunological and performance related
attributes of livestock such as pigs, poultry, horses, as well as
rabbits, cats, dogs, fish and human.
[0013] It is a further object of the invention that the composition
will beneficially affect the immune response by altering the
expression of pro- and anti-inflammatory cytokines, leukocytes
population and expression of immunoglobulins, mucins and trefoil
factors.
[0014] Further objects of the invention include increasing the
production of volatile straight chained fatty acids and reducing
production of branched chain fatty acids (such as valeric,
isovaleric and isobutyric acids) by altering the microbiological
profile in favour of one that preferentially metabolises
carbohydrates as fermentation substrate.
SUMMARY OF THE INVENTION
[0015] According to a first aspect of the present invention, there
is provided a composition comprising at least one glucan, at least
one fucan, or at least one glucan and at least one fucan for use in
improving or maintaining the gastrointestinal health or function of
a progeny of a maternal animal by administration to the maternal
animal.
[0016] According to a second aspect of the present invention, there
is provided a method for improving or maintaining the
gastrointestinal health or function of a progeny of a maternal
animal, the method comprising administering a composition
comprising at least one glucan, at least one fucan, or at least one
glucan and at least one fucan to the maternal animal.
[0017] The composition may comprise at least one glucan. When the
composition comprises more than one glucan, each glucan may be the
same glucan or a different glucan. Optionally or additionally, the
composition may comprise at least one fucan. When the composition
comprises more than one fucan, each fucan may be the same fucan or
a different fucan. Optionally, the composition comprises at least
on glucan, at least one fucan, or a mixture or combination
thereof.
[0018] Optionally, the composition is administered to the maternal
animal perinatally, prenatally, and/or postnatally. By "prenatally"
is meant during the period of time extending from initiation
(fertilisation) to approximately 50% of the total gestational term.
Prenatal improvement or maintenance of the gastrointestinal health
or function of the progeny can occur during prenatal
administration. By "perinatally" is meant during the period of time
extending from approximately 50% of the total gestational term to
the time of birth. Perinatal improvement or maintenance of the
gastrointestinal health or function of the progeny can occur during
perinatal administration. By "postnatally" is meant during the
period of time extending from the time of birth, and is intended to
extend to the period post-weaning (the period following the time
that the progeny ceases to ingest maternal colostrum or milk).
Postnatal improvement or maintenance of the gastrointestinal health
or function of the progeny can occur during postnatal
administration.
[0019] By "progeny" is meant the offspring of a maternal animal,
and is intended to include offspring developing in utero during the
prenatal period, and offspring developing ex vivo during the
postnatal period.
[0020] By "glucan" is meant a polysaccharide molecule comprising at
least two saccharide monomers, optionally D-glucose monomers,
wherein each monomer is linked to an adjacent monomer by a
glycosidic bond. The polysaccharide molecule may be linear or
branched i.e. the polysaccharide molecule can be a straight-chain
polysaccharide or a branched chain polysaccharide. Optionally, the
glucan is a branched chain glucan. The glucan may be an alpha
glucan or a beta glucan. Optionally, the glucan is a beta glucan.
By "beta glucan" is meant a glucan comprising at least one beta
glycosidic bond. A glycosidic bond is intended to mean a glycosidic
bond, wherein a carbon atom of a first monomer forms a bond,
optionally a single order bond, with a carbon atom on an adjacent
monomer. A beta glycosidic bond is intended to mean a glycosidic
bond, wherein a functional group, optionally a hydroxyl group,
attached to a carbon atom of a first monomer extends above the
plane of the monomer (equatorially). Optionally, the C1 carbon atom
of a first monomer forms a bond, optionally a single order bond,
with the C6 carbon atom on an adjacent monomer. Further optionally,
the glucan comprises a beta (1.fwdarw.6) glycosidic bond,
optionally an oxygen-containing beta (1.fwdarw.6) glycosidic bond.
Optionally, at least one glucan is beta (1.fwdarw.3, 1.fwdarw.6)
glucan. Still further optionally, the glucan is laminarin.
[0021] By "fucan" is meant a polysaccharide, optionally a sulphated
polysaccharide, comprising at least two fucose saccharide monomers,
wherein each monomer is linked to an adjacent monomer by a
glycosidic bond. The polysaccharide molecule may be linear or
branched. Optionally, the fucan is a branched fucan. The fucan may
be an alpha fucan or a beta fucan. Optionally, the fucan is an
alpha fucan. By "alpha fucan" is meant a fucan comprising at least
one alpha glycosidic bond. A glycosidic bond is intended to mean a
glycosidic bond, wherein a carbon atom of a first monomer forms a
bond, optionally a single order bond, with a carbon atom on an
adjacent monomer. An alpha glycosidic bond is intended to mean a
glycosidic bond, wherein a functional group, optionally a hydroxyl
group, attached to a carbon atom of a first monomer extends below
the plane of the monomer (axially).
[0022] Optionally, the C1 carbon atom of a first monomer forms a
bond, optionally a single order bond, with either the C3 or C4
carbon atom on an adjacent monomer. Optionally, the fucan is
fucoidan.
[0023] Optionally, the glucan and/or the fucan is isolated from a
brown alga, optionally brown seaweed. Optionally, the brown alga is
a brown macroalga. Optionally, the brown macroalga, optionally
brown seaweed, is selected from Phaeophyceae, optionally selected
from Phaeophyceae Laminariales and Phaeophyceae Fucales. Further
optionally, the brown alga, optionally brown seaweed, is selected
from Laminariaceae, Fucaceae, and Lessoniaceae. Optionally, the
brown macroalga, optionally brown seaweed, is selected from
Ascophyllum species, optionally Ascophyllum nodosum and Laminaria
species, optionally Laminaria digitata, Laminaria hyperborea,
Laminaria saccharina, Laminaria japonica or Sargassum species.
[0024] Alternatively, the glucan and/or the fucan is isolated from
a red alga, optionally red seaweed. Optionally, the red alga is a
red macroalga. Optionally, the red macroalga, optionally red
seaweed, is selected from Florideophyceae, optionally selected from
Florideophyceae Gigantinales, optionally selected from
Gigartinaceae.
[0025] Optionally, the composition is administered daily to the
maternal animal.
[0026] Optionally, the composition is administered, optionally
daily, to the maternal animal in an amount such that about 3-50
milligrams of glucan per kilogram of body weight is administered to
the maternal animal. Further optionally, the composition is
administered, optionally daily, to the maternal animal in an amount
such that about 2-40 milligrams of fucan per kilogram of body
weight is administered to the maternal animal.
[0027] Optionally, the composition is administered, optionally
daily, to the animal in an amount such that about 3-50 milligrams
of glucan per kilogram of body weight is administered to the
animal. Further optionally, the composition is administered,
optionally daily, to the animal in an amount such that about 2-40
milligrams of fucan per kilogram of body weight is administered to
the animal.
[0028] Optionally, the animal is a monogastric animal. Further
optionally, the animal is selected from pigs, poultry, horses,
sheep, rabbits, fish, cats, dogs, and humans.
[0029] By "improving or maintaining the gastrointestinal health or
function" is meant improving the physiological function or
histology of the gastrointestinal tract and/or the microbiological
population of the gastrointestinal tract. Moreover,
gastrointestinal health or function can be improved or maintained
at the molecular level by improving the immunological state of the
host. The improvement or maintenance of gastrointestinal health or
function is intended to prevent or prophylactically treat disorders
associated with poor gastrointestinal health or function, such as
Crohn's disease, irritable bowel syndrome, and other such chronic
conditions. Other disorders associated with poor gastrointestinal
health are less serious and can include food-borne pathogens and
certain bacteria and viruses that often result in diarrhoea, poor
stool quality, low birth weight or weight gain, or other symptoms
of poor gastrointestinal health.
[0030] Optionally, the gastrointestinal health or function is
improved or maintained by increasing the concentration of
immunoglobulin, optionally Immunoglobulin G, in the colostrum or
milk of the maternal animal.
[0031] Optionally, the gastrointestinal health or function is
improved or maintained by increasing the concentration of crude
protein in the colostrum or milk of the maternal animal.
[0032] Optionally, the gastrointestinal health or function is
improved or maintained by decreasing bacterial, optionally
pathogenic bacterial, infection in the progeny. Further optionally,
the bacterial, optionally pathogenic bacterial, infection is an
Enterobacteriaceae infection, optionally selected from 20
Salmonella and Escherichia coli.
[0033] Optionally, the gastrointestinal health or function is
improved or maintained by increasing the expression of cytokines,
optionally selected from tumour necrosis factor alpha,
interleukin-1 alpha, interleukin-6, and trefoil factor 3.
[0034] Optionally, the gastrointestinal health or function is
improved or maintained by decreasing the concentration of volatile
branched-chain fatty acids, optionally selected from isobutyric
acid, valeric acid, and isovaleric acid.
[0035] Optionally, the gastrointestinal health or function is
improved or maintained by altering the concentration or activity of
phagocytes, optionally leukocytes, neutrophils, eosinophils,
monocytes, or lymphocytes, further optionally leukocytes,
eosinophils or lymphocytes. Further optionally, the concentration
or activity of leukocytes is increased and/or the concentration or
activity of lymphocytes is decreased and/or the concentration or
activity of eosinophils is decreased.
[0036] According to a further aspect of the present invention,
there is provided a composition comprising at least one glucan, at
least one fucan, or at least one glucan and at least one fucan for
use in improving or maintaining the gastrointestinal health or
function of an animal by administration to the animal in an amount
such that about 3-50 milligrams of glucan per kilogram of body
weight is administered, optionally daily, to the animal; or by
administration to the animal in an amount such that about 2-40
milligrams of fucan per kilogram of body weight is administered,
optionally daily, to the animal.
[0037] According to a still further aspect of the present
invention, there is provided a method for improving or maintaining
the gastrointestinal health or function of an animal, the method
comprising administering a composition comprising at least one
glucan, at least one fucan, or at least one fucan and at least one
fucan to the animal in an amount such that about 3-50 milligrams of
glucan per kilogram of body weight is administered, optionally
daily, to the animal; or by administration to the animal in an
amount such that about 2-40 milligrams of fucan per kilogram of
body weight is administered, optionally daily, to the animal.
[0038] Optionally, the composition further comprises a sugar,
optionally a disaccharide, optionally selected from lactose,
sucrose, lactulose, and maltose. Further optionally, the
composition further comprises sugar, optionally a disaccharide,
optionally selected from lactose, sucrose, lactulose, and
maltose.
[0039] Optionally, the gastrointestinal health or function is
improved or maintained by decreasing bacterial infection,
optionally Escherichia coli infection.
[0040] Optionally, the gastrointestinal health or function is
improved or maintained and prevents or prophylactically treats
diarrhoea.
[0041] Optionally, the gastrointestinal health or function is
improved or maintained by increasing the expression of cytokines,
optionally in the presence of antigen. Further optionally, the
antigen is bacterial antigen, optionally bacterial
lipopolysaccharide. Optionally, the cytokine is selected from
interleukin-6 and interleukin-8.
[0042] Optionally, the gastrointestinal health or function is
improved or maintained by increasing the expression of mucins,
optionally mucin-2 and/or mucin-4.
[0043] Optionally, the gastrointestinal health or function is
improved or maintained by decreasing the concentration of
circovirus or parvovirus, optionally porcine circovirus or porcine
parvovirus. Further optionally, the porcine circovirus is type-2
porcine circovirus.
[0044] Optionally, the gastrointestinal health or function is
improved or maintained by increasing the concentration of
straight-chain volatile fatty acids.
[0045] The inventors have developed a composition consisting of a
formulation of laminarin and/or alpha-fucans that has altering
effects on: (I) gut histology, (II) gut microbiology, (III) pro-
and anti-inflammatory cytokine expression, (III) neonatal serum
immunoglobulin levels, (IV) mucin production, (V) trefoil factor
production, (VI) nutritional and immunological composition of
colostrum and breast milk and (VII) performance indices. In
addition, there were clear detrimental effect on intestinal
Enterobacteria populations which has associated benefits in reduced
morbidity and mortality rates from reduced infection and
inflammation.
[0046] Accordingly, the present invention provides use of a
composition comprising beta-glucans and/or alpha-fucans in a method
of improving neonatal and weanling gastrointestinal health and
immunity through pre- and postnatal supplementation of the maternal
diet. In preferred embodiments, beta-glucans and alpha-fucans may
be derived from more than one source including seaweed and some
echinoderms. The seaweed may be from the group consisting of
Laminariaceae, Fucacea, Gigartinaceae or Lessoniaceae.
[0047] The invention also provides use of a composition comprising
beta-glucans and/or alpha-fucans: [0048] in a method of producing a
maternal dietary supplement or feedstuff, for reducing
gastrointestinal bacterial populations in neonates and weanlings;
[0049] in a method of producing a maternal dietary supplement or
feedstuff, for reducing morbidity and mortality rates in neonates
and weanlings; [0050] in a method of producing a maternal dietary
supplement or feedstuff, for improving digestive histology by
increasing the villus height, reducing the crypt depth or
increasing the overall villus height:crypt depth ratio in neonates
and weanlings; [0051] in a method of producing a maternal dietary
supplement or feedstuff, for improving performance in the progeny
of livestock such as pigs, poultry, horses, as well as rabbits,
fish, cats, dogs and humans including an increase in average daily
gain, an increase in average daily feed intake and an improvement
in feed efficiency; [0052] in a method of improving
gastrointestinal health by encouraging beneficial microflora,
reducing pathogenic microflora and improving performance in
neonates and weanlings, by supplementing maternal diets [0053] in a
method of upregulating the production of mucins and trefoil factors
by epithelial cells as a means of enhanced physical protection of
the gastrointestinal epithelium.
[0054] In a further aspect the invention provides methods of
achieving the above-mentioned effects by feeding a composition
comprising beta-glucans and/or alpha-fucans to humans, non-human
animals or poultry.
[0055] In a still further aspect, the invention provides: [0056] a
dosing regimen for preventing bacterial or viral infection and
inflammation in livestock such as pigs, poultry, horses, sheep as
well as rabbits, fish, cats, dogs and humans by directly
supplementing the diet or by supplementing the maternal diet in
pre- and postnatal periods with a composition comprising
beta-glucans and alpha-fucans; [0057] a dosing regimen for
improving the nutritional quality and increasing the immunoglobulin
levels of colostrum and breast milk by supplementing the maternal
diet in the pre- and postnatal periods with a composition
comprising beta-glucans and/or alpha-fucans; [0058] a dosing
regimen for increasing neonatal serum immunoglobulin levels by
in-utero transfer of beneficial immunostimulatory compounds across
the placental membrane by supplementing the maternal diet in the
pre- and postnatal periods with a composition comprising beta
glucans and/or alpha fucans; [0059] a dosing regimen for increasing
neonatal serum immunoglobulin levels through an increased uptake in
colostrum or breast milk by supplementing the maternal diet in the
pre- and postnatal periods with a composition comprising beta
glucans and/or alpha fucans; [0060] a dosing regimen for reducing
Enterobacteria, including E. coli, populations in the digestive
tracts of neonatal pigs, poultry, horses, as well as rabbits, fish,
cats, dogs, humans and other monogastric subjects by supplementing
the maternal diet in the pre- and postnatal periods with a
composition comprising beta glucans and/or alpha fucans; [0061] a
dosing regimen for alleviating functional intestinal disorders
associated with weaning by supplementing the maternal diet in the
pre- and postnatal periods with a composition comprising beta
glucans and/or alpha fucans; [0062] a dosing regimen for
encouraging a healthy intestinal microbiological profile in
neonates and weanlings by selectively encouraging a dominant ratio
of beneficial bacteria and selectively inhibiting the growth of
pathogenic bacteria in the period of bacterial colonisation of
intestine immediately after birth by supplementing the maternal
diet in the pre- and postnatal periods with a composition
comprising beta glucans and/or alpha fucans.
[0063] The dosing regimen for administration of laminarin may be a
daily dosage administered at greater than 3 milligrams of laminarin
per kilogram of body weight per day to a maximum of 50 milligrams
per kilogram of body weight per day.
[0064] The dosing regimen for administration of alpha fucans may be
a daily dosage administered of greater than 2 milligrams per
kilogram of body weight per day to a maximum of 40 milligrams per
kilogram of body weight per day.
[0065] The dosing regimen for administration of a combination of
laminarin and alpha fucans may be a daily dosage of laminarin
administered greater than 3 milligrams per kilogram of body weight
per day to a maximum of 50 milligrams per kilogram of body weight
per day in combination with a daily dosage of alpha-fucans greater
than 2 milligrams per kilogram of body weight per day to a maximum
of 50 milligrams per kilogram of body weight per day.
[0066] The invention also provides use of a composition comprising
beta-glucans and/or alpha-fucans in a method: [0067] for increasing
straight chain volatile fatty acid production in-vivo; [0068] for
reducing branched chain volatile fatty acid production in-vivo and
their excretion; [0069] for increasing long chain polyunsaturated
fatty acids production in-vivo; [0070] for improving immune status
and response in immune-challenged livestock such as pigs, poultry,
horses, as well as rabbits, fish, humans and other monogastric
subjects. [0071] for improving the immune status by increased
expression of pro- and anti-inflammatory cytokines, mucins and
trefoil factors.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] Embodiments of the present invention will now be described
by the use of non-limiting examples, and reference made to the
accompanying drawings, in which:
[0073] FIG. 1 illustrates PCV2-specific antibody titres of piglets
fed a basal diet;
[0074] FIG. 2 illustrates PCV2-specific antibody titres of piglets
fed a basal diet supplemented with LAM+FUC;
[0075] FIG. 3 illustrates PCV2-specific antibody titres of piglets
fed a basal diet supplemented with LAM+FUC and WPI;
[0076] FIG. 4 illustrates the percentage lymphocyte population for
piglets weaned onto different diets and subsequently challenged
with PCV2 and PPV;
[0077] FIG. 5 illustrates the average percentage eosinophil
population in piglets fed different diets and subsequently
challenged with PCV2 and PPV (Day 14 PI);
[0078] FIG. 6 illustrates the average terminal weight (Kg) of pigs
weaned onto different diets and then challenged with PCV2 and
PPV;
[0079] FIG. 7 illustrates the average PCV2 DNA copy number detected
in faeces of piglets weaned onto different diets and subsequently
challenged with PCV2 and PPV; and
[0080] FIG. 8 illustrates PCV2 DNA copy number of pigs weaned onto
different diets.
THE EXAMPLES
[0081] The examples given are results of investigative research on
the effects of laminarin and/or fucoidan supplementation on porcine
subjects as a model for all monogastrics including humans, animals
and poultry. The examples shown include trials carried out using
seaweed extract containing laminarin and fucoidan in combination
(hereafter referred to as SWE) or each of the compounds
individually as laminarin (hereafter referred to as LAM) or
fucoidan (hereafter referred to as FUC).
Example 1
Materials and Methods
Animals and Treatment
[0082] 40 pregnant sows were assigned to 1 of 4 dietary treatments
(n=10 sows/treatment): (T1) basal lactation; (T2) basal
lactation+100 g/day fish oil (F.O.); (T3) basal lactation+1.8 g/day
SWE; and (T4) basal lactation+100 g/day F.O.+1.8 g/day SWE from day
109 of gestation until weaning at 26 days. SWE contained daily
doses of laminarin (1 g) and fucoidan (0.8 g). For the test
subjects, diets were top-dressed daily with the experimental
supplement. At birth, weight was recorded and 3 piglets were
selected to represent the mean birth weight of the litter. These
were weighed weekly until weaning. At weaning, 120 mixed sex pigs
(3 pigs per litter; average weight=8.05.+-.0.46 Kg) were selected
and offered starter diets for 21 days. Feed and water were
available ad libitum throughout the experiment. Pigs were
individually weighed on the day of weaning (day 0) and thereafter
at 7, 14 and 21 days post weaning. Feed intake recorded on a daily
basis.
Sample Collection
[0083] 30 ml of colostrum and milk was collected from sows on days
0 and 12 post farrowing. Blood samples were collected from the
jugular veins of 2 piglets/litter on day 5 and 12 of lactation for
immunoglobulin analysis. Crude protein was determined in accordance
with the Association of Official Analytical Chemists (AOAC,
1995).
Quantification of Immunoglobulins
[0084] Immunoglobulin assays were performed using specific pig
ELISA quantification kits (Bethyl Laboratories, Inc., Montgomery,
Tex., USA). Porcine assays were performed on sow colostrum and milk
and piglet serum as described by Ilsley and Miller (2005).
Analysis of Selected Microbial Populations
[0085] Digesta samples were aseptically removed from the caecum and
colon of each pig post-slaughter. Populations of Bifidobacterium,
E. coli and Lactobacillus spp. were selectively isolated and
enumerated according to Pierce et al. (2006).
Volatile Fatty Acid (VFA) Analysis
[0086] Samples of digesta from the caecum and colon were recovered
for VFA analysis by a gas chromatographic method following the
procedures of Pierce et al. (2006).
Histological Analysis
[0087] Sections of duodenum, jejunum and ileum were aseptically
removed, excised and fixed in 10% phosphate-buffered formalin.
Cross sections at 5 .mu.m thickness of each intestinal segment were
stained with haemotoxylin and eosin. Villus height and crypt depth
were measured using a light microscope fitted with an image
analyser (Image Pro Plus; Media Cybernetics, Bethesda, Md.,
USA).
Phagocytosis Estimation of Blood Cells by Flow Cytometry
[0088] The PHAGOTEST.RTM. kit (Orpegen Pharma, Heidelberg,
Germany), measuring the uptake of unopsonised, FITC-labelled E.
coli, was used to measure the phagocytosing activity in whole blood
cells. Samples were analyzed using a Dako Cyan-ADP flow cytometer
(Dako, Glostrup, Denmark).
Ileum and Colon Gene Expression--RNA Extraction and cDNA
Synthesis
[0089] Tissue samples were collected from the ileum and colon,
rinsed with ice-cold PBS and immediately placed into tubes
containing RNAlater.RTM. (Ambion Inc, Austin, Tex.). Total RNA was
extracted using a Gene Elute Mammalian Total RNA Miniprep Kit
(Sigma-Aldrich) and quantified using a NanoDrop-ND1000
Spectrophotometer (Thermo Fisher Scientific Inc. MA, USA). Purity
was assessed by determining the absorbance ratio at 260 and 280 nm.
Total RNA was reverse transcribed (RT) utilising a First Strand
cDNA Synthesis Kit (Fermentas) using oligo dT primers.
Quantitative Real-Time PCR (qPCR)
[0090] Quantitative real-time PCR (qPCR) assays were performed on
cDNA samples on a 7900HT ABI Prism Sequence Detection System (PE
Applied Biosystems, Foster City, Calif.) using SYBR Green PCR
Master Mix (Applied Biosystems). All primers used for RT-PCR
(IL-la, IL-6, IL-10, TNF-.alpha., MUC2, TFF3, GAPDH, B2M, ACTB,
PPIA and YWHAZ) were designed using Primer Express.TM. software.
Amplification was carried out in 10 .mu.l SYBR PCR Mastermix, 1
.mu.l forward and reverse primer, 8 .mu.l DPEC treated water and 1
.mu.l of template cDNA. Dissociation analyses of the PCR product
was performed to confirm the specificity of the resulting PCR
products.
Results
Colostrum and Milk Composition
[0091] Colostral IgG levels were significantly higher in SWE
supplemented sows (p<0.01). Supplementation increased protein
concentration in sow's milk on day 12 (p<0.05).
TABLE-US-00001 TABLE 1 Effect of dietary treatment on total solid,
crude protein, crude fat and immunoglobulin concentrations of sow
colostrum and milk FO 0 g/day 100 g/day p-value SWE - 1.8 g/day No
Yes No Yes SEM SWE FO SWE x FO Colostrum Total solids % 24.78 25.20
25.69 24.89 1.422 0.897 0.834 0.673 Crude protein % 13.27 13.90
14.72 14.31 1.145 0.925 0.423 0.656 Fat % 6.27 6.10 5.93 5.04 0.737
0.478 0.352 0.632 IgG (mg/ml) 62.52 70.11 64.01 69.56 2.557 0.010
0.844 0.681 IgA (mg/ml) 10.30 8.93 10.50 8.69 1.022 0.105 0.980
0.819 IgM (mg/ml) 4.08 4.91 3.86 4.11 0.444 0.238 0.263 0.513 Sows
milk Total solids % 20.11 20.22 19.48 19.77 0.486 0.687 0.273 0.851
Crude protein % 5.17 5.42 5.17 5.36 0.109 0.050 0.837 0.761 Fat %
9.02 8.80 8.51 8.36 0.546 0.731 0.388 0.940 IgG (mg/ml) 0.37 0.44
0.45 0.49 0.056 0.297 0.204 0.772 IgA (mg/ml) 3.59 3.72 4.03 3.83
0.331 0.914 0.370 0.584 IgM (mg/ml) 1.56 1.56 2.10 1.56 0.419 0.519
0.514 0.526
Suckling Piglet Immunoglobulins
[0092] Piglets suckling SWE supplemented sows had significantly
higher serum IgG concentrations on day 5 (p<0.01) and day 12 of
lactation (p<0.05) and enhanced serum IgA concentrations on day
5 of lactation (p>0.05).
TABLE-US-00002 TABLE 2 Serum immunoglobulin concentrations in
piglets suckling supplemented sows. FO 0 g/day 100 g/day p-value
SWE - 1.8 g/day No Yes No Yes SEM SWE FO SWE x FO Immunoglobulins
(mg/ml) Day 5 IgG 17.63 23.02 20.98 22.80 1.253 0.006 0.213 0.160
IgA 3.00 3.24 2.02 3.02 0.278 0.033 0.036 0.174 IgM 1.52 1.61 1.41
1.39 0.222 0.881 0.468 0.805 Day 12 IgG 9.91 12.47 10.07 11.62
0.880 0.025 0.689 0.570 IgA 0.35 0.27 0.41 0.39 0.090 0.598 0.324
0.776 IgM 0.53 0.61 0.54 0.63 0.051 0.098 0.789 0.929
Suckling Piglet Performance
[0093] Piglets suckling SWE supplemented sows had significantly
lower average daily gains (p<0.05) during week 1 of lactation.
There were no significant differences in daily gains between birth
and weaning. Litter size, litter weight, piglet birth weight and
weaning weight were not influenced by sow dietary treatments.
TABLE-US-00003 TABLE 3 Effect of maternal SWE supplementation on
litter size, litter weight, piglet live weight and average daily
gain (ADG) +FO 0 100 p-value +SWE 1.8 g/day No Yes No Yes SEM SWE
FO SWE x FO Litter size, n 12.40 12.40 12.40 12.35 0.710 0.968
0.958 0.968 Litter weight (Kg) 14.59 15.80 16.04 14.7 0.958 0.924
0.861 0.154 Birth weight (Kg) 1.25 1.28 1.28 1.27 0.061 0.828 0.921
0.656 Piglet BW (Kg) Day 7 3.37 2.92 3.20 2.91 0.161 0.016 0.546
0.612 Day 14 5.41 4.73 5.16 4.80 0.233 0.021 0.697 0.462 Day 21
7.31 6.58 6.67 6.52 0.280 0.093 0.185 0.274 Day 26 8.73 7.85 7.85
7.76 0.340 0.127 0.131 0.209 ADG (Kg/day) Day 0 to 7 0.230 0.195
0.236 0.216 0.014 0.045 0.319 0.589 Day 8 to 15 0.282 0.263 0.274
0.259 0.013 0.185 0.635 0.804 Day 15 to 21 0.286 0.277 0.237 0.260
0.018 0.674 0.053 0.353 Day 21 to 26 0.264 0.254 0.240 0.266 0.022
0.694 0.779 0.384 Day 0 to 26 0.277 0.256 0.250 0.254 0.012 0.501
0.236 0.294
Post-Weaning Piglet Performance
[0094] Piglets from SWE supplemented sows had significantly higher
ADG from day 7-14 (p<0.05) and day 0-21 (p=0.063) and feed
intake (p<0.05) between days 7-14 post weaning.
TABLE-US-00004 TABLE 4 Effect of maternal dietary supplementation
with SWE and FO from day 109 of gestation until weaning (day 26) on
post-weaning performance. SWE FO p-value Treatment No Yes SEM No
Yes SEM SWE FO ADG (Kg/day) Day 0 to 7 0.091 0.104 0.018 0.089
0.106 0.018 0.634 0.518 Day 7 to 14 0.282 0.335 0.017 0.278 0.340
0.017 0.042 0.016 Day 14 to 21 0.450 0.476 0.019 0.485 0.441 0.017
0.351 0.115 Day 0 to 21 0.275 0.308 0.012 0.284 0.299 0.012 0.063
0.403 ADFI (Kg/day) Day 0 to 7 0.169 0.174 0.013 0.167 0.175 0.013
0.781 0.691 Day 7 to 14 0.366 0.424 0.017 0.394 0.396 0.017 0.025
0.932 Day 14 to 21 0.669 0.669 0.050 0.655 0.713 0.050 0.669 0.417
Day 0 to 21 0.401 0.433 0.019 0.405 0.428 0.019 0.186 0.288
Gain:feed ratio Day 0 to 7 0.444 0.532 0.080 0.456 0.519 0.080
0.439 0583 Day 7 to 14 0.764 0.779 0.030 0.699 0.844 0.030 0.719
0.002 Day 14 to 21 0.692 0.741 0.032 0.755 0.678 0.032 0.289 0.107
Day 0 to 21 0.634 0.692 0.030 0.639 0.686 0.030 0.258 0.407
Microbiology
[0095] In the colon, maternal SWE supplementation resulted in a
significant decrease in Bifidobacteria populations (p<0.01).
Furthermore, SWE supplementation had a tendency to decrease E. coli
and Lactobacillus populations in the colon compared to the control
(p=0.09).
TABLE-US-00005 TABLE 5 Effect of maternal dietary supplementation
with SWE and FO from day 109 of gestation until weaning on selected
intestinal microflora in the 9 day old weaned pig. FO (g/day) 0 100
p-value SWE (1.8 g/day) No Yes No Yes SEM SWE FO SWE x FO Caecum
(Log.sub.10 CFU/g digesta) Bifidobacteria spp. 8.52 8.57 8.54 8.30
0.211 0.652 0.563 0.506 Lactobacilli spp. 8.15 8.14 8.41 7.93 0.328
0.466 0.926 0.486 E. coli 4.89 3.67 3.37 3.78 0.387 0.311 0.081
0.048 Colon (Log.sub.10 CFU/g digesta) Bifidobacteria spp. 8.91
8.53 9.32 8.11 0.276 0.008 0.998 0.148 Lactobacilli spp. 8.50 8.33
8.99 8.01 0.322 0.087 0.775 0.222 E. coli 5.51 4.62 5.16 4.38 0.473
0.093 0.535 0.917
Cytokine Gene Expression
[0096] In the ileum of the post weaned pig, maternal SWE
supplementation induced a significant increase in the expression of
the pro-inflammatory cytokine TNF-.alpha. (p<0.01). A
significant increase in TFF 3 gene expression was also observed in
the colon (p<0.05).
TABLE-US-00006 TABLE 6 Effect of maternal dietary supplementation
with SWE from day 109 of gestation until weaning on selected gene
expression in the ileum and colon of the weaned pig. SWE FO p-value
Treatment No Yes SEM No Yes SEM SWE FO Ileum IL-1.alpha. 0.216
0.215 0.034 0.224 0.206 0.034 0.984 0.741 IL-6 0.212 0.166 0.032
0.197 0.181 0.032 0.325 0.747 TNF-.alpha. 0.164 0.575 0.102 0.264
0.475 0.106 0.010 0.182 IL-10 0.127 0.075 0.023 0.085 0.116 0.023
0.122 0.371 MUC 2 0.518 0.724 0.132 0.635 0.608 0.132 0.281 0.859
TFF 3 0.585 0.708 0.076 0.664 0.629 0.076 0.266 0.766 Colon
IL-1.alpha. 0.150 0.132 0.025 0.099 0.182 0.025 0.632 0.029 IL-6
0.170 0.124 0.026 0.102 0.193 0.102 0.236 0.024 TNF-.alpha. 0.242
0.206 0.026 0.214 0.234 0.026 0.338 0.592 IL-10 0.132 0.077 0.022
0.089 0.121 0.022 0.092 0.324 MUC 2 0.490 0.508 0.095 0.616 0.381
0.095 0.733 0.182 TFF 3 0.371 0.565 0.068 0.536 0.400 0.068 0.045
0.111
Volatile Fatty Acid (VFA) Analysis and pH measurement
TABLE-US-00007 TABLE 7 Effect of maternal dietary treatment with
SWE and FO from day 109 of gestation until weaning on VFA
composition of intestinal contents of the 9 day old weaned pig. FO
(g/day) 0 100 p-value SWE (1.8 g/day) No Yes No Yes SEM SWE FO SWE
x FO Caecum (mmol/g digesta) Total VFA 181.7 168.0 170.4 183.2
11.20 0.968 0.865 0.249 Acetic acid 0.660 0.645 0.675 0.665 0.011
0.289 0.124 0.801 Propionic acid 0.228 0.245 0.240 0.245 0.010
0.298 0.539 0.542 Butyric acid 0.093 0.089 0.063 0.074 0.008 0.664
0.009 0.371 Isobutyric acid 0.003 0.003 0.004 0.002 0.001 0.308
0.949 0.295 Valeric acid 0.011 0.012 0.012 0.010 0.002 0.823 0.686
0.492 Isovaleric acid 0.005 0.006 0.006 0.004 0.001 0.582 0.572
0.264 Acetic:propionic acid 2.94 2.68 2.84 2.75 0.158 0.287 0.897
0.624 BCFAs* 0.020 0.021 0.022 0.016 0.003 0.482 0.623 0.226 pH
6.17 6.27 6.41 6.07 0.189 0.511 0.922 0.255 Colon (mmol/g digesta)
Total VFA 151.6 146.0 128.1 168.7 12.62 0.177 0.974 0.080 Acetic
acid 0.658 0.631 0.672 0.663 0.014 0.209 0.113 0.521 Propionic acid
0.216 0.229 0.281 0.232 0.356 0.617 0.344 0.386 Butyric acid 0.087
0.010 0.087 0.077 0.012 0.799 0.268 0.269 Isobutyric acid 0.007
0.008 0.012 0.006 0.002 0.080 0.317 0.043 Valeric acid 0.011 0.016
0.019 0.012 0.002 0.705 0.471 0.038 Isovaleric acid 0.012 0.013
0.021 0.010 0.002 0.053 0.222 0.028 Acetic:propionic acid 3.07 2.79
3.18 2.95 0.184 0.166 0.462 0.883 BCFAs* 0.030 0.036 0.052 0.028
0.005 0.127 0.195 0.009 pH 6.28 6.17 6.48 6.44 0.119 0.554 0.063
0.783 *BCFAs, branched chain fatty acids
Histology
[0097] In the ileum, there was a significant effect of SWE
supplementation on villus height and villus height to crypt depth
ratio (p<0.05). Results from the duodenum also showed a
beneficial effect emulating from SWE supplementation on crypt depth
(p>0.10)
TABLE-US-00008 TABLE 8 Effect of maternal dietary supplementation
with SWE and fish oil (FO) from day 109 of gestation until weaning
(day 26) on villus height, crypt depth and villus height to crypt
depth ratio in the 9 day old weaned pig. Fish oil (g/d) 0 100
p-value SWE (1.8 g/d) No Yes No Yes SEM SWE FO SWE x FO Villous
height (.mu.m) Duodenum 419.4 415.9 430.1 421.5 5.62 0.291 0.183
0.645 Jejunum 384.2 396.2 395.4 382.8 5.00 0.952 0.843 0.022 Ileum
215.0 233.0 238.7 232.6 6.00 0.328 0.063 0.055 Crypt depth (.mu.m)
Duodenum 328.8 314.3 316.0 315.4 4.40 0.097 0.216 0.122 Jejunum
288.6 280.3 291.7 288.1 6.87 0.392 0.458 0.731 Ileum 178.0 172.4
167.9 171.7 4.75 0.853 0.270 0.333 Villous:crypt depth ratio
Duodenum 1.28 1.31 1.36 1.32 0.02 0.788 0.049 0.164 Jejunum 1.33
1.43 1.36 1.33 0.03 0.288 0.177 0.034 Ileum 1.21 1.36 1.42 1.35
0.04 0.444 0.015 0.013
Phagocytosing Capacity
[0098] SWE supplementation exerted a suppressive effect on total
eosinophil numbers (p<0.01) in suckling piglets. Dietary SWE
supplementation resulted in a higher percentage of E. coli
phagocytosing leukocytes (p<0.05) and a lower percentage of E.
coli phagocytosing lymphocytes (p<0.01) compared to non
SWE-supplemented diets.
TABLE-US-00009 TABLE 9 Effect of dietary treatment on the
phagocytosing activity (total number and % positive phagocytosis)
of piglet whole blood cells at weaning SWE FO p-value No Yes SEM No
Yes SEM SWE FO Leukocytes 22475 20912 6637 20896 22492 1637 0.595
0.365 Positive % 57.6 64 2.2 57.1 64.5 2.2 0.046 0.024 Lymphocytes
6650 5127 672 6292 5485 672 0.116 0.575 Positive % 13.3 10.1 0.834
10.5 12.9 0.834 0.008 0.050 Monocytes 2641 2578 83 2575 2644 83
0.627 0.614 Positive % 74.1 77.7 2.8 72.7 79.1 2.8 0.369 0.112
Neutrophils 8650 9489 883 7977 10161 883 0.407 0.076 Positive %
91.1 92.1 1.2 91.4 91.9 1.2 0.564 0.796 Eosinophils 512 338 61 384
466 61 0.002 0.297 Positive % 26 21.8 2.1 23.2 24.6 2.1 0.163
0.653
Example 2
Experiment 1
Materials and Methods
Experimental Design and Diets
[0099] Experiment 1 was designed as a complete randomised design
comprising of five dietary treatments as follows: (T1) 0 g/Kg SWE
(control), (T2) 0.7 g/Kg SWE, (T3) 1.4 g/Kg SWE extract, (T4) 2.8
g/Kg SWE extract and (T5) 5.6 g/Kg SWE extract. The SWE contained
LAM+FUC. All diets were formulated to have identical concentrations
of net energy and total lysine. The amino acid requirements were
met relative to lysine (Close, 1994). Chromic oxide was added at
the time of milling to all diets at the rate of 150 ppm for the
determination of ash digestibility.
Animals and Management
[0100] 30 finishing boars with an initial live weight of 51.+-.3.4
Kg were used in the experiment. The pigs were blocked on the basis
of live weight and randomly allocated to one of five dietary
treatments. The pigs were allowed a 14-day dietary adaptation
period after which time they were weighed and transferred to
individual metabolism crates. Animals were allowed a 5-day
acclimatisation period, followed by a 5-day collection period to
facilitate an apparent digestibility and nitrogen balance study.
The daily feed allowance (DE intake=3.44.times.(live
weight).sup.0.54 (Close, 1994) was divided over two meals. Water
was provided with meals in a 1:1 ratio. Between meals, fresh water
was provided ad libitum. The metabolism crates were located in an
environmentally controlled room, maintained at a constant
temperature of 22.degree. C. (.+-.1.5.degree. C.).
Coefficient of Total Tract Apparent Digestibility (CTTAD) and
Nitrogen Balance Study
[0101] During collections, urine was collected in a plastic
container, via a funnel below the crate, containing 20 ml of
sulphuric acid (25% H.sub.2S0.sub.4). To avoid nitrogen
volatilisation, the funnel was sprayed four times daily with weak
sulphuric acid (2% H.sub.2S0.sub.4) solution. The urine volume was
recorded daily and a 50 ml sample was collected and frozen for
laboratory analysis. Total faeces weight was recorded daily and
oven dried at 100.degree. C. A sample of freshly voided faeces was
collected daily and frozen for nitrogen analysis and pH
measurement. At the end of the collection period, the faeces
samples were pooled and a sub-sample retained for laboratory
analysis. Feed samples were collected each day and retained for
chemical analysis. All 30 pigs remained on their respective dietary
treatments until slaughter.
Example 2
Experiment 2
Materials and Methods
Experimental Design and Diets
[0102] This experiment was designed as a 2.times.2 factorial design
comprising four dietary treatments: (T1) control diet, (T2)
control+300 ppm LAM, (T3) control+238 ppm FUC, (T4) control+300 ppm
LAM+238 ppm FUC. All diets were standardised for net energy (9.8
MJ/Kg) and total lysine (10 g/Kg). Amino acid requirements were met
relative to lysine (Close, 1994).
TABLE-US-00010 TABLE 10 Composition and analysis of diets -
experiment 1 (as fed basis). Treatment 1 2 3 4 5 Ingredients (g
kg.sup.-1) Laminaria hyperborea extract 0 0.7 1.4 2.8 5.6 Wheat
704.3 703.6 702.9 701.5 698.7 Soybean Meal 265 265 265 265 265 Soya
Oil 5.7 5.7 5.7 5.7 5.7 Mineral and Vitamin.sup..dagger. 2.5 2.5
2.5 2.5 2.5 Limestone 15 15 15 15 15 Dicalcium phosphate 7.5 7.5
7.5 7.5 7.5 Analysed Composition (g kg.sup.-1) Laminarin 0 0.075
0.150 0.300 0.600 Fucoidan 0 0.059 0.119 0.238 0.476 Dry Matter
869.1 871.8 886.5 884.4 878.2 Crude Protein (N x 6.25) 215.7 203.2
199.5 200.8 195.1 Neutral Detergent Fibre 119.7 97.8 91.8 96.4 98
Acid detergent fibre 39.0 35.2 30.7 32.7 35.7 Crude Ash 45.7 48.4
47.7 51.2 52.6 Lysine 9.9 9.9 9.9 9.9 9.9 Methionine and
cysteine.sup.$ 6.0 6.0 6.0 6.0 6.0 Threonine 7.0 7.0 7.0 7.0 7.0
Tryptophan 1.9 1.9 1.9 1.9 1.9 Calculated composition (g kg.sup.-1)
Digestible Energy.sup.# 13.9 13.9 13.9 13.9 13.8 Calcium 7.29 7.29
7.30 7.32 7.35 Phosphorus 4.04 4.04 4.04 4.04 4.04
.sup..dagger.Provided per kg of complete diet: 3 mg retinol, 0.05
mg cholecalciferol, 40 mg alpha- tocopherol, 90 mg copper as copper
II sulphate, 100 mg iron as iron II sulphate, 100 mg zinc as zinc
oxide, 0.3 mg selenium as sodium selenite, 25 mg manganese as
manganous oxide and 0.2 mg iodine as calcium iodate on a calcium
sulphate/calcium carbonate carrier.
Animals and Management
[0103] 28 finishing boars with an initial live weight of 55 Kg were
used. Pigs were blocked on the basis of live weight and were
randomly allocated to one of four dietary treatments. The pigs were
allowed a 28-day dietary adaptation period after which time they
were weighed and slaughtered.
Microbiology and Apparent Digestibility of Ash in the Proximate
Caecum and Colon
[0104] Digesta was aseptically removed from the proximal caecum and
colon of each animal after slaughter. Chromic oxide was used as
marker to determine ash digestibility in the caecum and colon.
Bifidobacteria spp., Lactobacillus spp. and Enterobacteria were
isolated and counted according to the method described by O'Connell
et al., (2005).
Volatile Fatty Acid Sampling and Analysis
[0105] Samples of digesta from the caecum and the proximal and
distal colon of individual pigs were taken for VFA analysis. VFA
concentrations in the digesta were determined using a modified
method of Porter and Murray (2001) according to O'Connell et al.
(2005).
Results
Experiment 1--Microbiology Study
TABLE-US-00011 [0106] TABLE 11 Effect of SWE concentration on
microbial ecology and pH in the caecum and colon Treatment 1 2 3 4
5 L. hyperborea extract (g/Kg) 0 0.7 1.4 2.8 5.6 s.e.m. Linear
Quadratic Proximal Caecum Bacterial Populations (CFU/ml digesta)
Enterobacteria spp. 6.94 7.15 6.65 6.42 6.70 0.196 ns *
Bifidobacteria spp. 8.33 8.45 8.41 8.25 7.86 0.174 ** ns
Lactobacilli spp. 8.67 8.85 8.73 8.84 8.62 0.140 ns ns Proximal
Colon Bacterial Populations (CFU/ml digesta) Enterobacteria 6.95
6.72 6.34 6.49 6.85 0.245 ns * Bifidobacteria spp. 8.37 8.62 8.77
8.57 8.16 0.118 ns ** Lactobacilli spp. 9.10 9.15 9.07 8.90 8.83
0.113 * ns Caecum pH 5.63 5.90 6.42 5.49 5.69 0.125 ns *** Colon pH
5.94 6.11 6.18 5.85 5.94 0.079 ns ** * = (p < 0.05), ** = (p
< 0.01), *** = (p < 0.001), ns = non significant (p >
0.05), $ = (p < 0.1)
Experiment 1--Apparent Ash Digestibility
TABLE-US-00012 [0107] TABLE 12 Effect of SWE concentration on
apparent nutrient digestibility and nitrogen balance. Treatment 1 2
3 4 5 L. hyperborea SWE 0 0.7 1.4 2.8 5.6 s.e.m. Linear Quadratic
Average Daily Feed 2158 2168 2138 2214 2209 * Intake (g/d)
Laminarin Intake (mg/d) 0 162.6 320.7 664.2 1325.4 Fucoidan Intake
(mg/d) 0 127.9 254.4 526.9 1051.4 Water Intake (Kg/d) 5.11 4.65
5.44 5.80 6.08 0.043 * ns Urine Output (Kg/day) 2.803 3.256 3.654
3.445 4.273 0.320 * ns Nitrogen Intake (g/day) 64.72 61.48 60.48
62.90 60.51 0.691 * ns Digestibility Coefficients Neutral Detergent
Fibre 0.66 0.55 0.56 0.58 0.56 0.012 ns * Nitrogen 0.90 0.90 0.89
0.90 0.89 0.006 ns ns Dry Matter 0.89 0.89 0.89 0.89 0.89 0.002 ns
ns Organic Matter 0.91 0.91 0.91 0.91 0.91 0.003 ns ns Ash
Digestibility Coefficients Caecal Ash 0.49 0.45 0.40 0.35 0.43
0.037 ns ** Colonic Ash 0.56 0.49 0.51 0.50 0.51 0.016 ns ** Total
Tract Ash 0.57 0.62 0.56 0.62 0.62 0.010 ** ns Nitrogen (N) Balance
Faecal N Excretion 6.93 6.93 7.02 7.34 7.16 0.417 ns ns (g/day)
Urinary N Excretion 29.18 28.91 29.61 28.79 34.27 1.07 ns * (g/day)
Total N Excretion (g/day) 36.14 35.89 36.84 35.69 41.60 1.12 ns * N
Retention (g/day) 25.87 26.12 25.18 26.31 20.41 1.17 ns * * = (p
< 0.05), ** = (p < 0.01), *** = (p < 0.001), ns = non
significant (p > 0.05)
Experiment 1--Volatile Fatty Acid Study
TABLE-US-00013 [0108] TABLE 13 Effect of SWE concentration on
concentration & molar proportions of VFAs Treatment 1 2 3 4 5
L. hyperborea SWE 0 0.7 1.4 2.8 5.6 s.e.m. Linear Quadratic
Proximal Caecum (mmol/L) VFAs 281.2 241.7 378.1 197.9 227.8 13.91 *
** Acetic acid 0.626 0.656 0.653 0.621 0.629 0.011 ns ** Propionic
acid 0.210 0.197 0.172 0.202 0.195 0.008 ns ** Isobutyric acid
0.014 0.011 0.017 0.014 0.005 0.002 * * Butyric acid 0.107 0.101
0.111 0.105 0.118 0.005 ns ns Isovaleric acid 0.020 0.017 0.023
0.027 0.014 0.002 ns ** Valeric acid 0.019 0.016 0.021 0.019 0.014
0.002 ns ns Acetic:Propionic 2.86 3.22 3.83 3.16 3.35 0.155 ns ***
BCFAs 0.052 0.044 0.062 0.060 0.034 0.006 ns * Proximal Colon
(mmol/L) VFAs 342.3 376.8 371.7 281.0 369.0 45.31 ns ns Acetic acid
0.579 0.575 0.569 0.574 0.579 0.013 ns ns Propionic acid 0.201
0.196 0.200 0.206 0.195 0.004 ns ns Isobutyric acid 0.023 0.027
0.025 0.025 0.026 0.005 ns ns Butyric acid 0.123 0.132 0.133 0.130
0.126 0.006 ns ns Isovaleric acid 0.034 0.038 0.038 0.034 0.039
0.003 ns ns Valeric acid 0.030 0.035 0.033 0.028 0.033 0.005 ns ns
Acetic:Propionic 2.801 3.032 2.839 2.784 2.869 0.099 ns ns BCFAs
0.088 0.101 0.097 0.088 0.099 0.016 ns ns * = (p < 0.05), ** =
(p < 0.01), *** = (p < 0.001), ns = non significant (p >
0.05)
Experiment 2--Microbiology Study
TABLE-US-00014 [0109] TABLE 14 Effect of LAM and FUC on the
concentration and molar proportions of VFAs Treatment 1 2 3 4
Significance Control LAM FUC LAM/FUC s.e.m. LAM FUC LAM X FUC
Proximal Colon (mmol/L) Total VFA 168.7 173.6 195.5 197.9 7.925 ns
** ns Acetic acid 0.618 0.576 0.599 0.647 0.011 ns * *** Propionic
acid 0.213 0.268 0.251 0.217 0.011 ns ns *** Isobutyric acid 0.009
0.003 0.004 0.003 0.001 ** * * Butyric acid 0.124 0.125 0.118 0.114
0.004 ns ns ns Isovaleric acid 0.017 0.011 0.011 0.008 0.002 * * ns
Valeric acid 0.017 0.017 0.014 0.011 0.002 ns ** ns
Acetic:propionic 2.94 2.17 2.44 3.02 0.159 ns ns *** BCFAs 0.042
0.031 0.026 0.022 0.004 * ** ns Distal Colon (mmol/L) Total VFA
126.02 136.7 172.6 159.5 9.34 ns *** ns Acetic acid 0.597 0.571
0.599 0.636 0.012 ns ** ** Propionic acid 0.195 0.203 0.186 0.181
0.005 ns *** ns Isobutyric acid 0.026 0.022 0.021 0.020 0.001 ns **
ns Butyric acid 0.118 0.134 0.139 0.113 0.007 ns ns ** Isovaleric
acid 0.040 0.034 0.034 0.032 0.002 * * ns Valeric acid 0.024 0.023
0.021 0.018 0.001 ns *** ns Acetic:propionic 3.08 2.76 3.23 3.52
0.128 ns *** * BCFAs 0.089 0.078 0.076 0.070 0.003 * ** ns
Experiment 2--Volatile Fatty Acid Study
TABLE-US-00015 [0110] TABLE 15 Effect of LAM and FUC concentration
on concentration and molar proportions of VFAs Treatment 1 2 3 4
Significance Control LAM FUC LAM/FUC s.e.m. LAM FUC LAM x FUC
Proximal Colon (mmol/L) Total VFA 168.7 173.6 195.5 197.9 7.925 ns
** ns Acetic acid 0.618 0.576 0.599 0.647 0.011 ns * *** Propionic
acid 0.213 0.268 0.251 0.217 0.011 ns ns *** Isobutyric acid 0.009
0.003 0.004 0.003 0.001 ** * * Butyric acid 0.124 0.125 0.118 0.114
0.004 ns ns ns Isovaleric acid 0.017 0.011 0.011 0.008 0.002 * * ns
Valeric acid 0.017 0.017 0.014 0.011 0.002 ns ** ns
Acetic:Propionic 2.94 2.17 2.44 3.02 0.159 ns ns *** BCFAs 0.042
0.031 0.026 0.022 0.004 * ** ns Distal Colon (mmol/L) Total VFA
126.02 136.7 172.6 159.5 9.34 ns *** ns Acetic acid 0.597 0.571
0.599 0.636 0.012 ns ** ** Propionic acid 0.195 0.203 0.186 0.181
0.005 ns *** ns Isobutyric acid 0.026 0.022 0.021 0.020 0.001 ns **
ns Butyric acid 0.118 0.134 0.139 0.113 0.007 ns ns ** Isovaleric
acid 0.040 0.034 0.034 0.032 0.002 * * ns Valeric acid 0.024 0.023
0.021 0.018 0.001 ns *** ns Acetic:Propionic 3.08 2.76 3.23 3.52
0.128 ns *** * ratio BCFAs 0.089 0.078 0.076 0.070 0.003 * ** ns *
= (p < 0.05), ** = (p < 0.01), *** = (p < 0.001), ns = non
significant (p > 0.05)
Example 3
Experimental Design and Diets
[0111] This experiment was carried out over two consecutive periods
of 25 days. 240 piglets were selected after weaning at 24 days and
assigned to one of four dietary treatments. Pigs in period 1 and 2
had initial live weights of 7.2 Kg and 7.8 Kg (.+-.0.9 Kg),
respectively. This experiment was designed as a 2.times.2
factorial. During the experiment (days 0-25) piglets were offered
the following diets: (T1) 150 g/Kg lactose; (T2) 150 g/Kg
lactose+SWE; (T3) 250 g/Kg lactose (T4) 250 g/Kg lactose+SWE. SWE
was included at 2.8 g/Kg and derived from Laminaria digitata. It
contained laminarin (112 g/Kg), fucoidan (89 g/Kg) and ash (799
g/Kg).
Animals and Management
[0112] Pigs were housed in groups of 4 (n=15/treatment) and weighed
at weaning (day 0), day 7, 14 and 25. Pigs were fed ad libitum.
Fresh faecal samples were collected on days 10 to 15 for
determination of nutrient digestibility and VFA analysis. Fresh
faecal samples were collected on day 10 for enumeration of E. coli
and Lactobacilli (O'Connell et al., 2005).
Microbiology
[0113] 1 g of faecal sample was serially diluted in maximum
recovery diluent (MRD; Oxoid, Basingstoke, UK) and plated on
selective agars. Lactobacillus spp. were isolated on de Man Rogosa
Sharp agar (MRS, Oxoid). The API 50 CHL (BioMerieux, France) kit
was used to confirm suspect Lactobacilli spp. E. coli species were
isolated on MacConkey agar (Oxoid). Suspect colonies were confirmed
with API 20E (BioMerieux, France).
Results
Performance
TABLE-US-00016 [0114] TABLE 16 The effect of lactose and SWE on
piglet performance Treatment Significance 1 2 3 4 SEM Lactose SWE
Lactose x SWE Lactose (g/Kg) 150 250 SWE - + - + Average Daily Gain
(ADG) (Kg/day) Day 0-7 0.100 0.148 0.146 0.183 0.018 * * ns Day
7-14 0.302 0.303 0.325 0.387 0.021 * ns ns Day 14-25 0.438 0.427
0.388 0.455 0.020 ns ns * Day 0-25 0.275 0.293 0.287 0.350 0.013 *
** ns Average Daily Feed Intake (ADFI) (Kg/day) Day 0-7 0.242 0.257
0.239 0.271 0.015 ns ns ns Day 7-14 0.415 0.426 0.450 0.472 0.019 *
ns ns Day 14-25 0.682 0.663 0.683 0.737 0.025 ns ns ns Day 0-25
0.446 0.449 0.458 0.502 0.014 * ns ns Gain: Feed ratio (Kg/Kg) Day
0-7 0.413 0.558 0.589 0.659 0.062 * ns ns Day 7-14 0.747 0.705
0.729 0.832 0.055 ns ns ns Day 14-25 0.633 0.636 0.569 0.622 0.027
* ns ns Day 0-25 0.603 0.633 0.619 0.691 0.062 ns * ns Probability
of significance; * P < 0.05; ** P < 0.01, ns P > 0.05
Coefficient of Total Tract Apparent Digestibility (CTTAD)
TABLE-US-00017 [0115] TABLE 17 The effect of dietary treatment on
the coefficient of total tract apparent digestibility Treatment
Significance 1 2 3 4 SEM Lactose SWE Lactose x SWE Lactose 150 250
(g/Kg) SWE - + - + Digestibility (%) DM 87.75 91.65 91.34 95.25
0.500 *** *** ns OM 89.16 92.52 92.24 95.82 0.535 *** *** ns N
83.69 89.59 86.86 92.34 1.038 * *** ns Ash 53.30 70.60 72.80 83.08
2.140 *** *** ns GE 85.93 90.93 90.22 94.46 0.698 *** *** ns NDF
37.55 65.01 61.91 74.60 2.970 *** *** * Probability of
significance; * p < 0.05; ** p < 0.01, *** p < 0.001, ns p
> 0.05
Microbiology and VFAs
TABLE-US-00018 [0116] TABLE 18 Effect of dietary treatment on
Lactobacilli and Escherichia coli populations Treatment
Significance 1 2 3 4 SEM Lactose SWE Lactose x SWE Lactose (g/Kg)
150 250 SWE - + - + Bacterial populations (Log.sub.10 CFU/g faeces)
Lactobacilli 8.46 8.63 8.19 8.84 0.12 ns *** * Escherichia coli
6.30 5.80 5.70 4.50 0.42 * * ns Probability of significance; * P
< 0.05; ** P < 0.01, ns P > 0.05
Example 4
Experimental Design and Diets
[0117] One hundred and ninety two piglets were weaned at twenty
four days of age, with an initial live weight of 6.4.+-.0.785 Kg
and assigned to one of four dietary treatments or 21 days post
weaning. The dietary treatments consisted of (T1) basal diet, (T2)
basal diet with 300 ppm LAM, (T3) basal diet with 236 ppm FUC, (T4)
Basal diet with 300 ppm LAM and 236 ppm FUC. Diets were formulated
to have identical concentrations of digestible energy (DE) (16
MJ/Kg) and ileal digestible lysine (14 g/Kg). All amino acid
requirements were met relative to lysine (Close, 1994). Chromium
III oxide was added to the diets for determination of nutrient
digestibility. The LAM and FUC were derived from Laminaria
hyperborea.
Management
[0118] Piglets were housed in groups of 4 and offered feed twice
daily. Water was supplied ad-libitum. Any pig displaying symptoms
of illness was treated appropriately and recorded. Pigs were
weighed on day 0 (day of weaning), 7, 14 and 21. Feed intake was
monitored weekly. Fresh faecal samples were taken on day 10 and
were analysed for E. coli and Lactobacilli concentrations. Faeces
samples were collected from each pen on day 12-17 and were retained
for chemical analysis. Fresh faecal samples were removed on day 14
and were frozen and retained for volatile fatty acid analysis.
Fresh faecal samples were taken on day 17 for pH determination.
Faeces Scoring and Morbidity
[0119] Pigs were observed for clinical signs of diarrhoea from day
0-21. A scoring system was applied to indicate its presence and
severity. The following scoring system was used: 1=hard, 2=slightly
soft, 3=soft, partially formed, 4=loose, semi-liquid and 5=watery,
mucous-like.
Microbiology
[0120] A sample was serially diluted (1:10) in 9.0 ml aliquots of
maximum recovery diluent (MRD, Oxoid, Basingstoke, UK), and spread
plated (0.1 ml aliquots) onto selective agars. Lactobacillus spp.
were isolated on de Man, Rogosa, Sharp agar (MRS, Oxoid) with
overnight incubation at 37.degree. C. in 5% CO2. The API 50 CHL
(BioMerieux, France) kit was used to confirm suspect Lactobacilli
spp. E. coli species were isolated on MacConkey agar (Oxoid),
following aerobic incubation at 37.degree. C. for 18-24 hours.
Suspect colonies were confirmed with API 20E (BioMerieux,
France).
Results
Performance
[0121] Pigs fed LAM supplemented diets had an increased ADG (0.344
v 0.266, p<0.01) during days 7-14 and during the entire
experimental period (0.324 v 0.232, p<0.01) compared to pigs
offered diets with no LAM. Pig fed LAM supplementation had improved
gain:feed ratio during days 7-14 (0.763 vs. 0.569, p<0.001) and
during the entire experimental period (0.703 v 0.646, p<0.05)
compared to unsupplemented LAM diets. There was a significant
interaction (p<0.05) between LAM and FUC supplementation on ADG
during days 14-21. Pigs offered the FUC diet had a significantly
higher ADG than pigs offered the basal diet, however there was no
effect of FUC when added to a LAM diet. There was no effect of LAM
or FUC inclusion on average daily feed intake.
TABLE-US-00019 TABLE 19 The effect of seaweed extract on pig
performance post weaning. Treatment Significance T1 T2 T3 T4 SEM
LAM FUC LAM x FUC LAM - + - + FUC - - + + No of pens 12 12 12 12
Daily Gain (g/day) D 0-7 181 178 166 185 0.025 ns ns ns D 7-14 268
320 265 368 0.022 ** ns ns D 14-21 418 459 475 430 0.016 ns ns * D
0-21 288 319 302 328 0.012 * ns ns Food Intake (g/day) D 0-7 256
263 253 257 0.020 ns ns ns D 7-14 449 464 477 457 0.027 ns ns ns D
14-21 604 686 673 619 0.024 ns ns ns D 0-21 436 471 467 444 0.017
ns ns ns Gain to feed ratio (Kg/Kg) D 0-7 0.666 0.646 0.646 0.679
0.055 ns ns ns D 7-14 0.579 0.707 0.561 0.818 0.049 *** ns ns D
14-21 0.716 0.673 0.708 0.697 0.039 ns ns ns Days 0-21 0.654 0.675
0.638 0.732 0.024 * ns ns Probability of significance; * = (P <
0.05), ** = (P < 0.01), *** = (P < 0.001).
Faecal pH, DM, Faecal Score
[0122] Pigs offered diets supplemented with LAM had an increased
faecal DM content (28.64 v 26.24; p<0.05) compared to
unsupplemented LAM diets. Pigs offered diets supplemented with LAM
had a decreased faecal score during days 7-14 (2.05 v 2.57;
p<0.05). There was a significant interaction between LAM and FUC
inclusion on faecal score during the entire experimental period
(days 0-21) (P<0.05). Pigs offered the combination of LAM and
FUC had a reduced faecal score compared to pigs offered the FUC
alone diet. However, there was no effect of LAM inclusion on faecal
score compared to the basal diet.
TABLE-US-00020 TABLE 20 Effect of dietary treatment on faecal dry
matter and faecal score Treatment Significance T1 T2 T3 T4 SEM LAM
FUC LAM x FUC LAM - + - + FUC - - + + No of pens 12 12 12 12 Faecal
DM (g/Kg) 272.8 290.1 252.2 282.8 10.2 * ns ns Faecal pH 6.42 6.25
6.19 6.31 0.109 ns ns ns Faecal score Days 0-7 2.45 2.61 2.49 2.07
0.149 ns ns ns Days 7-14 2.62 2.22 2.53 1.88 0.196 * ns ns Days
14-21 1.58 1.93 1.77 1.62 0.119 ns ns ns Days 0-21 2.22 2.25 2.26
1.85 0.110 ns ns * Probability of significance; * = (P <
0.05)
Microbiology and Volatile Fatty Acids (VFAs)
[0123] Pigs offered LAM diets had a reduced faecal E. coli
population compared to pigs offered diets with no LAM
supplementation (7.22 vs. 7.84; p<0.05). There was a significant
interaction (P<0.01) between LAM and FUC on faecal Lactobacilli
populations. Pigs offered the FUC diet had increased Lactobacilli
numbers compared to pigs offered the basal diet (9.22 v 8.93)
however there was no effect of FUC on faecal lactobacilli
populations when included with LAM. There was no significant effect
of treatment on volatile fatty acid concentrations.
TABLE-US-00021 TABLE 21 The effect of dietary treatment on faecal
Lactobacilli and Escherichia coli populations and faecal molar
proportions of volatile fatty acids Treatment Significance T1 T2 T3
T4 s.e.m LAM FUC LAM x FUC LAM - + - + FUC - - + + E. coli 8.04
7.41 7.67 7.05 0.217 * ns ns Lactobacilli 8.93 9.18 9.22 9.06 0.076
ns ns ** Total VFA (mmol/L) Molar Proportions 141.4 134.5 130.1
110.4 9.197 ns ns ns Acetic Acid 0.568 0.568 0.590 0.588 0.014 ns
ns ns Propionic Acid 0.210 0.209 0.288 0.219 0.007 ns ns ns
Isobutyric acid 0.018 0.021 0.018 0.022 0.001 ns ns ns Butyric Acid
0.144 0.135 0.152 0.123 0.011 ns ns ns Isovaleric Acid 0.034 0.038
0.034 0.043 0.002 ns ns ns Valeric Acid 0.037 0.040 0.038 0.038
0.003 ns ns ns Probability of significance; * = (p < 0.05), ** =
(p < 0.01).
[0124] Pig offered LAM supplemented diets had improved Average
Daily Gain (ADG) and gain to feed ratio (GFR) compared to pigs
offered the unsupplemented diets. This positive response to LAM may
be due to the reduced E. coli population in the gut of these pigs.
Diets supplemented with LAM resulted in pigs having a reduced
faecal E. coli population which resulted in reduced faecal DM and
less diarrhoea (lower faecal score) during days 7-14, compared to
pigs offered diets containing no LAM. Inclusion of LAM in the diet
resulted in reduced Enterobacteria population in the gut of the
pig. Thus the improved performance seen with pigs fed laminarin
diets could be due to the associated antimicrobial properties of
LAM, which may result in an improved health status and reduced
coliform load in the gut of the pig. Modulation of mucosal immunity
by the binding of LAM to the specific receptors of immune cells may
provide beneficial effects on pig health through preventing the
colonization and proliferation of bacteria and therefore the
subsequent damage of the intestinal wall. The proliferation of
Lactobacilli spp. in FUC supplemented diets would suggest that a
proportion of the supplemented FUC is escaping hydrolysis in the
foregut and passing into the colon for bacterial fermentation.
Saccharolytic species of bacteria such as Lactobacilli spp. take
part in the breakdown of complex carbohydrates. FUC is soluble in
water making it a rapidly fermentable carbohydrate source.
Lactobacillus spp. have been reported to ferment a number of
monosaccharides which included L-fucose. In the current study, it
was found that the concentration of Lactobacillus spp. in the colon
increased with the inclusion of FUC. Despite the increase in the
Lactobacilli population, there was no dietary effect on VFA
concentration or profiles. The quantity of VFA produced in the
large intestine depends on the amount and composition of the
substrate and on the microflora present (MacFarlane and MacFarlane,
2003). However, faecal VFA concentrations may not be a totally
accurate way to demonstrate fermentation intensity in the large
intestine.
[0125] The combination diets of FUC+LAM were most effective at
reducing post weaning diarrhoea. This could be attributed to a
number of reasons. Firstly, it could be due to an immune response
from feeding the combination diets. Secondly, there was a numerical
decrease in faecal E. coli numbers with the combination treatment.
Pigs that express the symptoms of diarrhoea harbour massive numbers
of haemolytic E. coli. Therefore, a reduction in the numbers of E.
coli present in the gut would reduce the severity of diarrhoea and
ultimately reduce piglet morbidity post weaning.
[0126] Overall, the reduction in faecal E. coli population and the
increase in ADG and GFR suggest that LAM may provide a dietary
means to improve gut health post weaning. However, a combination of
LAM and FUC is more effective at reducing diarrhoea.
Example 5
Experimental Design and Animal Diets
[0127] 21 pigs with an initial weight of 17.9.+-.2.2 Kg were
assigned to one of the 3 dietary treatments: (T1) control; (T2)
basal diet+300 ppm LAM; (T3) basal diet+600 ppm LAM. Experimental
feeding continued for 21 days ad libitum. Diets were formulated to
have similar digestible energy (DE) (14.4 MJ/Kg) and ileal
digestible lysine (12.5 g/Kg).
Microbial and Volatile Fatty Acid (VFA) Analysis
[0128] Post-slaughter, digestive tract was removed by dissection
and digesta was removed from the ileum. Each digesta sample was
serially diluted in maximum recovery diluent (MRD, Oxoid,
Basingstoke, UK), and spread plated onto selective agars.
Bifidobacteria, Lactobacilli and Enterobacteria species were
isolated according to the methods described by Pierce et al.
(2005). Digesta samples used to measure VFA concentration were
collected from the caecum and the same location in the ileum and
colon. VFA analysis was performed using gas liquid chromatography
(GLC) according to the method described by Pierce et al.,
(2005).
Collection of Tissue Samples and Tissue Challenge Procedure
[0129] Ileal and colonic tissues were sampled from the same
location as described for digesta samples. Excised tissues were
emptied by dissecting them along the mesentery and rinsing them
using sterile phosphate buffered saline (PBS) (Oxoid). Tissue
sections 1 cm.sup.3, which had been stripped of the overlying
smooth muscle were cut from each tissue. Two sections from each
tissue were placed in 1 ml of Dulbecco's Modified Eagle's Medium
(DMEM) (Gibco), one in the presence of bacterial lipopolysaccharide
(LPS) (Sigma Aldrich) at a concentration of 10 .mu.g/ml. The other
tissue sample was used as a control and incubated in sterile DMEM
in the absence of LPS. Both challenged and unchallenged tissues
were incubated at 37.degree. C. for 90 minutes before being
removed, blotted dry and weighed. Approximately 1-2 g of porcine
ileum and colon tissues were cut into small pieces and collected in
15 ml of RNAlater.RTM. (Applied Biosystems, Foster City, Calif.).
RNAlater.RTM. was removed before storing the samples at -80.0 until
used for RNA extraction.
Preparation of Unchallenged Tissue for Quantitative Real Time PCR
(qRT-PCR)
[0130] Ileal and colonic tissues was stabilised in RNAlater.RTM.
solution and stored overnight at 4.degree. C. The following day,
RNAlater.RTM. was removed and samples were stored at -86.degree. C.
prior to RNA extraction.
RNA Extraction and cDNA Synthesis
[0131] Tissue samples for RNA extraction were removed from
-86.degree. C. and homogenised. 500 .mu.l of lysis solution/2-ME
was added to each sample and these were mechanically disrupted
using one 5 mm stainless steel bead per sample. These were then
placed in a Tissue Lyser (Qiagen) and lysates were homogenised for
3 mins and transferred to a GenElute Filtration Column (Sigma
Aldrich) and RNA was extracted. 1 .quadrature.g of total RNA was
used for cDNA synthesis using oligo(dT).sub.20 primer in a final
reaction volume of 20 .mu.l with Superscript.TM. III First-Strand
synthesis system for reverse transcriptase-polymerase chain
reaction (RT-PCR) (Invitrogen Life Technologies, Carlsbad, Calif.).
At the last step of cDNA synthesis, treatment with E. coli RNase H
(Invitrogen Corp.) was performed to digest the remaining RNA/mRNA
template, resulting in the production of the single-stranded cDNA
template for subsequent qRT-PCR reactions.
Quantitative Real-Time PCR (qPCR) and Normalization of qPCR
Data
[0132] All porcine primers for the cytokine genes interferon gamma
(IFN-.gamma.), interleukin-1.alpha. (IL-1.alpha.), IL-6, IL-8,
IL-10, IL-17, tumour necrosis factor (TNF-.alpha.), the mucin genes
(MUCs 1, 2, 4, SAC, 12, 13 and 20) and three reference genes,
.beta.-actin (ACTB), Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and Peptidylprolyl isomerase A (PPIA), were designed using
Primer Express.TM. (PE Applied Biosystems, Foster City, Calif.) and
synthesised by MWG Biotech (Milton Keynes, UK). These reference
genes were previously validated for use in porcine tissue and qPCR
was then carried out on the cDNA using the ABI PRISM 7900HT Fast
sequence detection system for 96-well plates (Applied Biosystems,
Foster City, Calif.). All samples were prepared in duplicate using
SYBR Green Fast PCR Master Mix (Applied Biosystems, Foster City,
Calif.), cDNA as template and specific primers for the genes
selected. For each reaction 5 .mu.l cDNA, 1.2 .mu.l (forward and
reverse primer mix, 5 .mu.M), 10 .mu.l Fast SYBR Green PCR Master
Mix (PE Applied Biosystems, Foster City, Calif.) was added and made
up to a final volume of 20 .mu.l. The two step PCR program was as
follows: 95.degree. C. for 10 minutes for 1 cycle followed by
95.degree. C. for 15 seconds and 60.degree. C. for 1 minute for 40
cycles. The raw Ct values for the reference genes were converted to
relative quantities using the formula Q=E.sup..DELTA.Ct where E is
the PCR efficiency of the assay and .sup..DELTA.Ct is the value
calculated for the difference between the lowest Ct value for each
gene minus the Ct value of the sample in question. The relative
quantities of the endogenous controls were then analysed for
stability in geNorm (Vandesompele et al., 2002). The stability `M`
value generated by the geNorm application for the selected
endogenous controls (ACTB, GAPDH and PPIA) indicated their
suitability as endogenous controls for these intestinal samples.
The geometric mean of the relative quantities for ACTB, GAPDH and
PP/A (normalisation factor) was then calculated using geNorm. The
relative quantities of each target gene were divided by the
normalisation factor (obtained in geNorm) for that sample to give
the final normalised relative expression.
Results
[0133] In this study, LAM from Laminaria digitata did not affect
performance, nutrient digestibility or selected bacteria in the
ileum, but it did decrease Enterobacteriaceae in the colon. Of
particular interest was the impact of LAM on cytokine gene
expression in the ileum and colon following in-vitro challenge with
lipopolysaccharide (LPS).
Animal Performance and Nutrient Digestibility
[0134] There was no effect on performance (food intake, daily gain
or food conversion ratio) or nutrient digestibility coefficients
(DM, OM, ash, N or GE) with increasing LAM.
[0135] Microbiology and Volatile Fatty Acids (VFAs)
[0136] Increasing the level of LAM from 0-600 ppm had no effect on
the Bifidobacteria, Lactobacilli or Enterobacteriaceae Populations
in the Ileum (p>0.05). There was a Decrease in
Enterobacteriaceae populations with increasing LAM. The potential
to reduce harmful Enterobacteriaceae strains, without influencing
Bifidobacteria and Lactobacilli numbers is of great significance as
pathogenic bacteria increase mortality rates. In context, results
indicate that the optimum LAM inclusion rate is 300 ppm.
TABLE-US-00022 TABLE 22 The effect of increasing LAM on selected
microbial populations in the ileum, proximal and distal colon and
total VFAs in the ileum, caecum and proximal colon of the pig.
Significance LAM 0 ppm 300 ppm 600 ppm SEM Linear Quadratic
(Log.sub.10 CFU/g) Ileum Bifidobacteria 4.94 5.44 5.51 0.708 ns ns
Lactobacilli 4.14 5.15 5.40 0.565 ns ns Enterobacteriacae 2.24 3.35
2.86 0.839 ns ns Colon Bifidobacteria 7.37 7.29 7.46 0.365 ns ns
Lactobacilli 7.89 8.16 8.19 0.221 ns ns Enterobacteriacae 5.42 3.87
4.24 0.358 * * Total VFAs Ileum 10.47 14.84 14.26 2.60 ns ns Caecum
173.5 189.2 194.4 9.70 * ns Colon 185.4 146.4 161.8 13.79 ns ns
[0137] Probability of significance: * P<0.05, ** P<0.01, ***
P<0.001, ns=non-significant P>0.05 There were no significant
effects of increasing dietary inclusion levels of LAM on total
VFAs, in the ileum or colon. There was a significant increase in
total VFAs with increasing levels of LAM in the caecum (p<0.05),
the main site of VFA production. There was no significant
alteration in digesta pH recorded from any the intestinal
region.
Cytokine Gene Expression
[0138] There were no effects of LAM in unchallenged ileum or colon
tissue for any of the cytokines analysed. This overall lack of an
effect on these inflammatory markers implies that the presence of
LAM in the diet did not elicit any negative effects. To mimic the
response of the ileal and colonic tissues of animals exposed to LAM
to a microbial challenge, these tissues were subsequently incubated
with LPS ex-vivo. While no effect was observed in the ileum, a
significant challenge effect was observed for IL-6 and IL-8 gene
expression in the colon of LPS-challenged tissue. LAM inclusion
levels at 300 ppm lead to an increase in IL-6 expression
(p<0.05), whilst a linear increase in IL-8 gene expression was
observed (p<0.05). These data suggest that dietary LAM could
enhance the pro-inflammatory response to microbial challenge. The
potential benefit of this enhanced gene up-regulation of IL-6 and
IL-8 cytokines following the LPS challenge are significant for the
host as IL-6 is a pro-inflammatory cytokine that plays an important
role in acute inflammation in the early immune response. Similarly
the chemokine IL-8 also plays an important role in inflammation and
is responsible for neutrophil recruitment and activation to the
initial site of infection. While exposure to LAM alone did not
stimulate pro-inflammatory cytokine production in the gastric
mucosa, it enhanced the LPS induced pro-inflammatory cytokine
production.
TABLE-US-00023 TABLE 23 The effect of increasing LAM from Laminaria
digitata on the immune response in unchallenged ileum and colon
tissues. Significance LAM 0 ppm 300 ppm 600 ppm SEM Linear
Quadratic Ileum IFN-.gamma. 1.000 1.278 0.841 0.233 ns ns
IL-1.alpha. 1.000 1.141 0.597 0.148 ns ns IL-6 1.000 1.681 0.908
0.252 ns ns IL-8 1.000 1.003 0.475 0.245 ns ns IL-10 1.000 1.128
0.646 0.229 ns ns TNF- .alpha. 1.000 1.023 0.805 0.133 ns ns Colon
IFN-.gamma. 1.000 1.217 1.148 0.286 ns ns IL-1.alpha. 1.000 0.716
0.851 0.144 ns ns IL-6 1.000 1.579 1.788 0.434 ns ns IL-8 1.000
1.245 1.137 0.224 ns ns IL-10 1.000 1.029 0.843 0.285 ns ns
TNF-.alpha. 1.000 1.400 1.446 0.301 ns ns Probability of
significance: * p < 0.05, ** p < 0.01, *** p < 0.001, ns =
non-significant p > 0.05
TABLE-US-00024 TABLE 24 The effect of LAM from Laminaria digitata
on immune response in the ileum and colon following an ex-vivo LPS
tissue challenge. Significance LAM 0 ppm 300 ppm 600 ppm SEM Linear
Quadratic Ileum IFN-.gamma. 1.000 0.927 1.098 0.223 ns ns
IL-1.alpha. 1.000 1.072 1.039 0.161 ns ns IL-6 1.000 1.352 1.143
0.266 ns ns IL-8 1.000 1.186 0.903 0.362 ns ns IL-10 1.000 1.198
1.076 0.160 ns ns TNF- .alpha. 1.000 0.819 0.921 0.108 ns ns Colon
IFN-.gamma. 1.000 2.051 1.614 0.385 ns ns IL-1.alpha. 1.000 0.983
1.242 0.199 ns ns IL-6 1.000 1.846 0.830 0.272 * * IL-8 1.000 1.590
1.948 0.303 * -- IL-10 1.000 0.936 1.039 0.256 ns ns TNF- .alpha.
1.000 1.557 0.938 0.250 ns ns Probability of significance: * p <
0.05, ** p < 0.01, *** p < 0.001, ns = non-significant p >
0.05
Mucin Gene Expression
[0139] Dietary factors such as fibre, protein and anti-nutritional
factors are known to directly influence the synthesis and secretion
of mucin from goblet cells and the recovery of mucin in digesta
(Montagne et al., 2004). All 7 mucin gene transcripts were reliably
detected in the porcine colon but only five of the seven were
accurately quantifiable in the ileum. An increase in MUC2 was
observed in the ileum of pigs supplemented with LAM at 300 ppm
(p=0.05) relative to the control animals. This increased MUC2
expression was not observed at the higher dietary inclusion level
(600 ppm). LAM supplementation had no effect on the remaining
detectable mucins (MUC4, MUC12, MUC13 and MUC20) in the ileum. In
the colon, dietary supplementation with LAM at an inclusion level
of 600 ppm, significantly increased MUC2 (quadratic; P<0.05) and
MUC4 (quadratic; P<0.05) expression but had no effect on the
expression of any of the remaining mucin genes at this site. Diets
containing beta-glucans also affect the quality and quantity of
mucin production of the jejunum, ileum, caecum and colon in the
murine model (Deville et al., 2007).
TABLE-US-00025 TABLE 25 Effect of LAM from Laminaria digitata on
mucin gene expression in ileum and colon. Significance LAM 0 ppm
300 ppm 600 ppm Linear Quadratic Ileum MUC2 1.000 0.000 * * MUC4
1.000 0.000 ns ns MUC12 1.000 0.000 ns ns MUC13 1.000 0.000 ns ns
MUC20 1.000 0.000 ns ns Colon MUC1 1.000 0.902 1.270 ns ns MUC2
1.000 0.726 1.207 * * MUC4 1.000 0.981 1.351 * * MUC5AC 1.000 2.134
0.285 ns ns MUC12 1.000 0.957 1.120 ns ns MUC13 1.000 0.924 1.077
ns ns MUC20 1.000 1.107 1.063 ns ns Probability of significance: *
p < 0.05, ** p < 0.01, *** p < 0.001, ns = non-significant
p > 0.05
Optimum Inclusion Level
[0140] 300 ppm LAM is sufficient to `prime` the immune system in an
ex-vivo LPS-challenge.
Example 6
[0141] Immune capacity can be modulated by nutritional
interventions with LAM and/or FUC leading to a reduction in Porcine
circovirus type 2 (PCV2) viral load in experimentally infected
snatch farrowed pigs and ameliorating the effects of post-weaning
multisystemic wasting syndrome (PMWS) in pigs.
Results
Immunofluorescent Detection of PCV2 Antigen in Tissues
[0142] PCV2 antigen was detected in tissue sections from necropsied
animals (liver, lung, kidney, spleen, ILN, MLN) by
immunofluorescence using PCV2-specific monoclonal antibody. [0143]
In the basal diet, 5 of 6 animals were euthanized. [0144] In the
basal diet+LAM and FUC treatment, one of six animals was
euthanised. [0145] In the basal diet with LAM and FUC and WPI, one
of the six pigs was euthanised during of the experiment. Tissues
from this animal contained high levels of PCV2 antigen. The
remaining five animals appeared healthy at the end of the
experiment. They had seroconverted and gained weight.
Determination of PCV2-Specific Antibody Titre
[0146] Referring to FIG. 1, there is shown the PCV2-specific
antibody titre of sera, which was determined by IPMA.
Intra Group Analysis
[0147] Basal diet: 5 of 6 piglets gave a poor PCV2 antibody
response and all had PCV2 antigen indicative of disease in analysed
tissue sections. The remaining animal (Tag 10) seroconverted to a
reasonable PCV2-specific antibody titre. This animal remained
healthy throughout the duration of the experiment. [0148] Basal
diet+LAM and FUC treatment: 1 of 6 pigs had to be euthanised before
the end of the experiment (Tag 30). This animal had the lowest
PCV2-specific antibody titre of all animals in this group and PCV2
antigen levels in tissues were indicative of PCV2 associated
disease. However, the antibody titre of this animal was higher than
that of piglets that developed disease in Group 1. [0149] Basal
diet+with LAM and FUC+WPI: One of the six pigs was euthanized
before the end of the experiment (Tag 26). All of the remaining
five animals were healthy at the end of the experiment, had
seroconverted and gained weight. Two animals (Tag 22 and 25) had
high levels of PCV2 antigen in its tissues.
Inter Group Analysis
[0150] At 21 and 28 days post-infection (PI), the mean
PCV2-specific antibody titre of animals in treatment 2 (+LAM and
FUC; See FIG. 2) and 4 (+LAM and FUC+WPI, See FIG. 3) were
significantly higher (p<0.05) than animals fed the basal diet
and piglets fed the basal+WPI. These results suggest that LAM and
FUC supplementation of pig feed, alone or in conjunction with WPI,
boosts the humoral response of PCV2 infected pigs.
Lymphocyte Numbers
[0151] Referring to FIG. 4, it can be seen that by day 28
post-infection (PI), the basal diet feed group had significantly
lower percentage lymphocyte cell population than the three
supplemented diets. Neither supplement was significantly different
from each other. At 14 days PI, piglets fed a diet supplemented
with LAM+FUC had significantly greater (p<0.05) percent
eosinophils than all the other groups, as illustrated in FIG. 5.
After this time, no significant difference was detected.
Analysis of Animal Weights
[0152] The weights of pigs were recorded weekly. With reference to
FIG. 6, it can be seen that the average terminal weights of piglets
in groups 2 (+LAM and FUC), 3 (+whey protein isolate) and 4 (+LAM
and FUC+whey protein) were greater than those fed the basal diet.
Animals in groups 2 and 4 were significantly heavier than piglets
fed the basal diet.
Analysis of Animal Body Temperatures
[0153] Body temperatures were also monitored throughout the study.
At 17 days PI, animals in group 3 (+whey protein isolate) and group
4 (+LAM and FUC and whey protein isolate) had significantly lower
mean temperatures (38.33 and 38.02.degree. C., respectively) than
piglets fed the basal diet (39.82.degree. C., p<0.05). No
significant difference in mean body temperatures were observed
between the individual feed groups at 24 days PI.
Analysis of Viral Shedding
[0154] Quantitative PCR was performed on all faeces samples to
estimate viral load and shedding. As illustrated in FIG. 7, by day
10 PI, the average PCV2 DNA copy number detected in animals in
Group 3 (+whey protein isolate) was significantly lower (p<0.05)
than the 3 other feed groups. On 35 days 20 and 24 PI, piglets fed
the LAM and FUC supplemented diet had significantly lower PCV2 DNA
copies than piglets in Groups 1 and 3. Animals fed LAM and FUC and
whey protein isolate contained significantly lower PCV2 DNA copy
numbers than those in the basal diet (p<0.05). By day 27 PI, all
supplemented diets had significantly lower copies of PCV2 DNA
(p<0.05) than those on the basal diet. The lowest average PCV2
DNA copy number was detected in the faeces samples of piglets fed
LAM+FUC, as seen in FIG. 8. These results indicate that
supplementing pig feed with LAM+FUC, WPI, or both in combination
led to a significant reduction in viral shedding of PCV2 under
these experimental conditions.
TABLE-US-00026 TABLE 26 Immunofluorescence detection of PCV2
antigen in tissue sections. Tag Group Disease PM Number No No Liver
Lung Kidney Spleen ILN MLN status Basal diet 07-11887 3 1 1+ 3+ 2+
3+ 3+ 2-3+ Y 07-11872 5 1 3+ 4+ 2-3 4+ 1+ 2+ Y 07-11893 7 1 2-3+ 4+
2-3+ 3+ 4+ 4+ Y 07-11894 10 1 2+ 3-4+ 1-2+ 3+ No tissue 2-3+ Y
07-11881 20 1 1+ 1+ 1-2+ 1+ 2+ 2-3+ N 07-11889 35 1 2+ 2-3+ 1-2+
1-2+ 3+ 3+ Y Basal diet + 1 g/Kg LAM and FUC 07-11888 2 2 +/- +/-
1+ 1+ 2+ 1-2+ N 07-11886 8 2 -ve -ve +/- 1-2+ 1+ 1-2+ N 07-11871 14
2 +/- +/- 1+ 1+ 1-2+ 1+ N 07-11878 24 2 -ve -ve +/- 1+ 1+ 1-2+ N
07-11890 30 2 2-3+ 3+ 2+ 2-3+ 4+ 3+ Y 07-11882 31 2 +/- +/- 1+ 1+
1-2+ 2+ N Basal diet + 80 g/Kg whey protein isolate 07-11869 9 3 1+
1-2+ 1+ 2+ 3+ 3-4+ Y 07-11884 15 3 2-3+ 3-4+ 3+ 3+ 3-4+ 3-4+ Y
07-11863 19 3 1+ 1+ 2+ 1+ 2+ 3+ Y 07-11892 27 3 2-3+ 4+ 2+ 2-3+ 4+
3+ Y 07-11859 29 3 -ve +/- 2+ 2+ 3+ 3-4+ Y 07-11858 38 3 -ve -ve
-ve +-1+ 1+ 1+ N Basal diet + 1 g/Kg LAM and FUC + 80 g/Kg whey
protein isolate 07-11861 1 4 +/- + 1+ + 2-3+ 1-2+ N 07-11870 4 4
-ve -ve 1-2+ 1+ 2+ 2+ N 07-11867 6 4 -ve -ve 1-2+ +/- 1-2+ 2+ N
07-11862 22 4 +/- + 1+ 1-2+ 2-3+ 3+ Y 07-11860 25 4 2+ 3+ 3+ 2-3+
4+ 4+ Y 07-11895 26 4 2-3+ 3-4+ 2+ 3+ No tissue 2+ Y Scores
.gtoreq.3+ = high level of viral antigen indicative of
PCV2-associated disease.
Cytokine PCR Results
[0155] Cytokine mRNA for IL-2, TNF-.alpha. and IL-4 were quantified
using an established two-step reverse transcription PCR (rtPCR)
assay. Results indicated that there were no significant differences
in the profile of these cytokines between the different feed
treatments.
Quantification of PCV2 DNA in Tissue Homogenate
[0156] Tissue homogenate pools (10% w/v) were prepared from the
liver, lung, spleen, kidney, mesenteric and inguinal lymph nodes of
each animal. PCV2 DNA was quantified using an established
quantification PCR method (qPCR). The highest amounts of PCV2 DNA
were detected in animals that received the basal diet (Group 1).
Pigs fed the diet supplemented with WPI (Group 3) also had higher
quantities of PCV2 DNA than groups that received the basal diet
supplemented with either LAM+FUC (Group 2) or LAM+FUC in
combination with WPI (Group 4). These results compare favourably
with the levels of the immunofluorescence-based detection of PCV2
antigen in the animal tissues. The least amount of PCV2 antigen was
detected in animals fed a diet supplemented with LAM+FUC alone or
in conjunction with WPI compared to the other two groups (i.e.
basal/basal+WPI).
[0157] This invention reduces Porcine circovirus type 2 (PCV2)
viral load in experimentally infected snatch farrowed pigs and
ameliorates the effects of post-weaning multisystemic wasting
syndrome (PMWS) in pigs.
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