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IMMUNE SYSTEM STIMULATION
AND GROWTH PERFORMANCE IN S m
A Thesis
Presented to
The Faculty of Graduate Studies
The University of Gueiph
by
LOUIS DIONISSOPOULOS
In partial fùlfiiment of requirements
For the degree of
Master of Science
Decernber. 1997
Q Louis Dionissopoulos, 1997
Acquisitions and Acquisitions et Bibliographie Senrices services bibliographiques
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ABSTRACT
IMMUNE SYSTEM ACTIVATION AND GROWTH PERFORMANCE IN SWNE
Louis Dionissopoulos University of Guelph, 1997
Advisor: Dr. C . F. M. de Lange
In a performance mdy, clear effects of health management strategies during
rearing were obsemed on growedfinisher (GE) pig performance. The order of best
performance was Minimal Disease (MD)> Conventionab Segregated Early Weaned
(SEW) ( P -05). Growth depressions coincided with increased antibody titres to some
common swine pathogens and increased levels of indicators of immune system activation
( P -05). Ln a subsequent shidy, the effects of b l o c h g the response to interleukin- 1 (IL-
I ) were evaiuateci with the addition of interleukin-1 receptor antagonist (IL-Ira) in a
mode1 of i2.@opkzwzu hyopneumonzue and porcine reproductive and respiratory
syndrome virus (PRRSV)-induced sub-dinical disease in young pigr. Disease depresseci
insulia-like gowth factor 1 (IGF-I ) levels only in the gilts ( P -10). IL- 1 $ and Wa
levels in the sick - IL-Ira group were lower than in the sick conml group (P - -10) and
similar to the healthy controls ( P -10). In addition, IL-Ira improved g o ~ h rates and
carcass protein retention compareci to heaithy and sick control pigs (P . .IO). These
resdts support theories involving irnmuno-endocrino logical modulation of growt h
performance, protein retention and the pro-inflammatory cytokine cascade following
exposure to disease.
To my fiancée Karen:
Througb the good times and beyond.
This work was supported by fun& from Ontario Pork and Ontario iMinistq of
Agriculture, Food and Rural Affairs.
1 would fmt and foremost like to thank my cornmirtee members. Drs. Atkinson,
Friendship, and Maches. I especiaiiy wish to thank my supervisor and mentor. Dr. de
Lange for his support and guidance throughout the preparation of this Thesis. Thanks
should also go out to Dr. V. Parker and AMGEN for their generous donation of the IL- 1 ra,
M. Quinton for statistical anaiysis, L. Trouten-Radford for technical assistance in
preparation of the porcine IGF-L assay, C. Feng, for dam analysis and blood sarnpling,
Dr. C.E. Dewey and J.P. Walton for surgery, and J. Blair, E. McNeilage, G. fic. B.
Bloomfield, and A. Deckert for their help during surgery and for pig handling. 1 also
wish to express appreciation to the Mycoplasmology iab at the University of Guelph for
their preparation of the M. hyopneumniae. We would also like to thank the efforts of the
Ponsonby general animal facility, in particular the efforts of B. Mutter and D. Hindley. in
addition, we would like to thank the National Hormone and Pituitary Program, the
National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute
of Child Heaith and Human Development, and the U.S. Department of Agriculture for
their generous donation of the ad-Somatomedin-C amisera (loMJB2-495) used in the
generation of resuits from the IGF-1 analysis.
Table of Contents
................... CRAPTER 2: SWINE HJULTH MANAGEMENT SIRATEGIES 2
3 DISESE MANAGEMENT IN THE ONTARIO SWLNE ~JDuSTRY ........................................................................
AN IMMUNE CHALLENGE .............................. ........................ 7
........................................................................................................................................... ~ O D U C I I O N -7
............................................................................................................................... The Hypothalamus -8
.......................................................................................................................................... The cytokines 9
............................................ EFFECTS OF IMMUNE SY S~FM STIMULATION ON NUTRCENT P A R T ~ O M N G 1 5
.......................................................... MODULATING THE RESPONSE TO IMMUNE SY!STEM STfMULATION 16
............................................................................................................................................. CONCLUSION 17
REsEARCH HYPOTHESIS AND OBJECTIVES ..........., ..... 18
CHAP'IER 4: EFlFECT OF HEALTH MANAGEMENT STRATEGY DURING REARING
ON GROWER/FIMSHER PIG PERFORMANCE AND SELECTED INDICATORS OF
IMMUNE SYSl''EM ACTIVATION .......................................o...................................................... ........ 20
................................................................................................................................................. ABSTRACT 20
........................................................................................................................ Animais and H o u s h g -22
EvaZuation of Smut . Gut. and Lmg Lesions .................... .... ..................................................... 26
......................................................................................... General Observationî ................... ...... -30
Growth Perjionnance and Carcass Quality .......................................................................................... 30
......................................................................... indicarors of Disease and Immune System Activation 32
............................................................................................................. SIGNIFICANCE TO THE ~NDUSTRY -39
CHAPTER 5: L I R A INCREASES LEAN MASS AND DECREASES THE CYTOKINE
RESFONSE IN A MODEL OF SUB-CLINICAL DISEASE IN GROWING PIGS , ..... -. ........,...... 45
................................................................................................................................................. AI~STRAC~ 45
.......................................................................................................................................... I ~ r ~ o ~ u c n o ~ 47
...................................................................................................................... MATERIALS AND MEMODS -49
.......................................................................................................................... Animais and Housing 4 9
................................................................................................... Surgery and Treatments .......... .... -51
.......................................................................................................................... Observations ......... .-.. 53
............................................................................................................................................. CONCLUSION 62
CHAPTER 6: SUMMARY AND GENERAL DISCUSSION ........................ ,., .., .......*....... ,w,L....... ......... 69
iii
List of Figures
FELIRE 1 . w- 1 B (E- 1 p) LMas iN PIGS FROM 3 TREATMENI CROUPS* ..................................... 67
............. . ~ G U R E 2 -UR NECROSIS FACTOR a (TNF-U) LEvELS üü PlGS FROM 3 TREA- GROüPSt .68
Chapter 1: Introduction
The enhancement of lean growth rates and increased feed efficiency are important
goals of the pork industry. The efficiency of pork production is influenced by many
factors including feeding management, housing, health management (eg., medication.
sanitation, etc.). and genetics (Schinckel. 1994). Clinical and sub-clinical disease is
important, since immune system stimulation is known to decrease performance in
grower/fiinisher (GIF) pigs, and hence can be very costly to the industry (Klasing, 1988:
Fong et al., 1989; Klasing et al., 1991; Johnson. 1997a).
There are various molecular and endocrinologicai mec hanisms that ei ther enhance
or inhibit growth in mammals. Insulin-like growth factor-1 (IGF-l), for example, is
known to stimulate nuaient uptake by a variety of ceU types (Guyton, 1991). Similarly,
somaîostaîin is known to inhibit this process. Yet the rnechanisms that relate immune
system stimulation to a decrease in performance in G/F pigs are not clear.
Hence, this thesis will examine some of the immunological and endocrinological
factors that are responsible for, or are associated with, growth depressions and overail
poor carcass characteristics that are typical following chronic immune system stimulation.
In addition, this thesis will examine if SEW is an effective means to control leveis of
disease and maintain g w d levels of performance in G/F pigs.
Chapter 2: S w h Health Mmagemmt Strateghs
Disease Management in the Ontario Swine Industry
There is a generai maikation that disûase whether clinical or s u k l h c a l results in
sipiilfiant decreases in p w t h performance (Klasing, 1988: Klasing et al., 199 1 : Porter and
Barratt, 1993; Williams et al., 1994; Schinckel, 1995: Johnson, 1997a). It appcars thar not
only disûase processes such as gut damage from infectious orgaaisms like Lawsonia
intracellularis. or Sepdinu sp. are important in causing reduced feed intake and reduced
p w t h rate (Harris and Lysons, 1992), but perhaps the process of mounMg successful
immunity also cornes at a pnce with regard to growth rate. A few studies in poultry and
swine have demonsaated quite clearly that vaccination will reduce performance in the
absence of disease challenge (Klasing et al.. 199 1 ; Williams et ai.. 1994).
In the swine industry in Ontario, there have traditionally exisred nvo types of
commercial famis: 1) farrow-to-f-h and 2) famis which produce 20-25 kg feeder pigs
and famis which purchase these pigs in order to raise hem to market weight. For the most
part, these systems have operated on a continuous flow basis. When herd size was very
sinaii, di production stages were in the same air space. As herd size has increased,
production stages have been partition4 off because of the different feed and enviromentai
n e a b of each stage (Dial et al., 1992). Those famers operahg a pwedf*her bam
(which in Ontario bas tended to be about 1000 head) have had two choic. when it came to
stocking their units: 1) buy fmm 1 source and therefore continuously add pigs on a weekly
bais or 2) fU the barn dl ai once by buyïng h m multiple sources. The second choice has
the advantages of d-daii-out management, allowing phase feeding and better monitoring
(Scheidt et al.. 1995). but is dangeruus because of the possible spread of disease (Clark et
al., 1991; Dial et al., 1992). Mixing pigs at about 10 weeks of age h m diffe~nt somces
means that some animalg may be sick or disease carriers and that others hm7e had no
previous exposure to disease and hence no active or passive immunity (Clark 1991; Pijoan.
1995). Therefore. most herds in Ontario have operami G/F bams as continuous fiow
operations out of necessity. However. there is clear evidence that pigs p w much better if
they enter a barn that has b e n previously disinfected and devoid of p i g (Scheidt et al..
1995; Clark, 199 1 ).
One methoci to d u c e the impact of disease in a continuous flow unir is to create a
herd or population of p i p thai is free of the specinc swioe pathogens such as enzootic
pneurnonia, mange. and swiue dysentery which are bciieved to be important in reducing
performance (Mimats and Jol, 1978; Mandrup and Madsen. 1980; Diai et al., 1992). For
many yem, the technological know-how has existtd to do thïs and has been referred to as
the specifk pathogen free (SPF) program (Mïniats, 1978). The SPF program has not been
widely adopteci, partiy because of the ease with which reintrcxiuction of diseases that have
been eliminaîed by the expensive process of depopulaùon/repopulation can rake place.
More recently, other schemes have been developed which allow ali-inlall-out flow
of the GE barns. One such system is segregated early weaning (SEW) with multi-site
production (MSP) (Clark, 1990). This system has been widely accepted by the North
American swine indust~y so that 25-3096 of a i l pigs are produceci in this way. The
difference between SEW-MSP and traditional rearing of pigs is thar comminglùig of
weaned piglets occurs at 2 weeks instead of 10 weeks of age. There are several reasons why
this is done. The concept of reducing weanuig age in order to reduce sow-piglet disease
transmission was first proposed by Alexander et al. (1980) as a means of establishing a
genetic nucleus herd without the necessity of cesarianderived piglets. Weaning at the
critical age of 7-14 days d o w s piglets to benefit from lactogenic immunity but minimises
the chance that they wili contract disease from the f'wing herd because of the short time
spent within the herd. During this p e n d the piglets are protected by colos~al-denved
antibodies (Clark et al., 1990; PeNgrew et al., 1995). The piglet's immune system develops
during the fust few weeks of Life and is not fdiy functional untii the pigiet is 5 or 6 weeks
of age. Meanwhile, passive immunity d e c m dramaùcally for many diseases during the
second or third week of life. About 14 days is thought to be an optimal time ro wean
because passive immunity is still present. but the pig's immune system is becoming
~asonably capable of mounting resistance to an invading pathogen (Pijoan, 1995). Hence,
the main objectives of SEW include decreasing clinicai signs of disease, increasing
performance, increasing the sow herd inventory, increasing finisher turnover rate. and
improving general grower-finisher productivity (Dial et al., 1995; Dufresne, 1995; Pettigrew
et al., 1995; Pijom, 1995). Evidence suggests that SEW pigs are healthier and grow faster
than theû conventionally raised counterparts (Pettigrew et al.. 1995). In fact. pigs that have
not experienced a high level of immune system activation grow 2942% faster and
commonly have 24-55% better protein:fat ratios (Williams et al., 1994). Moreover, such
high health pigs routinely reach slaughter weight without showing clinicai signs of disease
(Pijoan, 1995).
Another technique which is less widely used is to create farrowing groups- ie.,
farrowing once per month instead of weekly (English and Edwards, 1996). This may create
large enough numben to fd GIF rooms in an all-idall-out rnanner and could be a means for
the smaller fmw-to-finish operation to utilise d-in/all-out methodology.
The question in the swine industty is how these management techniques compare
for the 100 to 200 sow operator who is presently rearing pigs fmow-to-finish and has a
typical disease profile which likely includes emootic pneumonia and porcine reproductive
and respiratory syndrome (Pm). Should such a producer depopulate and repopulate with
SPF pigs and continue to operate in a continuous flow, weekly farrowing system? Should
the herd get involvecl with SEW-MSP by joining with other producers to commingle piglets
at 2 weeks of age and fd the GE barns in an all-in/all-out manner? Should the producer
rearrange the f m w i n g schedule to create larger batches of pigiets that couid be farrowed in
an aii-in/all-out m e r , raised ail-in/all-out in preexisting on-farm nurseries and then
raised in pre-existing on-farm aii-in/all-oui GE mm? No doubt as new information
cornes about on the mechanics of d u c h g disease by management saategy, producers wiii
be able to make more inforrned decisions on the adaptation of the these systerns of raising
p i s -
In the past few years, under cornpetitive pressure, producers have had to increase the
rearing intensity and size of their operations. Increasing intensity and size results in an
increased risk of disease. There is much to be learned yet in tems of the way disease affects
performance. Different management strategies affect the expression and the transmission of
disease. However, with the advent of specific management strategies, the health status of
entire herds may be manipulated for the better, making an improved product available to the
consumer at a more equitable pnce for the producer. Clearly, the hiture will brÏng with i r an
understanding of immune system stimulation and its relationship to feed intake and growth.
The next chapter w i l examine what is currently known about immune system stimulation
and nutrient partitioning.
Chapter 3: lmm11l10mdnlation of Nutrient Partitionhg During an Lmmune Challenge
Introduction
In response to an immunological challenge. the body initiates a sequence of events
that ultimately Iead to immune, haernatological, and rnetabolic changes. Many, if not al1 of
these changes have been shown to be the effect of mediators derived from activated
mononuclear ceils (Rouzer and Cerami, 1980). During the immune response, factors such
as cytokines. complement, and prostaglandins are released and act systemically to enhance
the response to the chaiienge. Specificaiiy, the cytokines initiate rnetabolic adaptation via
changes in carbohydrate. fat, and protein metabolism. Clinicaliy, the infected indîvidual
will dispIay fever, muscle wasting, and hypotension, transient hypergiycaemia followed by
hypoglycaemia, with a concomitant increase in vascular permeability (Moiioy et al., 1993).
The immune system exists as part of a network of regdatory and effector centres
that include the neural, the endocrine, and the neuroendocrine systems (Baternan et al.,
1989). It seems logical then that the immune system is modulated by the other systems and
that the other systems are under a reciprocal influence by the immune system.
The characteristic metabolic alterations that take place after an immunological
chaiienge have been previously described (Molloy et al.. 1993). Metaboiic alterations are
7
dependent upon the shifang of endocrinological homeostasis within the body. These
mechanisms are under the control of the higher centres of the brain and the collective
conml mechanism has corne to be bown as the immune-hpthalamic-piniitary-adrenal
(MPA) axis (Bateroan et al., 1 989).
This chapter will examine the characteristic shihs in carbohydrate, fat, and protein
metabolhm that take place foIlowing an immune challenge. The major effectors of the
IKPA axis WU be looked at within the context of their ability to enhance or inhibit
metabohc functions within the body.
The HvuothalamtLiF
In looking at the components of the MPA axis, one must realise the essentid role of
the hypothalamus. The hypothalamus is probably the most active regulatory centre in the
body, receiving highly diversified information which it tums into commands that lie central
to the functions of behaviour and metabolism (Guyton, 1992). The hypothalamus is Linked
to the endocrine system via its physical association with the pituitary gland with its array of
effector hormones. In this way, the hypothalamus is able to integrate signals from ali over
the body and hence modulate for exampie. blood flow, heart rate, or nutrient intake.
The most signiscant effector systems of the hypothalamus and pituitary as far as
nutrient partitionhg is concerned are the adRnocoiticotropincorticotropin releasing factor
(A(3TH-CRF), and p w t h hormone releasing hormone (GKE2H)-growth hormone (GH)-
insulin-like p w t h factor 1 (IGF-1) strategies (Bateman et al., 1989). The pituitary relies
on the hypothalamus for alterations in the release of A m ; it is under the control of CR-F,
which ultimately leads to the secretion of glucocorticoids. The mechanisms whereby the
body achieves this goal have been reviewed previously (Antoni, 1986; Rivier and Plotslq,
1986)'.
The central role of the hypothalamus in the regulation of merabolic homeostasis has
corne parllally from snidies in rats. Immunologicdy chaüenged rats show increased
activig of certain specific hypothdamic neurones which correspond wiih the tirnecourse of
immune Bceii stimuiation and antibody production (Besedovsky et al., 1977).
The cytokines
During an immune response, the antigen cornes in contact with resident tissue
monocytes andor macrophages. These phagocytic ceiis become subsequently activated and
are stimulateci to secrete a series of hormones that initiate the cascade of events leading to
widespread immune system activation. Such cytokines include interleukin- 1 (IL- 1 ),
interleukin-6 (IL&, and tumour necrosis factor-a (TNF-a) (Bateman et ai.. 1989; Roitt,
1994). These cytokines prime the immune system into widespread activation; the activated
lymphocytes and other phagocytes in him secrete a wide variety of other factors. Their
form and function is very complex and is beyond the scope of this Thesis. Hence, this
chapter WU focus on the pro-infiammatory cytokines IL- 1, L6, and TNF-a since they have
been shown to be major players in the ùnmunomodulatory response to infectious challenge
and affect whole animal nutrient metabolism (Klasing, 199 1; Moiioy et al., 1993; Roitt,
1994). A general outhe of the cytokines and hormones involved in the immunomodulation
of metaboikm is outlined below, Table 1.
Interleukin-1: IL-1 is a 17 kDa polypeptide produced by stimulated
monocytes/macrophages in mponse to injury, although endothelid ceiis, B-ceiis, epithelial
ceils, keratinocytes, microglia. md astrocytes c a . also secrete IL4 upon stimulation
(Benveniste, 1992). IL-1 coordinates many immune and biological aspects of stress and
infection (Bateman et al., 1989). Endotoxin infusion into experirnental animals resuits in
increases in IL1 (van Dam et al., 1992).
Two forms of IL4 exist IL la and IL- Ip, which generally serve the same
fimctions. Although the synthesis of these two analogues is controiled by different genes
(Benveniste, 1992), they bind to the same target receptor. IL- 1 is the main activator of T-
ce11 and B-ceil expansion via its enhancement of IL2 production. Hence, antigen
chailenged immune celis rely on this switch to respond to an infectious agent (Benveniste,
1 992).
10
Table 1. Factors involved in outrient partitioning foilowing immune system stimulation. Data
Site of Action
Hypothalamus
-
Whofe animal
Hypothalamus
I997a and DinarelIo. 1996.
Main Actions
Stimulates CRF release
Stimulates CRF release
Stimutates CRF release
S timuiates ACïH release
Wevares ACTH and gfucocortïcoid
levek
Stimulaus ACïH secretion
inhibits secretion and synttiesis of
CRF
Site of b o n p 4 a . L c &
Causes monocytopenia; inhibits IL- 1
secrction and synthesis
Recent reports indicate that resident brain macrophages (the amoeboid mimglia)
also produce L I (Guilm et al., 1986; Kidron et al., 1989). For a comprehensive review of
IL 1 function, see DinarelIo ( 1996). As stated above, the importance of IL- 1 is not oniy as
an immune system stimulaior, but aiso as a modulaor of the metabolic response.
BesedovsQ et al., (1986) reporteci that vkdly-challenged mice showed increases in ACTH
and corticosterone Ievels. Evidence that IL-I was indeed the mediator in this cascade was
indicated by the administration of anti-IL- 1 antibodies (affectiog only systemic IL- 1 ), which
resulted in suppression of A m and corticosterone secretion. Moreover, injection of IL- 1
itself resulted in increased levels of circulaihg ACïH and corticosterone. IL- la infusion in
rats resulted in si@cant hypergIycemia, ldc-acidemia and increased hepatic glucose
output with concomitant iocreases in plasma insulin (Comeii and Schwartz. 1989).
glucagon, and corticosterone (Kidron et al., 1989: Sander et al.. 1989; Lang et ai.. 1993).
These results indicate that one of the hallmarks of the immune response is an increase in
general glucose availability either via gluconeogenesis or by giycolysis. Hence, following
immune system stimulation. large amounts of glucose are liberated into the general
circulation.
Further insight into the interactions between IL4 and prostagiaudins came h m the
same group (Lang et ai., 1993) who infüsed purifieci prostaglandin Ez into rats. The same
results were obtained as when IL4 alone was hfwe& hyperglycemia and increases in
plasma i n s u and corticosterone. In another snidy. researchers administered the
cyclooxygenase blocker indomethacin to rats prior to IL4 infusion (ComeiI. 1989). They
found that indomethacin infusion attenuated the IL-1-mediated increases in insulin and
ACTH. Hence, the glucose response to IL-1 is mediated at least by the E-type
prostaglandins. It may ais0 be possible that other prostaglandins are involved such as the D
and F types. Evidence to support the involvement of these other mediators is yet unknown.
Injection of IL-1 and subsequent blood sampling from the hypothalamic-
hypophysiai ponal system showed increased CW levels (Sapolsb et al., 1987). Katsuura
et al. (1988) observed that in order to elicit the same increases in ACTH and corticosterone
output as that observed when IL-1 was injected directiy into the hypothalamus.
intraperitoneal IL1 injeaion had to be increased more than 1 0 fold. These results
strengthen the view that the hypothalamus is the main site involved in regulating the
merabolic response to IL 1.
Interleukin-6: IL-6 also has an influence on the regulation of the immune response. EL-6 is
secreted by fibroblasts, monocytes, B-celis, endotheliai ceiis, T-ceUs. microglia, and
astrocytes. It can stimulate the liver to produce proteins of the acute phase response and is
largely responsible for Bxell maturation foiiowing an immune challenge (Roitt. 1994).
Tumour necrosis factor-a: TNFa is a 17 kDa polypeptide secreted by activated
monocytes/macrophages in response to an immune challenge. It induces the formation of
IL 1. IL-6. and iwlf and promotes the formation of leukocyte-rich inflarnmatory infiltrates
at injured sites (Pohlman et al.. 1986). For a comprehensive review of TNF-01 function, see
Johnson (19974b). TNFu promotes the breakdown of skeletai muscle (Warren et al.,
1987; Fong et al., 1989), the initiation of viglycende release and the inhibition of fatty acid
synthesis (Molioy et al.. 1993), which results in the partitioning of proteins and fats from the
periphery to the liver during an immunological challenge.
Having bnefly explored some of the main functions of I L I . IL-6, and TNF-a the
next section will examine the effects of chronic immune system stimulation on nutrient
partitioning in m m . More importantly, the next sections will look at what is currently
14
h o w n about the endocrinological mechanisms responsible for nutrient partitioning
following an immune challenge.
E f k t s of Immune System Stimulation on Nutrient Partitionhg
Immunological stress brings about a series of metabolic changes that serve ro
enhance the immune response via the provision of nutrients. Such changes include
increased glucose metabolism. increased protein turnover. and increased maintenance
energy requirements (GeIfand et al., 1984; Del Rey and Besedovsky. 1987: Hargrove et al..
1988; Lang and Dobrescu, 1989; Johnson, 1997a). Warren et al-, ( 1987. 1997) and Webel
et al.. (1997) have examined the d e of cortisol (a metaboiïc regdatory hormone) following
an immune challenge. They found subsequent increases in cortisol and other evidence to
support the endocrinological Link between disease and merabolism following general
immune system stimulation or after iniracerebroventricular ( 1 0 injection of TNF-a
Another important hormone has recently gained attention for its ability to regdate
protein homeostasis in skeletal muscle. IGF-I is known to stimulate nutrient uptake by
skeletal muscle ceils (Guyton, 1991; Dimitriadis et al., 1992) perhaps tbrough the high
number of IGF-1 receptors present the skeletal muscle ceil surface (Livingston, et al., 1988).
IGF-1 has been shown to attenuate skeietai muscle breakdown in cachexic mimals
(Douglas et al., 199 1). Since immune system stimulation is known to result in skeietal
muscle wasting, it may be that a deficiency in this homone during the diseased state may be
responsible for the characteristic s h i . in nitrogen balance.
Glucagon is secreted in response to an immune challenge (Gelfand et ai.. 1984).
With respect to metabolism, the main effects of glucagon are increased hepatic
glycogenolysis and gluconeogenesis (Guyton, 199 1). These effects increase systemic
energy availabiiity in order to support the immune response.
Modulating the Response to Immune System Stimulation
Attempts to modulate the response to an infectious challenge have led to the
development of therapeutic strategies thaî are designeci either to block the cytokine cascade
or to block the response of the body to the infectious challenge itself, since abnormaiiy hi@
ievels of circulating cytokines have ken shown to lead to peripherai wasthg (Molloy et al.,
1993). The idea of blocking the metaboiic response to an immunologicai challenge has
only recently been explored. Breuillé et al., (1993) used a TNF-a receptor antagonist
(Pentox@lline, Hoechst, Regina, Sask) to successfuiiy lessen the severity of body weight
loss and muscle protein wasting characteristic of the disease state.
Promising experiments using the IL-1 receptor antagonist (IL-lra, AMGEN,
Thousand Oaks, CA) have dernonsaated its ability to lessen the degree of hypotention and
tissue damage typical of septic shock in rabbits, (Wakabayashi et al., 1991), baboons, and
humans (Fischer et al., 1992; Van Zee et al., 1995) and increased the survival tirrie of
patients in advanced septicaemia et al., 1994). Justification for these experiments
came h m early studies which demonstrateci the negative effects of c h ~ c cytokine release
on whole body nutrient metabolism, hedth status, and f d intake (McCarthy et al., 1985;
EUasing, 1988; Lang and Dobrescu, 1989; Romagnani, 1994). However. the
endocrinological mechanisms underlying these changes have only lately been addressed. To
the authors knowledge, no snidies have as of yet been perfomed to evaluate the metabolic
effêcts of blocking the response to I L 1 secretion.
Conclusion
IL- 1, IL-6, and TNF-a have been shown to be the main cytokines that lùik immune
system stimulation to nutrient metabolism, and hence animal performance. In addition,
evidence has been provided to support hduigs that the rnetabolic response to immune
system stimulation (ie., the effects of disease) on performance can be reduced.
Although the metabolic alteraiions have been descr iw their effects are moderated
by infiammatory mediaton such as IL-1, IL-6, and TNF-a which support the proliferation
of immune ceiis during the immune response, greatly increasing their numbers. Hence, the
number of celis in ne& of simultaneous nutriture incrases drarnatically, and in this way,
nutrients are proportioned to a i l ceils that require them. Depending on the magnitude of the
17
immune response, Ieukocyte pools may increase to such an extent as to cause significant
nuüient proportioning, leading to peripheral malnutrition and systemic wasting (Klasing,
1988; Johnson, 1997a).
Research Hypothesis and Objectives
Exposure to disease is reported to decrease growth performance in grower/finisher
(GF) pigs (Dial et al.. 1992; Schinckel. 1994; Williams et al.. 1994). Activation of the
immune system coincides with a reduction in feed intake. reduced lean growth. and a
mobilisation of nutrients in order to mount an appropriate immunological defence
(Penigrew et al., 1995). The rnechanisrn by which this occurs is SU unknown. However,
several cytokines have been suggested to play major roles (Johnson, 1997a). To combat
the detrimental effects of disease on pork production. health management suategies have
been developed such as AIAO, SEW, and MD which aim for reducing the exposure to
disease.
1 hypothesise that chronic, sub-clinical disease is responsible for growth
depressions and poor carcass quality in growing pigs. From a closer perspective, 1 feel
that there will be an induction in circulating cytokines which will in tum lower insulin-
like growth factor 1 (IGF-1) levels following the induction of sub-clinical disease. By
blocking the response to the cytokine interleukin- 1 (IL- 1). 1 feel that the catabolic effects
of chronic, sub-clinical disease can be attenuated.
The research reporteci in this thesis is meant to determine the causative agents
responsible for the enhancement or inhibition of growth performance in grower finisher
pigs. This thesis has a two-part objective; 1) to determine if SEW is an effective means
to control levels of disease and maintain good levels of performance in GE pigs. and 2)
to determine if IL1 is one of the main cytokines that influence the release of growth
regdahg hormones such as IGF- I and cytokines such as TNF-CL
Chapter 4: Effect of Heaith Mimagement Strategg Ihving Rearing on GrowerIFWsher K g Performance and Selecîed Indicators of Immune System Activation
The e k t s of health management strategy during early rearing on subsequent
growth performance and indicaton of immune system activation in grower/finisher (G/n
pigs were evaluated. At approximately 25 kg body weight (BW), pigs were sourced from
three different management systems: (1) Conventional health farrow-to-finish herd with
identified respiratory problems in the nursery and G/F barn, (2) Segregated Early Weaned
(SEW), and (3) Minimal Disease (MD). These pigs were housed under identicai
conditions until shipped to market. During this period, various production and
immunologicd indicators of disease were measured. In the grower phase (up to
approximately 60 kg body weight), the MD group had the highest average daily gain
(ADG) (Pc.05). while growth was similar in the SEW and Conventional groups (P>.fO) .
The lowest average dady feed intake (ADFI) was seen in the SEW group (Pc.05); it was
similar in the Conventional and MD groups (P>.10). Feed:gain (F:G) was similar in the
MD and SEW groups (P>.IO), and was poorest in the Conventional group (Pc.05) .
D u ~ g the f ~ s h e r phase however. the SEW group had the lowest ADG . ADFL and the
20
poorest F:G (Pe.05). These values were similar in the Conventional and MD groups
(P>.IO). G/F performance of pigs denved from a MD source was better than that of
animais from Conventional and SEW systerns. Performance depressions appear to be
related to the presence of lesions due to disease or indicators of immune system activation
such as the degree of antenor-ventral lung atelectasis, antibody titres to Porcine
Reproductive and Respiratory Syndrome virus (PRRSV) and Mycoplasma
hyopneumoniae, thymus size. insulin-like growth factor L (IGF- I ) levels, stomach
ulceration. nasal turbinate damage, and microbial burden.
Introduction
In commercial pork production, several different management strategies have
k e n developed to reduce the exposure to disease. These include segregated early
weaning (SEW), ail-in/ali-out ( AIAO) movement of animals, and maintenance of
minimal disease health status (MD) (Did et al.. 1992). To date, few controlled studies
have been conducted to detemiine the effects of such alternative strategies during early
rearing on subsequent performance in the grower-finisher period. Therefore, the
objectives of this snidy were: 1) To compare the effect of management strategy or hedth
stahis during early rearing (MD. SEW, Conventional farrow to finish) on growerlfinisher
pig performance and carcass quality and, 2) to monitor indicators of immune system
activation in these three groups of pigs which may aiiow for an estimation of the extent to
which performance potentials are reduced due to disease. To eliminate potential
confounding effects between hedth status, management, and environmental conditions,
pigs were managed under identical conditions during the GE phase.
Materials and Methods
Ani& and Housing
Forty-eight pigs (24 gilts and 24 barrows) were sourced from each of three
different management systems at approximately 25 kg body weight (BW) which were
confumed to be fiee of the foiiowing swine pathogens: Actinobacillus pleuropneumoniae
(APP), S e r p u l i ~ hyodysenteriae, mange and lice. The segregated early weaned (SEW)
group of pigs came from one isolated commercial SEW nursery. These pigiets originated
from 6 different sow herds and were commingled when they entered the SEW nursery at
approximately 14 days of age. Comrningling of newly weaned piglets from different
farrowing units is typical of most Ontario SEW operations. The Conventional health
status pigs originated from a farrow-to-finish operation with chnical signs of porcine
reproductive and respiratory virus (PRRS ) and Streptococcus suis infection in the nursery
and pneumonia in the grower/finisher barn. PRRS virus and M. hyopnewnoniae were
confimeci to be present as a clinical problem in the continuous flow. growerfinisher
(G/F) barn of this producer. On this commercial farm, the farrowing rooms and the
nurseries were operated in au A-idail-out basis and pigs were weaned at approximately
28 days of age. The MD group originated from a breeding stock multiplier wirh a defined
health status. On the basis of clinical signs and serologic testing of those animais. the
LW group was also confumed to be fiee of the major swine pathogens. toxigenic
Pasteurefia multocida-type D, and Mycopinrmn h y o p n e w n i a e . AU pigs in rhis study
had the sarne genetic background (Yorkshire x Landrace; sourced from a large
multinational pig breeding cornpany) and had medium to hi@ Iean growth potentials.
Upon amival, the different groups of pigs were kept in three separate but identicai
rooms at the Ponsonby research station of the University of Guelph. In each room, sexes
were penned separately with 8 pens of 6 pigs each. The pens were 1.82111 by 3.05m (6ft
by IOft) and they had solid concrete fiooring. Pen partitions were consmicted of solid
concrete walls for haif the pen and galvanised steel railing for the other haif. Each room
had separate heating, ventilation, and manure handling systems. Room temperames were
mainrained at approximately 20 O C during the grower phase, and at approximately 18 OC
during the fuiisher phase.
To prevent the transfer of disease between rooms, access to facilities was
restricted, rooms were aiways inspected in the same order: MD, SEW, and Conventional,
disinfectant foot bath were placeci at the entrance of each r o m and staE changed boots
and coverails prior to entering each roorn. Blood samples and body weights were taken
on separate days, and aLi the sampling equipment was thoroughly disinfecteci wirh a
commercial power spray disinfectant before king moved between rooms.
Pigs had free access to water from nipple drinkers and were fed a high quaiity
grower diet ad libitum from single space feeders. up to approximately 60 kg BW.
followed by a high quality finisber diet up to market weight ( 1 10 kg). Both dieü were
corn-soybean med based die& thai contained 3% fishmeal and 2% whey and had a
calculated DE content of 3470 kcal/kg. The calculated lysine contents were 1.30% and
1.10% in the grower and finisher feeds. respectively. The vitamin and mineral levels
were in excess of NRC (1988) requirements. These diets were designed so that the pigs
were able to Mly express their performance potentials (Schinckel. 1994).
Observations
Daily obsemations were d e t~ evaiuate animai behaviour and weii k i n g and to
monitor any clinical signs of disease according to guideiines set by the Canadian Council
on Animal Care (RowseU, 1991). Nasal, tonsiUar, and rectal swabs were taken from 2
randomly selected animais per pen at the beginnuig of the trial and at slaughter for culture
of nomial bacterial microflom Blood sarnples were taken at the beginning of the trial,
monthly thereafter. and at slaughter £rom the orbital sinus of every animal (Moldovan and
May, 1975) for determination of IGF- 1 and IL- 1 B levels, and antibody profdes. Total
antibody (IgG) was determined for the f m and last blood samples on every animal. Pig
BW, average daily gain (ADG), and average daily feed intake (ADFI), were determined at
two-week intervals. At slaughter. carcass weight and the weight of dissccted h m were
determined (Aker et al., 1996). In addition. observations on lungs, stomachs. snouts. and
thymuses were made on al1 pigs Uicluding cuils. Carcass lean yield and carcass index
were estimated based on measurements of backfat thickness and loin depth according to
the Canadian Carcass Grading System (Aker et al.. 1996).
Calcuiation of Performance
Growth performance data were evaluated separately for the p w e r and finisher
period, and for the cornbined growerffinisher p e n d In order to account for pigs rhat
were culled M m the experiment, ADFI was calculated as:
ADFI = (Total wr m Feed Uswd ( pig days per pen over the period that performsoce was m e a s d )
w here,
pig days = Z(# pigs per pen x number of days that pigs spent in a pen). ADG was
calculated based on observations h m pigs that reached at least 60 kg BW (grower phase)
or at least 95 kg BW (finisher phase). Feed:gain was calculated as the total per pen feed
consumption divided by the total weight gained for dl animals in the pen.
Evaluation ofSnout. Gut. and Lwin Lesions
The degree of Atrophic Rhinitis (AR) was estimateci by a single-blind evaluation
of snout turbinate atrophy according to the method of Straw et al., ( 1986). The scale used
was O to 5; O having no damage, while a score of 5 indicated total turbinaie atrophy.
Enzootic pneunonia lesions in the lung were scored by single-blind evaluation on the
basis of colour and texture of iung damage according to the method of Straw et al..
(1986). Stomachs were examined and scored for the degree of damage according to the
method of M a c h et al. (1997). Stomach scoring mged h m a normal stomlrh = O to
severe ulceration involving more than half of the pars esophagea king scored a 3.
Plasma IGF-1 levels were detefmined by standard radioimmunoassity (RIA)
according to the method of Elsasser et ai.. ( 1989). Plasma IL- 1 levels were rneasured
by using the CYTokit ~ e d ~ I p kit (CYTImmune Sciences, Inc., College Park -ID);
absorbance values were cdculated on a TitreTek Multiskan Plus ELISA reader- This
human IL- 1p kit was validateci for use in our study by standard recovery. For example. a
known amount of IL-@ was added to a sample weil containhg semm whos
concentration of IL-1 had been previously determined. Recovery was the amount of
known IL-IB that could be "recovered over the amount of IL-@ that had been
previously determineci. Antibody ievels ro porcine reproductive and respiratory syndrome
v ins (PRRSV), transmissible gastro-enteritis virus (TGEV), A. pleuropnewnoniue (APP)
types 1, 5, and 7, and Mycoplarma hyopneumoniae were rneasured by Exposure Semm
Antibody Profile ( E S A P ) at Oxford Laboratories (Worthington. Minn.). Total antibody
levels (IgG) were measureâ according to the method of Mancini et al. f 1965). Nasal and
tonsillar swabs were plated on chocolate and blood agar with 0.0 1 5% NAD' according to
Quinn et ai. ( 1994). Rectal swabs were plated on blood and McConkey agar, isolates
were M e r characteriseci by standard microbiologicd methods (Quino et al.. 1994). The
thymus index was calculami by dividing the wet thymus weight by the hot carcass
weight.
27
The data were aaalysed using the General Linear Models procedure (proc GLM)
procedure in SAS ( 1995) using the foilowing model:
where
q = treatment room (Conventional, SEW, MD)
S, = sex
pk = pen (nested within sex and treatment room)
1 = individual animal within pen
For the analysis of F:G, ADFI and carcass characteristics, pens rather than individuai
animals were the experimentat unit. In the grower phase, fmai BW was included as a
covariate in the statistical analysis. For the f ~ s h e r phase and combined G/F phase.
initial B W was included as a covariate in the statistical analysis. Treatment meam were
separated by the ciifferences in least square rneans (LSD; SAS, 1995). .4U p values less
28
than 0.05 (R.05) were considered simcant. Between p values of 0.05 and 0.10
(.OScp<.lO), matment means were said to have foliowed a trend. Any cornparison of
treatment means whose value was greater than 0.10 (P>. iO) was considered not
significant.
Results and Discussion
General Observations
Pigs appeared generally healthy upon arrivai at the facility. However. within 2
weeks after d v a l some coughng and diarrhoea was noted in the SEW and Conventional
pigs. Two pigs in the Conventional group were treated with a broad spectmm antibiotic
(Borgal, Hoecbst, Regina, Sask) but no pigs were removed from this group prior to
reaching slaughter weight. In the SEW group, 5 pigs were treated with Borgal, and 6
animals were rernoved from the trial when they displayed signs of clinical disease
(coughing, scouring, or lameness) and lost more than 10% of their BW. No clinical signs
of disease were observed in the MD pigs and no pigs were removed from this group pnor
to reaching slaughter weight.
Growth Performance and Carcass Ouality
The ADR, ADG, and F:G of the three groups of pigs are presented in Table 2.
Grower phase. ADG was highest in the MD group (P<.O5) but was sirnilar in the SEW
and Conventional groups (P>.lO). ADFI was lowest in the SEW group (Pc-0.5) but was
simiiar in the MD and the Conventionai group (P>.10). F:G was similar in the MD and
the SEW group (P>.IO) and was poorest in the Conventional group (Pe .05 ) . The
Conventional and SEW pigs had growth rates and F G values that wele substantially
better than those reported by NRC (1988), indicating that the pigs had irnproved lean
growth potentids.
Finisher phase. During this t h e period, SEW pigs had the lowest ADG (Pc .05) but it
was similar in the Conventional and MD groups (P>.IO). The Iowest ADFI was seen in
the SEW group (Pc.05). and was sirnilar in the MD and Conventional groups (P>.lO).
F:G was poorest in the SEW group (Pc.O5), intermediate in the Conventional group, and
best in the MD group (P>.IO). The performance hierarchy that was seen dunng the
grower phase was lost during the f ~ s h e r phase. presumably due to the greatest degree of
immune system activation in the Sm and Conventional groups. These results agree
with published data indicating growth depressions and reductions in efficiency following
immune system stimulation (Kiasing et al., 199 1 ; Schinckel, 1995; Johnson, 1997a).
Performance in the combined grower-finisher p e n d foiiowed the same trend as that in
the finisber period.
Unlike Wiiiiams et d.. (1993), we did not observe any differences in carcass
quality between the three groups (Tabie 3). This may be amibuted to the fact that high
quality diets were fed; in spite of differences in ADFI, the daiiy lysine intake was unlikely
to limit lean growth rate in any of the treatment groups.
Indicators of Disease and Immune System Activation
Lung scores were highest in the SEW group (18.25%) foilowed by Conventional
(8.90%) and MD (0.34%) (Pe.01) (Table 4). The majority of the lesions were anterior
ventrai atelectasis typical of enzootic pneurnonia Although SEW has been proposed as a
method of eiiminating clinicai signs of enzootic pneumonia (Dial et al.. 1992). the SEW-
reared pigs in this experiment had active infections as weii as PRRSV infection as
indicated by increased titres and lung lesions. W e believe the multi sourcing of SEW pigs
and active PRRS virus infection led to the acute respiratory disease in these SEW pigs.
Ross ( 1996) and Clark et al., ( 199 1 ) report high incidences of disease in GE swine in
which pigs are commingled. As expected, the MD group had the lowest average lung
score.
The least amount of stomach ulceration was seen in the MD group (1.32, Pc.01)
whereas the levels in the Conventional (2.08) and SEW (2.07) groups did not differ
(Pz. 10, Table 4). Stomach lesions have been related to diet particle size (Wondra et al..
1995). However, in our snidy, ail pigs were fed the same diet with a mean particle size of
880 microns; thus diet particle size did not contribute to merences among treatmeuts.
Our data are consistent with previous studies which showed that stomach ulcer severity is
correlated with depressions in growth performance in growedfinsher swine (Ayles et al..
1996). Others have noted a correlation between lung lesions and stomach scores (Senk et
al., 1994). It has been speculated that during acute respiratory disease, histamine is
released Histamine is a known promoter of acid secretions in the stomach (Guyton,
1991). In one snidy, injecting pigs with histamine produced ulcen in the pars esophagea
region of the stomach similar to those found in this study (Zamora et al., 1980).
However, as stomach scores are confounded with many other aspects of animal health
and decreased feed intake, differences in stomach scores are only one of many factors that
contributed to treatment-related differences in performance.
The highest snout scores for AR were seen in the SEW group (0.81, P c . 0 1 ) .
There were no differences between the two other groups (P>.10). Presumably, the
toxigenic strains of P. muitocida responsible for turbinate damage were not present in
either the Conventional or MD groups. The higher level of turbinate damage observed in
the SEW group was consistent with the isolation of Bordetella bronchiseptica and
Pasteurella multocida from this group of pigs (Table 4).
The MD group had the highest thymus index (Pc .01); it was similar in the SEW
and Conventional groups (P>.lO). High healih status pigs typicaiiy have larger thymuses
than low health status pigs (Harris et al.. 1990). The srnailest thyrnuses were seea in the
SEW and Conventional groups, which carried the largest levels of disease. The thymus
index generaiiy correlated weil with other measurements of performance and immune
s ystem activation.
IL-1 B is a cytokine released during the acute phase response of the immunological
challenge. In addition, ILL$ affects nutrient homeostasis throughout the body via its
neuroendocrine actions on the hypothalamic-pituitary-adrenal axis (Roitt, 1994; Johnson.
1997a). Initiaiiy, IL-1B values were similar among the three groups of pigs. After
approxirnately 90 days, the highest levels of IL-1p were seen in the MD group (Pc.05;
Table 4). This is inconsistent with the known effkcts of an immunological insult on IL4
levels and the fact that the MD group had the Iowest levels of disease and the best
performance. However, the half-life of IL-If3 is approximately 4 hours (Roin. 1994).
Since we sarnpled eveiy 30 days, the observed results of L I S levels may not accurately
reflect values over long t h e periods. This indicates that monthly blood sarnpling for IL-
lp may not provide an accurate assessrnent of health status.
IGF-I is a hepatic hormone under the control of the hypothdamic-pituitary-
adrenal axis. It is mainly responsible for increasing protein synthesis throughout the
body, mobilising and enhancing the breakdown of adipose stores, and decreasing the rate
of glucose utilisation (Guyton, 1991). Until approximately 60 days into the trial, the MD
pigs consistentiy had the highest levels of IGF-1 (Pc.05) while it did not ciiffer (P>.10)
between the SEW and Conventional groups. At approximately 90 days ùito the trial, the
Conventional and MD groups were similar in IGF- 1 values (P,. 1 O), but the lowest
(P1.05) levels were seen in the SEW group. The reduction in IGF-I levels seemed to
correspond to the onset of disease in the SEW group. IGF-1 levels aiso fell in the MD
34
group. That IGF-1 levels are related to immune system activation has been suggested by
Schinckel (1995). Antigenicaily chdenged pigs have lower levels of IGF- 1 than their
healthier counterparts. In addition. IGF-1 levels are related to age (Schinckel. 1995).
body size, and nutrient accretion (Lowe, 1991). We saw the highest levels of IGF- 1 at 60
days into the trial, which was at approximately 70 kg BW. This finding is consistent with
results demonstrating growth rates of various pig genotypes. Schinckel ( 1994) indicated
that the highest protein accretion rates are seen at approximately 55-70 kg BW. This BW
range falls within the time frame of maximal plasma IGF- 1 levels in the current study.
Pigs in the MD and Conventional groups were vaccinated for PRRSV. Hence.
titres for PRRSV in these two groups were initially high and fell over time. In the SEW
pigs, which were not vaccinated for PRRSV, titres for PRRSV were initiaüy low, but
increased with time and by slaughter had surpassed levels in the other groups (Pc.05;
Figure 4), suggesting îhey were actively infected. Over tirne, SEW and Conventional
pigs developed enzootic pneumonia as determined by serological and pathological
confirmation at slaughter (Pc.05, Table 4). Titres for M. hyopneumoniae in the MD
group did not increase with time (P>.10, Table 4). in this study, there was no evidence of
Actinobacillus pleuropneumoniae serotypes 1 , 5 , and 7 (APP 1, 5, 7) or transmissible
gasîro-enteritis virus (TGEV) infection. M. hyopnewnoniae titres were seen to rise in the
SEW and Conventional groups which is consistent with infection (Table 4). We expected
to observe M. hyopneumoniae in the Conventional pigs since we knew that they
originated h m a herd which had tested positive for M. hyopneumonioe. Onginally,
SEW was thought to be a means of eliminating the spread of enzootic pneumonia This
study combineci with field experience shows that not only is pneumonia not eliminated
but can sometimes be made worse in the SEW system because of the commingling of
immunologicaiiy naïve and sick pigs (Diai et al., 1992). Receni evidence has pointed to
the possible relationship between infections such as PRRS and M. hyopneumoniae. Ross
(1996) suggested that pnor or concurrent PRRSV iafection predisposes the pig to M.
hyopnewnoniae infection and subsequently enzootic pneumonia Since the SEW group
developed PRRS (titres for PRRSV feu over t h e in the Conventional group). the
development of PRRS and the multi-sourcing of pigs in this SEW group is likely why we
saw a twofold increase in mycoplasmal-associated lung damage in the SEW group as
compared to the Conventionai group despite the fact that both groups were sero-positive
for M. hyopneumoniae.
Extensive micro floral characterisation of the three di fferent groups of animals was
done. In Table 5, the organisms identified from nasal, tonsil, and rectal swabs are listed
in relative order of abundance and the paîhogens are highlighted in bold. Marked
ciifferences were noted between the different groups and there was a shift in the
microflora over t h e in dl groups. The MD animals had the lowest microbial burden both
in terms of absolute numbea of organisms and numbers of different types of organisms.
The conventional group had the highest apparent microbial burden including NO
important pathogens: S. suis and ?? d t o c i d a . However. these organisms were detected
ody upon culture at the final bleed The S E W - r e d animais a h c a r i d a relatively
large microbial burden compared to the MD pigs. In addition to S. suis and P. mrlzocidn.
the SEW animals canied a third paihopn, B. bronchzkeptica. Again, rhere was a shfi in
the organisms that could be isolateci at the difTerent times. B. bmnchïseptica was only
deteaed in cultures of nasal swabs of animais h m the SEW group taken at the 9me of
the final blood sampling. Dritz et al.. (1996) report s d a r rates of B. bmnchrreprica
isolation from both SEW and Conventional h d i b herds. These data therefore. point to
the limitations of simply using culture techniques for deteminine the presence of
particular pathogens. Given the high levet of biosecurity ir is Likely that the S. suis and P.
mulrocidn were not introduced during the course of the snidy. but rather. were present
h m the beginning and simply not detected.
As a measure of general acquired immune system stimulation. total
immunoglobuh-G (QG) levels were measured in sera obtained h m all p i e when the
animais &t enterai the snidy and at slaughter. These data are summarised in Table 6.
Trends were detected in the total levels of IgG in the Merent bpups. The total Ib@3
Ievels reflected the microbial blrrden and titers to specific diseases in the different hmups
(Conventional > SEW > MD). In aU groups, the antibody levels approximately doubled
between the time of initial sarnpling and slaughter. h addition. it shouid be noted that
there was considerable animal to animal vanabon. This finding is important because it
indicates that within a group of animals raised under the same conditions there can be
differences in the level of immune activation (and presumably protection).
General Discussion
The growth-repressive effects of disease have k e n well documented et
al., 1994; Pettïgrew et al., 1995; Johnson, 1997a). Iadeed, an immunological challenge
leads to the re-partitionhg of nutrients as well as existing metabolic stores away from
growth (Klasing, 1988; Bahnson and Dial, 1995). At the beginning of the trial. the pigs
in the three treatment p u p s performed as we expected: generally, the best performance
and hedth status was seen in the MD group followed by the SEW group and
Conventional group. However, during the finisher phase, growth performance was
poorest in the SEW group, with the other two groups remaining in the same order.
Differences in performance between groups of pigs were also larger during the finisher
than during the grower period. These differences between groups and relative changes in
performance with increasing BW coincide with the signs of ciinical disease that
developed in the Conventional and SEW groups. This was conf ied by titres to specific
pathogens and coincided with increases in lung damage. stomach ulceration. and
decreases in thymus index as weil as Merences in microbial burden and total plasma
immunoglobulin levels. Because a strict health protocol was foilowed in order to prevent
the rwm-to-mm -fer of disease, we suggest that the outbreaks of disease were
s p ~ a d from pig to pig within ~OOIIIS from animals that carried these pathogens into the
facilities.
In addition, of the health status indicators tested it appears that IGF-1. t h p u s
index, lung scores, and antibody titres are indicaton of the presence of disease. More
observations are required to assess whether rhey cm be used to pRdict the extent to
which reductions in gowth performance and carcass quality are related to disease
exposure.
Signifcance to the Indway
This smdy clearly demonsuates the implications of disease and its associated l q e
metabolic cost in G/F pigs. The fmancial benefits of reducing the exposure to disease are
quite substantid. This supports the need for M e r observations on the effects of specific
diseases on performance- In addition. this snidy outluies a need for a means by which the
extent of immune system activation (and hence disease challenge) cm be rnonitored in
pigs mana@ under different commercial conditions in order to predict depressions in
performance.
Table 2. Effect of health management strategy during rearing on G/F pig performance
BW Initial (kg)
BW Intermediate (kg)
BW Final (kg)'
Grower Phase (Initial to Inunnediate BW)' (25-60 kg)
Finisher Phase (Intermediate to Final BW )' (60- 1 08 kg)
Cornbined Grower/Finisher Phase (Initial to Final BwJ (25- 108 kg)
Conv.
8
30.69.
65.58'
109.04=
0.77'
1 .Tla
2-25.
0.97.
2-85'
2.94'
0.87"
2.22'
SEM
0.26
0.88
1 -69
0.0 1
0.03
0.03
0.02
0.10
0.09
0.0 I
0.02
' ~ h e length of time on trial differcd by group. '~orrected for final BW. 3~orrected for initial B W. '~orrected for initiai BW. ab%eans * SEM within a row lacking o common supencript differ (Pc0.05).
Table 3. Effect of heaith management stratev during rearing on carcass characteristics - -
Treatment Groups
Conv SEW MD P 8 8 8
Ham W. 6.94d. 12 7.0420.09 6.924.08 0.6 1 (kg)
Hot carcass 8 4 - 0 . 7 85.8d.5 86.5I0.5 0.23 wt (kg)'
96 Lean 6 1 -2d.4 62.010.3 6 1 - 8 4 . 3 0.28
* Values indicate those for which initial body weight was significant and therefore treated as a covariate.
Means SEM within a row lacking a common superscript differ (Pc0.01).
Tabie 4. Effect of health management saategy on health evaluation parameters? Treatment Groups
Item
Lungs Score
S tomach Score
Snout Score
Thymus Index
IL- 1 p. day O (n@mL)
IL- 1 BI day 90 (ng/mL)
IGF- 1 ,&y O (ng/rnL)
IGF- 1 ,day 30 (ng/mL)
IGF- l .&y 60 (ng/rnL)
[GF- 1 .day 90 (ng/rnL)
PRRS Titres day O
PRRS Xcres day 30
PRRS Titres day 60
PRRS Titres day 90
M. hyopnewnoniae 'Ctm day O
M. hyopnewnoniae Eues day 30
M. hyopnewnoniae 'litres day 60
M. hyopnewnaniae
Conv
8.9W.38'
2.08I0.2V
O. 1810.13a
I.55I0.1 la n d 7
0.204I0.013
0.506d. i 26'
109.717.0 n=42
137.611 1.2 n=36
151 .5*12Aa
152-4k7.7"
678.4139.3LW
2&4.4150.2u
303.a72.3'
235.6140.6a
MW
Xues day 90
Lb%eans r SEMwithin a row lacking a cornmon superscript differ (P405). t n 4 8 unless indicated otherwise, w;LY%eans i SEM within a column for each factor (PRRS and M. hyopneumoniae titres) lacking a common superscript di£€& (k0.05).
Table 5. Microflc < -a of conventional health status, MD, and SEW swine? ? Ncissem sp. Smp. sp. P ~ u r c l l a SQ.
€ coli (mr,pah) Pediac~tcys q. R-km Gr-vc &ciIIus sp. Pmteus sp. CampyIobaner coti Moruulh sp.
HcisxecM sp. Strep. sp (mt .suis) e coli Pmrcu~ sp. P~rliococcu~ sp.
Neisseria sp. Smp. sp. (Mt suis) Staphylococclu sp. Monurlùt sp.
Neissem sp. Stmp sp. (mt suis) Smp. SUU Pltsfct~~1lo sp. S&phylococcus sp. RmPItocido e col? P e d i o c ~ ~ ~ r ~ sp. Crp B. S a p
-
Neïssenu sp. Smp. sp. (wt sur) M o r a u l h sp. E coli Srop~lococcw sp.
N e k e m sp. Moraul la sp. tileruûucrer sp. StaphyloC~clK sp. ErhcPnFr1ell0 unla & coli
Neisseria sp. Smp. sp (mt mir) Acifutobucl~r sp. S c ~ ~ p h y l o c m ~ sp. E culi s r ~ m ~ l l ~ ~ o c ~ l u sp. MorazcIlu sp.
Neisserta sp. Pareur~l lu sp. M o m h sp. E culi streptomyces sp. Enterobucrer sp. srrcprococcur sp.
Nciucnci sp. NAD kp. (mi APP) Str iphyloct lc~~~ sp. B. brotukirrpiccr P. modroOda E coli Momxelh sp. Srrep. sp. ( mt suit). Srrtp- JW
Smp. sp. (not S u i s )
E coli (4 types) Actnewba~~er *p. Citmbactcr sp. Prrn~umllri sp Budlw sp. Stmpto~ccs sp.
E coli Stmp* sp. (mi surs sirep. suis AcUvw&cwr sp. DYcmbucter sp.
S m . rp. (mt r u ) E coli &iriUw sp. Sraphvloc~cui sp.
E cnli Slmp. rp. lm suis) Sfaphyloc~cru sp.
- --
& culi (H*) srrrp. sp. imi surs) Pmrew sp. Ertnurdrieliu sp. Sraphvlococcvr sp. Y-t
& cd1 Smp. sp. (not sus) Con.bucrenwn sp. M o m u l h sp.
tOrganisms in bold type indicate a greater relative abundance
Table 6. Total IgG Ievels in sera from conventional health status, SEW, and MD swine as
d
Initial IgG levels' (mg/100 ml) Final IgG levels* (mg/100 ml)
Conventional Hedth Stritus 101 1 I 1 15 1976 t- 871
Segregated Early Weaning 840 187 1951 I 654
Minimal Disease 736 205 1149 k464 %lues indicate Means 4 SEM
Chapter 5: L l r a Increases Lean M a s and Deceases the Cytokine Respouse in a Model of SubClinicai Disease in Gmwing Pigs
There is a growing body of evidence to suggest direct cytokine involvement in the
initiation of skeletai muscle catabolism and depressions in growth performance as a result
of disease. Since TNF-a and IL-6 are presumably under the control of IL- 1, we atternpted
to delineate whether growth depressions in a mode1 of sub-clinical disease were due to
increased IL-lp levels, and to determine whether an IL- 1 receptor antagonist, IL- ha.
could reduce these negative effects in growing pigs. In this snidy, 24 crossbred barrows
and gilts were randomly assigned to three treatments: 1) healthy conuol. saline infusion;
2) sick control, infection with Mycoplarma hyopnewnoniae and Porcine reproductive and
respiratory syndrome Virus (PRRSV) and saline infusion; 3) Sick plus infusion with IL-
Ira. The sick control group had growth performance similar to that of the hedthy pigs
(P>.lO) and the highest levels of circulating cytokines ( P c . 10). The sick + IL- lra group
also had growth performance similar to the other groups (P>.10), but had the best carcass
characteristics (Pc.10). and similar levels in circulating cytokines as the healthy control
group (P>.10). We saw no treatment-induced Merences in IGF-1 levels (P>.10), but
did observe a ltreatment x sexl interaction; in the gilts, we consistently saw highest levels
of IGF-I in the L l r a and healthy control groups, and the lowest in the sick control group
(Pc.10). This relationship was not seen in the barrows. In this snidy. we show that
infusion with IL-Ira in a mode1 of sub-ciinical disease helps attenuate the catabolic
effects of immune system stimulation.
Introduction
In mammals, interleukin- 1 (IL- l p) is a cytokine released durhg the initial stages
of the immune response to foreign insult or injury (Roia. 1994). It is thought that die
peaistence of this cytokine among othen, resuits in reductions in appetite, whole body
protein wasting, and general repartitioning of nutrients favouring the immune respouse
(Klasing, 1988; Johnson, 1997a). This response to an immunological insult is part of a
complex cascade of events that is thought to involve the immune. the endocrine. and the
neuroendocrine systems of the body and is commonly referred to as the immune-
hypothalamic-pituitary-adrenal axis (MPA) (Bateman et al., 1989).
Recent evidence of the involvement of the IHPA in growth and whole body
nutrient partitionhg has corne from snidies examining AIDS-related wasting in the
chronically ill (Mulligan et al., 1993; Frost et ai.. 1996: Steele. 1996), h m studies
involving high and low hedth starus pigs (Schinckel 1995; Dionissopoulos et al.. 1997).
and endocrinological studies in the rat (Kakucska et al., 1993). In one study, blocking the
response to one of the infkmmatory cytokines, tumour necrosis factor-a (RIF-a). has
had promising effects in terms of reducing the body weight loss and muscle wasting that
is typical of chronic disease (Breuillé et al., 1993).
T'en years ago, an IL- 1 receptor antagonist (IL- 1 ra) was identifed (Seckinger et
al., 1987) and was recently found to attenuate many of the symptorns associated with
47
septic shock in rabbit models and human studies (Wakabayashi et ai.. 199 1; Fisher et al.,
1994; van Zee et al., 1995). To our knowledge, IL-Ira has not been used to reduce the
effects of chronic disease on skeletal muscle wasting or nuaient partitioning. The goals
of the curent study were to define the eHect of chronic sub-clinical disease on lean
growth performance of pigs, and to determine whether IL- Ira reduces the negative effects
of immune system stimulation on nutrient partitioning. In this study, 24 crossbred
barrows and gilts were randody assigned to three treamients: 1) healthy control, saline
infusion; 2) sick control, infection with Mycoplasma hyopnewnoniae and Porcine
reproductive and respiratory syndrome V h s (PRRSV) and saline infusion; 3) Sick plus
infusion with IL- lra.
Materiais and Methods
A total of 30 pigs (half gilts, haif b m w s ) with an initiai body weight (BW) of
approximately lOkg were denved h m a minimal disease herd with no apparent health
problerns. On the basis of clinical signs and serologic testing of those animds. the minimal
disease pigs were confirmai to be free of the foiiowing swine pathogens: Actinubaciih.s
pleuropneumoniae, SerpuiuUr hyodysenteriae. toxigenic Pasteurella multocida-type D.
mange, lice, porche respiratory and respiratory syndrome v i n s (PRRSV) and
Mycoplamur hyopneumoniae (M. hyopneumoniae). AU pigs in this snidy were of the same
genetic background (Yorkshire x Landrace) and had medium to high lean growth potentials.
Upon arrival, the pigs were split into three treatrnent groups (healthy control, sick
control, and sick + IL-Ira) and were kept in three separate but identical rooms at the
Ponsonby research station of the University of Guelph (See Chapter 4). In each room,
pigs were assigned to pens at random with i pig per Pen. Each room had separate heating,
ventilation, and manure handiing systems; room temperatures were maintained at
approximately 20 OC during the trial. Six pigs were slaughtered immediately at the
initiation of the trial in order to detemine initial body composition for an estimation of
retained carcass nitrogen, ash, and lipid during the experiment.
49
In orùer to prevent the transfer of disease between rooms, access to facilities was
restricted, and rmms were always inspected in the same order: Healthy control, sick
control, and sick + IL- lra; disinfectant foot bath were placed at the entrance of each
roorn. and staff changed boots and coveraiis prior to entering each room. Blood samples
and body weights were taken on separate days for each room, and dl the sampling
equipment was thoroughty disinfected with a commercial power sprayer disinfectant
before king moved between rooms (See Chapter 4).
Pigs had free access to water h m nipple drinkers and were fed a high quality
grower diet (Dionissopoulos e t al., 1997) in two equal meds daily (at 08:ûûh and
16:OOh) at 2.5 t h e s their maintenance energy requirement according ro the following
equation (NRC, 1988):
Daily Feed AUowance (kg) = (2.5 x 110 x BW(kg)A0.75)/DE content of diet (KcaUkg)
These feeding levels are weil below ad libifwn intake in healthy growing pigs. The pigs
were fed at a restricted intake level to ensure that feed intake levels were similar across
the three treatments.
The corn-soybean meal-based diet contained 3% fishmed and 2% whey, and had a
calculated DE content of 3470 kcaYkg. The calculated lysine content was 1.30%. The
vitamin and minerai contents were in excess of NRC (1988) requirements. Diets were
designed so that none of the essentiai nutrients limited the expression of performance
potentials.
Surnery und Treutments
Surgery was conducted in order to fit extemal jugular catheten for the routine
sampbg of blood and to insert inm-peritoneal osmotic pumps (mode1 WMLA. Alza
Corporation, Pa10 Aito, CA) for the delivery of saiine or IL-lra (AMGEN. Thousand
Oaks, CA) to the pigs. Haif of the pigs in each treatment group received jugular caiheters
whereas al1 pigs received intra-pentoned pumps. Sex was balanced across aii treatments.
The pigs were @en a pre-mix anaesthetic compriseci of 5mL atropine (O.Smg/mL), 2mL
Demeroi (1ûûrng/mL), and 1mL acepromazine (IOmg/mL) at a dose of O.lmi/kg body
weight Foilowing anaesthetic induction, the pigs were given oxygen with 1% consmr
halothane via face mask, and extemal jugular catheterisation was performed as described by
de Lange et al., (1990). The catheter was anchored to supporting tissues and passed
dorsally through to the postenor portion of the neck, where it was secured to the animal via
a faûly loose fining coliar. Pumps were implanted intra-pentoneally via mid-line
abdominal incision. AU sub-dermal tissue layers were sutured with #Y0 chromic catgut;
demial layers were sutureci with W0 silk. All surgical procedures lasted less than 30
minutes. The pigs regaineci full consciousness within 30 minutes post surgery.
Immediately foiiowing surgery, the pigs in the healthy control group were given 5mL liquid
saline intra-nasally and 2.5mL saline intra-muscularly, while the sick control and sick + IL-
lm pigs, were infected inira-nasaily with 5mL of a live culture of Mycoplasma
hyopneumoniae (M. hyopnernoniae) and intra-muscdarly wiih 2SmL Porcine
reproductive and respiratory syndrome virus (PRRSV) vaccine.
A preliminary study using 2 pigs of similar weight and genetic background to those
in the main study showed thai infection with M. hyopnemniae and PRRSV vaccine in this
manner resulted in an increase in circuiating IL-lp levels from 2 ng/rnL to 18 ng/mL
between days 1 and 14 pst infkction and resulted in ciinical s i p of disease (coughing and
lethargy).
In the sick + IL- lm treatment group, 0.2 mg/kg/hr IL- l ra was delivered from the
intra peritoneal osmotic pumps. The delivery rate of the IL-Lra was constant over the
duration of the trial. A second preliminary study using 2 pigs of sirnilar weight and
genetic background showed that given this rate of delivery, IL-lra levels rose to
approxirnately 220 ng/mL by &y L of the trial and stay fairly constant over time.
According to Arend et al., (1990), this infusion rate was sufficient to block the response
to IL- 1.
Daily observations were made to evaluate animal behaviour and well k i n g and to
monitor any clinical signs of disease according to guidelines set by the Canadian Council
on Animal Care (Rowseii. 199 1). Daily blood samples were taken for the fmt week of the
trial h m cathetensed pigs, and every week thereafter h m d pigs until the end of the 28
&y trial. Those pigs that were not fitted with catheters had blood drawn by retro-orbital
sinus venipuncture according to the methoci of Moldovan and May ( 1975). Approxirnately
5rnL of blood was taken for the determination of insulin-like growth factor 1 (IGF- 1), IL- 1
p, and TNFu levels in normal (senun) tubes, and 5mL in EDTA tubes for white blood cell
(WBC) evaluaîion. Foilowing blood sampling, caiheters were Bushed distaily with 5rnL of
2% heparin-saline to prevent cloning. After day 8 of the trial due to diffîculty in obtaining
sufficient blood samples, blood was obtained fkom each pig on a weekly b a i s by retro-
orbital sinus venipuncture. Pig BW, average daily gain (ADG) (kgMay), and feed intake
(FI) (kgfday), were determined at weekiy intervals for 4 weeks after the start of the
infusions. Immediately foliowing the last weighing, the pigs were kilied by elecaical
sninning and exsanguination. The pigs were slaughtered according to standard
procedures (Mohn and de Lange, 1998). The carcasses including head, feet. skin and
hair, muscle and bone, but excluding blood, viscera, organs, and gut-fill, were weighed 24
hours after slaughter and were h z e n for fat, protein, dry matter, and ash analysis.
Plasma IGF- L levels were determined by standard radioirnrnunoassay according to
the method of Elsasser et al., ( 1989). Plasma IL1 levels were measured by using the
CYTokit R# L kit (CYTlmmme Sciences, Inc., Coiiege Park, MD, (see Chapter 4);
TNF-a levels were measured hy TNF-cY ELISA (Endogen, Inc., Cambridge, MA) and
absorbantes were calculated on a TitreTek Multiskan Plus ELISA reader. WBC's were
counted and differentiated according to the method of Odink et al. ( 1990).
The h z e n carcasses were completeiy ground and analysed for protein. fat. and
ash content according to the method of Mohn and de Lange (1998).
Statisîkal AM lvsis
The data were analysed using the General Linear Models procedure (proc GLM)
in SAS (1995) using the foilowing modei:
y i j ~ = CL + Ti + Sj + Qij + pik(ij) + q g k j
w here,
'Ci = treatmendroorn
S, = sex
mi=teraction between treatment and sex
For the analysis of data, pigipen was the experimentai unit and LSD separated treatment
means. Given the small number of animais used in this experiment, the treatrnent effects
and the ciifferences between means were considered to be statistically significant when
P d O . In these analyses, treatment was confounded with rooms. It was therefore
assumed that there were no room effects per se. In addition, correlation analysis was
perforrned on main effects variables using the error sum of squares matrk (SAS. 1995).
Only those correlations which were simcant (k.05) are reponed.
e trial, aU pigs appeared generally he er day 5. pigs
in the sick control and sick + IL- lm groups displayed signs of disease (coughing and
lethargy), which continuai for the duration of the trial. Pigs in the heaithy control groups
were without clinical signs of disease for the duration of the trial. Two pigs fiom the sick
+ L l r a group and one pig fiom the healthy control group had to be removed ftorn the
via1 due to AcrUromyces pyogenes-related descendhg catheter infections as determined by
pst-mortem analpis.
There was a treatment-induced difference in fmal BW, total weight gained. and
ADG (Table 7). The sick + IL-lra group showed signifcantly bemr growth performance
than the sick controi group (Pd@. For the hedthy control group, these values were
intermediate to the sick control and sick + IL- 1 ra group. but did not differ (P> . 10) from
either group (Table 7). Although no significanr difference was seen in te- of f e d
eficiency, h r e was noted better feed efficiency in the IL-lra group over the other two
groups, (Table 7).
Results for carcass analyses are presented in Table 8. Carcass weight was greatest
in the IL- 1 ra group (P<. 10) but was similar in the healthy conuol and sick connol groups
( P X IO). Protein, iipid, and ash represented 15.0. 6.9. and 2.6 8 of carcass weight for the
initiai slaughter group (n4). These values were then used to estimate carcass protein.
lipid, and ash retention rates by the pigs in the 3 treatrnent groups. The carcass protein
retention rate was Iower in the sick conwl group than in the sick + IL- lra group (Pc-10) .
No apparent differences were seen between the healthy control group and sick control
group, or between the healthy control group and sick + IL- l ra group ( P X IO), except for
carcass protein accretion rates which were higher (Pe .10) in the sick + IL- Ira group.
Treatment-induced differences in senun IL- 1 became apparent only after &y 15
of the trial (Figure 1). IL-1$ values after day 8 indicaie that the sick + IL- lra p u p
56
consistentiy had either the lowest levels or levels equal to the healthy control group. The
highest levels of IL- 1 B were seen in the sick control group (Pc. 10: Figure 1).
As c m be seen in Figure 2, ueatment induced differences in serum TNFa became
apparent only after day 8. For the sake of clarity, aiI data points between days 1 and 8
have b e n omitted and are placed in appendix 1. In general, the lowest levels of TNF-a
were seen in the healthy control group, foilowed by the IL-lra and sick conml groups.
However, statistical differences were only observeci on day 15 and &y 29 of the triai.
Throughout the duration of the trial. there were no treatrnent-induced differences
in IGF-1 levek (P>.IO; Table 9). However, a ltreatment x sexl interaction was noted on
day 29 of the trial. On day 29 in the f a d e s , the healthy conml and the sick + IL-Ira
groups had consistentiy higher ( P d U ) IGF-I values than the sick conmol group. Data
for the males in this trial follows an almost opposite pattern as the femaies.
In tenns of blood ce11 counts. the only discernible differences were seen in the
levels of circuiating monocytes (Table 10). The lowest levels of total circulaùng
monocytes were seen in the sick + IL-lra group and the healthy control group, and the
highest levels were seen in the sick conml group (Pc.10).
Correlation analysis indicated thaî IL-1P levels for days 8 and 15 of the tr iai were
negahvely correlated with IGF- 1 levels (r-û.9093, P=0.0120, day 8; I=-0.7880.
P=0.0626, day 15). In addition. TNF-a levels negaîiveiy correlated with protein
deposition (r=-û.8297, W.0411, &y 29) and positively correiared with IL- 1 levels
(d.8299, P=û.0409, day 15).
Discussion
Moiloy et al., (1993) suggested that the effects of disease on n u ~ e n t partiuoning
rnay be modifieci by blocking the response to selecttd cytokines. and so cm lessen some
symptoms of chronic disease. This suggestion was based on eariier findings which
demonsiraied the negarive effects of disease-causing organisms on circulating cytokine
levels in blood, whole body nutrient metabohm, health status. and feed intake (McCarthy
et al., 1985: Klasing, 1988; Lang and Dobrescu. 1989: Romagnani. 1994). Indeed.
immunologicid saess brings about a series of endocrinologie and metaboiic changes char
serve to enhance the immune response via the provision of nutrïenrs. Such changes include
increased glucose metabiism, increased protein tunover. and increased maintenance
energy requirements (Gelfand et al., 1984; Del Rey and Besedovsky, 1987; Hargove et al..
1988: Lang and Dobrescu, 1989; Johnson, 1997a). Since IL- 1. TNF* and IL-6 have
previously been shown to be eievated in the diseased state, their presence rnay exert a k t
effect on nutrient partitionhg (Johnson, 1997a). The idea of blocking the metabolic
~~sponse to these cytokines following an inununological challenge has only recently been
explored. In rats. Brefle et al., (1993) used a cytokine receptor antagouist (Pentoxrfviline,
Hoehcst, Regina, Sask-) to successfdiy lessen the decreases in body weight loss and muscle
protein wasting characteristic of sepsis. However, the endocrinological mechanisms
underlying these changes have only laîely been addressed (Warren et al., 1997; Webel et al..
1997). In a previous study, our group showed decreases in IGF-1 levels which
correspondeci with pends of maximal immune system stimuiation (Dionissopoulos et al.,
1997). This finding. dong with data pmsented here, suggests a Link between cytokine
release foliowing immune system stimulation and IGF- 1 levels.
L l r a was chosen as IL1 release is known to stimulate the production of IL6
(De Simoni et al., 1995) and presumably TNF-a (Roitt, 1994). Furthemore, experiments
using IL-lra have demonstrated its ability to lessen endotoxemic symptoms in the
development of septic shock (Wakabayashi et al., 199 1 ; Fisher et al., 1992; Fisher et al.,
1994; Van Zee et al., 1995). It was thought that IL4 blocka.de rnight partially or
completely reverse the catabolic consequences of disease. In our mode1 of chronic, sub-
cihical disease, the sick control group had the highest levels of circulating IL-lp and
TNF-a as compared to the healthy control and IL-lra groups beyond day 8 of the trial
(Figures 1 and 2). The differences in IL- 1 and TNF-a levels between the healthy control
and the sick control groups was as expected. That immunologicalIy chdenged pigs grow
slower and retain less nitrogen has been demonstrated by Williams et al., (1993).
However, in the c m n t study, performance in the heaithy pigs was poorer than expected
59
and was in fact, similar to the sick control group ( P x I O ) . The reasons for this
discrepancy are not immediately clear and may be due to the small sample size in these
growth perfoxmanœ parameters.
However. we showed that by blocking the response to the immunotogical
challenge, we reduced circulating IL-if3 and TNF-a levels. reduced the nurnber of
circulaihg monocytes, and increased carcass protein retention (Pc.10) . IL- i B levels
were positively correlated with TNF-a Ievels ody on day 15 of the trial (Pc.O.5). III fact.
in the IL i ra group we saw the greatest amount of carcass protein retention. rhe fastest
body weight gain, and the highest rotai amouot of body weight gained (Pc.10). la
particular. the improved rate of carcass protein retention may result in increases in overail
muscle m a s and agrees with studies by Breuillé et ai.. (1993) and Lang et al. ( 1996). Ln
fact, TNF-a levels negatively comlated with carcass protein retention rates panicularly
by &y 29 of the niai, (P<.05) supporting the view that TNF-a is a cachexic factor. Other
studies such as those by McHugh et al., (1994) pointed to the si@icance of partial
attenuation of anorexia folïowing acute disease via ueatment with IL-Lra because
centrally administered IL-1 is believed to be one of the primary mediators of anorexia
(McCarthy et al., 1985; Johnson. 1997a). Because the sick + IL- Ira group had cytokine
levels similar to those of the healthy control group, we suggest that in our model, the
increased cytokine production is at least partidy responsible for the growth depressions
and poorer carcass characteristics such as those seen in the sick control group.
60
We decided to test the hypothesis that the intended growth increases would be in
part due to decreases in cytokine production. which would in tum decrease serum IGF-1
secretion. Recent evidence suggests that IL- lra reverses the negative effects of immune
system stimulation on growth hormone secretion (Peisen et al., 1995) and that wasting in
the chronically ill is mediated by reduced levels of growth hormone and IGF-I (Frost et
al., 1996). In addition, many of the metabolic effects of growth hormone are mediated by
IGF-1 (Guyton, 199 1). However, in our model. we saw no treatment-induced differences
in IGF-1 secretion, though we did see trends in the female pigs; the differences becoming
statistically significant ( P d O ) by the final blood sampling time. In this female group.
those pigs in the sick control group had coasistently lower serurn levels of IGF- 1 than did
their counterparts in the hedthy control and sick + IL- lra groups, which incidentaily had
the best growth rates, feed efficiencies, and retained carcass protein. These differences
may in fact be due to inherent variability in circulathg IGF-1 levels. When considering
individual animals rather than group means, IGF- I levels negatively correlated with IL- 1
levels on days 8 and 15 of the trial (Pc.05 and P<.IO respectively). These correlation
results strengthen the view that carcass protein retention is mediated in part by IGF-1
who's secretion may be negatively affected by IL- 1.
Our results for total circulating leukocytes, platelets, neutmphils, and lymphocytes
indicate that there was no real treatment-induced effect of our model of sub-clinical
disease on the parametes tested (P>.IO). These resuits agree with those obtained by
61
O d M et al., (1990) that in general, pigs with pneurnonia typically do not display
pronounced differences in W C numbers compared to healthy pigs. However, we did
observe an interesting trend in the to tai number of circulating monocytes (Tabie 1 0). W e
generally found that the highest levels of circulating monocytes were seen in the sick
control group (P<. 10). while results were simila. for the healthy control and sick + IL- 1 ra
groups. That the sick + IL- lra group had suppression of IL- 1 effects suggests that IL- 1 is
a monokhe, since generally the lowest monocyte numbers were seen in the sick + IL-1 ra
group. However, we did not see any identifiable patterns in total lymphocyte counts
among the three groups, despite the fact that IL-1 is a known stimulator of B and T-ce11
proHeration (Roitt, 1994). Since B and Tcell maturation occurs over a period of two to
three weeks, the time course of our sampling procedure was more than likely insufficient
to detect real differences in these lymphocytes. Both the high ievels of TNF-a and IL- 1
in the sick contml group. and the low levels of TNF-a and IL- 1 B in the sick + IL- 1 ra and
healthy control groups generally correspond to the levels of circulating monocytes,
neutrophils, and platelets (Pc. IO).
Conclusion
In Ehis study, we demonstrated that a mode1 of sub-clinical disease causes
iocreases in IL- 1 P and TNFu levels in plasma of young pigs. We ais0 demonstrated that
the negative effects of chronic immune system stimulation (reduced ADG. reduced final
%W. increased monocyte counts, increased ILL$, and increased TNF-a levels) were
shown to be partially anenuated by constant infusion of IL-Ira. These data combined
with previously published data from others, suggest a possible role for IL-lra in the
treatrnent of wasting attributed to chronic disease.
Further snidies using a larger nurnber of pigs are required to c o n f i these
observations. Furthemore, studies need to be conducted over longer tirne periods to
more closely represent the effects of chronic disease or immune system stimulation on
growth performance in growing pigs.
Table 7. Growth ~erfonnance for three mou~s of D~ES.
Item Treatment Groups Healthy Control Sick Control Sick + IL- 1 ra P
n 7 8 6 Initial BWNg) 10.7CM.38 1 0.24fi.32 9.89a.43 0.5832 Final BW(kg) 1 6.22&66* 15.76M5 la 17.72d.73~ 0.0190 Total Weight Gained (kg) 5.95+0.66* 5 . 4 8 4 5 ta 7.45&.73b 0.0264
(kg/day) 0.2 110.02* 0.20I0.02* 0.2710.03~ 0.0264 ( k l i m w 053I0.02 051d.Ot 0.52I0.02 0.3585
F:G 2.6 1 d . 4 1 3.m0.3 1 2.09dl.45 0.1 117
Means 2 SEM within a row lacking a common superscript differ significantly (Pc. 1 O).
Table 8. Carcass characteristics for three groups of pigs. d
Treatment Groups Item Health y Sick Controi Sick + IL- ln P
ConaoI
n 7 7 6 Carcass Weight (CW) (kg) 12.15M.28' 12.2M.26" 13.6m.30b 0.0001 CW:BW 0.724.0 1 0.7 1 i0.0 1 0.72Al.O 1 0.2 1 12 Protein Mass (5% of CW) 17.93M.21 17.94M.19 17.9 14.19 0.9268 Lipid Mass (% of CW) 5.6 1 H.54 554S.54 6.16M.6 1 0.5 275 Lipid:Protein 0.33H.03 0.33s-O3 0.37dl.04 0.5269 Retained Carcass Cmde h i n (giday) 34.5412.8ga 32.35e.89' ~ . 3 4 + 3 . 2 7 ~ 0.0240 Recained Carcass Lipid (g/day) -1.-.13 -1.79d.13 5 -84-ct.55 0.1509 Retained Carcass Ash (glday) 6.922 1.37 6.4111.37 6.06+156 0.733 I
Means t S E M withui a row lacking a common superscrÏpt differ (P<. 10).
Table 9. Insulin-like growth factor 1 levels for three groups of pigs showing the ltreatment x sexl interaction.
Treatment Groups
Item IGF- 1, day 1 ( ng/mL) IGF- 1, day 8 IGF- 1, day 15 IGF- 1.
Sick Control 5.8 1 ~ 8 . 6 2 ~
n=8
Sick + IL- 1 rat 1 32.42142.8ga
n=8
Barrows Gilts Healthy Control Sick Control Sick + IL- 1 ra Healthy Control Sick Control Sick + IL- 1 ra
IGF- 1. 87.2&1 0.B8 1 15.621 10.05~ 4 1 -97d.24' 88.1Ck7.29 38.85k8.d 78.91+5.10' 0.0194 day 29
ab*c Means I: SEM within a row lacking a common superscript differ ( P c - I O ) . '~nterleukin- l Rece ptor Antagonis t
Table 10. White blood celi differentials.
Sick Control Item Healthy Control Sick + Lira
Leukocytes, day 8 ( x l o k ) Leukocytes, day 15 kukocytes, day 22 Leukocytes, day 29 Platelets, Ray 8 ( ~ 1 0 % ) Platetets, day 15 Plate lets, day 22 Platetets, day 29 Segs, day 8 ( x 1 o g / ~ ) Segs. &y 15 Segs, &y 22 Segs, day 29 Bands, day 8 (X 109/~) Bands, day 1 5 Bands, day 22 Bands, day 29 Tot Lymphocytes, day 8 (x 1 6 ) ~ ) Tot Lymphocytes, &y 15 Tot Lymphocytes, day 22 Tot Lymphocytes. day 29 Tot Monocytes, day 8 ( ~ 1 0 ~ ~ ) Tot Monocytes, day 15 Tot Monocytes. day 22 Tot Monocytes, day 29
a,b,c Means & SEM within a row lacking a common superscript differ (Pc.10).
Figure 1. Interleukin- 1 fi ( h l p) levels in pigs fiom 3 treatment groups*.
IL4 beta levels for three groups of
+ Sick Control
-t Healthy Control
Day of Trial
"',' Means within a colurnn lacking a common superscript differ ( P<. 1 0 ). f Three treatments were used: 1 ) sick + infuçion with interleukin-l receptor antagonist ( IL- 1 ra); infection with :lL hopnemonrue and porcine reproductive and respiratory syndrome MIUS (PRRSV), 2) sick conmi: infection with iL/. &opneumoniue and PRRSV, saline infusion, 3 ) healthy control; infusion with saline.
Figure 2. Tumour Necrosis Factor a (Ma) levels in pigs from 3 treatment groupsi.
TNF- levels over time for three groups of pigs
500 -
+ Sick +IL4 ra
+ Sick Contro l
-t Healthy Control
- A- L -
-200 -
Day of Trial
".b" Means within a column lacking a common superscript differ (P-c IO). tThree treatrnents were useci: 1 ) sick + infusion with interleukin- l receptor antagonist (IL- l ra); infection with M. hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV), 2) sick coatrot; infection with M. &opnezanoniue and PRRSV, saline infusion, 3) heaithy control; infusion with saline.
Chapter 6: Smnrirary and General Dscossioa
In a growing-f~shing pig performance study, clear effects of health management
strategy during rearing on f a d efficiency (F/G), average daily gain (ADG). and average
daily faed intake (ADFI) were observed. At approxirnately 25 kg body weight (BW), pigs
were sourced fiom three dinerent management systems (Conventional, Segregated Early
Weaned ( S m , and Minimai disease (MD)) and housed under identical conditions until
shipped to market. In the grower phase, the best perfonning group was the MD. foUowed
by the SEW, and Conventional groups. respectively. In the f ~ s h e r phase (when
respiratory disease was noticeable, particuiariy in the SEW group), performance levels in
the SEW group were lower than in the other rwo groups. The SEW group came from a
common SEW nursery but had piglets sourced h m 6 individual sow herds. The negative
effects of commingling have been established (Dial et ai.. 1992). InW the risk of
enzootic pneumonia increases greatly when pigs from different sources are commingled
(Jorsal and Thornsen, 1988). It seems that commingling pigs may resdt in the corning
together of pigs with different serological profiles even if they are commingled at
approximately 14 days of age. Since some pigs may inherently be immunologically naive
to the incoming paihogens, disease outbreak occurs. This is consistent with our results of
a significantly decreased h d t h s t a t u and performance of the SEW group compared with
the others. These growth depressions coincided with increased antibody titres to the
common swine pathogens Mycoplamin hyopneumonine and porcine reproductive and
respiratory syndrome virus (PRRSV), and to mycopiasmal-associated lung damage.
atrophic rhinitis (AR), and to decreased thymus size. In addition. performance
depressions in the SEW pigs conesponded with reductions in serurn insulin-like growth
factor L (IGF-1) levels but not with changes in serum inrerleukin-l (IL4 p) levels.
However, since IGF-1 levels are known to be affected by a range of factors including feed
intake, these resulu rnust be approached with caution. Hence, IGF- 1 may not be an
appropriate indicator or predictor of performance reductions following disease. Chapter 4
clearly demonstrated the effects of immune system stimulation on G/F pig performance.
Chapter 4 also demonstrated that antibody titres and slaughter checks are good indicatoa
of expusure to disease and hence to performance reductions.
In a subsequent study (Chapter 5). the response to blocking the immune response
to interleukin- 1 (IL- 1) was evaluated. These pigs were of the same genetic background
and had a body weight (BW) of approximately IOkg. In this study, pigs were infected
with M y c u p l l a v ~ hppnewtoniae and porcine reproductive and respiratory syndrome
virus (PRRSV). Sub-clinical disease was induced and the growing pigs were either
untreated (sick control), or treated with an IL- l receptor antagonist (IL-lra) (sick + IL-
Ira). In contrast to the expected outcorne. exposure to PRRSV and M. hyopneurnoniae
did not resdt in reductions in growth performance. This may be due to the fact that in
Chapter 4, exposure to PRRSV and M. Hyupnemniae negatively affected _mwth
performance after 60 days into the triai. The duration of the subsequent trial was only 28
days. This may mean that differences in growth performance would probably have been
seen had the trial been extended past 60 days. However, the intra-pentoneal pumps used
had a maximum duration of 28 days, thus obviating the need to limit the duration of the
expriment. Yet exposure to disease did result in increases in IL-@ and T N F u Pigs in
the sick + IL-Ira group performed the best and had the best carcass characterisucs. This
fiading couid not be amibuteci to IGF-1 levels since we observed consistent depressions
in IGF- 1 levels in the sick conml group and incrûases in IGF- L in the IL- 1 ra group of
gdts only. However, this relationship was not apparent in the barrows. The circulating
cytokines IL-lp and TNF-a were lowest in the IL-lra group, which were simiiar to
heaithy controls. These resuits corroborate the theones involving L I regdation of
cytokine synrhesis and the cytokine synthesis cascade. In fact, it was shown that IL- l a levels were correlated with TNF-a levels on day 15 of the tnal when tested on individual
animals instead of treatment means. In addition. TNF-a levels were negatively correlated
with EF- 1 levels on day 29 of the trial, and IL- lp levels were negatively correlated with
IGF-1 levels on days 8 and 15 of the trial which corresponded to the penod of maximal
immune system stimulation. These correlation resdts strengthen the view that carcass
protein retention is mediated in pari by IGF-1.
The toxicological side effects (if any) of high circulating levels of IL-lra have not
been established Therefore, preceeding the use of this drug for therapeutic use should be
7 1
the determination of toxic levels of IL-lm This finding also supports immune systern
regulation of the immune-hypothalamic-pituiq-adrenal axis (IHPA), since reductions in
IGF-I levels were seen in the sick control group. Moreover. due to the fact that
circulating TNF-a levels change predictably with health status, its routine use and
sarnphg may be useful in p r e d i c ~ g the overail level of immune system stimulation in a
herd, and hence, performance reductions due to disease.
Now that a link between cytokine levels, growth performance. and IGF-1 levels
has been demonstrated, future research should address the elucidation of the mechanistic
relationship between TNF-a and IL-1 and growth via modulation of IGF-1 secretion
through the IHPA axis. If these pathways involving chronic immune system stimulation
can be detemiined, perhaps therapeutic strategies could be designed that limit growth
depressions and yet do not immuno-compromise the host.
Tumour Nerosis Factors levels for t h e groups of pigst Day of Heaithy Sick Control Sick - P Trial IL1mtSEM2 - -- . . .... C-t-YEM - -- -*SEM . -. . -. -. . - . - - - .-A
?Values are given in pgimL "-%leans within a row lacking a common superscript differ ( P . -10).
Interteukin- 1 beta levels for three groups of pigs? Day Healthy Sick Sick P of ControlISEM ControkSEM + a- 1 raISEM
?Values are given in ngImL abMeans within a row Lacking a common superscript differ (Pc. 10).
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l MAGE EVALUATION TEST TARGET (QA-3)
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