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This is to certify that the

thesis entitled

Antibiotics inhibit in vitro but not in vivo
cartilage degradation '

presented by

Tonia L. Peters

has been accepted towards fulfillment
of the requirements for

 

 

 

 

Animal
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ANTIBIOTICS INHIBIT IN VITRO BUT NOT IN VIVO CARTILAGE
DEGRADATION

By

Tonia L Peters

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Animal Science

2000

ABSTRACT

ANTIBIOTICS INHIBIT IN VITRO BUT NOT IN VIVO CARTILAGE
DEGRADATION

By

Tonia L Peters

Recently, certain antibiotic families utilized in the poultry industry have been
found to adversely affect bone formation and cartilage metabolism in dogs, rats, and
humans. Therefore, the objectives of this study were to 1) determine if certain antibiotics
inhibit in vitro cartilage degradation and 2) determine if antibiotics that inhibited in vitro
cartilage degradation would induce tibial dyschondroplasia (TD) in growing broilers.
Ten antibiotics were studied using an avian explant culture system that is designed to
completely degrade embryonic tibia over 16 days. Lincomycin, tylosin tartrate,
gentarnicin, erythromycin, and neomycin sulfate did not inhibit cartilage degradation.
Doxycycline (200 ug/ml), oxytetracycline (200 ug/ml), enrofloxacin (200 and 400
ug/ml), cefiiofur (400 ug/ml) and salinomycin (10 ug/ml) significantly decreased
proteoglycan and nitric oxide concentrations (markers of cartilage breakdown) in
conditioned media. These antibiotics plus chlortetracycline (known not to inhibit in vitro
cartilage degradation) and thirarn (known to induce TD) were then administered to day
old broiler cockerels at 25, 100, and 400% of recommended dose levels. At 22 days of
age the birds were killed and inspected for TD lesions in both proximal tibia. Incidence
of TD did not differ between the antibiotic treatment groups and control birds. These
data show that although some antibiotics inhibit in vitro cartilage degradation, their

administration to growing broilers does not induce TD lesions.

This thesis is dedicated to husband, daughter, mom and dad. Without their love and
support, I would not have been able to complete this degree.

iii

ACKNOWLEDGMENTS

I would like to thank graduate assistants, technicians, student help, farm manager
and the farm crew: Jenifer Fenton, Dana Dvoracek-Driksna, Kim Brown, Jennifer
Hawkins, and Angelo Napolitano. Without their help and support I could not have
completed these projects. I would also like to thank my committee for their support and
guidance: Dr. Kevin Roberson, Dr. Richard Balander, and Dr. Richard “Mick” Fulton. I
especially want to thank Dr. Michael Orth for giving me the opportunity to earn this
degree. Finally, I would like to thank my husband and parents who were always

supportive.

iv

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. vi
LIST OF FIGURES ................................................................................. vii
LIST OF ABBREVIATIONS ..................................................................... ix
INTRODUCTION ................................................................................... 1
CHAPTER 1
LITRATURE REVIEW ............................................................................. 3
Introduction .................................................................................. 3
Avian Growth Plate ......................................................................... 4
Extracellular Matrix ............................................................... 7
Proliferative Zone .................................................................. 8
Hypertrophic Zone ................................................................. 9
Tibial Dyschondroplasia .................................................................. 11
Antibiotics and Bone Metabolism ...................................................... 15
Tetracyclines ...................................................................... l 7
F luoroquinolones ................................................................. 1 9
Cephalosporins .................................................................... 21
Aminoglycosides .................................................................. 22
References .................................................................................. 24
CHAPTER 2
THE EFFECTS OF ANTIBIOTICS ON IV VITRO CARTILAGE DEGRADATION... 31
Introduction ................................................................................. 3 1
Materials and Methods .................................................................... 32
Results ....................................................................................... 39
Discussion .................................................................................. 41
References .................................................................................. 57
CHAPTER 3
THE EFFECT OF ANTIBIOTICS ON BODY WEIGHT, FEED CONVERSION, AND
TD SCORES OF 3-WEEK-OLD BROILER COCKERELS ................................. 61
Introduction ................................................................................. 61
Materials and Methods .................................................................... 63
Results ....................................................................................... 67
Discussion .................................................................................. 70
References .................................................................................. 72
CHAPTER 4
SUMMARY AND CONCLUSION ............................................................. 74

LIST OF TABLES

TABLE 1. Composition of medium used to induce cartilage degradation in the
embryonic chick tibia explant culture system ................................................... 35

TABLE 2. Description of treatments for the tibia explant culture experiments ........... 37

TABLE 3. Total release of PG or NO into conditioned media between days 6 and
16 of culture. The tables are broken into three groups in order to compare the treatments

to the control group within its experiment ...................................................... 40
TABLE 4. Diet Composition for 0-3 week old broiler cockerels ......................... 65
TABLE 5. Treatment groups within the 3-week broiler experiments ....................... 66

TABLE 6. Body weight, feed conversion and tibial dyschondroplasia incidence for 3-
week-old male broilers treated with several levels of seven antibiotics or thiram (a
fungicide) ............................................................................................. 68

vi

LIST OF FIGURES

FIGURE 1. Diagram representing the location and organization of the primary growth
plate (physis) .......................................................................................... 6

FIGURE 2. Twelve-day embryonic chick tibia containing bone marrow, a bony sheath,
and two cartilage ends. The dotted areas depict the proliferative zone and dashed areas
show the hypertrophic zone ....................................................................... 34

FIGURE 3. Figure 3. (Next page) Proteoglycan and nitric oxide release into conditioned
media from chick tibia explants with two tetracycline treatments (n=2). Panel A depicts
the proteoglycan release into conditioned media throughout the 30-day culture period.
Decreased proteoglycan concentrations were found for oxytetracycline (200 ug/ml) and
doxycycline (200 ug/ml) between day 6 and 16 of culture compared to the control (p=
0.008, p= 0.01). Panel B depicts the nitric oxide production released into conditioned
media throughout the 30-day culture period. Decreased nitric oxide concentrations were
found for oxytetracycline (200 ug/ml) and doxycycline (200 ug/ml) between day 6 and
16 of culture compared to the control (p= 0.002; p=0.001) ................................... 48

Figure 4. (Next page) Proteoglycan and nitric oxide release into conditioned media
from chick tibia explants with enrofloxacin treatments (n=2). Panel A depicts the
proteoglycan release into conditioned media throughout the 30-day culture period.
Decreased proteoglycan release was found for enrofloxacin at the 200 ug/ml (p= 0.003)
and 400 ug/ml (p= 0.004) concentrations between day 6 and 16 of culture compared to
the control. A trend for decreased proteoglycan release existed for the 100 ug/ml
enrofloxacin treatment (p= 0.09). Panel B depicts the nitric oxide production released
into conditioned media throughout the 30-day culture period. Decreased nitric oxide
concentrations were found for enrofloxacin at the 200 ug/ml (p= 0.007) and 400 ug/ml
(p= 0.006) concentrations between day 6 and 16 of culture compared to the control. A
trend for decreased nitric oxide production existed for the 100 ug/ml enrofloxacin
treatment (p= 0.06) ................................................................................. 50

Figure 5. (Next page) Proteoglycan and nitric oxide release into conditioned media
from chick tibia explants with ceftiofur treatments (n= 2). Panel A depicts the
proteoglycan release into conditioned media throughout the 30-day culture period.
Decreased proteoglycan concentrations were found only for the high dose of ceftiofur
(400 ug/ml) between day 6 and 16 of culture compared to the control (p= 0.007). Panel
B depicts the nitric oxide production released into conditioned media throughout the 30-
day culture period. Decreased nitric oxide concentrations were found for the same
concentration of ceftiofur between day 6 and 16 of culture compared to the control (p=
0.005) ................................................................................................. 52

Figure 6. (Next page) Proteoglycan and nitric oxide release into conditioned media
from chick tibia explants with salinomycin treatments (n=3). Panel A depicts the

vii

proteoglycan release into conditioned media throughout the 30-day culture period.
Decreased proteoglycan concentrations were found for salinomycin at 10 ug/ml between
day 6 and 16 of culture compared to the control (p= 0.03). A trend for decreased
proteoglycan release existed for the 1 ug/ml salinomycin treatment (p= 0.06). Panel B
depicts the nitric oxide production released into conditioned media throughout the 30-day
culture period. Decreased nitric oxide concentration was found for 10 ug/ml salinomycin
between day 6 and 16 of culture compared to the control (p= 0.03) ........................ 54

FIGURE 7. Tetracycline molecule. Dashed box highlights major metal binding site.

The four-ring structure and this location are needed for inhibition of collagenase. R1 and
R2 are the sites where the tetracycline derivatives differ in molecular structure .......... 56

viii

LIST OF ABBREVIATIONS

CM — Conditioned media

C02 - Carbon Dioxide

ECM — Extracellular matrix

FGF —— Fibroblast growth factor
IGF — Insulin-like growth factor
LPS - Lipopolysaccharide

MMP - Matrix metalloproteinases
NO — Nitric oxide

NOS — Nitric oxide synthase

PG —- Proteoglycan

ppm — Parts per million

QAP — Quinolone arthropathy
TCCS —- Tissue culture chick serum
TD — Tibial dyschondroplasia

TGFB - Transforming growth factor-beta

ix

INTRODUCTION

Tibial dyschondroplasia (TD) is one of the major metabolic skeletal disorders
to affect the long bones of fast growing meat producing birds (broilers, turkeys, and
ducks). A developmental abnormality of the tibiotarsal bone, TD originates in the growth
plate where it disrupts the delicate balance between cartilage synthesis and degradation.
Cells within a TD lesion fail to differentiate into mature hypertrophic chondrocytes and
remain in a pre-hypertrophic state. Tibial dyschondroplasia affected birds accumulate an
avascular, non-mineralized cartilage plug in the metaphyseal region of the tibia.
Lameness, decreased feed efficiency, and increased mortality, culls, and condemnations
at slaughter can result from TD. Producers see a peak in clinical TD cases at
approximately half the market age of the bird, 3 weeks for broilers and 9 weeks for
turkeys.

Understanding the etiology of such a debilitating metabolic disorder would
benefit the poultry industry. Experimental conditions and compounds that induce TD
include: copper deficiency; calciumzphosphorous imbalance, vitamin D deficiency;
fusarochromanone, thiram, and antabuse toxicity; excessive dietary levels of cysteine,
homocysteine and histidine; and metabolic acidosis. Genetics and environmental factors
such as time of year and rearing conditions also induce the disease. Many experimental
theories have been presented, but the pathogenicity of TD in commercial industry is still
elusive.

Recently, some antibiotics in the tetracycline family have been shown to

inhibit cartilage degradation of the embryonic chick tibiae. Other antibiotics implicated

in the disruption of bone metabolism include tobrarnycin, ceforanide, and quinolones. In
vitra data on the effects of tetracyclines and other antibiotics, which interfere with bone
metabolism, show that further investigation into the concentrations of growth promoting
antibiotics used in the poultry industry is needed.

The use of antibiotics as grth promotants began at about the same point in
time that TD prevalence increased. Modern broilers have an 18-63% incidence of TD.
Although genetic changes were also prevalent over this time period, recent data support
the hypothesis of the studies reported herein that certain antibiotics might induce TD
lesions in viva. Therefore, the objectives of this research were: 1) To determine if
antibiotics commonly used in the poultry industry inhibit growth plate cartilage
degradation in vitro and 2) To determine if the antibiotics that inhibited degradation in
vitra would induce TD in the proximal tibial growth plate of broilers in viva.

The following chapters of this thesis describe research projects that address
these objectives. Chapter 1 provides background information on normal and TD growth
plate cartilage physiology as well as mechanisms of antibiotics used in this research.
Chapter 2 is the data, methodology and results of the embryonic chick tibiae project and
demonstrates which antibiotics inhibited in vitra cartilage degradation. Chapter 3
describes the in viva experiments using antibiotics from the previous chapter to determine
if they induce TD lesions in growing broilers. In conclusion (Chapter 4), I discuss the
effects of antibiotics on in vitra compared to in viva cartilage degradation and their

implications to the poultry industry.

Chapter 1

Literature Review

Introduction

Poultry skeletal disorders have been estimated to cause losses of approximately
$160 million per year to the United States broiler and turkey industries (Sullivan, 1994).
One of the major metabolic skeletal disorders that affects the long bones of fast growing
meat producing birds (broiler chickens, turkeys, and ducks) is tibial dyschondroplasia
(TD). A developmental abnormality of the tibiotarsal bone, TD originates in the growth
plate where it disrupts the delicate balance between cartilage synthesis and degradation.
Growth plates of birds affected with TD undergo cell proliferation at a normal rate
whereas hypertrophic chondrocytes within these lesions only reach 40% for their normal
size (Hargest et al., 1985). Due to inhibited degradation, an avascular cartilage plug
accumulates in the metaphyseal region of the tibia, which may caused impairment of
long bone growth and clinical TD lesions. Lameness, decreased feed efficiency, and
increased mortality, culls, and condemnations at slaughter can result from TD.

Modern broilers have an 18 to 63% incidence of TD (Praul et al., 2000;
Roberson, 1999; Elliot and Edwards, 1997). Knowing the etiology of such a debilitating
metabolic disorder would benefit the poultry industry. Havenstein et al. (1994) reported
that when broilers were fed a 1991 commercial diet, birds with 1991 genetics had a
48.6% incidence of TD, whereas birds with 1957 genetics had a 1.2% incidence of TD.
However, genetics is not the only factor involved with TD. Other experimental

conditions and compounds that induce TD include: copper deficiency;

fusarochromanone, thiram, and antabuse toxicity; excessive dietary levels of cysteine,
homocysteine and histidine; calcium and phosphorus imbalance, and metabolic acidosis
(Orth and Cook, 1994). Environmental factors such as time of year and rearing
conditions also induce TD (Orth and Cook, 1994). Many hypotheses have been
presented, but the cause of TD in the commercial industry is still not known.

Recently, some antibiotics in the tetracycline family have been shown to inhibit
cartilage degradation in embryonic chick tibiae (Orth et al., 1997). Other antibiotics
implicated in the disruption of bone metabolism include tobramycin (Murakami et al.,
1996), ceforanide (Smith et al., 1987), and quinolones (Vormann et al., 1997). These
and possibly other antibiotics routinely used in the industry may cause TD cases in the
field. This hypothesis is circumstantially supported by the parallel increase in standard
feeding of growth promoting antibiotics with the rise in commercial TD incidence.
Further investigation may reveal these antibiotics to be the common link between many

commercial TD incidences.

Avian Growth Plate

Endochondral ossification is the process by which cartilage is calcified and bone
is formed. Growth plates, thin discs of cartilage located between the epiphyseal and
metaphyseal regions of long bones, are responsible for the longitudinal growth rate and
the ultimate length of bones. The primary center of ossification (growth plate) is
responsible for longitudinal growth; the secondary center of ossification is responsible

for latitudinal growth and is located in the epiphysis. In the chick tibia, the proximal end

has only a primary center of ossification whereas the distal end has both the primary and
secondary center of ossification.

Two main zones exist within the growth plate, the proliferative zone, which is the
proximal portion of the plate and the hypertrophic zone (Fig 1). Although chondrocytes,
the cells of the growth plate, appear to move through the growth plate, they stay at a
spatially fixed location as bone is formed and new chondrocytes are produced.
Chondrocytes start out as flat cells in the proliferative zone. These cells are arranged in a
column fashion and as they mature, they proliferate and produce extracellular matrix
(ECM) proteins including proteoglycans and collagens. The chondrocytes are
surrounded by ECM, which gives the growth plate its structural strength as well as its
cushioning support. In the hypertrophic zone, the cells enlarge and become round as they
differentiate. As they enlarge, hypertrophic chondrocytes synthesize proteinases that
degrade the ECM surrounding them (pericellular matrix) (Brighton et al., 1973).
Columns are no longer apparent in the growth plate once chondrocytes reach the
hypertrophic zone. Although these zones are defined by their metabolic function, the
transitions that occur between them are gradual. The rates of synthesis and degradation
must be in equilibrium if the growth plate is to properly elongate and ossify.

The avian growth plate is not as highly organized as the mammalian; avian cells
are more numerous and are randomly oriented into longer disarrayed columns (Pines and
Hurwitz, 1991). Approximately 200 cells per column (Howlett, 1979) are contained in a
4-7 week old chicken, in contrast to the rat growth plate that contains 25 cells (Kember,

1960). In comparison, Leghorn chickens have a more orderly transition than broilers

Figure 1. Diagram representing the location and organization of the primary growth
plate (physis).

   
 
 
 
 
 
 
  
  

 

 

 

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between the proliferative and hypertrophic zone and a more regular vascularization of

hypertrophied cells (Reiland et al., 1978).

Extracellular Matrix

The extracellular matrix undergoes synthesis, reorganization, and eventually
degradation. The ECM is composed of 50% water, 25% proteoglycan (PG) complexes,
and 25% collagen. The high concentration of water and PG content gives cartilage its
gel-like appearance and cushion feature. Aggrecan, the primary PG in cartilage, is a
macromolecule consisting of a protein core covalently linked to long side chains of
glycosaminoglycans. Aggrecan molecules bind to a hyaluronic acid backbone via link
proteins. A collagen network provides structural support for PGs. Collagen type II and
smaller amounts of type VI, IX, X and XI are the major collagens found in growth plate
cartilage. Orth ( 1999) published an extensive list of proteins and cell signals found in the
growth plate.

Type II collagen is synthesized mainly by proliferative chondrocytes and early
hypertrophic chondrocytes; it is the primary collagen in the growth plate that provides
tensile strength. Covalently cross-linked to type II collagen is type IX collagen, which is
thought to limit the collagen fibril diameter along with collagen type XI (Mendler et al.,
1989). Type IX collagen is distributed throughout the matrix (Muller-Glauser et al.,
1986) and potentially facilitates the interactions of glycosaminoglycan side chains to type
II collagen fibrils (Van Der Rest and Garrone, 1991). A protein unique to the
hypertrophic zone is type X collagen, which is 45% of the total collagen for that zone.
This collagen is thought to be involved with mineralization or possibly facilitates matrix

degradation near the chondro-osseous junction, but the exact function is unknown

(Schmid et al., 1986). Type VI collagen is thought to stabilize the main collagen fibril
network to the chondrocytes (Van Der Rest and Garrone, 1991).

The structural organization of the ECM changes throughout the proliferative and
hypertrophic zones of the growth plate. As the chondrocytes mature, the matrix is
reduced to thin longitudinal and transverse septae due to the enlargement of the
chondrocytes in the lacunar spaces. Eggli et a1. (1985) described the ECM as having
three compartments: the pericellular, territorial, and interterritorial. The pericellular
matrix compartment immediately surrounds the chondrocyte and is rich in PGs. A
collagen fiber network surrounds the chondrocytes to compose the territorial
compartment. The interterritorial compartment consists of parallel collagen fibers that
connect the chondrocyte columns through the longitudinal matrix septa. The collagen
content of the ECM increases as the columns of cells enter the hypertrophic zone. The
degradation of the ECM by matrix metalloproteinases (MMP) facilitates growth plate

remodeling, vascularization, and maturation into bone.

Proliferative Zone
The proliferative zone is characterized by disk shaped chondrocytes, mitotic cell
division, and synthesis of ECM. Proteoglycan content is highest in the proliferative
zone. Collagen fibrils are distributed randomly; collagen type II is the main collagen that
appears in this zone as well as types IX and XI (Pines and Hurwitz, 1991). Active cell
division occurs in the upper proliferative zone, which is the progenitor layer for
longitudinal growth; chondrocytes are in a germinal stage having few mitochondria and

appear dense due to a large amount of rough endoplasmic reticulum (RER) (Howlett,

1979). Maturing proliferative cells have ample glycogen stores and extensive RER to
promote high rates of aerobic glycolysis and protein synthesis. The longitudinal columns
of disk-like chondrocytes become disarranged as the cells begin to hypertrophy and
become separated by ECM (Howlett, 1979). Golgi apparatus also become more
numerous as the chondrocytes enter the hypertrophic zone.

Nutrients, oxygen, and growth factors are supplied to the proliferating
chondrocytes by the epiphyseal arteries. Chondrocytes are under direct regulation by
hormones such as vitamin D metabolites (Corvol et al., 1977) and growth factors. Basic
fibroblast grth factor (bFGF), insulin-like growth factor 1 (IGF-1), transforming
growth factor-B (TGFB), and platelet-derived growth factor (PDGF) have been primarily
associated with mitogenic activity in chondrocytes of the proliferative zone (Rosselot et

al., 1994).

Hypertrophic Zone
Proliferating chondrocytes eventually differentiate and hypertrophy. As

chondrocytes enter the hypertrophic zone, the RER becomes irregularly shaped, the
number and size of organelles in the cytoplasm decrease. Hypertrophic chondrocytes are
metabolically active even though the area surrounding then becomes anaerobic and
lacking in nutrients. The chondrocytes become spherical and increase 5-10 times the size
of the cells in the proliferative zone. They synthesize extracellular proteins such as
alkaline phosphatase, osteocalcin, osteopontin, and bone sialoprotein. Proteolytic
enzymes such as collagenase are expressed which allow the breakdown of collagen

facilitating cell expansion (by degrading the matrix) and vascularization. An important

autocrine regulator, TGFB is a growth factor responsible for chondrocyte differentiation
and maturation (Rosen et al., 1986;Rosen et al., 1988). However, terminal differentiation
and matrix mineralization appears to be inhibited by TGFB. It inhibits matrix vesicle
phospholipase A2 activity, an enzyme involved in calcification (Schwartz and Boyan,
1988; Schwartz et al., 1993).

In the degenerative zone of the hypertrophic area, calcification of the matrix and
vascularization occur. The aggregated structure of PGs and the irregular distribution of
matrix vesicles assist in inhibiting mineralization in other zones of the grth plate.
Once in the distal end of the hypertrophic zone the vesicles merge (Poole and Pidoux,
1989) and PGs disaggregate (Anderson, 1989) to initiate calcification. Rapid turnover of
broiler chick growth plate cartilage allows for little calcification to occur. However,
nutrients cannot penetrate the increasingly calcified matrix and cells closest to the
chondro-osseous junction undergo apoptosis (Hatori et al., 1995). Phagocytic cells
originating from the metaphyseal blood supply remove apoptotic cells (Howlett, 1980).
Capillary sprouts penetrate into the lacunae of apoptotic cells at the last transverse septa
of the chondrocyte columns to provide a vascular network. A portion of chondrocytes do
not degenerate in avians due to metaphyseal vascular penetration (Howlett, 1979).
Fibroblast growth factor is released from terminal hypertrophic chondrocytes and may
act as an angiogenic signal for metaphyseal blood vessel vacularization (Twal et al.,
1994). Nutrients are supplied through vacularization to matrix vesicles as well as other
components of cartilage to support normal function and mineralization (Nie et al., 1995).
Therefore, angiogenesis supports cartilage calcification and endochondral bone

formation. The majority of matrix vesicles, containing calcium phosphate, are in the

10

lower hypertrophic zone due to their participation in mineralization. The first mineral
crystals appear within matrix vesicles and spread outward between columns by accretion
of calcium salts. The cartilage matrix degradation and calcification provides a
framework for invading osteoblasts, which deposit bone on the calcified cartilage. This

bone is resorbed and replaced by trabecular bone.

Tibial Dyschondroplasia

Tibial dyschondroplasia, a metabolic disorder of the growth plate in rapidly
growing meat-type birds, disrupts the formation of endochondral bone by impairing the
resorption of cartilage. An avascular, uncalcified cartilaginous plug accumulates in the
proximal metaphyseal region of the tibial grth plate. The rapid growth rate of broilers
may contribute to a disarrangement of chondrocytes during bone formation, supporting
the formation of the plug and therefore predisposes these birds to TD. In severe lesions,
the metaphyseal region may weaken resulting in bowing of the long bone and lameness.

The growth plate of TD birds consists of similar components and concentrations
as those of a normal bird. However, TD chondrocytes tend to aggregate and have a
condensed morphology in comparison to normal chondrocytes (Rath et al., 1997).
Dyschondroplastic growth plates also have a decreased level of S and K in the ECM of
the upper hypertrophic zone (Hargest et al., 1985). Collagen type II expression
(normally a characteristic of proliferative chondrocytes) in the hypertrophic zone of TD
growth plates suggests abnormal cell proliferation (Pines et al., 1998). Other researchers
have shown that chondrocyte differentiation and hypertrophy are interrupted before

maturation is complete (Bashey et al., 1989). Collagenase-gelatinase activity is

11

significantly decreased in TD cartilage compared to normal cartilage (Rath et al., 1997).
Reduced MMP activity may decrease ECM turnover, inhibit vascularization, and lead to
the retention of avascular cartilage. Chondrocytes within TD lesions only reach 40% of
normal hypertrophic chondrocyte size (Hargest et al., 1985), and undergo premature
necrotic changes. These areas within the transitional zone of the growth plate contain
large numbers of apoptotic chondrocytes; a decrease in DNA content and an increase in
DNA fragmentation indicate cell death in this region (Rath et al., 1997). Maturation of
the chondrocytes is therefore inhibited. The inability of phagocytic cells to enter
cartilage and remove apoptotic cells may contribute to the accumulation of an avascular
plug, leading to the arrest of endochondral bone formation and the pathogenesis of TD.
Matrix vesicles, which are responsible for mineral formation in endochondral
ossification, are decreased in TD lesions and only vesicles on the perimeter of the lesion
mineralize. They primarily lack a functional nucleational core and provide insufficient
mineral ions (calcium and phosphorus specifically) to form normal matrix vesicles (N ie
etaL,1995)

Levels of proteins associated with bone formation are below normal in the TD
growth plate. Type X collagen, calmodulin, alkaline phosphatase, and basic Fibroblast
Growth Factor (bFGF) are decreased, suggesting mineralization and vascularization may
be impaired (Twal et al., 1996; Bashey et al., 1989). Skeletal tissue collagenase activity
can be increased by FGF (Ries and Petrides, 1995). Varghese et al. (1995) reported that
FGF facilitates angiogenesis. Therefore, a decreased level may not provide a signal for
invading capillaries. The exact roles of Type X collagen and alkaline phosphatase are

unknown but their presence precedes calcification and proposed roles include stimulation

12

of angiogenesis (Kwan et al., 1997) and targeting cells for osteoclast resorption
(Linsenmeyer et al., 1991). Kwan (1997) also suggested that Type X collagen synthesis
was not reduced but a defect in incorporation into the matrix occurred resulting in
decreased levels. However, Wardale and Duance (1996) reported that TD cartilage had
reduced levels of Type II and Type XI collagen but had increased levels of Type X
collagen. They hypothesized that since the chondrocytes were in an arrested state of
hypertrophy they would produce the proteins associated with that stage of maturation.

Accumulated cartilage in the metaphysis has increased collagen and non-
reducible collagen cross-link concentrations (Orth et al., 1991).
Hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) cross-links assist in the
stabilization of the collagen fibril network as well as increase the hydrophobicity of
collagen (Eyre et al., 1984). Increased cross-linking makes the collagen more resistant to
enzymatic breakdown (Vater et al., 1979). The distal area cartilage of the TD lesion
contains over a 10-fold increase in HP concentration (Orth et al., 1991). These
abnormalities are likely involved in the increase of time for cartilage resorption from less
than 24 hours to weeks (Thorp, 1988). Increasing the time for cartilage resorption will
alter the balance of synthesis and degradation of growth plate cartilage, which facilitates
impaired bone growth.

The accumulated cartilage can be resorbed and replaced by bone as the growth
rate of the bird slows. Rath et a1. (1997), reported that TD affected cartilage has healthy
as well as apoptotic areas, which may allow for the replacement of TD tissue as the bird
ages. However, in fast growing birds such as broilers and turkeys, the rate of growth

does not slow enough for the cartilage to be resorbed properly. It is during the first half

13

of growth that the greatest changes in proportional growth occur (Marks, 1979).
Therefore producers see a peak in clinical TD cases at approximately half the market age
of the bird (broiler 3 weeks, turkey 9 weeks), even though the birds have only achieved
40% or less of their final body weight (Lilburn, 1994). Broiler tibia and femur
diaphyseal ash data indicate that the first 7 days of growth are the most critical to overall
skeletal development; this data may provide a physiological window in which to
manipulate TD incidence and severity (Lilbum et al., 1989).

The etiology of TD is not well known in the poultry industry although many
factors and experimental conditions can induce dyschondroplastic lesions including:
copper deficiency; fusarachromanone, thiram and antabuse toxicity; cysteine,
homocysteine deficiency; metabolic acidosis; calcium and phosphorous imbalance;
vitamin D deficiency; genetic selection and environment. One study showed that in
vitra, lesion chondrocytes have the ability to terminally differentiate and mineralize
suggesting that ECM interaction, vascularization or other regulatory factors are
contributing to the etiology of TD in viva (Farquharson et al., 1995). Recently, Kestin et
al (1999) found that Ross (208 and 308 lines) and Shaver broilers had an increased
incidence of TD compared to the Cobb 500 broiler line. How these experimental models
relate to commercial TD incidences is unclear. They also may induce TD lesions by
different mechanisms. Thiram, a thiocarbamate fungicide, was shown to induce TD at
nonlethal dietary concentrations (Edwards, 1985; Wu et al., 1993). In vitra,
chondrocytes exposed to less than 5 uM of thiram by 72 h of culture showed a decreased
level of cellular alkaline phosphates, acid phosphatase, and LDH activity (Rath et al.,

1995). This effect may be seen since thiram has the ability to create a “leaky” cell

14

membrane. Thiocarbamates are metabolized to carbon disulfide in viva (World Health
Organization, 1979), which may alter cell-membrane proteins (DeCaprio et al., 1992).
Thiram is also highly lipophilic which would decrease its clearance rate and allow for
retention in adipose tissue and synovial spaces, which may assist thiram’s action on
developing cartilage. Rath et a1. (1995) hypothesized that these properties may allow
thiram to exert cytotoxic effects and modify cell membranes to induce TD.

Tibial dyschondroplasia is widespread across the United States as well as
worldwide in fast growing poultry (Hemsley, 1970; Laursen-Jones, 1970; Riddell et al.,
1971; Itakura and Gato, 1973). A common link must exist in order to have such wide
spread occurrence. One such common management practice within each species is the
addition of grth promotants to diets to improve performance and to create an
economic advantage. Antimicrobial agents, which include antibiotics, are the most

widely used growth promotants.

Antibiotics and Bone Metabolism

Antibiotics are derived from cultures of microorganisms or produced
synthetically. They interfere with the development of bacteria by the following general
mechanisms: inhibiting bacterial cell wall synthesis, microbial DNA translation and
transcription, or essential metabolite synthesis, and altering membrane permeability.
Antibiotic feed additives given at sub-therapeutic levels alter the gut microflora in a way
that ultimately enhances nutrient uptake by the animal. This alteration in natural flora
concentration suppresses bacteria that cause mild infections, reducing immune stress and

freeing energy for growth. Microfloral production of vitamins and other nutrients is

15

increased and nutrient utilization by the bacteria is reduced. The primary benefit from
antimicrobial supplements is an improved feed conversion. Dr. Tomas Jukes discovered
in 1949 that chlortetracycline, fed at low levels to chicks or pigs, improved growth and
feed efficiency (Swick, 1996). Other benefits include reduced mortality, resistance to
disease challenge, and improved pigmentation and litter quality. Depending on grow-out
conditions, savings from an improved feed conversion ratio range from 2 to 12 fold on
product return (Swick, 1996).

Antibiotics have minimal effects in a new, clean house due to an absence of
microbial challenge to the animal. This response depends on animal management,
cleaning procedures and downtime between flocks, age of housing facility, and feed
quality. Therefore, growth promotant levels of antibiotics are used primarily in houses
with less than sanitary conditions.

The poultry industry uses antibiotics worldwide for growth promotion as well as
for disease treatment. Broilers and turkeys are exposed to sub-therapeutic levels of
growth promoting antibiotics throughout most of the grow-out period. The potential
effects of chronic exposure to sub-therapeutic doses are not known on skeletal
development. However, a single therapeutic dose of certain antibiotics during a critical
postnatal growth period have been documented to cause deleterious effects on bone
formation in humans, rats, and dogs (Gale et al., 1981;Papick, 1998;Vormann et al.,
1997;Walker, 1992). Growth plate chondrocytes may be affected in the same manner
since they are rapidly turning over and are the foundation for bone growth. According to
the 1996 Feed Additive Compendium, the following antibiotics are approved as grth

promotants for poultry in the United States: Zinc bacitracin, bacitracin methylene

16

disalicylate, bamberrnycins, chlortetracycline, lincomycin, oxytetracycline, penicillin,
tylosin, tiamulin, and virginiamycin.

Recently, some antibiotics in the tetracycline family have been shown to
inhibit cartilage degradation of the embryonic chick tibiae. Other antibiotics implicated
in the disruption of bone metabolism include tobramycin, ceforanide, and quinolones. In
vitro data on the effects of tetracyclines and other antibiotics, which interfere with bone
metabolism, show that further investigation into the use of growth promoting antibiotics

in the poultry industry is needed.

Tetracyclines

Tetracyclines are broad spectrum antibiotics that have the ability to inhibit protein
synthesis as well as chelate cations. Tetracycline derivatives (tetracycline, doxycycline,
oxytetracycline, minocycline, and chlortetracycline) were shown to have an anti-
metalloproteinase property that is independent of their antimicrobial property (Golub et
al., 1984). The minimum requirement for anti-metalloproteinase activity by the
tetracyclines is the 4-ring structure and the oxygen molecules located on the B and C
rings, at carbons-11 and 12. These sites are responsible for the primary site of cation
binding at physiological pH. Whereas, the removal of the dimethylamino group from the
carbon-4 position of the A ring results in loss of only the antimicrobial activity.

We know from several sources that the tetracycline family affects the metabolism
of bone growth (Golub et al., 1991; Cole et al., 1994; Orth et al., 1997). In humans,
tetracyclines are not recommended for children or pregnant women, especially

doxycycline, minocycline and tetracycline due to their lipophilic nature (high rate of

17

diffusion into tissue). The tetracycline family has been found to cross the placental
barrier where they bind calcium, interfering with dental and bone growth in the fetus.
This adverse effect of the tetracycline family as well as permanent discoloration of teeth
in infants and children persists until 8 years of age, but many drug authorities suggest the
avoidance of tetracyclines until 12 years of age.

In vitra, tetracyclines have been investigated for their effects on bone formation.
At 20-40 ug/ml of doxycycline, MMP activity was reduced, PG loss was prevented, and
cell death and deposition of type X collagen was decreased (Cole et al., 1994).
Tetracyclines shown to inhibit MMP activity can do so by chelating cations (Golub et al.,
1991). By binding Ca2+ and more specifically Zn”, tetracyclines inhibit the activity of
collagenase and gelatinase, which require Ca2+ as a cofactor and Zn” at the active site.
These MMPs degrade the collagen matrix and allow cartilage to be remodeled and
vascularized. In vitra data have shown that 20 ug/ml minocycline, 40 ug/ml
doxycycline, 80 ug/ml tetracycline, and 80 ug/ml oxytetracycline inhibit cartilage
degradation, while chlortetracycline did not inhibit cartilage degradation at any
concentration tested (Orth et al., 1997). Tetracyclines easily diffuse into tissue due to
their lipophilic nature. Orth et al. (1997) correlated the lipophilic nature of the
antibiotics to the inhibitory property they possess; the more lipophilic the antibiotic, the
more it inhibits cartilage degradation.

Tetracyclines inhibit matrix degradation in several ways: inhibiting MMPS,
impairing vascularization, and decreasing protein synthesis. Doxycycline at low levels (5
ug/ml) decreases type X collagen synthesis as well as gelatinase and collagenase activity

in hypertrophic chondrocytes (Davies et al., 1996). Degradation of the ECM may release

18

matrix-bound activating factors of angiogenesis (Brown et al., 1993; Fisher et al., 1994;
Hirshman and Dziewiatkowski, 1996). Suomalainen et al. (1992), found that
doxycycline and minocycline inhibited tumor-induced angiogenesis in rabbit cornea,
possibly due to the anticollagenase action of the tetracyclines. Therefore, inhibiting
MMPS, which degrade the ECM, may impede penetration of new vessels. Furthermore,
tetracyclines inhibit protein synthesis by penetrating intracellular spaces of cells and
binding to ribosomes (Chopra, 1985).

Tetracyclines may interfere with the release of cytokines such as interleukin-1
and tumor necrosis factor (McNamara et al., 1997), which are natural stimulants of
cartilage degradation. Tetracyclines may also affect skeletal development by decreasing
nitric oxide (N 0) production through the inhibition of inducible nitric oxide synthase
mRN A expression and protein synthesis (Amin et al., 1996). Nitric oxide participates in
matrix degradation by up-regulating MMP activity (Murrell et al., 1995); when NO
production is reduced, MMPs may not be activated. Doxycycline at 20-50 [.1ng inhibits
collagenase and gelatinase activity, PG degradation, and NO production, as well as

decrease cell death associated with PG release (Amin et al., 1996; Cole et al., 1994).

F luoroquinolones
F luoroquinolones have good tissue penetration ability and target a broad spectrum
of gram-negative and gram-positive bacteria. Since they attain high concentrations in the
urine, quinolones are used to treat urinary tract infections. Side effects in humans and
animals include gastrointestinal disturbances and at high concentrations central nervous

system adverse reactions. In humans, tendenitous and tendon ruptures may also occur.

19

In young, rapidly growing animals, quinolones can induce arthropathy (Gough et al.,
1992). Arthropathy is defined as a chondrocyte toxicity causing abnormal conditions
within a joint and in severe cases lameness. The severity of symptoms increase with
increasing dosages. Quinolones are not recommended for children, adolescents, or
pregnant and nursing women due to this chondro-toxic effect.

The mechanism of action for quinolone-induced arthropathy may be related to
their magnesium (Mg2+) chelating property. Juvenile rats (3-6 weeks of age) deficient in
Mg2+ were shown to have identical lesions as those induced by quinolone treatment
(Vormann et al., 1997). Vormann et al (1997) found reduced Mg2+ concentrations in
hyaline cartilage of rats receiving a magnesium deficient diet between 3-5 weeks of age
compared to the rats fed the same diet at 8-11 weeks of age, suggesting a lower Mg2+
turnover in aged rats. By inducing cartilage lesions only at 3-5 weeks postnatally, the
Mg” deficiency correlates to the sensitive time for young rats to develop chondro-toxic
effects from quinolone treatment. Ciprofloxacin is a quinolone used in the treatment of
bone and joint infections. Further investigation into the use of this antibiotic was needed
after animal studies showed chondro-toxic effects. Ciprofloxacin has been shown to
decrease chondrocyte proliferation at 0.5 and 50 mg/l, which correspond to therapeutic
and toxic serum levels respectively. There was no effect on PG synthesis due to
ciprofloxacin. These data suggest that at increasing concentrations, ciprofloxacin affects
newly differentiated cells by inhibiting DNA synthesis (Mont et al., 1996).

Enrofloxacin is a more lipophilic quinolone than ciprofloxacin (Papick, 1998) but
ciprofloxacin is slightly more active than enrofloxacin. Oral treatment (25 mg/kg

Baytril®) of 15-28 week old growing puppies induced abnormal carriage of the carpal

20

joint and weakness in the hindquarters compared to no signs of joint limitation in puppies
29-34 weeks of age at the same dose level (Baytril® product information sheet, June
1997). According to the drug label, Baytril® is not recommended for small or medium
breeds of dogs during their rapid growth period (2-8 months of age). Large and giant
breeds have not been studied, but their growth phase can last until 12-18 months of age

and therefore limited use of enrofloxacin should be considered.

Cephalosporins

There are four generations of synthetic cephalosporins. Each generation differs in
their spectrum of activity against various gram-negative bacteria, in their susceptibility to
beta-lactamases, and in their ability to overcome bacterial resistance when other drugs
fail. Cephalosporins inhibit bacterial cell wall synthesis by tricking the peptidoglycan net
of the cell wall. They readily cross the placental barrier and should not be administered
to pregnant women. Rarely are side effects associated with these antibiotics.

Ceforanide, a long acting second-generation cephalosporin, was investigated with
respect to its ability to inhibit cartilage degradation of bacterial-induced arthritis. Smith
et al. (1987) found that glycosaminoglycan loss was inhibited only when the antibiotic
was administered before induction of arthritis. When given one day after arthritis
induction, decreased collagen loss occurred, but PG loss was only slowed. Proteoglycan
tissue concentration was the same as the control by day 21 (Smith et al., 1987). If
ceforanide can inhibit collagen loss in articular cartilage, it may also inhibit collagen loss

in grth plate cartilage, which would be detrimental to bone development.

21

Second-generation cephalosporins reach the synovial fluid. However, whether
third or fourth-generation compounds reach the synovial fluid is not clear. Ccfliofur, or
Naxcel®, is a third-generation cephalosporin. This antibiotic has a longer half-life than
most third-generation cephalosporins and is therefore used once daily and administered
less frequently. In the poultry industry, turkey poults may be given an injection of
Naxcel® at day one to decrease the incidence of poult enteritis. Research regarding bone
health has not been studied for this antibiotic. Anemia and thrombocytopenia have been
reported in dogs that received 3-5 times the approved daily dose of 2.2 mg/kg (Papick,

1998).

Aminoglycosides

Aminoglycosides include gentamicin, streptomycin, tobramycin, and neomycin.
These drugs inhibit protein synthesis and inaccurately translate mRNA at the ribosome.
Streptomycin has a site-specific protein inhibition whereas the others in this group act at
multiple sites to either inhibit synthesis or mistranslate the message. Side effects include
nephrotoxicosis, ototoxicosis, and vestibulotoxicosis.

In vitra, tobramycin was found to be toxic when embryonic chick tibiae where
exposed to concentrations higher than 0.5 mg/ml, and was shown to inhibit glucose
metabolism and matrix synthesis in bone (Murakami et al., 1996). Additionally, it
lowered the pH of the bone microenvironment, which inhibits bone formation. Protein
and collagen synthesis were inhibited and did not return to control values even after

administration of the antibiotic was discontinued. Murakami et al. (1996) showed that

22

exposure to high concentrations of tobramycin has damaging, long lasting effects on

osteoblast function.

23

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growth factor in avian tibial dyschondroplasia. Poultry Science 1996, 75, 130-
134.

Van Der Rest, M.; Garrone, R. Collagen family of proteins. The F ederatian of American
Sacietiesfor Experimental Biology Journal 1991, 5, 2814-2823.

Varghese, S.; Ramsby, M. L.; Canalis, E. Basic fibroblast growth factor stimulates
expression of interstitial collagenase and inhibitors of metalloproteinases in rat
bone cells. Endocrinology 1995, 136, 2156-2162.

Vater, C. A.; Harris, E. D., Jr.; Siegel, R. C. Native cross-links in collagen fibrils induced
resistance to human synovial collagenase. Biochemical Journal 1979, I81, 639-
645.

Vorrnann, J .; Forster, C.; Zippel, U.; Lozo, E.; Gunther, T.; Merker, H. J .; Stahlmann, R.
Effects of Magnesium Deficiency on Magnesium and Calcium Content in Bone
and Cartilage in Developing Rats in Correlation to Chondrotoxicity. Calcified
Tissue International 1997, 230-238.

Walker, R. D. Pharrnocokinetic Evaluation of Enrofloxacin Administered Orally to
Healthy Dogs. American Journal of Research 1992, 53, 2315-2319.

Wardale, R. J .; Duance, V. C. Collagen expression in chicken tibial dyschondroplasia
1996.109, 1119-1131.

World Health Organization. Environmental health criteria 10: carbon disulfide. Geneva:
World Health Organization 1979.

Wu, W.; Cook, M. E.; Chu, Q.; Smalley, E. B. Tibial dyschondroplasia of chickens
induced by fusarochromanone, a mycotoxin. Avian Diseases 1993, 3 7, 302-309.

30

Chapter 2

The effect of antibiotics on in vitra cartilage degradation

Introduction

Endochondral ossification, which occurs in the epiphyseal grth plate, is the
process by which long bones develop. Chondrocytes undergo a tightly regulated order of
proliferation, differentiation and hypertrophy. Subsequent to chondrocyte growth, the
extracellular matrix (ECM) is mineralized, vascularized, and finally invaded by
osteogenic cells from the bone marrow. This delicate balance of synthesis and
degradation (turnover) determines the ultimate health of long bones. Changes in the
sequence may result in pathological skeletal disorders.

Tibial dyschondroplasia (TD), which may result in lameness, is characterized by
an abnormality of the growth plate in which an avascular, uncalcified cartilage plug
accumulates near the metaphysis of the proximal tibiotarsi. The lesion contains more
matrix than normal cartilage (Thorp et al., 1991) and has an increased collagen and non-
reducible collagen cross-link concentration (Orth et al., 1991). Proteoglycans (PG), a
group of matrix proteins, interact with collagen to provide integrity and also regulate
matrix cation concentration (Kuettner, 1992). Chondrocytes of TD cartilage have a
decreased ability to synthesize matrix PG (Rosselot et al., 1994).

Many factors induce TD, some examples utilized in experimental studies include
nutrition imbalances, fungicides, and environmental conditions. The etiology of TD in
commercial poultry production remains unknown. Recently, some antibiotics in the
tetracycline family have been shown to inhibit cartilage degradation in an embryonic

chick tibia explant culture system (Orth et al., 1997). Other antibiotic families implicated

31

 

in the disruption of bone metabolism include aminoglycosides (Murakami et al., 1996),
cephalosporins (Smith et al., 1987), and quinolones (Vormann et al., 1997).
Furthermore, the increase in the standard feeding of grth promoting antibiotics
parallels with the rise in commercial TD incidence. Antibiotic families implicated in the
disruption of bone formation include tetracyclines, fluoroquinolones, cephalosporins, and
aminoglycosides. The poultry industry commonly utilizes antibiotics from these families
to treat disease and promote growth. In the present study, we determined whether certain
antibiotics inhibited in vitra cartilage degradation by quantifying indicators of cartilage

catabolism in an embryonic chick tibiae culture system.

Materials and Methods

Dulbecco’s modified Eagle’s medium (DMEM): nutrient mixture F-12 (Ham) and
the Penicillin/streptomycin additive were purchased from Gibco Laboratories (Grand
Island, NY). Fibroblast growth factor and human-recombinant insulin-like grth factor
were purchased from R&D Systems (Minneapolis, MN). Enrofloxacin (Baytril®) and
ceftiofur (Naxcel®) were purchased from the Michigan State University Veterinary
Teaching Hospital Pharmacy (East Lansing, MI). Salinomycin was purchased from ICN
Biomedical Research Products (Costa Mesa, CA). All other chemicals were purchased
from Sigma (St. Louis, MO).

Explant Cultures

Chick tibiae were isolated from 12-day-old leghom embryos. The bone was

isolated from the articular cartilage cap and muscle tissue (Cole et al., 1992). At this

stage of development, the tibiae contain two cartilaginous ends, a bony sheath, and bone

32

marrow (Fig 2). The cartilage ends contain a proliferative and hypertrophic zone similar
to that of growth plate cartilage (Schmid and Linsenmeyer, 1985).

Each treatment consisted of either two or three wells in a 24-well culture plate
(Becton Dickinson, Lincoln Park, NJ). Each of the wells contained three tibiae and 780
uL (260 ul/tibia) of Dulbecco’s modified Eagle’s medium (Table 1) supplemented with
growth factors (fibroblast growth factor 100 ng/ml and insulin-like growth factor-I 100
pg/ml), ascorbate (50 ug/ml) and treated with varying concentrations of antibiotics (Table
2). Tissue culture chick serum (5%) and lipopolysaccharide (10 ug/ml) were added to
stimulate cartilage catabolism. Treatments are listed in Table 2. Explants were
maintained in a humidified incubator with 7% C02 at 37 °C. Medium was replaced every
two days for 30 days or until the cartilage ends of the tibiae were degraded. All samples
were stored at 4 °C until analyzed for proteoglycan and nitric oxide concentrations; these
compounds have been found to be indicators of cartilage degradation in this explant
system (Cole et al., 1994; Orth et al., 1999). Each experiment was replicated to validate

results.

33

 

Proliferative Zone

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I Marrow Cavity
. . ;.l 'I 7,1, .7 1, ‘3:51;33:3}:33gziifif:13:35:32, :5” -----------
/
Bony Sheath

 

 

 

 

 

HypertrophicZone

 

 

 

Figure 2. Twelve-day embryonic chick tibia containing bone marrow,
a bony sheath, and two cartilage ends. The dotted areas depict the proliferative
zone and dashed areas show the hypertrophic zone.

34

 

Table 1. Composition of medium used to induce cartilage degradation in the embryonic
chick tibia explant culture system.*

 

 

 

 

 

 

 

 

 

 

Component Concentration
DMEM:F12 (1:1) ............
Sodium bicarbonate 44 mM
Lactalbumin hydrolysate 2 ug/ml
Sodium selenite 1 pg/ml
Manganese sulfate 169 ng/ml
Fibroblast grth factor 100 ng/ml
Insulin-like grth factor-I 100 pg/ml
LPS 10 ug/ml
TCCS 5%

 

 

 

 

* Amino acids were supplemented to enhance chondrocyte viability as outlined in
Rosselot et al. (1992)

LPS=lipopolysaccharide; TCCS=tissue culture chick serum; DMEM= Dulbecco’s
modified Eagle’s medium

35

Experiments

The effects on in vitra cartilage degradation of lincomycin hydrochloride (Sigma
lot # 096H09775), tylosin tartrate (Sigma lot # 085H10166), gentamicin (Sigma lot #
O28H2309), neomycin sulfate (Sigma lot # 075H09875), erythromycin (Sigma lot#
026H09865), doxycycline (Sigma lot# 59H0954), oxytetracycline (Sigma lot # 96H0571)
enrofloxacin (Baytril® injectable), ceftiofur (N axcel®), and salinomycin (ICN lot#
0050053) were determined. All antibiotics were tested at three concentrations, ranging
from 50 to 400 ug/ml. However, salinomycin had a testing range of 100 ng/ml to 10
ug/ml. A control group (with or without penicillin/streptomycin solution depending on
the time of year) was included in each experiment. Treatments are listed in Table 2.

In the months (April-July) that yeast contamination was expected, all treatment
groups were supplemented with a penicillin (10 U/ml)/streptomycin (10 ug/ml) mixture.
Yeast is a seasonal contaminant ubiquitous in the air, able to penetrate the filtration
system of the biological hoods used in these experiments. Preliminary studies showed
that 10 U/ml penicillin/ 10 ug/ml streptomycin mixture was an optimal concentration for
this tissue culture system and prevented contamination. The addition of
penicillin/streptomycin to the antibiotic treatment being tested did not affect PG or NO
release from the explants. Therefore, an interaction was not found to occur in this
system. These results agree with Murakami (1996), who also reported the addition of an
antibiotic solution routinely added to medium such as penicillin/streptomycin does not

interact with additional antibiotic treatment.

36

Table 2. Description of treatments for the tibia explant culture experiments.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Concentration Tested
Grows
Antibiotic-free
Control“ """
Doxycycline 50, 100, 200 ug/ml
Oxytetracycline 50, 100, 200 ug/ml
Lincomycin HCl 100,200,400 11ng
Tylosin tartrate 100, 200, 400 ug/ml
Gentamicin 100, 200, 400 ug/ml
Neomycin sulfate 100, 200, 400 ug/ml
Erythromycin 100, 200, 400 ug/ml
Enrofloxacin 100, 200, 400 ug/ml
Ccfiiofur 100, 200, 400 ug/ml
Salinomycin 1'0’ 1186:2211 and

 

* A 10 U/ml Penicillin/ 10 ug/ml streptomycin mixture was used in treatments
when yeast contamination was expected (April-July); HCl=hydrochloride

37

Analyses

Proteoglycan (PG) content of the conditioned media (CM) was determined using
the dimethylmethylene blue assay described by Chandrasekhar (1987). Chondroitin
sulfate (3 proteoglycan) from bovine cartilage was used as the standard. To determine
the PG content of the CM, the sulfated glycosaminoglycan content (pg PG/ well) was
measured at absorbencies of 540 and 595 nm using a SpectraMax 300 plate reader
(Molecular Devices, Sunnyvale, CA).

Nitric oxide (N O) was measured in the CM using the method outlined by Blanco
et al. (1995). Quantitative measurements of nitrite, a stable end-product of nitric oxide
metabolism, were measured via the Greiss reaction at an absorbency of 540 nm using a
SpectraMax 300 plate reader (Molecular Devices, Sunnyvale, CA). A standard curve of

sodium nitrite was used and results are expressed as nmol NO/ well.

Statistical analysis
Data were analyzed using the repeated measures option of SAS (1999) PROC
MIXED statistical software. The total amount of PG or NO released into the CM
between days 6 and 16 of culture was analyzed to determine significance between
treatment groups. The first two collection days were not analyzed to allow for
equilibration of tibiae to treatments. Treatments were compared using the least squares
mean difference procedure. Significance was considered at p S 0.05 and a trend was

considered at p S 0.10.

38

Results

Antibiotics shown to inhibit in vitro cartilage degradation included doxycycline,
oxytetracycline, enrofloxacin, cefliofur and salinomycin (Table 3). Lincomycin, tylosin
tartrate, gentamicin, neomycin sulfate, and erythromycin did not alter the cartilage’s
catabolic metabolism at any concentration tested (100, 200, or 400 ug/ml). In all
treatment and control groups, complete cartilage degradation (visual examination)
occurred by day 16 of culture.

Doxycycline at 200 ug/ml inhibited cartilage degradation (Fig 3); PG ( pg 0.01)
and NO (p5 0.001) release were decreased when compared to the control.
Another tetracycline, oxytetracycline (200 ug/ml) also showed significant inhibition of
degradation. Oxytetracycline inhibited release of PG into the CM Q35 0.008; Fig 3A) as
compared to the control. Nitric oxide release into media was less than the control (p_<_
0.002; Fig 3B). The cartilage ends of the tetracycline-treated tibiae remained intact for
the entire culture period whereas the control tibiae were degraded by day 14-16 of
culture. The bones of the tetracycline treated groups were discolored (brown tinted) by
day 8 of culture.

Enrofloxacin inhibited PG release in the CM at 200 (p5 0.003) and 400 ug/ml (p5
0.004) compared to the antibiotic-free treatment (Fig. 4A). At the low dose of
enrofloxacin, 100 ug/ml, PG release was decreased (p_<_ 0.08). Nitric oxide concentration
in the CM at the highest and intermediate doses of enrofloxacin was lower than in the
control (p_<_ 0.007 and p5 0.006, respectively; Fig 48). Once again, the lowest dose only

decreased NO concentrations (135 0.06). Cartilage ends in all three treatment groups

39

Table 3. Total release of proteoglycan or nitric oxide into conditioned media between
days 6 and 16 of culture. The tables are broken into three groups in order to compare the
treatments to the control group within its experiment. Observations without p-values were

not different from controls.

 

 

 

Treatment Group (n=2) Total [PG] Total [NO]
pg PG/ well 11M NO/ well
Control 509 :t 62 118 :1: 14
200 ug/ml Doxycycline 214 i 26 (p: 0.01) 16 :l: 3 (p5 0.001)

 

 

200 ug/ml Oxytetracycline

 

194 at 21 (p5 0.008)

 

21 :t 3 (1350.002)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Group (n=2) Total [PG] Total [NO]
pg PG/ well pM NO/ well
Control 711 d: 25 103 i 5
100 ug/ml Enrofloxacin 480 i 78 (p5 0.08) 42 :1: 6 (p5 0.06)
200 ug/ml Enrofloxacin 340 i 57 (p5 0.003) 17 d: 3 (p_<_ 0.006)
400 ug/ml Enrofloxacin 169 i 20 (p5 0.004) 22 i 1 (p5 0.007)
100 ug/ml Ceftiofur 725 i 45 141 i 14
200 ug/ml Ceftiofur 511 :1: 38 53 i 9
400 ug/ml Ceftiofur 224 :t 20 (p5 0.007) 14 :L- 3 (p5 0.005)
Treatment Group (n=3) Total [PG] Total [NO]
ug PG/ well 11M NO/ well
Control 755 i 89 143 :t 13
100 ng/ml Salinomycin 319 i 70 65 :1: 17
1 ug/ml Salinomycin 456 i 72 Q35 0.06) 70 i 15
10 ug/ml Salinomycin 244 d: 11 (p5 0.03) 24 i 3 (p: 0.03)

 

 

 

40

 

 

 

appeared intact after 30 days of culture, indicating inhibited cartilage degradation at all
concentrations tested.

Only at 400 ug/ml did ceftiofur inhibit PG release (p5 0.007; Fig 5A) and NO
production (p5 0.005; F ig 5B) when compared to the control. At the high dose, ceftiofirr
had intact cartilaginous ends at the end of the culture period (day 30). The cartilage ends
of the intermediate dose remained intact through day 22 of culture, and the low dose was
completely degraded by day 18 of culture.

The last antibiotic to inhibit cartilage degradation was salinomycin. This
antibiotic was lethal to chondrocytes at concentrations 2 100 [.1ng (data not shown);
therefore, lower concentrations were tested. An additional control was added to these
experiments because salinomycin had to be dissolved in methanol. The methanol control
group was not significantly different from the control for either CM analysis performed.
Salinomycin at 10 ug/ml had the same inhibitory effect as the previous antibiotics tested;
PG release was lower than the control (p: 0.03; Fig 6A) and NO production was
decreased (p5 0.03; Fig 63). At 1 11ng salinomycin, a trend for decreased PG release
was seen (p5 0.06), but NO was not reduced. However, at 100 ng/ml, salinomycin
appeared to increase the rate of cartilage degradation relative to the control but did not

differ significantly in PG release or NO production.

Discussion
These results show that some of the antibacterial agents used in the poultry
industry inhibit in vitra embryonic chick cartilage degradation. Antibiotics are used in

the poultry industry to treat disease, increase disease resistance, reduce mortality,

41

increase feed efficiency, and promote growth in less than ideal environmental conditions.
Although they are used as a management tool to increase the livability and economic
value of the animal, certain antibiotics may add to the cull and condemnation rates
associated with skeletal disorders.

Tetracyclines are broad-spectrum, rapidly absorbed antibiotics that have recently
been found to have an anti-metalloproteinase property that is separate from its
antibacterial property. The site for metal binding on the four-ring structure is located in
the B and C rings, specifically the oxygen molecules at the 11 and 12-carbon-position
(Fig. 7) (Golub et al., 1991; Duarte et al., 1999). The ability of tetracyclines to bind
metal ions (Zn2+, Ca2+ and Mg”) may be the primary mechanism by which tetracyclines
inhibit cartilage degradation. Matrix metalloproteinases (MMP) are enzymes responsible
for the degradation of ECM in many body organs; they require zinc at their active site
and calcium as a cofactor. Tetracyclines have been reported to inhibit some of these
enzymes including: skin collagenase (Golub et al., 1983), cartilage, synovial, and corneal
collagenase (Golub et al., 1991; Greenwald et al., 1992; Burns et al., 1989) neutrophil
collagenase (MMP-8) (Suomayor et al., 1992; Smith et al., 1996), gelatinase A (MMP-2)
(Lu et al., 1991) and gelatinase B (MMP-9) (Nip et al., 1993). The derivative,
concentration, and experimental procedure differed between the aforementioned studies.
Smith et al. (1999) found that the sensitivity of the MMP to tetracyclines depends on the
MMPs structure and whether it can be altered by the binding of tetracyclines to an
enzyme-associated Ca2+. Matrix metalloproteinases that have a hemopexin-like domain
of MMP-13 or a catalytic domain of MMP-8 show significant inhibition against type II

collagen when cultured with concentrations of doxycycline, whereas the MMP-1

42

structure is resistant to tetracycline reconfiguration (Smith et al., 1999). Smith’s results
seems logical since in the present explant culture model calcium was present in excess;
there would be enough calcium available for MMP activity even if the tetracyclines
bound 100%. However, the tetracyclines could have bound zinc and rendered the MMP
inactive.

The present study showed that two tetracyclines, oxytetracycline and doxycycline,
were able to prevent cartilage degradation of embryonic chick tibiae. This result agrees
with previous reports on the tetracycline family (Orth et al., 1997; Cole et al., 1994).
Proteoglycan and nitric oxide were decreased in the tissue treated with tetracyclines when
compared to the control. Doxycycline was more potent than oxytetracycline, which
agrees with earlier studies showing that the more lipophilic the tetracycline, the greater
the inhibition of cartilage degradation (Orth et al., 1997).

Nitric oxide is a multifunctional mediator produced by nitric oxide synthases
(NOS). Nitric oxide participates in the inflammatory and autoimmune mediated response
of tissue degradation. Explant tibiae in the present study were stimulated with the
endotoxin, LPS, to initiate cartilage degradation. When treated with tetracyclines, NO
production was inhibited compared to the control. Attur et al. (1999) reported that
doxycycline blocked NO in LPS-stimulated bovine cartilage as well as human
osteoarthritis cartilage. Doxycycline and minocycline inhibit expression of inducible
NOS (Amin et al., 1996). Nitric oxide is thought to activate MMPs (Golub et al., 1991;
Orth et al., 2000) and therefore, if down-regulated, cartilage degradation would decline

due to decreased enzyme activity.

43

Proteoglycan release into media is one way of quantifying cartilage matrix
degradation. In the current study, PG release was significantly inhibited by doxycycline
and oxytetracycline. In many studies a reduced PG release correlated with inhibition of
MMP activity. Cole et al. (1994) found that 40 ug/ml of doxycycline prevented PG loss
from the matrix, cell death and deposition of type X collagen and increased the
hypertrophic region of the cartilage.

Fluoroquinolones, which also chelate cations, have broad spectrum antibacterial
properties (they inhibit DNA gyrase, which is responsible for DNA supercoiling) and
excellent tissue penetration. Like the tetracyclines, quinolones are ringed structures. In
young, rapidly growing animals it is known that these drugs cause arthropathy, a
condition characterized by vesicles forming on the articular cartilage surface causing
lameness in the affected animal (for a complete review see Gough et al., 1992).
Quinolone arthropathy (QAP) may not occur in adults because of the drugs inability
affect mature tissue. An experimental trial on racing pigeons showed that 800 ppm
enrofloxacin given over a long time period did not induce abnormalities in the adult
birds, but increased embryo mortality in eggs of treated birds. Chicks raised from these
birds had joint lesions such as arthropathy (Allen, 1998).

In the present study enrofloxacin was tested. It prevented PG loss and NO
production in the embryonic chick explant model. Beluche et al. (1999) reported that
high concentrations of enrofloxacin (>1000 ug/ml) inhibited synthesis and increased
degradation of PG in equine articular cartilage explants whereas low doses (2 and 10

ug/ml) did not affect PG metabolism. These high doses of enrofloxacin also were

44

associated with chondrocyte toxicity. In our work, enrofloxacin did not cause cytotoxic
effects (data not shown).

Enrofloxacin is partially metabolized to ciprofloxacin (Allen, 1998).
Ciprofloxacin is slightly more active than enrofloxacin, but enrofloxacin is more
lipophilic. Mont et al. (1996) showed that ciprofloxacin did not inhibit PG synthesis of
human chondrocytes, but inhibited cell proliferation at therapeutic serum concentrations.
Others have reported that quinolones inhibit glycosaminoglycan synthesis initially and
DNA synthesis secondarily (Kato and Onodera, 1988a; Kato and Onodera, 1988b; Kato
et al., 1995; Takada et al., 1994; Takayama et al., 1995). One proposed mechanism is
the quinolones ability to form chelates with magnesium. Magnesium is needed for cell
proliferation and cell-matrix interactions. Juvenile rats have been shown to develop QAP
(age 5 5 wks) whereas adults (age > 8 wks) do not develop lesions (Mayer, 1987; Kato
and Onodera, 1988b). Voorrnan et a1 (1997) reported that rats less than 4 weeks of age
have lower magnesium concentrations and only at this age can cartilage lesions be
induced by feeding magnesium-deficient diets. How these drugs affect growth plate
chondrocytes is not known

Cephalosporins are widely used in human as well as veterinary medicine. They
are broad spectrum, fast acting drugs that inhibit bacterial cell wall synthesis and are
known to reach synovial fluid. Ceftiofur is a cephalosporin with a relatively long half-
life; it is used to treat most gram-negative urinary tract pathogens as well as systemic
infections. To my knowledge, no studies have linked cephalosporins to cartilage
abnormalities. However, Smith (1987) showed that a therapeutic dose of ceforanide (15

mg/kg), another member of the cephalosporin family, given before inoculation with

45

bacteria prevented PG loss in rabbit articular cartilage. In the present study only the high
dose of the cephalosporin (400 ug/ml ceftiofur) inhibited PG loss and NO production.
Cephalosporins are also a ring structure, but have not been reported to bind cations.

Salinomycin is a polyether antibiotic that has ionophoric properties and is known
to transport monovalent cations, especially sodium and potassium, across biological
membranes (Dobler, 1981), which may explain why it killed chondrocytes in the present
explant model at concentrations over 100 ug/ml. This antibiotic increased the rate of PG
release at 100 ng/ml yet inhibited PG loss at 10 ug/ml. Why this occurred in not
understood. Possibly cell metabolism was increased due to the excess activity of
sodium/potassium channels at the lowest dose, yet at the highest dose it interfered with
MMP activity similar to other antibiotics tested. Salinomycin could also interfere with
intracellular Ca2+ concentrations, leading to changes in chondrocyte metabolism.
Ionophore use has been associated with increased leg problems (Leeson and Summers,
1988). However, monensin (another ionophore) fed at 121 ppm to broilers reduced
mortality due to leg abnormalities (Chapman et al., 1995).

These data confirm published results concerning tetracyclines, fluoroquinolones,
cephalosporins and other categories of antibiotics regarding inhibition of cartilage
degradation. For certain diseases such as osteoarthritis, diabetes and periodontal
disorders the ability to stop cartilage degradation is utilized for treatment. However,
growth plate cartilage develops into bone by tightly regulating cartilage turnover. If this
is disrupted, skeletal disorders such as TD could arise. These antibiotics all inhibited PG
release and NO production in an embryonic chick explant culture system. However, they

may all act by different mechanisms and possibly affect development at critical growth

46

periods. The ability to chelate cations is a recurring theme that needs further
investigation regarding these families of antibacterial agents. Future research should
determine if these antibiotics inhibit in viva growth plate cartilage degradation at doses

relevant to the poultry industry.

47

Figure 3. (Next page) Proteoglycan and nitric oxide release into conditioned media from
chick tibia explants with two tetracycline treatments (n=2). Panel A depicts the
proteoglycan release into conditioned media throughout the 30-day culture period.
Decreased proteoglycan concentrations were found for oxytetracycline (200 ug/ml) and
doxytetracycline (200 ug/ml) between day 6 and 16 of culture compared to the control
(p= 0.008, p= 0.01). Panel B depicts the nitric oxide production released into conditioned
media throughout the 30-day culture period. Decreased nitric oxide concentrations were
found for oxytetracycline (200 ug/ml) and doxycycline (200 ug/ml) between day 6 and

16 of culture compared to the control (p= 0.002; p= 0.001).

48

 

 

ug PG/well
§

200

 

 

150

+ control

A + 200 ug/ml doxycycline
-O- 200 ug/ml oxytetracycline

 

LII
O
n

   

 

 

 

/\

 

 

 

 

6 8 10 12 14 16 18 20

Days in culture

 

 

 

M NO /well

 

+ control

 

40

30

+ 200 ug/ml doxycycline
-O- 200 ug/ml oxytetracycline

 

 

 

 

 

 

 

 

 

8 10 12 14 16 18 20
Days in culture

 

49

 

 

Figure 4. (Next page) Proteoglycan and nitric oxide release into conditioned media
from chick tibia explants with enrofloxacin treatments (n=2). Panel A depicts the
proteoglycan release into conditioned media throughout the 30-day culture period.
Decreased proteoglycan release was found for enrofloxacin at the 200 ug/ml (p= 0.003)
and 400 ug/ml (p= 0.004) concentrations between day 6 and 16 of culture compared to
the control. A trend for decreased proteoglycan release existed for the 100 ug/ml
enrofloxacin treatment (p= 0.09). Panel B depicts the nitric oxide production released
into conditioned media throughout the 30-day culture period. Decreased nitric oxide
concentrations were found for enrofloxacin at the 200 ug/ml (p= 0.007) and 400 ug/ml
(p= 0.006) concentrations between day 6 and 16 of culture compared to the control. A
trend for decreased nitric oxide production existed for the 100 ug/ml enrofloxacin

treatment (p= 0.06).

50

 

 

 

 

 

 

  
   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

+control g
200 R -I- lOOug/ml enrofloxacin
= + ZOOug/ml enrofloxacin
; 150 - --400ug/ml enrofloxacin
2
on 100 -
1 |
50
O I I I I I I I I I I I I I I I
2 4 6 810121416182022242628
Days in culture
30
+control
= + 100 ug/ml enrofloxacin
; 20 - I +200 ug/ml enrofloxacin
5 r -- 400 ug/ml enrofloxacin
7‘ l
2
:1.

 

 

 

2 4 6 810121416182022242628

Days in culture

 

51

 

Figure 5. (Next page) Proteoglycan and nitric oxide release into conditioned media
from chick tibia explants with ceftiofur treatments (n= 2). Panel A depicts the
proteoglycan release into conditioned media throughout the 30-day culture period.
Decreased proteoglycan concentrations were found only for the high dose of ceftiofur
(400 ug/ml) between day 6 and 16 of culture compared to the control (p= 0.007). Panel
B depicts the nitric oxide production released into conditioned media throughout the 30-
day culture period. Decreased nitric oxide concentrations were found for the same
concentration of ceftiofur between day 6 and 16 of culture compared to the control (p=

0.005).

52

 

 

 

 

 

  

 

  
  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

200
+control
_ g -I- 100ug/ml ceftiofur
35 150 + 200ug/ml ceftiofur
E -- 400ug/rnl ceftiofur
E ’/ ‘W \ / \'
it 50 4
O I I I I I I I I I I I I 1 I
246810121416182022242628
Days in culture
50
A +control
40 -I- 100 ug/ml ceftiofur _
-O- 200 ug/ml cefliofur
E —-— 400 ug/ml ceftiofur
E 30
o I" '\ / All
Z
7 .1
0 I I I I
246810121416182022242628

Days in culture

 

53

 

 

 

Figure 6. (Next page) Proteoglycan and nitric oxide release into conditioned media
from chick tibia explants with salinomycin treatments (n=3). Panel A depicts the
proteoglycan release into conditioned media throughout the 30-day culture period.
Decreased proteoglycan concentrations were found for salinomycin at 10 ug/ml between
day 6 and 16 of culture compared to the control (p= 0.03). A trend for decreased
proteoglycan release existed for the 1 ug/ml salinomycin treatment (p= 0.06). Panel B
depicts the nitric oxide production released into conditioned media throughout the 30-day
culture period. Decreased nitric oxide concentration was found for 10 ug/ml salinomycin

between day 6 and 16 of culture compared to the control (p= 0.03).

54

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

250 + control
I k A -I- 100 ng/ml salinomycin
200 -O- 1 ug/ml salimomycin
__ A ‘\Q\ -- 10 ug/ml salinomycin
15
a; 150
on 100-
:3.
50-
0 I I I I I I I I I I
2 4 6 8 10 12 14 16 18 20
Days in culture
50 + control
-I- 100 ng/ml salinomycin
40 ' +1 11ng salinomycin
'7: -- 10 ug/ml salinomycin
E 30
O
Z220
a (.2 g
10-
0 I I I I I I

 

 

2 4 6 8 10 12 14 16 18

Days in culture

 

55

 

 

 

 

Figure 7. Tetracycline molecule. Dashed box highlights major metal binding site.
The four-ring structure and this location are needed for inhibition of collagenase. R1
and R2 are the sites where the tetracycline derivatives differ in molecular structure.

 

56

 

References

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1998.

Amin, A. R.; Attur, M. G.; Thakker, G. D.; Patel, P. D.; Vyas, P. R.; Patel, R. N.; Patel, 1.
R.; Abramson, S. B. A novel mechanism of action of tetracyclines: effects on

nitric oxide synthases. Proceedings of the National Academy of Sciences of the
United States of American 1996, 93, 14014-9.

Attur, M. G.; Patel, R. N.; Patel, P. D.; Abramson, S.B.; Amin. A. R. Tetracycline Up-
Regulates COX-2 Expression and Prostaglandin E2 Production Independent of its
Effect on Nitric Oxide. Journal of Immunology 1999, 162, 1360-3167.

Beluche, L. A. ; Bertone, A. L.; Anderson, D. E.; Kohn, C. W.; Weisbrode, S. E. In
vitra dose-dependent effects of enrofloxacin on equine articular cartilage.
American Journal of Veterinary Research 1999, 60, 577-582.

Blanco, F.J.; Ochs, R.L.; Schwarz, H.; Lotz, M. Chondrocyte apoptosis induced by nitric
oxide. American Journal of Pathology 1995, 146, 75-85.

Burns, F .; Stack, M.; Gray, R.; Paterson, C. Inhibition of alkali-induced corneal

ulceration and perforation by a thiol peptide. Investigative Ophthalmology and
Visual Science 1989, 30, 1569-1575.

Chandrasekhar, A.; Esterrnan, M.A.; Hoffman, H.A. Microdeterrnination of PCs and
glycosaminoglycans in the presence of guanidine hydrochloride. Analytical
Biochemistry 1987, 161, 103-108.

Chapman, M. E.; Chapman, H. D.; Wideman, R. F.; Huff, W. E.; Hacker, A. H.; Rath, N.
C.; Balog, J. M. Does pulmonary hypertension syndrome (ascites) occur more

frequently in broilers medicated with monensin? Poultry Science 1995, 74, 1591-
1596.

Cole, A. A.; Luchene, L. J .; Linsenmeyer, T. F.; Schmid, T. M. The influence of bone
and marrow on cartilage hypertrophy and degradation during 30-day serum-free
culture of the embryonic chick tibia. Developmental Dynamics 1992, 193, 277-
285.

Cole, A. A.; Chubinskaya, S.; Luchene, L. J .; Chlebek, K.; Orth, M. W.; Greenwald, R.
A.; Kuettner, K. E.; Schmid, T. M. Doxycycline disrupts chondrocyte
differentiation and inhibits cartilage matrix degradation. Arthritis and
Rheumatism 1994, 37, 1727-1734.

57

Dobler, M. Ianaphares and Their Structures; John Wiley & Sons: New York, 1981.

Duarte, H. A.; Carvalho, S.; Paniago, E. B.; Simas, A. M. Importance of Tautomers in the
Chemical Behavior of Tetracyclines. Journal of Pharmaceutical Sciences 1999,
88, 111-120.

Golub, L. M.; Lee, H. M.; Lehrer, G.; Nemiroff, A.; McNamara, T. F .; Kaplan, R.;
Ramamurthy, N. S. Minocycline reduces gingival collagenolytic activity during

diabetes: preliminary observations and a proposed new mechanism of action.
Journal of Periodontal Research 1983, 18, 516-523.

Golub, L. M.; Ramamurthy, N. S.; McNamara, T. F. Tetracyclines inhibit connective

tissue breakdown: new therapeutic implications for an old family of drugs.
Critical Reviews of Oral Biological Medicine 1991, 2, 297-322.

Gough, A.W.; Kasali, O.B.; Sigler, R.E. Quinolone arthropathy- acute toxicity to
immature articular cartilage. T axicalagical Pathology 1992, 20, 436-447.

Greenwald, R. A.; Moak, S. A.; Ramamurthy, N. S.; Golub, L. M. Tetracyclines
suppress matrix metalloproteinase activity in adjuvant arthritis and in combination

with flurbiprofen, ameliorate bone damage. Journal of Rheumatalagy 1992, 19,
927-938.

Havenstein, G. B.; Ferket, P. R.; Scheideler, S. E.; Larson, B. T. Growth, livability, and
feed conversion of 1957 vs 1991 broilers when fed "typical" 1957 and 1991
broiler diets. Poultry Science 1994, 73, 1785-1794.

Kato, M.; Onodera, T. Effect of ofloxacin on the uptake of [3H]thymidine by articular
cartilage cells in the rat. Toxicology Letters 1988a, 44, 131-142.

Kato, M.; Onodera, T. Morphological investigation of cavity formation in articular

cartilage induced by ofloxacin in rats. Fundamental and Applied Toxicology
1988b, 11,110-119.

Kato, M.; Takada, S.; Ogawara, S.; Takayama, S. Effect of levofloxacin on
glycosaminoglycan and DNA synthesis of cultured rabbit chondrocytes at
concentrations inducing cartilage lesions in viva. Antimicrobial Agents and
Chemotherapy 1995, 39, 1979-1983.

Kuettner, K. The biochemistry of articular cartilage in health and disease. Clinical
Biochemistry 1992, 25, 155-163.

Leach, R. M., Jr. Poultry industry should reconsider if bigger is better. F eedstufis 1996,
68, 10.

58

Leeson, S.; Summers, J. D. Some nutritional implications of leg problems with poultry.
British Veterinary Journal 1988, 144, 81-92.

Lu, L. J .; Jr, G. S.; Hasty, K.; Brandt, K. Doxycycline inhibits type XI collagenolytic
activity in human osteoarthritic cartilage. Journal of Rheumatalagy 1991, 18,
1450-1452.

Mayer, D. G. Overview of toxicological studies. Drugs 1987, 34 (suppl 1), 150-154.

Mont, M. A.; Mathur, S. K.; Frondoza, C. G.; Hungerford, D. S. The Effects of
Ciprofloxacin on Human Chondrocytes in Cell Culture. Infection 1996, 24, 151-
155.

Murakami, T.; Murakami, H.; Ramp, W. K.; Hanley, E. N. J. Interaction of tobramycin
and pH in Cultured Chick Tibiae. Journal of Orthopaedic Research 1996, 742-
748.

Nip, L.; Uitto, V.; Golub, L. Inhibition of epithelial cell matrix metalloproteinases by
tetracyclines. Journal of Periodontal Research 1993, 28, 379-385.

Orth, M. W.; Martinez, D. A.; Cook, M. E.; Vailas, A. C. Nonreducible crosslink
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homocysteine. Biochemical and Biophysical Research Communication 1991, 1 79,
1582-1586.

Orth, M. W.; Chlebek, K. A.; Cole, A. A.; Schmid, T. M. Tetracycline derivatives inhibit
cartilage degradation in cultured embryonic chick tibiae. Research in Veterinary
Science 1997, 67, 11-14.

Orth, M.; Fenton, J.; Chlebek-Brown, K. Biochemical characterization of cartilage
degradation in embryonic chick tibial explant cultures. Poultry Science 1999, 78,
1596—1600.

Orth, M.; Peters, T.; Chlebek-Brown, K. Cartilage Turnover in Embryonic Chick Tibial
Explant Cultures. Poultry Science 2000, 79, 990-993.

Rosselot, G.; Sokol, C.; Leach, R. M. Effect of lesion size on the metabolic activity of
tibial dyschondroplastic chondrocytes. Poultry Science 1994, 73, 452-456.

SAS: SAS/STAT User's Guide, Version 8.00. Cary, NC: SAS Institute Inc., 1999

Schmid, T. M.; Linsenmeyer, T. F. Developmental acquisition of type X collagen in the
embryonic chick tibiotarsus. Developmental Biology 1985a, 107, 373-381.

59

Smith, R. L.; Schurman, D. J .; Kajiyama, G.; Mell, M.; Gilkerson, E. The Effect of
Antibiotics on the Destruction of Cartilage in Experimental Infectious Arthritis.
The Journal of Bone and Joint Surgery 1987, 69-A, 1063- 1068.

Smith, G. N., Jr.; Brandt, K. D.; Hasty, K. A. Activation of recombinant human
neutrophil procollagenase in the presence of doxycycline results in fragmentation

of the enzyme and loss of enzyme activity. Arthritis and Rheumatism 1996, 39,
235-244.

Smith, G. J .; Mickler, E.; Hasty, K.; Brandt, K. Specificity of inhibition of matrix
metalloproteinase activity by doxycycline. Arthritis and Rheumatism 1999, 42,
1140-1146.

Suomayor, K.; Sorsa, T.; Ingman, T.; Lindy, O.; Golub, L. Tetracyclines inhibition
identifies the cellular origin of interstitial collagenase in human periodontal
diseases in viva. Oral Microbiology and Immunology 1992, 7, 121-123.

Takada, S.; Kato, M.; Takayama, S. Comparison of lesions induced by intra-articular
injections of quinolones and compounds damaging cartilage components in rat
femoral condyles. Journal of Toxicological Science 1994, 42, 73-88.

Takayama, S.; Hirohashi, M.; Kato, M.; Shimada, H. Toxicity of quinolone antimicrobial
agents. Journal of Toxicology and Environmental Health 1995, 45.

Thorp, B. H.; Whitehead, C. C.; Rennie, J. S. Avian tibial dyschondroplasia: a
comparison of the incidence and severity as assessed by gross examination and
histopathology. Research in Veterinary Science 1991, 51, 48-54.

Vormann, J.; Forster, C.; Zippel, U.; Lozo, E.; Gunther, T.; Merker, H. J .; Stahlmann, R.
Effects of Magnesium Deficiency on Magnesium and Calcium Content in Bone
and Cartilage in Developing Rats in Correlation to Chondrotoxicity. Calcified
Tissue International 1997, 230-238.

60

Chapter 3
The effect of antibiotics on body weight, feed conversion, and tibial
dyschondmplasia scores of 3-week-old broiler cockerels

Introduction

Poultry skeletal disorders have been estimated to cause losses of approximately
$160 million per year to the United States broiler and turkey industry (Sullivan, 1994).
One of the major metabolic skeletal disorders that effect the long bones of fast growing
meat producing birds (broiler chickens, turkeys, and ducks) is tibial dyschondroplasia
(TD). A developmental abnormality of the tibiotarsal bone, TD originates in the growth
plate where it disrupts the normal balance between cartilage synthesis and degradation.
Growth plates of birds affected with TD undergo cell proliferation at a normal rate
whereas hypertrophic chondrocytes within these lesions only reach 40% for their normal
size (Hargest et al., 1985). Due to inhibited degradation, an avascular cartilage plug
accumulates in the metaphyseal region of the tibia, causing impairment of long bone
growth and clinical TD lesions. Lameness, decreased feed efficiency, and increased
mortality, culls, and condemnations at slaughter can be the results of TD.

Modern broilers have an 18 to 63% incidence of TD (Praul et al., 2000;Roberson,
1999; Elliot and Edwards, 1997). Knowing the etiology of such a debilitating metabolic
disorder would benefit the poultry industry. Experimental conditions and compounds
that induce TD include: copper deficiency; fusarochromanone, thiram, and antabuse
toxicity; excessive dietary levels of cysteine, homocysteine and histidine; calcium and
phosphorus imbalance, and metabolic acidosis (Orth and Cook, 1994). Genetics and

environmental factors such as time of year and rearing conditions also induce TD (Orth

61

and Cook, 1994). Many hypotheses have been presented, but the cause of TD in the
commercial industry is still not known.

Recently, some antibiotics in the tetracycline family have been shown to
inhibit cartilage degradation in an embryonic chick tibiae explant culture system (Orth et
al., 1997). Other antibiotic families implicated in the disruption of bone metabolism
include aminoglycosides (Murakami et al., 1996), cephalosporins (Smith et al., 1987),
and quinolones (Vormann et al., 1997). Antibiotics have not been documented to disrupt
growth plate cartilage metabolism directly. However, antibiotics routinely used in the
industry are derivatives or members of these antibiotic families and may cause TD in the
field. We have observed a parallel increase between the use of antibiotics and the rise in
commercial TD incidence.

In our laboratory, certain antibiotics were found to inhibit in vitra cartilage
degradation. Enrofloxacin, ceftiofur, doxycycline, oxytetracycline, and salinomycin
significantly inhibited proteoglycan (PG) release and nitric oxide (N 0) production from
embryonic chick tibia cartilage (Chapter 2). Therefore, the purpose of this research was
to determine if these antibiotics induced TD lesions in the proximal tibial grth plate
when supplemented to broilers (0-3 weeks of age) at 25, 100 and 400% above the

recommended use level.

62

Materials and Methods

Terramycin® 50 (50 g/ lb oxytetracycline HCl; Pfizer Inc., New York, NY) and
Aeuromycin® 50 (50 g/lb chlortetracycline HCl; Roche Vitamins, Inc., Parsippany, NJ)
were purchased from the Michigan State University feed mill (East Lansing, MI).
Naxcel® (50 mg/ml ceftiofur; SmithKline Beecham Corp., Philadelphia, PA) was
purchased from the Michigan State University Veterinary Teaching Hospital pharmacy
(East Lansing, MI). Bio-Cox® 60 (60 g/ton salinomycin) was generously donated by
Roche Vitamins (Gibsonville, NC). Concentrated liquid Baytril® (3.23% enrofloxacin;
Bayer, Shawnee Mission, KA) was used in this study. Thiram and doxycycline were

purchased from Sigma (St. Louis, MO).

Trials

To determine if certain antibiotics inhibit cartilage degradation and induce tibial
dyschondroplasia in growing broilers, day-old cockerels (Ross x Arbor Acres) were
purchased from Hoover Hatchery (Rudd, Iowa) and housed at the Michigan State
University Poultry Science Research and Teaching Center (East Lansing, MI). All
Animal Use and Care regulations were followed. The day-old chicks were randomly
wing-banded and placed in an electrically heated wire-floored battery brooder at eight
birds per pen with three pens per treatment. A control and three treatment groups per
antibiotic were established with 24 birds per group. The chicks were raised on a

continuous illumination schedule with both incandescent (in the room) and fluorescent

63

lighting (in the pen). Feed and water were available ad libitum and diets (Table 4) met or
exceeded all NRC requirements throughout the 21-day experimental period.

Recommended use levels for poultry of the antibiotics tested were obtained from
the 1999 Feed Additive Compendium. Oxytetracycline and chlortetracycline treatment
groups were administered antibiotics in their feed at 25%, 100%, or 400% above the
recommended use level (except salinomycin, which was administered at 75%, 300% and
1200% above recommended use levels due to a mixing error). Doxycycline was
administered at 15, 30, 60 and 200 g/ton in the feed to test its ability to induce TD.
Enrofloxacin was administered in the drinking water at 25%, 100%, and 400% above the
recommended use level. The three-ceftiofur treatments were injected sub-cutaneously in
the neck area of one-day-old chicks (25%, 100% and 400% above the recommended
dosage). The control group received no antibiotic treatment. Thiram, a fungicide known
to induce experimental TD, was also given at 20 and 40 ppm. The number of available
pens was limited; therefore, observations were divided into four separate experiments.
Each experiment consisted of a control and two antibiotic treatments (three
concentrations per antibiotic and three replicate groups of eight cockerels per
concentration). The four experiments, and the antibiotics tested in each, are detailed in
Table 5.

Weekly growth rates and feed consumption were tabulated by individual bird
weights and pen feed consumption. Tibial dyschondroplasia lesions (degree of physeal
thickening) were visually examined at the end of the 3-week trial. Birds were killed by

cervical dislocation at 22 days of age. Right and left proximal tibiotarsi were

64

Table 4. Diet Composition for 0-3 week old broiler cockerels.

 

 

 

 

 

 

 

 

 

 

Ingredient % of Diet
Ground yellow corn 53.75
Soybean meal (48%) 36.69
Choice white grease 5.14
Dicalcium phosphate (18.5% P) 1.57
Limestone l .33
Salt 0.45
DL- methionine 0.20
Vitamin premixl 0.25
Trace mineral premix2 0.25

 

 

 

I Supplied X mg/kg of diet (except where noted): vitamin A* (all-
trans-retinol acetate), 5000 IU; vitamin D3, 309 UCU, vitamin E*
(alpha-tocopherol acetate), 11 IU; biotin“, 0.3; choline Cl*, 600;
ethoxyquin, 125; folic acid, 3; menadione sodium bisulfite, 1.1;
nicotinic acid, 44; D-pantothenic acid*, 10; riboflavin“, 4.4; thiamin
mononitrate, 2.2; pryridoxine HCl (B6), 3; vitamin B12, 0.01.

2 Supplied X mg/kg of diet: cupric sulfate, 10; potassium iodide, 2.1;
ferrous sulfate, 60; manganese dioxide“, 120; sodium selenite, 0.1;
zinc oxide“, 100.

Vitamins and minerals purchased from ICN Pharmaceuticals Inc.
(Costa Mesa, CA) except noted by * those were purchased from
Sigma (St. Louis, MO).

 

65

 

Table 5. Treatment groups within the 3-wk broiler experiments.

 

 

 

 

 

 

 

 

 

Antibiotic Recommended Treatment Method of
Use Level Use Level Administration
. a 9.4, 15, 30
Oxytetracyclrne 7.5 g/ton g/ton Feed
- b 500, 800,
Chlortetracyclme 400 g/ton 1600 g/ton Feed
, M 15, 30, 60,
Doxycycline 200 g/ton Feed
. . a 180, 225,
Salrnomycrn 60 g/ton 360 g/ton Feed
0.25, 0.4,
Ceftiofurd 0.2 mg/chick 0.8 Injection
mg/chick Sub-cutaneous
. c 31.3, 50,
Enrofloxacrn 25 ppm 100 ppm Water
Thiramc 20 and 40 Feed
ppm

 

 

 

 

 

a= treatments administered in Experiment 1; b: treatments in Experiment 2;
°= treatments in Experiment 3; d= treatments in Experiment 4 (only the

200 g/ton doxycycline was tested in this experiment, the other 3 treatments were
tested in experiment 2)

66

longitudinally sectioned and growth plate lesions were visually scored. A scale of l-4
was used with 1 representing no lesion and 4 representing the most severe lesion as
previously described by Orth et al. (1992). The incidence of TD was determined using
the 2 to 4 scores as positive indicators of TD compared to the I scored indicating no TD
lesion.
Statistical Analysis

Body weight and feed conversion data were analyzed using the general linear
model procedure (SAS, 1999). Where appropriate, main effect means were separated by
Tukey’s studentized range test. The incidence of TD was examined by Chi Square
analysis using the PROC FRIC program of SAS, 1999. A score greater than 1 was
considered positive for tibial dyschondroplasia. Significance was determined only for the

presence of TD.

Results

No difference in the incidence of TD lesions was observed between the control
birds and those subjected to any of the antibiotics tested (Table 6). However, both thiram
treatments induced TD lesions in 3 wk old broilers when compared to the control group.
The 20 ppm treated birds experienced an 92% incidence rate (p5 0.0006) and the 40 ppm
treatment group experienced a 78% incidence rate (p_<_ 0.0004).

All of the salinomycin treated birds were lighter than the controls (p5 0.001;
Table 6C). Feed conversion (amount of feed consumed to the rate of gain) only differed
from the control at the 360 g/ton salinomycin treatment (p5 0.05). The 31.3 ppm

enrofloxacin treated birds were heavier than control birds (pg 0.04; Table 6C), but there

67

Table 6. Body weight, feed conversion and tibial dyschondroplasia incidence
for 3-week-old male broilers treated with several levels of seven antibiotics or

thiram (a fungicide).

A: Experiment 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Body Weight1 Feed TD
grams Conversionl incidence2
Ratio %
Control 574 i 56 1.83 i 0.04 13
9.4 g/ton oxytetracycline 628 i 42 1.70 i 0.24 8
15 g/ton oxytetracycline 602 i 53 1.37 i 0.28 0
30 g/ton oxytetracycline 587 i- 60 1.79 i0.21 0
180 g/ton salinomycin 434 i 12“ 1.94 i 0.14 4
225 g/ton salinomycin 387 i 3** 2.13 i0.10 0
360 g/ton salinomycin 232 i 9" 3.71 £062" 0
B: Experrment 2
Treatment Body WeightI Feed TD
grams Conversionl incidence2
Ratio %
Control 599 i 77 1.83 i 0.37 13
500 g/ton 683 i 21 1.62 i0.12 0
chlortetracycline
800 g/ton 693 i 27 1.62 i0.31 0
chlortetracycline
1600 g/ton 611:55 1.69:0.11 0
chlortetracycline
15 g/ton doxycycline 689 i 96 1.68 i 0.26 4
30 g/ton doxycycline 669 i 4 1.74 i 0.18 8
6O g/ton doxycycline 672i 32 1.53 10.06 0

 

 

 

 

68

 

 

C: Experiment 3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Body WeightI Feed TD
grams Conversion1 incidencez
Ratio %
Control 594 i 87 1.91 i0.44 15
20 ppm thiram 429 i 13 1.74 :0.11 92M
40 ppm thiram 235 i 42" 1.76 i0.18 78"
31.3 ppm enrofloxacin 637 i 51* 1.81 i0.47 4
50 ppm enrofloxacin 619 :60 1.91 i0.30 17
100 ppm enrofloxacin 676 i 56 1.73 :0.02 12
D: Experiment 4#
Treatment Body Weight1 Feed TD
grams Conversionl incidence2
Ratio %
Control 502 i 53 1.60 i 0.17 8
200 g/ton doxycycline 547 i 76 1.57 :0.19 0
0.25 mg cefiiofur/ chick 546 i 68 1.65 i0.14 0
0.4 mg ceftiofur/ chick 506 i 52 1.73 i0.16 0
0.8 mg cefiiofur/ chick 534 148 1.67 2"0.16 0

 

 

 

 

1= Mean of 3 pens; 2: TD incidence was determine by using a positive indicator
of a lesion (scores 2 to 4); # = Birds (same strain) were purchased from Townline

hatchery (Zeeland, MI)

* significantly different from control group within its experiment at p5 0.05
** significantly different from control group within its experiment at p5 0.001

69

 

 

was no difference in feed conversion when compared to control birds. Thiram treated
birds also did not differ from controls in feed conversion, but the 40 ppm treated birds

were lighter than control birds (p5 0.001).

Discussion

The antibiotics (oxytetracycline, chlortetracycline, doxycycline, enrofloxacin,
ceftiofur and salinomycin) tested in this experiment inhibited in vitra cartilage
degradation of embryonic chick tibiae (Chapter 2). However, they did not induce TD
lesions in 3-week-old male broilers. Tibial dyschondroplasia lesions were evident in
thiram treated birds, which agrees with other literature (Orth et al., 1994; Vargas et al.,
1983; Wu et al., 1990). Both treatment levels of thiram, 20 and 40 ppm, induced TD
lesion when compared to control birds.

The antibiotics may have inhibited cartilage degradation in vitra because the
antibiotic concentrations were higher than what the growth plate was exposed to in the
bird. In the in vitra trials, explants are directly exposed to treatments. However, in viva
the drugs have to travel through the body to concentrate in growth plate tissue.
Absorption factors could prevent the drugs from being active in the growth plate. If Ca“
or Zn2+ were in excess, the tetracyclines would bind these molecules and become less
available. Blood levels of antibiotics were not tested so possibly the absorption rates of
the antibiotics in this study were not as high as those published. Trying to achieve higher
concentrations of antibiotics to mimic in vitro concentrations would not be
pharmacologically relevant.

Another factor that may have influenced the results of this study are the

fluorescent lights used in the brooder where the chicks were housed. Edwards (1989,

70

1990) reported that birds fed adequate calcium and vitamin D3 had a low incidence of TD
when allowed access to ultraviolet light emitted by fluorescent lighting. In 1997, Elliot
and Edwards showed that rapidly growing broilers fed diets adequate in both calcium and
phosphorus and lacking sunlight or fluorescent lighting had a high incidence of TD,
which could not be reduced by supplementing 10 times the recommended dose of
cholecalciferol. They also stated that fluorescent lighting and supplementation with 10
jig/kg 1,25-(OH)2D3 (the active form of vitamin D3) are equally effective in reducing TD
deve10pment in broiler chicks. Thiram may act by a different mechanism than the
Vitamin D pathway and overcome the effects of fluorescent lighting to induce TD.
However, antibiotics that bind cations (tetracyclines and fluoroquinolones are thought to
act primary by this mechanism) may not be able to inhibit cartilage degradation in the
presence of fluorescent light.

Although skeletal disorders have been associated with tetracyclines,
fluoroquinolones, cephalosporins and aminoglycosides, this study showed that
doxycycline, oxytetracycline, chlortetracycline, enrofloxacin, ceftiofur, and salinomycin
did not induce TD lesions in growing broiler chicks. However, thiram (20 and 40 ppm)
significantly induced TD lesions in chicks raised in the same environment and fed the
identical basal diet. Orth et al. (1999) reported that dithiocarbamates, such as thiram,
inhibit in vitra cartilage degradation in this explant system. Dithiocarbamates and
calcium deficiency both inhibit in vitra and in viva cartilage degradation indicating that
our explant model may predict whether certain compounds or conditions will induce TD.
However, the results of this study show that the antibiotics tested are likely not involved

in the etiology of TD in the poultry industry.

71

References

Edwards, H. J. The effect of dietary cholecalciferol, 25-hydroxycholecalciferol and 1,25-
dihydroxycholecalciferol on the development of tibial dyschondroplasia in broiler

chickens in the absence and presence of disulfiram. Journal of Nutrition 1989,
119, 647-652.

Edwards, H. J. Efficacy of several vitamin D compounds in the prevention of tibial
dyschondroplasia in broiler chickens. Journal of Nutrition 1990, 120, 1054-1061.

Elliot, M.; Edwards, H. J. Effect of 1,25-Dihydroxycholecalciferol, cholecalciferol, and
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broiler chickens. Poultry Science 1997, 76, 570-580.

Hargest, T. E.; Gay, C. V.; Leach, R. M. Avian tibial dyschondroplasia: III. Electron
probe analysis. American Journal of Pathology 1985, 119, 199-209.

Havenstein, G. B.; Ferket, P. R.; Scheideler, S. E.; Larson, B. T. Growth, livability, and
feed conversion of 1957 vs 1991 broilers when fed "typical" 1957 and 1991
broiler diets. Poultry Science 1994, 73, 1785-1794.

Leach, R. M., Jr. Poultry industry should reconsider if bigger is better. F eedstufifs 1996,
68, 10.

Murakami, T.; Murakami, H.; Ramp, W. K.; Hanley, E. N. J. Interaction of tobramycin
and pH in Cultured Chick Tibiae. Journal of Orthopaedic Research 1996, 742-
748.

Orth, M. W., Bai, Y., Zeytun, I. 8., Cook, M. E. Excess levels of cysteine and
homocysteine induce tibial dyschondroplasia in broiler chicks. American Institute
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Orth, M. W.; Leach, R. M.; Vailas, A. C.; Cook, M. E. Non-reducible collagen cross-
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Orth, M. W.; Cook, M. E. Avian tibial dyschondroplasia: A morphological and
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Orth, M. W.; Chlebek, K. A.; Cole, A. A.; Schmid, T. M. Tetracycline derivatives inhibit

cartilage degradation in cultured embryonic chick tibiae. Research in Veterinary
Science 1997, 67, 11-14.

72

Orth, M.; F enton, J .; Chlebek-Brown, K. Biochemical characterization of cartilage
degradation in embryonic chick tibial explant cultures. Poultry Science 1999, 78,
1596-1600.

Praul, C. A.; Ford, B. C.; Gay, C. V.; Pines, M.; Leach, R. M. Gene expression and tibial
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Roberson, K. D. 25-Hydroxycholecalciferol fails to prevent tibial dyschondroplasia in
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Chapter 4

Summary and Conclusion

Poultry skeletal disorders have been estimated to cause losses of approximately
$160 million per year to the United States broiler and turkey industries. One of the major
metabolic skeletal disorders that effects the long bones of fast growing meat producing
birds (broiler chickens, turkeys, and ducks) is tibial dyschondroplasia (TD). An
avascular cartilage plug accumulates in the metaphyseal region of the tibia, causing
impairment of long bone growth and clinical TD lesions. Lameness, decreased feed
efficiency, and increased mortality, culls, and condemnations at slaughter can result from
TD.

If the etiology of this debilitating skeletal disorder could be found, the industry
would economically benefit from the solution. Antibiotics are incorporated into the diet
of meat-type birds to promote growth and increase feed efficiency. Many of the
antibiotics used in the poultry industry for growth promotion and disease treatment have
been found to disrupt normal bone formation. Therefore, the objectives of this study
were 1) to determine if antibiotics commonly utilized in the poultry industry inhibited in
vitra cartilage degradation and 2) determine if antibiotics that inhibited in vitra cartilage
degradation also induced TD in growing broilers.

An embryonic chick tibiae explant culture system was used to answer objective 1.
Lincomycin, tylosin tartrate, gentamicin, erythromycin, and neomycin sulfate did not
alter the cartilage’s catabolic metabolism at any concentration tested. This work

determined that doxycycline, oxytetracycline, enrofloxacin, cefiiofirr and salinomycin

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significantly inhibited proteoglycan and nitric oxide (indicators of cartilage degradation)
release into conditioned media. The mechanism by which these antibiotics decrease
cartilage degradation is not completely understood. However, binding cations (Caz:
Zn2+, or Mg“) or altering cation concentrations is a common theme. If these cations are
limited, the normal metabolism of the growth plate is altered and may cause impaired
turnover leading to skeletal disorders like TD.

Conversely, when these five antibiotics were administered daily to day-old male
broilers, TD lesions were not evident after 3 weeks of treatment. Thiram, a
dithiocarbamate, did induce TD lesions when administered in the same environment.
Dithiocarbamates have also been shown to inhibit cartilage degradation in the same
embryonic chick tibia system that indicated the five antibiotics inhibited cartilage
degradation. We believe that our in vitra model potentially predicts TD for certain
compounds. However, in viva the antibiotics tested were not involved in the etiology of

TD.

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