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THE USE OF TYLOSIN TO TREAT INTRAMAMMARY
INFECTIONS DURING THE NON-LACTATING PERIOD
OF HEIFERS AND DAIRY COWS

presented by
GENARO ANDRES CONTRERAS

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oftherequirementeforthe

MS degree in Large Animal Clinical Sciences

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April 24, 2008
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6/07 p:/C|RC/DateDue.indd-p.1

 

THE USE OF TYLOSIN TO TREAT INTRAMAMMARY INFECTIONS
DURING THE NON-LACTATING PERIOD OF HEIFERS AND DAIRY
cows
By

Genaro Andres Contreras

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

Department of Large Animal Clinical Sciences

2008

ABSTRACT

THE USE OF TYLOSIN TO TREAT INTRAMAMMARY INFECTIONS DURING
THE NON-LACTATING PERIOD OF HEIFERS AND DAIRY COWS

By
Genaro Andres Contreras

There are two types of non-lactating periods during the lifespan of a dairy cow.
The first type refers to heifers from infancy to puberty and gestation prior to the first
calving. The second is referred to as the dry cow period, and is defined as the time when
a mature cow is not milked between two lactations. During the non-lactation period
there is a risk of acquiring an intramammary infections (IMI) for both heifers and dairy
cows. This is the safest time to treat IMI, because antibiotic treatments can be
administered at higher a dose to increase effectiveness while reducing the risk of milk
residues. In the present thesis, systemic tylosin was used in mature dairy cows alone or
in combination with intramammary cephapirin, to treat IMI at the end of lactation.
While in dairy heifers, systemic tylosin was used 2 weeks prepartum to reduce IMI after
calving.

Tylosin was effective in reducing IMI caused by Gram-positive bacteria in dairy
cows when used alone or in combination with intramammary infiised cephapirin. In
dairy heifers, intramuscular tylosin reduced IMI caused by coagulase negative
staphylococci. The use of tylosin in the non-lactating period of dairy animals offers a
management option that may be useful on some farms, as a part of their udder health
program. Deciding whether to use tylosin should be based on sound evaluations of

environment, management and economic implication for the farm.

DEDICATION

To Samuel, Santiago, Monica, Genaro and Veronica, for all the dedication and
support during this time.

iii

ACKNOWLEDGMENTS

I would like to thank Dr. Philip Sears, for all his wise orientation, support, and
advice during my internship and my masters program. I do thank Dr Barbara E. Straw
for her support, her help during sample collection, and for her collaboration during
the manuscript elaboration.

I would like to thank all the members in my graduate committee. Dr Lou
Neuder, for his advice and active collaboration at Green Meadows Farms during the
heifer trial. Dr Ron Erskine, for his orientations and helpful advices. Finally, Dr
Lorraine Sordillo for improving my writing skills, and constructive criticism.

The antibiotic resistance tests were performed thank to the collaboration of Dr
John Kaneene and Katie May. I would like to thank Lisa Lipsey and all the personnel
at Green Meadow Farms for their help during sample collection for the heifer trial.

I would like to thank Mr. Tim denDulk and his family for all the support.
Thanks to Dr. Walter Guterbock, for his advice and orientation, during all this time
Thanks to all the personnel at denDulk dairies, especially Mike, Hector, Jacobo,
Scott, and Lucio for their help during my internship and the dry cow project.

The American Association of Bovine Practitioners through their research

assistantship granted the funding for the heifer trial

iv

TABLE OF CONTENTS

 

 

 

 

LIST OF TABLES ......................................................................................... VII
LIST OF FIGURES ...................................................................................... VIII
INTRODUCTION ............................................................................................ 1
CHAPTER 1 ................................................................................................... 3
Dry Period: The non-lactating period in dairy cows 3
Implications of the dry period ................................................................................... 5
Pathogens .................................................................................................................. 6
Dry Cow Treatment .................................................................................................. 8
Intramammary dry cow therapy ................................................................................ 8
Systemic therapy ....................................................................................................... 9
Combined therapy ................................................................................................... 10
Internal teat sealants ................................................................................................ 11
External teat sealants ............................................................................................... 12
Macrolides -- - -- l3
Tylosin .................................................................................................................... 1 5
Pharmacokinetics .................................................................................................... l 5
Non-lactating heifers 21
Marnmogenesis ....................................................................................................... 21
Prepubertal development ........................................................................................ 23
Puberty to Conception ............................................................................................. 25
Intramammary infections ........................................................................................ 25
Antibiotic treatment before calving ........................................................................ 27
Antibiotic treatment immediately after calving ...................................................... 28
Internal teat sealants ................................................................................................ 29
Tylosin use as systemic antibiotic in DCT and PCHT _ 29
CHAPTER 2 ................................................................................................. 31
Comparison of Systemic and Intramammary Dry Cow Treatments ................... 31
Summary ................................................................................................................. 32
Materials and methods ............................................................................................ 34
Results ..................................................................................................................... 37
Discussion ............................................................................................................... 39
CHAPTER 3 ................................................................................................. 45

Prepartum Systemic Antibiotic For Heifer Mastitis - - - 45

 

 

 

 

Interpretative Summary .......................................................................................... 46
Abstract ................................................................................................................... 47
Introduction ............................................................................................................. 48
Materials and methods ............................................................................................ 49
Results and discussion ............................................................................................ 51
Conclusion .............................................................................................................. 56
CHAPTER 4 ................................................................................................. 62
Conclusion - 62
REFERENCES ............................................................................................. 65
References Chapter 1 and Chapter 4 - -- 65
References Dry Cows ............................................................................................. 65
References Heifers .................................................................................................. 71
References Chapter 2 g - - 74
References Chapter 3 - 76

vi

LIST OF TABLES

Table 1. Summary of physico-chemical properties of tylosin (Paesen et a1. , l995abc;
McFarland et al., 1997; The Merck Index, 2001). ...................................................... 17

Table 2. Antimicrobial activity for tylosin. Reported minimum inhibitory
concentrations (MIC 90) for different bacterial isolates (Catry et al., 2003. Odlan et
aL,2002) ..................................................................................................................... 20

Table 3. Treatment designations, description and number of animals in each treatment
group. Number of cows infected at dry-off, percentage of cows with IMI at dry-off.
Number of cows cured after DCT and cure rate by treatment group. Number of cows
with new IMI after calving. Number of quarters enrolled, non-functional quarters,
number of quarters infected and percentage of Gram-positive bacterial infection of
total number of quarters at dry-off. Number and percentage of quarters cured after
calving. Number and percentage of bacterial infection by bacteria type at Dry-off in
each treatment group, and number of quarters cured and cure rate after calving by
bacteria type in each treatment. Values with the same letter are not different from
each other (p<0.05). .................................................................................................... 43

Table 4. Culture results at heifer level and quarter level at sample-1 and sample-2
including percent of spontaneous cure and percent of new infections, for control and
tylosin groups. Staphylococci and streptococci infections at sample-1 and sample-2
for control and tylosin groups, including spontaneous cure and new infections.
Percentages with different letters are considered to be different at P<0.05 ................ 57

Table 5. Minimum inhibitory concentrations (MIC) of ampicillin (AMP), ceftiofur
(CF), enrofloxacin (ENR), neomycin (N EO), penicillin (PEN) and tylosin (TY) for S.

chromogenes, S. simulans, Streptococci uberis, and Enterococcusfaecalis. ............. 60

Table 6. Speciation of representative isolates at sample 1, from cultures at sample 1
for control and tylosin groups. .................................................................................... 60

Table 7. Culture results for tylosin and control groups at sample 1 and sample 2 in
summer and spring seasons ......................................................................................... 61

vii

LIST OF FIGURES

Figure 1. Tylosin and associated metabolites. (Lewicki, 2006) ................................. 18

Figure 2. Mean values of logSCC at dry-off, first test and second test after calving for
each treatment group. .................................................................................................. 41

Figure 3. Somatic cell count reduction afier calving explained by the difference
between the log SCC at DHI dry-off test and log SCC at first DHI test after calving
for each treatment ....................................................................................................... 42

Figure 4. Means for somatic cell scores (SCS) at sample-1 and sample-2, SCS at 1St
and 2lrld DHI tests after calving, and projected 305d ME milk production at lSt and 2"d
DHI tests for control and tylosin group. ..................................................................... 58

Figure 5. Means for somatic cell scores (SCS) at sample-l and sample-2, SCS at lSt

and 2"d DHI tests after calving, and projected 305d ME milk production at 1St and 2nd
DHI tests for infected heifers. CNS and others ........................................................... 59

viii

INTRODUCTION

Mastitis is the most common disease of dairy cows and the single greatest cause
of economic loss to the dairy industry today. The economic impact on the dairy
industry attributed to mastitis is estimated at $2 billion a year in the USA or
approximately 6% of the value of production (DeGraves and Fetrow, 1993; Harmon,
1994; Wells and Ott, 1998). At the herd level, costs of mastitis are divided into four
main losses including: reduced milk quality, less efficient milk production due to
chronic subclinically infected cows, increased costs related to treatment, and
increased replacement rate or culling of cows at suboptimal time in lactation (Osteras,
2000; Halasa et al., 2007). An additional consideration includes the cost of mastitis
prevention that amounts to almost half of total costs of dairy herd health programs

(Miller et al., 1993).

Mastitis is a multifactorial disease, in which both the environment and the
animal play a role. This not only requires an understanding of animal physiology and
defense against pathogens, but also how the environment affects the animal responses

to pathogens when designing effective control programs.

Producers and dairy professionals have developed strategies to control mastitis
and reduce economic losses based on an understanding of animal-environment
interactions. One of these strategies is antibiotic treatment during the non-lactating
period to prevent clinical and subclinical mastitis in the subsquent lactation. There
are two types of non-lactating periods during the lifespan of a dairy cow. The first

type refers to heifers from infancy to puberty and gestation prior to the first calving.

The second type is referred to as the dry cow period, and is defined as the time when

a mature cow is not milked between two lactations.

Antibiotic treatment for prevention of mastitis in dry cows is called dry cow
therapy (DCT). It was first proposed in the 1950’s (Pearson, 1950), and is generally
practiced by dairy farmers around the world (Dingwell et al., 2003). Similar to DCT,
prepartum intramammary antibiotic treatment of heifers (Precalving Heifer
Treatment, PCHT) has been used from early pregnancy until 2-6 wk before expected
calving (Trinidad et al., 1990c). Antibiotics used in DCT and PCHT include beta-
lactams, while other families of antibiotics such as macrolides are available but are
less commonly used. The macrolide antimicrobials diffuse readily into the mammary
gland. Commercial products including, tylosin, tylmicosin and tulathromycin are

currently used in food animal practice but not in DCT or PCHT.

Tylosin has the advantage of being relatively inexpensive, safe and easy to use
when compared to the other members of the macrolide family. As a macrolide
antibiotic, it diffuses well into the udder compartment, because of its basic pK.
Furthermore; clinical research has proven its efficacy and it is approved in other

countries such as New Zealand to treat clinical mastitis during the lactating period
(McDougall, 2007). The efficacy of tylosin as systemic DCT or PCHT, however, has
not been well documented. We investigated the effectiveness of tylosin as a systemic

antibiotic in DCT and PCHT, and found that administration of systemic tylosin for

DCT and PCHT decreases the incidence of IMIs at parturition.

CHAPTER 1

Dry Period: The non-lactating period in dairy cows

The period of highest risk for new intramammary infections (IMI) in the
lactation cycle of cows is during the early non-lactating period (dry period). In order
to identify management programs that address this high-risk period, it is necessary to
understand the biological events of the dry period, as well as management practices
that address this risk. The following chapter will review the physiology and the
management practices of the dry period.

The ideal dairy cow should attain peak milk production at 60 days in milk
(DIM), become pregnant again by 100 DIM, dry off at 305 DIM and rest during the
dry period for 60 days. The dry period is important for lactation development, since
dairy cows without a dry period have at least a 20% decrease in total milk yield as
compared to herd-mates with a 45 to 60 days dry period (Remond et al., 1997;
Sorensen and Enevoldsen 1991). In a pioneer study, Smith et a1. (1966) compared
milk production by quarters in two cows. In each animal two quarters were milked
throughout the dry period phase (60 days) and two were not milked. Quarters without
a dry period produced 38% and 44% less milk in the subsequent lactation than their
counterparts that had a 60-day dry period.

The length of the dry period is also important in terms of milk components. Fat
and protein yield is maximized in a lactation that follows a 60-day dry period. In
contrast, dry periods of 20 days or less result in substantial losses in fat and protein
yield in the subsequent lactation (Kuhn et al., 2006). Udder health also is affected by

dry period length, with long dry periods (45 to 70 days) improving somatic cell count

in the subsequent lactation. In general the ideal dry period length is 45 to 60 days
(Kuhn et al., 2007), but as noted by Bachman and Schairer (2003), further research is
needed to determine the ideal length of the dry period for the modern dairy cow that

accounts for various management scenarios.

Involution of the mammary gland occurs in two stages during the dry period.
The first is triggered by local stimuli, such as accumulation of milk within the alveoli,
which initiates apoptosis in some milk producing cells, although re-initiating milk
removal can reverse this process (Furth et al., 1997). This process is accompanied by
a decrease in lactose and fatty acids secretion, an increase in lactoferrin and NAG
secretion, and a more active intracellular transport of immunoglobulins (Hurley,
1989). Differences among species are found in the second stage. Involution is
irreversible in mice and rats and is characterized by activation of proteases that
destroy the lobular-alveolar structure of the gland by degrading the extra-cellular
matrix and basement membrane, as well as a loss of alveolar cells (Walker et al.,
1989). In contrast, alveolar structure is maintained in dairy cows. Cows are usually
gestating at dry-off and pregnancy might explain the conservation of the mammary

structure (Lund et al., 1996; Capuco et al., 2002).

There are morphological changes in the gland after the cessation of milking.
Within 24 hours of the last milking, secretory vesicles and fat droplets accumulate
within the cytoplasm of alveolar cells. After 2 or 3 days, the vesicles fuse and large
vacuoles are formed. Accumulation of secretory products and vacuole formation is
consistent with inhibition of milk secretion (Capuco and Akers, 1999). Within the

first 48 hours after the last milking, many cells display a decrease in cytoplasmatic

organelles involved in milk synthesis. After two weeks, cells exhibit a reduction in
apparent secretory capacity, but they are still viable (Capuco and Akers, 1999).
During the dry period there is no net loss of mammary cells, but there is an enhanced
turnover of mammary epithelial cells, suggesting that this time of rest permits
replacement of damaged or senescent cells prior to the following lactation (Capuco et

aL,1997)

Implications of the dry period

Endevolsen and Sorensen (1992) demonstrated that dry periods of 7 weeks were
associated with the lowest risk of clinical health disorders when compared to periods
of 4 and 10 weeks. However, the susceptibility to new IMI is higher during the early
dry period and near calving (Oliver 1988). This increased incidence of new IMI
results in an elevated number of infected quarters at calving, and is responsible for the

high level of IMI during early lactation in many herds.

According to the National Mastitis Council guidelines (2001), the elevated rate
of new infections during the early dry period in the absence of DCT may be due to
one or several of the following: 1) flushing of colonized bacteria from the teat canal
during milking is terminated; 2) udder sanitation and teat dipping are discontinued; 3)
the teat canal becomes dilated and shortened due to udder fill at milk cessation, which
allows organisms to enter the udder; 4) phagocytes are involved in removing milk
components instead of bacteria; and 5) activity of lymphocytes is reduced. In

contrast, the rate of new infections during the mid-dry period is very low. Mammary

gland resistance during this time may be attributed to: l) a keratin plug forms in the
teat canal which prevents mastitis pathogens from entering the udder; and 2)
increased presence of antibacterial factors such as lactoferrin and immunoglobulins in
the udder. Inhibition of growth due to antibacterial factors may be associated with the
high rate of spontaneous elimination of some IMI (e.g., Corynebacterium bovis)
during the dry period (Oliver and Juneja, 1990). Susceptibility to infection again
increases near calving. This may be due to: 1) increased fluid volume and dilation of
the teat canal; 2) decreased lactoferrin concentration; 3) reduced leukocyte numbers

and phagocytic ability; and 4) utilization of milk components for bacterial growth.

Dingwell et al. (2003) outlined specific factors influencing susceptibility of
cows to IMI infections during the dry period; such factors are at the level of the
quarter, cow and herd. Quarter-level factors include, bacterial population at the teat
end, teat end integrity, and formation of teat canal keratin plug. Cow-level factors
include parity, milk production, and method of milk cessation. Herd-level factors are
housing, bedding materials, udder health management practices and nutritional status.

Pathogens

Mastitis pathogens are divided into contagious and environmental groups.
Contagious pathogens include Staphylococcus aureus, Streptococcus agalagtiae,
Mycoplasma bovis and Corynebacterium b0vis,. Environmental pathogens include
Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli, Klebsiella spp,
Enterobacter spp, Prototheca spp, Pastereulla spp, Arcanobacterium pyogenes and

Pseudomonas spp. (Hogan et al., 1999).

Contagious organisms, as well as some environmental bacteria, are transmitted
among cows and quarters during the milking process, and are the predominant
infections at the time of dry-off. Effective long-acting antibiotic therapy offers the
best opportunity to eliminate these existing infections. In contrast, exposure to
environmental pathogens is likely to continue throughout the dry period. These
organisms are primarily contracted from contamination of udders by fecal material
and bedding. Among environmental pathogens, infections with Klebsiella, and
Enterobacter occur more frequently early in the dry period. On the other hand, E.
coli infections tend to occur immediately before and afier calving (Leslie, 2001).
Infections with Streptococci are frequent throughout the non-lactating period, and

their incidence increases a few weeks before and around parturition (Eberhart, 1987).

Bradley and Green (2000) assessed the incidence of enterobacterial infection in
mammary glands during the non-lactating period. A rise in the incidence of
enterobacterial infections was detected around drying off and calving. Quarters that
acquired an infection were more likely to develop mastitis in the subsequent lactation.
The authors found that more than 70% of enterobacterial mastitis occurs following a

period of subclinical infection in the dry period.

Intramammary infections in the dry period increase the risk of clinical mastitis
in the following lactation, especially in the early stage. Furthermore, infections with
one organism during the dry period may increase susceptibility to infection caused by

another pathogen, resulting in clinical mastitis during lactation (Green et al., 2002).

Dry Cow Treatment

The DCT is defined as the use of antibiotics immediately after the last milking
of lactation. It can decrease the number of existing infections and prevent new
infection during the early weeks of the dry period (NMC, 2000). Dry cow therapy
has advantages over lactation therapy including higher cure rate, a reduction in new
IMI after calving, and a higher dose of antibiotic can be used with lower risk of milk
residues (Ruegg, 2005). Two approaches can be used to administer DCT in dairy
herds: 1) blanket DCT that involves the treatment of all cows at dry-off following the
last milking of lactation and 2) selective DCT only treats cows with high SCC or IMI
at dry-off. In general, DCT is a cost-effective measure compared with no treatment at
dry-off (Berry et al., 2004; Robert et al., 2006; Huijps and Hogeveen, 2007).
Intramammary dry treatment reduces IMI in herds with a high prevalence and high
risk of new infections. The most common route of DCT is intramammary infusion
into each productive quarter, however antibiotic therapy for DCT can be administered

by intramammary, systemic or as a combination of both routes.

Intramammary dry cow therapy

Intramammary dry cow treatment alone is widely used and very effective in
reducing IMI (Zwald et al., 2004; Dingwell et al., 2003; Berry et al., 2002; Dimmick,
2001; Sol et al., 1994). However, low efficacy against some coagulase negative
staphylococci has been demonstrated (Rajala-Schultz et al., 2004). The use of the

intramammary route started as early as 1950 when penicillin was used to prevent

bacterial infection in the dry period (Pearson, 1950). New formulations of antibiotics
were developed in the 1960’s, using cloxacillin and penicillin G to extend the
duration of effective concentration for several weeks after infusion (Smith et al., 1967
a, b). Currently, different antibiotics are used for intramammary treatment and there
are often differences within geographical regions regarding antimicrobials of choice.
For example, a survey of dairy herds in Pennsylvania found that drugs commonly
used in dry cow therapy were cephapirin (52%), novobiocin (27%), penicillin G
procaine (24%), and cloxacillin (15%) (Sawant et al., 2004). In contrast, a similar
survey was conducted in California where the most commonly used antibiotics were
cloxacillin, penicillin/streptomycin, and cephalosporin. Most dairies chose a single
type of antibiotic and used the same antibiotic over an extended period of time (Kirk,
2004). In general, DCT is used in 98% of conventional dairy farms in USA, 99% in
United Kingdom and 76.5% in Canada (Zwald et al., 2004; Berry et al., 2002;

Dimmick 2001 ).

Systemic therapy

Systemic dry cow therapy is less expensive and easier to administer than
intramammary but has been reported to be less effective and therefore, rarely used.
Nickerson (1999) administered systemic tilmicosin (5mg/kg b.w. subcutaneous) to
cows with at least one quarter infected with Staphylococcus aureus. Cure rates for
cows treated with systemic tilmicosin were 9.1%, which was much lower than the
ones achieved by intramammary tilmicosin (74%) and intramammary cephapirin

(78%).

In another study, Shpigel et al., (2006) used systemic cefquinome (1mg/kg b.w.
intramuscular) in cows with IMI caused by S aureus, reporting 28% cure rates for the
cefquinone group in contrast with 70% for the intramammary infused group (sodium
nafcillin, procaine benzylpenicillin and dihydrostreptomycin). The authors also
reported an increase of [MI due to S. aureus in the cefquinone treated group.
Norfloxacin nicotinate and oxytetracycline-HCL have also been reported as therapies

using this route (Soback et al., 1990).

Combined therapy

The use of systemic and intramammary antibiotics at the same time was initially
applied in treating subclinical mastitis (Owens, 1988). In this study, cows with
quarters subclinically infected with S. aureus were treated with intramammary
amoxicillin plus systemic penicillin or intramammary amoxicillin only. Cows treated
with the combination had a 51% cure rate compared to 25% of the intramammary
group. Based on this study, it was inferred that a combined therapy could increase
the effectiveness of intramammary treatment alone because it provided better
diffusion of certain systemic antibiotics into the mammary gland.

Combined therapy in DCT has been reported by O’Boyle et al., (2006) who
compared two combinations: 1) 12 g of oxytetracycline hydrochloride (OTC-HCl)
administered intramuscularly plus intramammary cephapirin 300 mg and 2) 20 g of
tylosin subcutaneously plus intramammary cephapirin 300 mg. A teat sealant was
used in combination with both treatments. Tylosin plus cephapirin gave better results

in reducing somatic cell counts (SCC) and intramammary infections after calving

10

than OTC-HCL plus cephapirin. Other combinations have been used. Erskine et a1.
(1994) compared efficacies of intramammary infusion of cephapirin benzathine alone
against infusion of the same antibiotic combined with intramuscular oxytetracycline
at 11 mg/kg once daily on days 7, 8, 9, and 10 after drying off for treatment of
Staphylococcus aureus mastitis. Systemic oxytetracycline, in combination with
intramammary dry cow treatment, did not improve the cure rate for S. aureus mastitis,
however, many of these infections were chronic and the effect on other pathogens
was not reported.

Combined therapy has advantages over systemic or intramammary
administration of DCT. The use of two or more antibiotics at the same time expands
the spectrum of the treatment and increases the possibility of synergism among the
antimicrobials. A combined DCT could be advised in certain herds with higher rates
of [MI or high SCC, but this recommendation must be based on a thorough analysis

of the udder health of the dairy herd.

Internal teat sealants

Internal sealants infused into the teat canal can occlude the entry of bacteria into
the gland. A commonly used sealant, bismuth subnitrate in paraffin base is infused
after the last milking of lactation alone or combined with antibiotic therapy. Bismuth
subnitrate was initially evaluated in the UK. Berry and Hillerton (2002) found fewer
new infections at calving in quarters of cows treated with teat sealant than in quarters
of untreated cows. The authors reported that the predominant pathogen causing new

infections was Streptococcus uberis. The teat-sealed group had fewer IMI caused by

11

this agent (9/27 quarters) than the control group (44/93 quarters). Similarly, Huxley
et a1. (2002) observed that quarters treated with teat sealant had fewer IMI caused by
Escherichia coli and all Enterobacteriaceae when compared to quarters treated with
intramammary antibiotic. Both studies noted that the use of internal teat sealant was

also useful in preventing new IMI around calving caused by Enterobacteriaceae.

In North America, Godden et a1. (2003) demonstrated a significant effect of
adding teat sealant to DCT, in which quarters sealed with bismuth subnitrate were
30% less likely to develop a new IMI between dry-off and calving. These quarters
were also 31% less likely to have an IMI present at 1 to 3 DIM, and were 33% less
likely to experience a clinical mastitis event between dry off and 60 DIM, with a
significantly lower linear score at l to 3 DIM and 6 to 8 DIM, as compared with
control quarters.

External teat sealants

Barrier teat dips with a germicide have been used in‘heifers and in cows to
prevent new IMI timing the dry period. However, no benefits of its use were
demonstrated on new infection rate at parturition (Edinger et al., 2000; McArthur et
al., 1984; Matthews et al., 1988). Duration of sealant adherence to the teat-end should
be considered when evaluating the impact of teat sealant treatment at drying off on
the level of infection after calving. Moreover, the use of new compounds with higher
adherence and also repeated teat dipping reduced IMI suggesting that the use of dry-
cow teat sealants have a beneficial impact on the level of infection at calving, if a

durable seal is formed and remains on the teat-end (Lim et al., 2007).

12

The use of DCT does not have to be limited to the classical intramammary
approach. There are other choices that include systemic antibiotics or a combination
of both options. Teat sealant can be added, to ensure protection to the mammary
gland when the risk of infection is higher at the beginning or the end of the dry
period. But general management practices such as clean environment, comfortable
hygienic bedding, and clean calving pens cannot be forgotten, since DCT will not

replace a poorly managed transition and calving barn.

Macrolides

Macrolides were first isolated in 1950 from a strain of streptomyces, a type of
bacteria found in soil. All of the antibiotics in this family share a macrocyclic lactone
in their structure. The family is named for this feature and each member is classified
according to the size of the macrocyclic ring with 12-, 14-, or 16- membered ring
macrolides. The lZ-membered drugs include methymicin, the 14-membered group
includes Erythromycin, and the l6-membered group includes tylosin. A subgroup
called triamilide includes new semisynthetic macrolides such as tulathromycin that
contains 3 additional amino groups (Lewicki, 2006).

Macrolides are generally bacteriostatic. Bactericidal activity may occur under
certain conditions such as increasing the dose or against specific microorganisms.
Their macrocyclic ring reversibly binds to the 23S ribosomal RNA (rRNA) in the SOS
subunit of susceptible organisms, and thus inhibits mRNA-directed protein synthesis.
Moreover, they stimulate the dissociation of peptidyl-tRNA during translocation,

suppressing RNA-dependent protein synthesis and inhibiting bacterial growth

13

(Blondeau et al., 2002). Macrolides diffuse very well into milk following systemic
administration with mammary tissue concentrations higher than plasma
concentrations. Their pK (pH at which concentrations of dissociated and
undissociated antibiotic are equal) is basic which results in a milk to plasma
concentration ratio of 5:1 (Riviere, 1999). Once the macrolides are in milk they are
virtually trapped inside the mammary gland (ion trapping) because of their basic pK.
Macrolides bear a unique cellular distribution. Scomeaux and Shryock (1999)
described tilmicosin distribution in cellular organelles, finding that 70 to 80% of the
drug was located in lysosomes. Furthermore, neutrophil antibacterial activity is based
on phagocytosis, a process in which lysosomes play a key role. Thus, the propensity
of macrolides for the lysosome may aid in bacterial killing (Paape and Capuco, 1997).

Macrolides are active against aerobic and anaerobic Gram-positive cocci such
as coagulase negative staphylococci (CNS). They are not effective against Gram-
negative anaerobes and some enterococci. The antimicrobial spectrum of macrolides
generally includes Gram-positive cocci such as Staphylococcus aureus, CNS, [3-
hemolytic streptococci, other streptococci species and some enterococci. Additional
activity has been documented, other agents including Haemophilus influenzae, some
pathogenic Neisseria species, Bordetella, Corynebacterium, Chlamydia,
Mycoplasma, Rickettsia and Legionella species. The modification of macrolide
structure has been shown to affect antibacterial activity. These changes also cause
differences in pharmacokinetics (Abu-Gharbieh et al., 2004).

Resistance to macrolides has been reported especially in the streptococci group

A, by two distinct mechanisms: 1) ribosomal modification resulting from the presence

14

of an errnmethylase and 2) drug efflux conferred by a membrane protein encoded by
the mefA gene. This resistance is specific for 14- and 15-member macrolides
(erythromycin, azithromycin and clarithromycin); l6-member macrolides (e. g.

josamycin, tylosin) are not affected (Weiss et al., 2001).

Tylosin

Tylosin is a mixture of four macrolide antibiotics produced by a strain of
Streptomyces fiadiae. The main component of the mixture (> 80%) is tylosin A,
although tylosin B, tylosin C and tylosin D may also be present (Lewicki, 2006). All
four components contribute to the potency of tylosin, which is not less than 900
IU/mg, calculated with reference to the dried substance (European Pharmacopoeia,

2004). A summary of tylosin physico-chemical properties is shown in Table 1.

Pharmacokinetics

Tylosin is adnrinistered to cattle either intramuscularly or subcutaneously.
Following [M administration of tylosin to calves (10 mg/kg body weight) at 12 hours
and 24 hours, the mean serum concentrations (:t SE) at 25 and 26 hours were 1.47 i
0.14 ug/mL and 1.25 :t 0.08, ug/mL (Burrows et al., 1986). Peak blood
concentrations of tylosin were reached in 2-4 hours following intramuscular injection
of tylosin base in 50% propylene glycol and the aqueous solution of the tartrate salt in
cows, ponies and pigs (Sauter et al., 1962; Gingerich et al., 1977). In another study,
peak serum concentrations in cattle following intramuscular injection of tylosin (12.5

mg/kg body weight) reached values of 2.5 ug/mL about 5-6 hours after injection with

15

systemic bioavailability of 70-80% of the administered dose (Ziv and Suhnan, 1973).
Saurit et a1. (2002) established peak concentration in cows for tylosin of 0.65 i 0.07
ug/mL although the dose reported (10 mg/kg body weight) was lower than normally
used.

With a pKa of 7.73 and featuring a high degree of lipid solubility, tylosin is well

distributed in tissues. Calculated theoretical tissue: plasma ratios (k 1 2/k2 l) of tylosin

in cows were 2.05 (Baggot and Gingerich, 1976). Milk to plasma ratio calculated in
milk samples was 5:1 (Riviere, 1999). Protein binding coefficients were 33.5-44% in
cows (Ziv and Sulman, 1972; Ziv and Sulrrran, 1973). Reported half-life values for
tylosin were 2.14, 1.62 and 2.24 hours (Ziv and Sulman, 1973; Baggot and Gingerich
1976; Saurit et al., 2002).

Primary metabolism occurs in the liver. Metabolites of tylosin include tylosin
A, N-desmethyl-tylosin A, tylosin D, N-desmethyl-dihydro-tylosin A, O-desmethyl-
tylosin, tylosin C and dihydrodesmycosin (JECFA, 1991). Tylosin is excreted
mainly through the liver. Its concentration in bile at 24 hours of IM administration
(7mg/ kg body weight) was 35.1ug/ml. Renal excretion is also important, urine
concentration of tylosin was 12.9 pig/ml 24 hours after IM administration (7mg/ kg

body weight), (Nouws and Ziv, 1977).

16

Table 1. Summary of physico-chemical properties of tylosin (Paesen et al.,
1995abc; McFarland et al., 1997; The Merck Index, 2001).

 

Molecular formula:
Molecular weight:

Appearance:
Melting point:

Solubility:

Stability:

pKa:

Log P (octanol-water):

UV Absorption:

c H Nol7

46 77

916.1

An almost white or slightly yellow crystalline
powder

128-132°C

5 mg/ml (water 250C), soluble in lower alcohols,
esters, ketones, chlorinated hydrocarbons,
benzene, ether, chloroform

Solutions are stable at pH 4-9 (most stable at pH
7);

Below pH 4 tylosin B (desmycosin) is formed as a
result of acid hydrolysis;

In neutral and alkaline pH — tylosin aldol (TAD) is
formed together with polar degradation products
of unknown identity;

When tylosin solution is exposed to daylight, a
photodegradation product - isotylosin A (isoTA) is
formed

7.73

1.63

1%
UV at 282 nm, extinction coefficient (E )
max. lcm

is 245 at 282 nm

 

l7

 

 

 

 

HO
CH3
0H
R3 CH3
CH3
0
H3C\ ,CH3 CH3
N O
9
R1 OH
R2 OCH3
Name Mol. R1 R2 R3
Formula
tyIOSill C45H77N017 osyl OCH3 CHO
A
tyIOSill C39H65N014 H OCH3 CHO
B
tyIOSIn C45H75N017 08y] OH CHO
C
tyIOSill C46H79N017 osyl OCH3 CH2 OH
D

 

Figure 1. Tylosin and associated metabolites. (Lewicki, 2006)

18

Cows receiving a single intravenous injection of tylosin tartrate (20 mg/kg b.w.)
had peak concentrations of tylosin in milk of 10 [Lg/m1. In contrast, corresponding
plasma concentrations of tylosin 4 hours after injection were only around 3.5 [lg/ml.
Lower peak values (approx. 6 [Lg/ml) were observed in cow’s milk 6 hours after a
single intramuscular injection of tylosin tartrate at the same dose. The ratio of peak
normal milk (pH = 6.5-6.8) concentrations to peak serum concentrations of tylosin
was approximately 2.5, while the ratio of peak mastitis milk (pH = 7.1-7.4) to peak
serum concentrations was 1.6 (Ziv and Sulman, 1973). In cows receiving tylosin base
intramuscularly (12.5 mg/kg b.w.) every 12 hours for 48 hours, the peak
concentration of tylosin in milk (approx. 7 ug/ml) appeared after 60 hours and then
rapidly decreased to 1.5 [lg/ml at 72 hours. Milkzserum ratios corrected for
differences in protein binding and calculated at various times ranged up to about 20:1
(Gingerich et al., 1977). Similar milkzserum ratios (up to approx. 17.5:1) were
observed in cows after a single intramammary infusion of 200 mg of tylosin/quarter.
When mastitic cows received repeated intramuscular injections of tylosin base at a
dose of 10 mg/kg b.w. every 12 hours for 5 days, peak milk concentrations increased
gradually up to 18 ug/ml at the fifth day after the onset of therapy (El-Sayed et al.,

1986)

19

Table 2. Antimicrobial activity for tylosin. Reported minimum inhibitory
concentrations (MIC 90) for different bacterial isolates (Catry et al., 2003. Odlan
et al., 2002)

 

BACTERIA MIC90 rig/ml
Staphylococcus chromogenes 1.00
Staphylococcus aureus 80329 0.5

Staphylococcus aureus A T C C2921 3 1-2
Streptococcus pneumoniae 80541 <0.125
Streptococcus pyogenes 80542 <0. 125
Escherichia coli 80001 >64
Escherichia coli AT CC25922 >64
Haemophilus influenzae 16

 

20

Non-lactating heifers

The dairy heifer in lactogenesis faces several risk periods. During these
periods, exposure to IMI can affect mammary development, and negatively influence
milk production over her lifetime. Understanding lactogenesis and mammary
development is essential in designing management protocols to reduce the risk of [MI
at calving and optimize milk quality and production.

Marnmogenesis

Mammary glands are exclusive of animals belonging to class Mammalia. These
exocrine glands are modified sweat glands, which are located in anatomically
different alignments among species. Each gland has a teat or nipple; this structure
differs in formation depending on the infraclass either Methateria or Eutheria.
Methateria animals (e.g. kangaroos) have nipples covered by a flap of skin forming
the pouch. Eutheria animals have the nipples uncovered and with a wide variation of
locations along the ventral line.

In the bovine embryo, mammogenesis follows six stages as described by Larson
(1978). The first stage starts at 30 days of gestation with the formation of the
mammary band, which is a thickening of the ventrolateral ectoderrn. Then with
further development, this group of epithelial cells becomes a mammary streak at 32
days. A mammary line is formed at 35 days, becoming a mammary crest by 37 days.
At 40 days of gestation the embryo bears the mammary hillock that is transformed at
43 days to a localized area of epithelial development from which a mammary gland

will arise regardless of species.

21

Larson (1978) also defined the pattern of development for the fetal mammary
gland. After a mammary bud has been formed, further differentiation continues.
Teats are formed at 65 days from a protruding structure of the mesenchyme
surrounding the sprout. The primary sprout is recognized by 80 days of fetal age.
This is a projection of the mammary bud into the surrounding mesenchyme. The
number of projections determines the number of openings arising from each teat, in
the cow there is only one, whereas humans have 15 to 25. By 90 days secondary
sprouts develop; these are secondary structures that will become mammary ducts, due
to a lack of a fatty pad, secondary sprout development is very limited in the male. At
later stages secondary sprouts will branch and appear as the tertiary sprouts. These
are the precursors of the secondary milk ducts. Canalization of the primary sprout is
visible by 100 days. As the circumference of the sprout increases, a lumen is formed.
This begins at the proximal end of the primary sprout (inner end) and proceeds in
both directions, forming the gland cistern. The most distal region of the lumen will
develop into the streak canal.

Larson (1978) also defined the complementary structures for the mammary
gland. The fatty pad, which is originated in the mesoderrn, is the adipose tissue
structure into which the entire mammary complex of ducts and lobule alveoli
penetrate. The pad provides space for growth and a source of energy for the epithelial
cells. The structural tissues vary depending of the specie, e.g. in the bovine
suspensory ligaments such as the central and lateral are developed by 200 days of

fetal age. Myoepithelial cells are contractile epithelial cells that originate in the

22

epithelial layer of the gland and differentiate from the secretory epithelial cells.
These cells are non-functional until lactation.
Prepubertal development

There are 2 stages of growth in the mammary gland of a female calf; isometric
that occurs for the first 2-3 months of age, and allometric growth, which initiates at
three months and reaches its plateau at 9 months of age (Hovey et al., 1999; Sinha
and Tucker, 1969).

Isometric growth means that the gland grows at the same rate as the entire body.
During this stage, there is no development of secretory lobular structures or secretory
tissues. The increase in the size of the gland is the result of growth of fat pad and
connective tissue (ansci.uiuc, 2006). In the female calf, a single primary duct extends
from the teat. The base of the duct forms the gland cistern and the distal portion
forms secondary and tertiary ducts to which mammary epithelium is related (Hovey et
aL,1999)

At about 3 months afier birth, allometric grth begins (growth rate faster than
the rest of the body). In the calf, this includes extensive growth and development of
the duct network, which invades the surrounding adipose tissue (fat pad). The
allometric growth phase lasts until about 1 year of age, when the mammary growth
rate returns to isometric growth (ansci.uiuc 2006). The allometric growth stage is
highly dependent on hormonal changes. Prepubertal mammary epithelium
proliferation is stimulated by progesterone and estrogen. The family of progesterone

receptors (PR) and estrogen receptors (ER) mediate their influence. The ERs include

23

ERa, ERB, and three estrogen-related receptors (ERRs) (Meyer et al., 2007; Connor et
al., 2005; Schams et al., 2003).

At 4 months of age, the mammary gland develops a limited ability to respond
to lactogenic stimuli (insulin, prolactin and EGF) if primed with steroid hormones.
By 7 months of age, priming with steroids is no longer necessary for histologic
changes in response to lactogenic stimuli. By 10 months of age, changes in histologic
appearance do not require in vivo steroid priming but functional differentiation and b-
casein mRNA expression is only achieved when glands are cultured in the presence
of the lactogenic hormones after steroid hormone priming. (Maple et al., 1998).

Other hormones and growth factors play a role in mammary gland development.
Growth hormone (GH) directs allometric grth in heifers, but requires the presence
of estrogen to promote growth. Local synthesis of various growth factors promotes
the allometric grth stage. Among these factors is TGF 131, which selectively acts
on the stromal compartment of the bovine mammary gland by increasing cell
proliferation and gene expression of extracellular matrix proteins. Fibroblast growth
factors are also involved, including FGF-l, IGF-l, TGF-or and HGF (Hovey et al.,
2002; Musters et al., 2004; Sinowatz et al., 2006).

Rearing heifers at an elevated level of nutrient intake especially a high energy
diet impairs mammary development (Meyer et al., 2006, Capuco et al., 2005).
Furthermore, leptin a hormone produced by adipocytes and elevated in serum of
animals fed high-energy diets inhibits mammary development by reducing DNA
synthesis in mammary gland associated epithelial cells (Silva et al., 2002). Its

physiological pathway is still unclear since bovine mammary epithelial cells have

24

negligible leptin receptor expression, and do not respond to leptin in vitro (Thorn et

aL,2006)

Puberty to Conception

At the onset of puberty, a 21-day estrous cycle begins in heifers, affecting
growth of mammary gland. During estrus mammary DNA synthesis increases 115%
and then decreases slowly through metestrus and diestrus until it reaches the lowest
level just before estrus (Sinha and Tucker, 1969). The fundamental developmental
unit in mammary tissue is the terminal ductule lobular unit (TDLU). After puberty it
grows at an isometric rate, and only reaches full alveolar development after
pregnancy (Hovey et al., 2002). Mammary development accelerates throughout
pregnancy, and reaches its peak during the last trimester of pregnancy, coinciding

with the most rapid period of fetal growth.

Intramammary infections

While mastitis in heifers has been recognized as a problem, most dairymen
view heifers at parturition as being free of intramammary infections. Mastitis in
heifers was first recognized in the 19403 by Palmer et a1. (1941). Schalm (1942)
traced IMI in heifers caused by Streptococcus agalactiae to suckling among calves.
More recently the prevalence of IMI in prepartum heifers has been investigated more
thoroughly. As many as 63% of heifers and 34% of their quarters may have
intramammary infections (IMI) at first parturition and this causes an increase in SCCs

and clinical mastitis cases after calving (Borm et al., 2006).

25

Intramammary infections affect dairy heifers from puberty throughout gestation
and around calving. Trinidad et al., (1990a) reported IMI in 87% of the quarters of
unbred heifers. Secretion samples from infected quarters had an increased SCC when
compared to normal quarters. In another study, Trinidad et al. (1990b) collected
mammary tissue samples from unbred heifers and found that secretory parenchyma
fiom infected quarters had more connective tissue than samples from uninfected
quarters. In addition, infected quarters exhibited greater leukocyte infiltration. Thus,
IMI in puberty can impair mammary growth and development and influence future
milk production.

The major problem associated with mastitis in heifers around calving is
increased SCC (Waage et al., 1999). During the weeks of subclinical IMI, infected
heifers would contribute elevated somatic cells to the bulk tank reducing the quality
of milk for the whole farm. These infections can become clinical and cause further
losses and premature culling. The heifer’s IMI rate is reflective of the mastitis
problem for the entire herd. A retrospective study (DeVliegher et al., 2004) using
117,496 four-weekly test-day records of 14,243 heifers revealed that heifers with a
high test-day value SCC (200,000 or more) on days 7 to 14 after calving were likely
to remain high during the whole first lactation. In the same study, the impact of early
lactation SCC on milk yield was analyzed. Heifers with SCC 500,000 and 1,000,000
produced 119 and 155 kg less milk respectively as heifers with SCC of 50,000
(DeVliegher et al., 2004, 2005). In another study, prepartum antibiotic-treated heifers
produced 531 kg more milk and also had lower SCC scores than the untreated control

group (Oliver et al., 2003).

26

The main causes of IMI in unbred and primigravid heifers are CNS (Fox et al.,
1995, Oliver et al., 2004, Trinidad et al., 1990). Some of the CNS are part of the
normal skin flora and include the species Staph simulans, Staph hyicus and Staph
epidermis; others are found in the environment such as novobiocin-resistant species.
The CNS are opportunists and infect the teat canal and the gland from skin sources.
Some may colonize the teat canal and persist for long periods (Aarestrup and Jensen
1997,Waage et al., 1999). Thus, while CNS are often grouped together, considerable
variation in CNS species is reported and some are more problematic than others
(Oliver et al., 2004). The duration of infection varies among Staph species, for
example, Staph chromogenes can be found in infected quarters beginning 4 weeks
before calving but its prevalence is reduced sharply after calving while other CNS
such as Staph simulans are present 2 weeks before calving but its infection persists

several weeks after calving (Aarestrup and Jensen, 1997).

Antibiotic treatment before calving

Prepartum antibiotic therapy has been evaluated in heifers. Trinidad et al.
(19900) evaluated intramammary antibiotics in breeding age and primigravid heifers
during different trimesters of pregnancy and demonstrated that this practice was
effective in reducing the prevalence of IMI at parturition. However, there were some
issues related to antibiotic residues in milk due to use of antibiotics formulated for
non-lactating cows.

Oliver et a1. (1992) used either intramammary Cloxacillin (200 mg) or

intramammary Cephapirin sodium (200 mg) 7 days before expected day of calving.

27

Both antibiotic formulations reduce [MI especially those caused by coagulase-
negative staphylococci. However antibiotic residues in milk were found at days 3 and
5 after parturition. More recently, lactating cow formulations have been used 7 to 14
d before expected day of parturition. These treatments reduced IMI in 60% to 75% of
quarters infected (Oliver et al., 2003; Borm et al., 2006).

Antibiotic infusion in mammary glands of pregnant heifers can reduce IMI
prevalence and can increase milk production. In a study of one herd, prepartum
antibiotic-treated heifers produced 531 kg more milk and also had lower SCC scores
than the untreated control group (Oliver et al., 2003). Bonn et al. (2006) analyzed
intramammary antibiotic infusion in heifers from 9 herds in 7 different locations. The
authors found that milk production and SCC from prepartum antibiotic-treated heifers
did not differ significantly from the untreated control group. However, there were

some differences depending on prevalence of IMI in each herd.

Antibiotic treatment immediately after calving

Some research groups have studied antibiotic treatment immediately after
calving. Kreiger et a1. (2007) used systemic (intramuscular) penethamate hydriodide,
in heifers from herds with a high prevalence of Staphylococcus aureus infections.
Heifers treated with antibiotics had fewer IMI and had higher milk production.
However, this study consisted of a small number of animals and required a 5-day
withholding for the milk produced by treated animals. This inconvenience did not
provide an economically attractive protocol. Oliver et a1. (2007) analyzed a different

approach, using either pirlymicin or penicillin-novobiocin an intramammary

28

antibiotic after the first milking post-calving. No significant differences were
observed when penicillin-novobiocin was compared with untreated control.
Significantly, fewer IMI during early lactation were observed in heifers treated with
pirlimycin compared to those in the untreated control group, although this treatment
did not appear to be as effective as prepartum antibiotic-treatment of heifer mammary

glands.

Internal teat sealants

Parker etal., (2007) tested a bismuth subnitrate teat-canal sealant 30 days before
expected calving in heifers. They found that it improved udder health, reducing the
risk of IMI caused by Streptococci uberis by 84% and clinical mastitis cases by 68%.
However, the use of teat sealants based on bismuth subnitrate has been linked to the
appearance of black spot defect (BSD) in Cheddar cheese. Lay et a1. (2007) related
this defect to residual levels of bismuth salt precursor unintentionally entrained within

the cheese milk.

Tylosin use as systemic antibiotic in DCT and PCHT

Metaphylaxis to prevent new and treat chronic IMI using DCT or PCHT has
been proven to be effective, however, effectiveness may vary depending on herd
health status, bacteria type, and herd management issues. Systemic administration of
antibiotics such as tylosin can be an alternative to DCT or PCHT alone or used in
combination with intramammary treatment. In the following chapters, the use of

tylosin alone or in combination with intramammary infusion of cephapirin and with

29

the addition of a teat sealant was tested in mature dairy cows at the time of dry off as
DCT or in primigravid heifers 2 wk before calving as PCHT. We hypothesize that
tylosin alone or in combination with intramammary antibiotic before calving as DCT

or PCHT will lower the occurrence of [MI and reduce SCC after calving.

30

CHAPTER 2

Comparison of Systemic and Intramammary Dry Cow Treatments

G. Andres Contreras DVM.
Department of Large Animal Clinical Science, Michigan State University,
East Lansing, Michigan

Walt M. Guterbock DVM, MS
DenDulk Dairy
Coopersville, Michigan

Philip M. Sears DVM, PhD
Department of Large Animal Clinical Science, Michigan State University,
East Lansing, Michigan

SYSTEMIC AND INTRAMAMMARY DRY COW TREATMENTS

Corresponding author: G. Andres Contreras
contrera@cvm.msu.edu
1642 Spartan Village, Apt D
East Lansing, Michigan, 48823
USA

31

Summary

The objective of the study was to compare four different dry cow treatments and
establish whether effectiveness at quarter level and by bacterial type increased when
intramuscular tylosin therapy was added to intramammary infusion of cephapirin, and
also evaluate tylosin alone intramuscularly as a systemic dry cow therapy (DCT). A
total of 278 cows at the end of lactation were randomly assigned to one of 4 dry cow
treatment groups: 1) Group CESE (n=89), cephapirin intramammary and teat sealant.
2) Group TYCESE (n=84), cephapirin intramammary, tylosin 12 g intramuscular and
teat sealant. 3)Group TYSE (n=86), tylosin 12 grams intramuscular and teat sealant;
4) Group TY (n=76) tylosin 12 g intramuscular only. Quarter milk samples for
culture were collected at dry-off and 1 and 2 wk after calving. Somatic cell counts
(SCC) were taken from Dairy Herd Improvement Association (DHI) tests at dry-off,
and the first two test days after calving. Milk production and health records were also
monitored. Bacteria cure rate for Gram-positive intramammary infections (IMI) for
group TYCESE was 93.6%, group CESE 78.9%, group TYSE 88.2%, and group TY,
78.1%. All four groups showed a decrease in the SCC at the first and second test after
calving. The difference in means for log SCC between dry-off and fresh was highest
for group TYCESE. Results demonstrated that use of systemic tylosin in combination
with the intramammary antibiotics increased the effectiveness of DCT. The inclusion

of this macrolide improves the cure rate of Gram-positive IMI.

32

Comparison of Systemic and Intramammary Dry Cow Treatments

Dry cow therapy (DCT) is defined as the use of antibiotics immediately after the
last milking of lactation. There are three different approaches to DCT. First,
intramammary dry treatment alone is widely used and very effective in reducing
intramammary infections (IMI) (Berry & Hillerton, 2002; Dimmick, 2001; S01 et al.,
1994; Zwald et al., 2004). However, low efficacy against some coagulase negative
staphylococci IMI has been demonstrated (Rajala-Schultz et al., 2004). Another
approach is systemic dry cow therapy (Soback et al., 1990; Nickerson et al., 1999),
which is relatively inexpensive and easy to use but has been reported to be less
effective and is therefore rarely used.

Finally a combination of systemic and intramammary antibiotics can also be
administered; this method could increase the effectiveness over that of intramammary
treatment alone because it provides advantages such as better diffusion of certain
systemic antibiotics into the mammary gland. O’Boyle et a1. (2006) compared two
combinations, Oxytetracycline hydrochloride (OTC-hcl) 12 g intramuscular plus
intramammary cephapirin 300 mg and tylosin subcutaneously 20 g plus
intramammary cephapirin 300 mg. A teat sealant was used in combination with both
treatments. Tylosin plus cephapirin gave better results in reducing somatic cell counts
(SCC) and intramammary infections after calving than OTC-hcl plus cephapirin.

The use of tylosin alone as a systemic DCT has not been tested before. This
antibiotic belongs to the macrolide family of antibiotics. One of the main advantages
of this type of antimicrobial is an excellent diffusion into the mammary gland related

to its basic pK (pH at which concentrations of dissociated and undissociated antibiotic

33

are equal), which results in a very high milk to plasma concentration ratio of 5:1
(Omura, 1984; Riviere, 1999). Once the macrolides are in milk they are virtually
trapped inside the mammary gland. Because of these features tylosin was used as
systemic DCT in this study.

Evaluation of combined DCT with tylosin as systemic antibiotic and cephapirin
as an intramammary antimicrobial has not been performed at quarter level. O’Boyle’s
et a1. (2006) results are based on composite samples; in this study, analysis of IMI
was based on quarter cultures.

The objective of this study was to compare four different DCTs and establish
whether effectiveness of DCT at quarter level increased when systemic
(intramuscular) tylosin therapy was added to intramammary infusion of cephapirin,
and also evaluate tylosin alone as a systemic DCT.

Materials and methods

A completely randomized design that included three treatments and a control
was used. Cows on a commercial farm in Michigan were selected. Every week,
starting from November 2005, and after confirming their gestation to be greater than
150 days, a group of mature Holstein cows due to go dry was assigned to one of four
DCT groups (Table 1).

Treatment CESE (n=89 cows), reflecting standard practice on the farm, was
used as control. Alter the last milking of lactation, animals were first infused in each
productive quarter with a commercial preparation of 300 mg cephapirin
(Tomorrow®, Fort Dodge, Ft Dodge, IA, USA) intramammary and then with 4 g of a

commercially available internal teat sealant containing 65% bismuth subnitrate

34

(Orbeseal®, Pfizer. New York, NY, USA). Treatment TYCESE (n=84 cows)
animals were first infused in each productive quarter with cephapirin intramammary
300 mg, then injected intramuscularly with tylosin (Tylosin, Agripharma. Westlake,
TX, USA) 12 g and finally were infused with teat sealant. Group TYSE (n=86 cows)
animals received tylosin 12 g intramuscular and then teat sealant was infused in each
productive quarter. Group TY (n=76 cows) animals received 12 g tylosin
intramuscularly. Intramammary infusions were administered by the partial insertion
technique. Prior to instillation, all teat ends were prepared following the farm’s
milking preparation routine that included a predip containing 0.1% iodine, after
which the dry teat end was disinfected with a cotton pad containing 70% isopropyl
alcohol. Intramuscular injections were given in the neck, splitting the dose between
two different sites. Tylosin dosage calculation was based in 18mg/kg dose for a 650
kg cow (average weight for mature cows on this commercial farm).

Quarter milk samples were taken following National Mastitis Council
guidelines (Hogan et al., 1999) from all functional quarters at dry-off and at 1 and 2
wks after calving. Samples were immediately processed at the dairy’s on-farrn
laboratory. A 10 uL aliquot from each sample was cultured on blood agar containing
5% esculin at 37°C for 24 h. Colonies were tentatively identified as staphylococci,
streptococci, Staphylococcus aureus, coliforms or others; a presumptive diagnosis of
staphylococci, streptococci, coliform, or other pathogens was made, based on colony
growth, morphology and appearance, pattern of hemolysis, and catalase reaction.
Staphylococcal isolates were tested for coagulase production with the tube coagulase

test. Grarn-negative bacteria were plated on MacConkey agar to facilitate

35

identification. Only staphylococci, streptococci and S. aureus were included in data
analysis based on the assumption that cephapirin and tylosin are indicated to treat
only Gram-positive infections. A cow was considered infected if at least one
productive quarter had an IMI. A quarter was considered infected if five or more cfu
of the same kind were identified, and was considered contaminated if three or more
different colony types were present. Post calving samples were used to establish
bacterial cure. A cow was considered cured if she had an IMI at dry-off and all
productive quarters were negative at both samples post-calving. A cow was not cured
if she had an IMI at dry-off and continued with the same quarter(s) infected with the
same species at first and second test after calving. A newly infected cow had no IMI
at dry-off and at least one quarter was positive at 1 and 2 wk after calving. A quarter
was considered cured if it was positive at dry-off and negative in first and second
post-calving samples. It was considered not cured if it was positive to the species
isolated at dry-off and again 1 and/or 2 wk after calving. A quarter was considered
newly infected if it was negative at dry-off and positive at 1 and 2 wk after calving.
Dairy Herd Improvement (DHI) tests were recorded to monitor SCC. The last
test of lactation was considered the dry-test, and then linear SCC scores from
subsequent monthly tests, were used to determine changes in SCC. Milk production
records were collected by DHI personnel and total production was established as
projected to 305 days mature equivalent (ME) by a herd management software
(DairyComp®, Herd Management software, Valley Agricultural Software, Tulare,
CA, USA) based on milk production at DHI test taken between 180 and 200 days in

milk (DIM) after calving following the DCT assigned.

36

Cows with less than 30 (1 dry were excluded because of antimicrobial residue
issues that required milk withdrawal. Cows with more than 100 (1 dry were also
excluded. Of the 335 cows enrolled in the dry cow treatment trial, 278 had complete
records and were included in the results. Culture results were analyzed using chi-
square and SCC were analyzed by one-way ANOVA proc mixed procedure in SAS®
(SAS Institute, 2003. Cary, NC, USA). Significance level was set at alpha=0.05.
Results

A total of 123 cows (44%) had IMI at dry-off. No differences in infection rate
were found when comparing all four groups. Percentages of cows with IMI within
each treatment group were as follows CESE 40%, TYCESE 39%, TYSE 45% and TY
53%. Cure rates at cow level were significantly higher for group TYCESE (75%)
when compared to group TY (46%). Group CESE had a (66%) cure rate and TYSE
(51%) and this difference was not significant. A summary of these results is shown in
Table l.

A total of 245 quarters (23% overall) were found infected with staphylococci,
streptococci or S. aureus. IMI rates by quarters at dry-off are shown in Table 1.
Group TY had 32.3% infected quarters and group TYSE had 24.6%, both were
significantly higher (p<0.05) than TYCESE 17.3% and CESE 19.7%. Staphylococci
accounted for 62.9% of the infections, streptococci for 32.6% and S. aureus for
4.5%. The staphylococcal infection rate was significantly higher in group TY when
compared to TYCESE and TYSE (p<0.05) (Table 1). Infections caused by S aureus
were found in 2 quarters of group CESE, 2 quarters of TYCESE, 6 quarters of TYSE

and 1 quarter of TY. With such a small number of infections it was not possible to

37

establish differences among groups. Further studies are needed to evaluate tylosin
efficacy alone or in combination against S. aureus IMI.

Bacterial cure rates for gram-positive bacteria by quarters are shown in Table 1.
TYCESE had the highest cure rate (93.6%) and was significantly different (p<0.05)
from TY (78.1%) and CESE (78.9%). No difference was found when TYCESE was
compared to Group TYSE (88.2%). New infections rates were low, group CESE
(1.8%), group TYCESE (2.6%), group TYSE (2.9%) and group TY (3.3%).

When comparing cure rates by bacteria type (Table 1) all four treatments had
good efficacy against streptococcal infections, but there were differences in cure rates
for staphylococcal infections. Group TYCESE had higher cure rates (92.5%) (p<0.05)
than CESE (73.17%) and TY (72.4%).

All four treatments resulted in a decrease in the SCC at the first and second test
after calving (Figure 2). When arithmetic means of logSCC (logarithm to base 2 of
raw SCC count) at dry-off, first and second test after calving were compared (Fig 3),
TYCESE had the greatest decrease in LogSCC between dry-off and first test (1.7) and
group TY the lowest (0.5), although differences among groups were not significant.

Milk production at test date between 180 to 200 DIM was analyzed using 305
ME value. Milk production for each treatment group was as follows: TYCESE 10999
i 2616 kg, CESE 10143 i 2348 kg, TY 10074 i 1968 kg and TYSE 10024 :t 1880
kg. When the difference between arithmetic mean of previous and current lactations

was calculated for all groups, no significant differences were found.

38

Discussion

The randomization was effective in distributing IMI among groups at cow level,
but was ineffective at quarter level generating a bias, observed in a higher number of
quarter infections and a lower cure rate for the TY group. An ideal randomization
would have included blocking alter culturing quarters but this process would have
represented major logistical problems on this commercial farm. However, all four
DCTs were effective in reducing IMI.
Results for the combination of intramuscular tylosin plus intramammary cephapirin
are similar to the results of O’Boyle et al. (2006). In his study cure rate for Grarn-
positive IMI was 87% when a combination DCT including tylosin was given
subcutaneously. Furthermore, reduction of SCC was greater in group TYCESE,
although not significant, similar to O’Boyle’s results. There is a greater reduction in
SCC when a DCT including systemic tylosin and intramammary cephapirin is used.

Bacterial cure rates for systemic tylosin (group TY 78.1%) are similar to those
obtained by McDougall et al. (2007) in New Zealand, who administered tylosin
intramuscularly to treat clinical mastitis during lactation and obtained 82% cure rates
for Gram-positive infections. Systemic tylosin as DCT is as effective as
intramammary cephapirin plus teat sealant (Group CESE 78.9%) to reduce IMI.
Tylosin’s effectiveness could be related to its distribution properties into the
mammary gland and also to its unique intracellular distribution in neutrophils. It is
known that udder immunological response is mainly mediated by neutrophils (Paape
& Capuco, 1997). Neutrophil antibacterial activity is based on phagocytosis, a

process in which lysosomes play a key role. The distribution in cellular organelles of

39

tilmicosin, another macrolide was described by Scomeaux & Shryock (1999), finding
that 70 to 80% of the drug was located in lysosomes. This effect may be similar to
that of tylosin by increasing the effectiveness of neutrophils in clearing IMI.

When analyzing cure rate by bacteria type, intramammary treatment alone could have
been limited in its ability to eliminate infections during the dry period (CESE 73.2%).
Because staphylococci are responsible for 49% of IMI in the dry period (Dingwell et
al., 2003), an increase in the cure rate towards this bacteria type obtained by the
addition of systemic tylosin to the intramammary DCT would be economically
important.

In general DCT remains a cost-effective measure compared with no treatment at
dry-off (Berry et al., 2004; Robert et al., 2006; Huijps & Hogeveen, 2007).
Inclusion of a combined systemic and intramammary treatment has to be based on an
economic analysis of the increased cost of such therapy versus its higher
effectiveness. The use of systemic tylosin in combination with the intramammary
cephapirin increased the effectiveness of intramammary DCT against Gram-positive
IMI. There were no differences between the use of tylosin plus teat sealant and
intramammary cephapirin plus teat sealant at dry-off. However, tylosin has the
advantage of being cheaper and easy to use. Adding the teat sealant to the systemic
treatment with tylosin at dry-off improved the response of the treatment. Further

studies are needed to validate this effect in other dairy herds.

40

 

 

 

 

 

 

 

+CESE
" .v-ATYCESE

TYSE
-- TY

 

 

 

 

 

 

 

 

 

 

 

 

DRY-OFF FIRST TEST SECOND TEST

 

 

 

 

 

 

Figure 2. Mean values of logSCC at dry-off, first test and second test after calving
for each treatment group.

41

 

 

 

 

 

Figure 3. Somatic cell count reduction after calving explained by the difference
between the log SCC at DHI dry-off test and log SCC at first DHI test after calving
for each treatment

42

Table 3. Treatment designations, description and number of animals in each
treatment group. Number of cows infected at dry-off, percentage of cows with IMI
at dry-off. Number of cows cured after DCT and cure rate by treatment group.
Number of cows with new IMI after calving. Number of quarters enrolled, non-
functional quarters, number of quarters infected and percentage of Gram-positive
bacterial infection of total number of quarters at dry-off. Number and percentage
of quarters cured after calving. Number and percentage of bacterial infection by
bacteria type at Dry-off in each treatment group, and number of quarters cured and
cure rate after calving by bacteria type in each treatment. Values with the same
letter are not different from each other (p<0.05).

43

TREATMENT GROUP

 

CESE TYCESE TYSE TY
Treatment description Cephapirin Tylosin 12 g intramuscular Tylosin 12 g Tylosin 12 g
300 mg Cephapirin 300 mg intramuscular intramuscular
intramammary intramammary Teat Sealant
Teat sealant Teat sealant
Cows Enrolled 75 71 72 60
Cows with IMI at dry- 30 28 33 32
off
% of cows with IMI at 40% 39% 45% 53%
dry-off
Cow cured after DCT at 20 21 17 15
dry-off
% of cows cured alter 66%ab 75%a 51%ab 46%b
DCT at dry-off
Cows with new IMI 4 4 6 5
after calving
Quarters Enrolled 289 271 276 226
Non-functional quarters l 1 13 12 14
Quarters infected at 57 47 68 73
dry-off
% of quarters with IMI 19.7%b 17.3%b 24.6%a 32.3%a
at dry-off
Quarters cured after 45 44 60 57
DCT at dry-off
% of quarters cured 78.9%b 93.6%a 88.2%a 78.1%b
after DCT at dry-off
Staphylococci 41 27 28 58
Number of quarters
infected at dry-off
Staphylococci 72%a 57%b 4 1 %b 79%a
% quarters infected at
dry-off
Staphylococci 73%a 92%b 89%b 72%a
% quarters cured after
DCT at dry-off
Streptococci 14 18 34 14
Number of quarters
infected at dry-off
Streptococci 24% 38% 50% 19%
% quarters infected at
dry-off
Streptococci 100% 94% 91% 100%
% quarters cured after
DCT at dry-off
S aureus 2 2 6 1
Number of quarters
infected at dry-off
S aureus 3% 4% 8% 1%
% quarters infected at
dry-off
S aureus 50% 100% 66% 100%
% quarters cured after
DCT at dry off

44

CHAPTER 3

Prepartum Systemic Antibiotic For Heifer Mastitis

A Practical Approach to Reducing the Incidence of Intramammary Infection in
Heifers by Using Prepartum Systemic Tylosin Therapy

G. A. Contrerasl and P. M. Sears

Department of Large Animal Clinical Sciences
Michigan State University

East Lansing, Michigan, 48824

Corresponding author

G. Andres Contreras

CVM, A15A

Michigan State University
East Lansing, Michigan 48824
517-353-6869

Fax 517-353-6869
contrera@cvm.msu.edu

 

' Corresponding author: contrera@cvm.msu.edu

45

Interpretative Summary

Reducing the incidence of intramammary infections in heifers by using
prepartum systemic tylosin. By Contreras and Sears. The effectiveness for 20 g of
tylosin administered intramuscularly to prepartum heifers 14 to 18 days before
expected calving was analyzed. A control group of heifers with no antibiotic
treatment was compared to a tylosin treated group for the prevention of
intramammary infection at calving. The use of tylosin reduced the infection rate of
intramammary infections due to coagulase negative staphylococci at quarter level.
The treatment of heifers prepartum should not be advised without first evaluating
udder health, management and economic implications on each individual dairy farm.

46

Abstract

Intramammary infusion of antibiotics has been studied as a method to prevent and
treat intramammary infections (IMI) in heifers. Systemic administration of
antibiotics could offer economic advantages related to drug cost and labor. In this
study, the effectiveness of intramuscular administration of tylosin (20 g) was
assessed. Heifers on a commercial farm in Michigan, due to calve within 14 to 18 d,
were assigned randomly to one of two treatment groups. The control group (n=108
heifers) received no antibiotic treatment or teat sealants to prevent IMI. Group
tylosin (n=112 heifers) animals were injected intramuscularly with 20 g of tylosin.
Quarter milk samples were taken in duplicate fi'om all functional quarters at 2 to 6
days (sarnple-l) and 7 to 15 days (sample-2) after calving for bacterial culture.
Representative isolates from sample 1 were speciated. Somatic cell counts and milk
production were recorded. At sample 1, 42% of the heifers, and 16.5% of the quarters
were infected. Coagulase negative staphylococci (CNS) infected 10.8% of the
quarters and streptococci 3.6%. No antibiotic residues were detected at either sample
1 or sample 2. At the heifer level, tylosin did not reduce IMI infection rate caused by
Gram-positive bacteria. At the quarter level, tylosin reduced levels of IMI caused by
CNS. No differences were observed in somatic cell count (SCC) and milk production
between tylosin treated animals and controls, uninfected heifers had a lower somatic
cell score (SCS). Tylosin administration to primigravid heifers 2 weeks before
expected calving should not be advised without first evaluating udder health,

management and economic implications on each individual dairy farm.

47

Introduction

While mastitis in heifers is recognized as a problem, most dairymen often view
heifers at parturition as free of intramammary infections (IMI) For example, in a
survey (Borm et al., 2006) that included farms in 7 states and l province, as many as
63% of heifers and 34% of their quarters had an IMI at calving. The predominant
pathogen causing IMI in heifers from breeding age to first parturition are the
coagulase negative staphylococci (CNS) (Trinidad et al., 1990a; Fox et al., 1995;
Millys, 1995; Wage et al., 1999). IMI during lactogenesis can impair mammary
development and cause a decrease in milk yield in the productive life at the heifer
level. At the herd level IMI could cause an increase in somatic cell counts (SCC) and
clinical mastitis cases (Trinidad et al., 1990b).

To prevent and treat IMI in heifers, infusion of antibiotics in the mammary
gland before calving was proposed (Trinidad et al., 1990c; Oliver et al., 2003; Born et
al., 2006). Intramammary antibiotics are usually effective in reducing IMI, but
economic benefits can differ among farms depending on prevalence (Born et al.,
2006). Furthermore, intramammary antibiotic infusions in heifers results in
additional labor. Antibiotic administration immediately after calving, either
intramammary (Oliver et al., 2007) or systemically (Kreiger at al. 2007) was
previously attempted, but issues regarding antibiotic residues in milk were reported.
Systemic administration to heifers before calving could offer advantages related to its
relative lower cost and easier to administer when compared to intramammary
infusion. Tylosin has been studied for use in systemic DCT (O’Boyle et al., 2006;

Contreras et al., 2007). The major advantage of this antimicrobial is an excellent

48

diffusion into the mammary gland related to its basic pK (pH at which concentrations
of dissociated and undissociated antibiotic are equal), which results in a very high
milk to plasma concentration ratio of 5:1 (Omura, 1984; Riviere, 1999). In this
study, we analyzed the effectiveness of tylosin injected intramuscularly at a dose of
20 g to prepartum heifers 14 to 18 d before expected calving in reducing IMI.
Materials and methods

Heifers on a commercial farm in Michigan, due to calve within 12 to 18 d, were
selected every week between March and July 2007. A completely randomized design
that included a systemic antibiotic treatment group and a negative control group was
used. Before randomization, animals were properly identified and their gestational
stage was confirmed. Heifers then were assigned randomly to one of two treatment
groups. The control group (n=108 heifers), reflecting standard practice on the farm,
received no antibiotic treatment or teat sealants to prevent IMI. Group tylosin (n=112
heifers) animals received 20 g of tylosin (Elanco, Greenfield, IN). Injections were
administered intramuscularly in the neck and the 20 g dose was divided between three
different sites.

Quarter milk samples were taken in duplicate following National Mastitis
Council guidelines from all functional quarters at 2 to 6 d (sample-1) and 7 to 15 d
(sample-2) after calving. Samples were stored in a container at 5°C and processed
within 3h after collected. A 10 uL aliquot fi'om each sample was cultured on blood
agar containing 0.1% esculin at 37°C for 24h. Colonies were tentatively identified at
24h and 48h as CNS, streptococci, Staphylococcus aureus, coliform or others. A

presumptive diagnosis was made based on colony growth, morphology and

49

appearance, pattern of hemolysis, and catalase reaction. Staphylococcal isolates were
tested for coagulase production using the tube coagulase test. Gram-negative bacteria
were plated on MacConkey agar to facilitate identification. For Speciation,
biochemical tests were performed on representative positive cultures from sample-l
of streptococci and staphylococci isolates by using the API20 Strep SYSTEM and the
API-Staph SYSTEM, (BioMerieux, Hazelwood, MO). A heifer was considered
infected if at least 1 quarter was infected at sample-1; considered newly infected if it
was negative for all quarters at sarnple-l but infected at sample-2; and was
spontaneously cured if it was infected at sarnple-l but negative at sample-2. Quarters
were considered infected if at sample-l, five or more cfu of the same kind were
identified, and were considered contaminated if three or more different colony types
were present on both duplicate samples. A new infection was considered when a
quarter was negative at sample 1 but was positive at sample-2. A quarter was
considered spontaneously cured if it was infected at sample 1 but negative at sample-
2.

Two composite milk (3ml) samples were collected during sarnpling-l and 2.
One of the samples was used to perform antibiotic residue test with the Delvotest®
(DMS food specialties, Parsippany, NJ) and the second sample was used to determine
SCC at the regional Dairy Herd Improvement (DHI) laboratory. Dairy Herd
Improvement tests were used to monitor SCC and milk production during lactation.
Milk production records were collected by DHI personnel and total production was

calculated as projected to 305 d mature equivalent (ME) by a herd management

50

software based on milk production at DHI test taken between 180 and 200 d in milk
(DIM) alter calving.

Minimal inhibition concentration (MIC) values for the following representative
isolates were determined using the Sensititre® system (Trek® diagnostics,
Cleveland,OH): 6 Streptococci uberis, 6 Enterococcus faecalis, 10 Staphylococcus
chromogenes, and 10 Staphylococcus simulans. The following antimicrobials were
included in the Sensititre® plate format: ampicillin, ceftiofur, chlortetracycline,
clindamycin, danofloxacin, enrofloxacin, gentamicin, neomycin, oxytetracycline,
penicillin, spectinomycin, sulphadimethoxime, trimethropin/sulphamethoxazole, and
tylosin.

Culture results were analyzed using chi-square and SCC scores were analyzed
as repeated measures using a mixed model procedure (PROC MDCED; SAS Inst. Inc.,
Cary NC) following the equation:

Yijk=u+Gi+Ij+Gi*Ij+Sk+Sk*Gi+Sk*Ij+Sk*Gi*Ij+Ezjk
Where Yg‘k is the dependent variable SCC score for a heifer in group i, with infection
status j, at sample k as repeated measure, and Eijk is the random error assumed to be
correlated. The Satterthwaite’s method for estimating degrees of freedom was used.
Significance level was set at alpha=0.05.

Results and discussion

Infection levels for heifer and quarter are shown in Table 4. A total of 92 (42%)
heifers were infected at calving. At sample-1, 50 (46%) heifers were infected at
sample-l in the control group and 42 (38%) in the tylosin group had an IMI. By the

second sampling 76 (34.5%) heifers were infected, 43 (40%) in the control group and

51

33 (29%) in the tylosin group. This prevalence is similar to previous observations
(Trinidad et al., 1990;Bom et al., 2006). Spontaneous cures were observed in 19
heifers (38%) for control group and 21 animals (50%) in tylosin group. In both
groups, 11% of the heifers acquired a new IMI in the first week after calving. At the
heifer level, tylosin administration to prepartum heifers in this farm did not reduce
IMI caused by gram-positive bacteria.

At the first sampling (Table 4), 145/879 quarters were infected. At sample 2,
97/879 (11.2%) quarters had an IMI. Quarter spontaneous cure rate for tylosin group
was 61% and 50% for control group. New infections accounted for 5% of the quarters
and no statistical difference was found between groups (Table 4).

Similar to previous studies, CNS were the principal agent causing IMI in heifers
(Trinidad et al. 1990b; Fox et al., 1995; Oliver et al., 2004). The tylosin group had
fewer CNS infections in sample-1, 36 quarters (8.1%) than the control group 59
quarters (13.5%) and this difference was significant (P<0.01), demonstrating that the
high efficacy of tylosin against CNS, could have decreased infection rate due to these
microbes (Table 4). At sample-2, CNS infected 25 quarters (5.6%) in tylosin group
and 47 quarters (10.7%) in the controls, this difference was significant also (P<0.01),
but could be related to the initial lower prevalence of CNS infection in tylosin group
at sample-1.

Spontaneous cure for CNS was 61% for tylosin and 49% for controls. New
infections accounted for 2.9% of quarters in tylosin group and 4.1% for the control

group. Further studies are needed to evaluate economic and physiological

52

implications of transient infections of CNS in the first week of lactation in heifers,
since at least half of the CNS infections will clear without antibiotic intervention.

Bacterial Speciation results are shown in Table 5. Among CNS Staphylococcus
chromogens -a natural inhabitant of teats skin flora, S. simulans and S. epidermis are
highly prevalent around calving (Aarestrup and Jensen, 1997). Aarestrup and Jensen
(1997) reported, a higher prevalence of S. Chromogenes (15% of the quarters) in the
first two wk after calving, reflecting the normal presence of this species on the skin.
Similar to their results, S. chromogenes was the most prevalent species among CNS
in this study, representing 41.2% of CNS infections. In contrast, S. simulans affected
25% of the quarters in this trial, and only 3% in Aarestrup’s trials (1997) but were
persistent after several weeks after calving.

Streptococci infections made up 32/ 145 (22%) of all IMI after calving. Over all
streptococci infected 3.6% of the quarters at sarnple-l and 2% at sample-2.
Streptococcal infection rate was equal for both groups (3.6%) at sample-1, but was
slightly higher at sample-2 for tylosin (2.9%) than for control (1.1%). Streptococcus
uberis was the most prevalent streptococcal infection, followed by Strep.
dysgalactiae. In contrast, studies with pasture-grazed heifers in New Zealand
(Compton at al., 2006) revealed a higher infection rate by Strep. uberis (10% of the
quarters) in the first week after calving, reflecting changes in IMI prevalence due to
housing systems. Spontaneous cures for streptococcal infection accounted for 43% of
the quarters in the tylosin group and 56% in the control group. Four new infections

were found in the tylosin group, and none in the control group. Contagious

53

pathogens, Streptococcus agalactiae and Staphylococci aureus were absent from the
study animals.

Effectiveness of systemic treatment with tylosin varies depending dose and time
interval between administration and calving. Parker et a1. (2008) using pasture-
grazed heifers in New Zealand, compared a commercial teat sealant, tylosin (5 g of
tylosin base I.M. for 3 d at 24-h intervals) or a combination of both. Teat sealant
reduced the risk of new IMI by 74% and reduced the risk on new IMI with Strep
uberis by 70%. Tylosin did not have any effect in reducing risks. Differences in
responses to tylosin treatment between this study and Parker’s (2008) could have
been related to the relative low dose used in New Zealand and timing of treatment (27
d before expected calving). A higher dose with a single injection was used in this
trial, and the treatment was administered close to the programmed calving date (14 d).
Tylosin’s peak rrrilk concentration was reported to be 10 ug/ml after a single injection
at a dose of 20 mg/kg b.w.) (Ziv and Sulman, 1973), and 18 jig/ml after three
repeated injections at a dose of 10 mg/kg b.w. (El-Sayed et al. 1986). In the present
work, tylosin’s dose was based on average calving weight for heifers (600kg) at
33mg/kg. Despite of the relative high dose used, antibiotic residues were not found in
the composite samples taken from sample-1 and sample-2.

It is presumable that peak milk concentration in tylosin treated animals was
higher than previously reported, therefore achieving bactericidal activity, which was
observed for macrolides when used in high concentrations (Diarra et a1, 1999).

However, tylosin activity against enterococci was poor, and although had an MIC90

54

0.5 rig/ml, for Strep uberis,, enterococci isolates were resistant (MIC90 32 ug/ml) to
this macrolide (Table 6). i

It is unknown if the use of tylosin as a systemic treatment to eliminate or
prevent IMI in heifers at calving is dose and time sensitive, thus further studies are
needed to evaluate of extended therapy at different doses with this macrolide to
improve its response. Because of the studies logistic limitations, multiple
administrations and dosage based on individual weight was not possible.

Quarter infection rate by season is summarized in Table 8. No differences were
observed for CNS infections in spring at any sampling for both groups. In contrast,
tylosin treated heifers had fewer infections caused by CNS in summer when
comparing to controls at sarnple-l and sample-2 and this difference was significant
(P<0.05). The use of systemic treatment might have a usefirl role in decreasing CNS
infections during warmer months.

Log scores for SCC results are shown in Figure 4. Treatment effect was not a
significant (P=0.l7) source of SCS variation. At sample-1, tylosin group had a higher
score (4.2i3) than control group (3.7i2.3), at sample-2, 1St and 2nd DHI tests both
groups had similar means for the somatic cell scores. As expected, IMI status had a
significant effect in SCS (P<0.0001). Infected heifers had a higher SCS than non-
infected. When separating IMI by bacteria type for SCS results (Figure 5), CNS
infected heifers had similar SCS values to animals infected with other pathogens.
Despite of the high spontaneous cure rates for CNS, the infection status was still

responsible of increased SCS.

55

Tylosin group had an average milk projection at 305 d of 9,515i1847 at the first
test and 10,556i1790 at the second test. Similarly the control group had 9,495i1658
at the first and 10,346: by the second test. No difference in milk yield was found
between the two groups.

Conclusion

Systemic administration of tylosin was effective in reducing levels of IMI in
heifers caused by CNS at quarter level, but routine use on this farm may not be
economically sound. Transient IMI occur early in lactations with at least half of the
infections cleared without antibiotic intervention. Tylosin, administration to
primigravid heifers 2 wk before parturition, should not be advised without a previous
analysis of udder health, management, and economic implications in each individual
farm. Furthermore, in this study 10% of the heifers acquire an IMI within the first
week after calving, therefore special attention has to be given to environmental and
housing issues. Antibiotic therapy will not replace poor management in a close-up,

calving or fresh pens.

56

Table 4. Culture results at heifer level and quarter level at sample-1 and sample-2
including percent of spontaneous cure and percent of new infections, for control and
tylosin groups. Staphylococci and streptococci infections at sample-l and sample-2 for
control and tylosin groups, including spontaneous cure and new infections.
Percentages with different letters are considered to be different at P<0.05.

 

 

PARAMETER CONTROL TYLOSIN TOTAL P value
HEIFERS 108 112 220

IMI 2- 6 DAYS 50 42 92 0.18
% INFECTED HEIFERS SAMPLE 1 46% 38% 42%

HEIFER IMI 7-14 DAYS 43 33 76 0.10

LEVEL %INFECTED HEIFERS SAMPLE 2 40% 29% 34.5%
SPONTANEOUS CURE 19 21 4o 0.24
%SPONTANEOUS CURE 38% 50% 44%

NEW INFECTIONS 12 12 24 0.60
% NEw INFECTIONS 11% 11% 11%
QUARTERS 436 443 879
IMI 2-6 DAYS 80 65 145 0.14

QUARTER %lNFECTED QUARTERS SAMPLE 1 18% 14.5% 16.5%

LEVEL IMI 7 - 14 DAYS 54 43 97 0.20
%INFECTED QUARTERS SAMPLE 2 12.3% 9.7% 11%
SPONTANEOUS CURE 4o 40 80 0.16
%SPONTANEOUS CURE 50% 61 % 55%

NEW INFECTIONS 18 18 36 0.85
% NEw INFECTIONS 5% 5% 5%

STAPH QUARTERS 436 443 879

INF IMI 2-6 DAYS 59 36 95 0.009
%INFECTED QUARTERS SAMPLE 1 13.5%a 8.1 %b 10.8%

IMI 7 - 14 DAYS 47 25 72 0.005
%INFECTED QUARTERS SAMPLE 2 10.7%a 5.6%b 8.2%
SPONTANEOUS CURE 29 22 51 0.9
%SPONTANEOUS CURE 49% 61 % 70%

NEW INFECTIONS 18 12 30 0.35
% NEW INFECTIONS 4.1 % 2.9%

STREP QUARTERS 436 443 879

INF IMI 2-6 DAYS 16 16 32 0.96
%INFECTED QUARTERS SAMPLE 1 3.6% 3.6% 3.6%

IMI 7 - 14 DAYS 5 13 18 0.06
%INFECTED QUARTERS SAMPLE 2 1.1% 2.9% 2%
SPONTANEOUS CURE 9 7 16 0.47
%SPONTANEOUS CURE 56% 43% 50%
NEW INFECTIONS o 4 4 0.055

 

57

 

 

 

 

 

4 K
3
2
1
I I I I
Sample-l Sample-2 DI-ll Test 1 DHI Test 2
8—8—4 01-.

Control Tylo sin

Figure 4. Means for somatic cell scores (SCS) at sample-1 and sample-2, SCS at
1“t and 2'"l DHI tests after calving, and projected 305d ME milk production at l“t
and 2"d DHI tests for control and tylosin group.

58

 

 

 

 

 

 

 

r
f w T \\
2
Sample-1 Sample-2 DHI Test 1 DHI Test 2
CNS OTHERS

Figure 5. Means for somatic cell scores (SCS) at sample-1 and sample-2, SCS at
1"t and 2"" DHI tests after calving, and projected 305d ME milk production at 1’t
and 2"" DHI tests for infected heifers. CNS and others

59

Table 5. Minimum inhibitory concentrations (MIC) of ampicillin (AMP), ceftiofur
(CF), enrofloxacin (ENR), neomycin (NEO), penicillin (PEN) and tylosin (TY) for S.
chromogenes, S. simulans, Streptococci uberis, and Enterococcusfaecalis.

 

 

Organism N AMP CF ENR NEO PEN TY
Cm Milk ug/ml 3.4 2147* 2.7 222* 0.385 10
S. chromogenes 10 MIC50 0.25 0.5 0.12 4 0.12 l
MIC90 0.25 0.5 0.12 4 0.12 1
MODE 0.25 0.5 0.12 4 0.12 1
RANGE <=0.2 0.25—0.5 0.12-0.5 4 0.12-8 0.5-2
5-16
S. simulans 10 MIC50 1 0.5 0.12 4 0.12 0.5
MIC90 4 0.5 0.5 8 8 6
MODE 0.25 0.5 0.12 4 0.12 0.5
RANGE <=0.2 0.25-1 0.12-2 4-8 0.12-8 0.5-
5-16 32
Streptococci 6 MIC50 16 0.25 0.5 16 0.5 0.5
uberis MIC90 16 0.25 0.5 16 8 0.5
MODE 0.25 0.25 0.12 16 8 0.5
RANGE 0.25- 0.25 0.25-0.5 4-16 0.5-8 0.5-
16 32
Enterococcus 6 MIC50 0.25 0.25 .5 8 .25 32
faecalis MIC90 l 8 1 32 2 32
MODE 0.25 0.25 1 8 0.25 32
RANGE <=0.2 <=0.25- 0.25-1 5-32 0.25-4 32
5-1.0 8

 

*CF intramammary infusion
*NEO intramammary infusion

Table 6. Speciation of representative isolates at sample 1, from cultures at
sample 1 for control and tylosin groups.

 

 

BACTERIA TYLOSIN CONTROL TOTAL %

S. Chromogenes 16 12 28 41.2%
S. Simulans 7 10 17 25%
S.Epidermis 0 1 1 1 .5 %
S. Xylosus 1 1 2 2.9%
Strep. Uberis 7 3 10 14.7%
Strep. Bovis 2 O 2 2.9%
Strep. dysgalactiae 3 1 4 5.9%
E. Faecalis 2 2 4 5.9%

 

60

Table 7. Culture results for tylosin and control groups at sample 1 and sample 2

in summer and spring seasons.

 

 

Sample Season Tylosin Control P-value
CNS
Culture Neg Staph Strep Neg Staph Strep
result
1 Spring 160 16 6 85 23 5 0.09
Summer 221 20 10 174 36 11 0.04
2 Spring 162 15 4 269 22 3 0.08
Summer 211 10 9 190 25 2 0.01

 

61

CHAPTER 4

Conclusion

Dairy cows have changed dramatically in the past 60 years with the average
milk production increasing from 4622 pounds in 1950 to 17800 pounds per year in
2000 (Lake, 1971; US census, 2002). At the same time susceptibility to some
infectious and metabolic diseases has increased proportionally (LeBlanc et al., 2006).
There is a decline in number of dairy farms and animals are being concentrated in
larger herds, where efficiency indexes have improved, but cows are put at a high risk
for disease. Although major dairying developments like dry cow therapy, teat dips,
and better milking systems have helped improve udder health, some things have not
changed fiom the 1950’s with mastitis still accounting for the single greatest
economic loss (Lake, 1971; Wells and Ott, 1998).

To prevent and treat mastitis, antibiotic therapy during the non-lactating period
was introduced in the 1950’s for cows (Pearson, 1950) and for heifers in the 1990’s
(Trinidad et al., 1990c). Recently, issues regarding reduced effectiveness of DCT
against certain pathogens like CNS were reported (Rajala—Schultz et al. 2004)
revealing the need to improve efficacy by not only changing antibiotics but also the
way they are administered. Although precalving heifer treatment (PCHT) using
intramammary infusion is effective in reducing IMI, it is labor intensive and not
practical in most dairy herds. This has stimulated discussion of alternative

approaches to reduce IMI in heifers.

62

The use of tylosin in DCT offers the advantages of better diffusion into the
mammary gland, increased effectiveness against CNS, a relatively low cost and a
synergistic effect when used in combination with other antibiotics. If included in
PCHT, tylosin administered systemically is less labor intensive than intramammary
infusion. In this thesis, we evaluated the use of systemic tylosin (12 g) in DCT alone
or in combination with intramammary cephapirin. We also evaluated the

administration of systemic tylosin at a dose of 20 g in PCHT.

The use of systemic tylosin in combination with intramammary cephapirin
increased the effectiveness of intramammary DCT against Gram-positive IMI,
specifically CNS. Systemic tylosin plus teat sealant was as effective as cephapirin
plus teat sealant in curing Gram-positive IMI. Similar to results observed in DCT,
tylosin reduced the prevalence of IMI caused by CNS in the first week after calving

when administered to heifers 14 to 18 days before expected calving.

Each farm has geographical and environmental conditions that when
combined with different management styles make them unique. Systemic use of
tylosin can help improve udder health in some herds, but should not be advised for all
farms. Its use as a systemic antibiotic alone or in combination with other drugs in
DCT or PCHT cannot be routinely advised. Tylosin is very effective against CNS,
but antimicrobial activity against enterococci and some streptococci can vary. Since
pasture grazed herds have a higher incidence of streptococcal infections (Parker et a1,
2007), tylosin administration might not be as effective as in confined operations.

Special attention has to be given to housing conditions for transition cows, fresh cows

63

and heifers. In the heifer experiment, a high rate of new infections was observed
during the first week of lactation, demonstrating a higher IMI susceptibility during

this early stage of lactation.

Further studies are required to test tylosin’s effectiveness against contagious
pathogens such as S. aureus and Strep. agalactiae. Although tylosin interactions with
teat sealant were evaluated recently in heifers (Parker et al., 2008), an analysis of
such interactions is required in dry cows. During the heifer trial a high rate of new
IMI during the first week of lactation was observed. Furthermore, this high
prevalence of IMI was followed by a similar high rate of spontaneous elimination by
the second week of lactation. Additional studies are needed to evaluate the

implications of this transient IMI in udder health and its economic impact.

The dairy industry must adapt to the changing requirements of the consumers.
Quality dairy products are possible because of high quality milk produced on well-
managed farms, where animal nutrition, preventive medicine and a good cow
environment is employed. Tylosin is just another tool that may have its place on some
farms as a part of their udder health program. The decision to use it has to be based

on sound evaluations of environment, management and economics for the farm.

64

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