Received Date : 05-Aug-2016

Revised Date : 12-Dec-2016

Accepted Article Accepted Date : 21-Feb-2017
Article type : Article

Effects of clarithromycin, azithromycin, and rifampicin on terbutaline-induced sweating

in foals

A. L. Stieler Stewart1, L. C. Sanchez1, M. F. Mallicote1, A. L Muniz1, M. S. Westerterp2, J. A.

Burrow1 and R. J. MacKay*1

1
Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of

Florida, PO Box 100136, Gainesville, Florida 32610, USA;

2
Faculty of Veterinary Medicine, Utrecht University, Postbus 80163, 3508 TD Utrecht, The

Netherlands.

*Corresponding author email: mackayr@ufl.edu

Keywords: horse; anhidrosis; rifampicin; macrolide; foal; sweat; Rhodococcus equi

Summary

Background: Erythromycin (ERY) induces anhidrosis in foals. Azithromycin (AZI) and

clarithromycin (CLA), often combined with rifampicin (RIF), are commonly used to treat

Rhodococcus equi infections, but effects on sweating have not been investigated.

Objective: To determine the effects of AZI, CLA and RIF on sweat responses in normal foals.

Study design: Each experiment was a blinded, duplicated, 6-foal × 3-period counterbalanced

within-subjects design (12 foals/experiment).

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/evj.12677
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 Methods: Antimicrobials were given orally for 5 days. In experiment 1, ERY (25 mg/kg, three
Accepted Article times daily), AZI (10 mg/kg, once daily), and CLA (7.5 mg/kg, twice daily) were given. In

experiment 2, ERY, RIF (10 mg/kg, twice daily), and ERY/RIF combination were used.

Quantitative intradermal terbutaline sweat tests were performed daily for 3 days before and 1, 2,

5, 9, 24, and 39 days after treatment. Data were analysed by repeated-measures analysis of

variance procedures. Significance was P<0.05.

Results: In experiment 1, all macrolides suppressed sweating although CLA and AZI were less

potent than ERY. In experiment 2, significant sweat suppression occurred in foals given ERY

with or without RIF but there was no effect of RIF alone. Rifampicin reduced sweat suppression

by ERY on day 1 of treatment but not thereafter.

Main limitations: Because ERY blood concentrations were not measured, effects of RIF on

ERY-induced anhidrosis could not definitively be ascribed to altered ERY bioavailability.

Conclusions: All macrolides commonly used to treat R. equi pneumonia, i.e. ERY, AZI, and

CLA, induce anhidrosis in foals. The potent anti-sudorific effect of ERY is delayed but not

substantially affected by concurrent RIF administration.

Introduction

Pneumonia caused by Rhodococcus equi remains an important cause of morbidity and mortality in

foals one to six months of age [1]. The treatment of choice for over 30 years has been the

combination of a macrolide antibiotic such as erythromycin (ERY) with rifampicin (RIF) [2].

Relatively higher bioavailability and superior tissue and cell penetration have made the newer

macrolides, clarithromycin (CLA) and azithromycin (AZI), preferred over ERY [3].

Adverse effects of ERY have been reported to occur within the first five days of treatment and

include mild diarrhoea, hyperthermia and respiratory distress [4; 5]. The authors previously

reported the use of a quantitative intradermal terbutaline sweat test (QITST) to demonstrate

suppression of sweating in healthy foals given ERY. Episodes of hyperthermia (rectal

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 temperatures >39.4 C) occurred during the first three days after treatment ended [6]. Whether or
Accepted Article not other macrolides including CLA and AZI can induce anhidrosis is unknown.

Rifampicin, a synthetic derivative of rifamycin, is a highly lipophilic, bactericidal drug

that acts by inhibiting bacterial DNA-dependent RNA polymerase and is effective against

intracellular organisms including R. equi [7; 8]. Administration of RIF with CLA resulted in

plasma and tissue concentrations of CLA less than 10% of those found when the drug was given

alone [9]. The effect of RIF co-administration on sweat responses in foals given macrolides has

not been investigated.

The main objectives of this study were first, to look for a class-wide anti-sudorific action

of macrolides in foals by examining the effects on sweating of CLA and AZI, and second, to

determine whether the reported marked reduction in bioavailability of CLA by RIF predicts a

similar effect on the ability of ERY to induce anhidrosis. We hypothesised that all macrolides

administered would cause anhidrosis and that co-administration of RIF would improve the sweat

response in foals given ERY.

Materials and Methods

Experimental animals and design

Twelve pony and pony-cross foals aged one month at enrolment were used for each experiment.

Ponies used for Experiment 1 (4 females and 8 males) weighed 19 to 63 kg and those used for

Experiment 2 (9 females and 3 males) weighed 43.5 to 100 kg. Experiments were performed

from April to November of a single year. Foals were considered healthy on the basis of normal

results of physical examinations and complete blood counts performed 2 weeks before

experiments. Each experiment consisted of 3 periods; periods were separated by a treatment-free

washout interval of at least 34 days to allow sufficient time for full return to free sweating [4]. At

the completion of experiments, foals and their dams were returned to research herds. For

Experiment 1, foals were given ERY, CLA, or AZI at each period. To achieve a completely

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 balanced, within-subjects, repeated-measures design for these 3 treatments, 2 foals were assigned
Accepted Article randomly to each of the 6 possible treatment orders (i.e. ABC, ACB, BAC, BCA, CAB, CBA,

where A, B, C are the 3 treatments ERY, CLA, AZI). For Experiment 2, foals were given ERY,

RIF, or the combination of ERY and RIF at each period, with the treatment order fully balanced

among foals as for Experiment 1. Each period was 42 days. A summary of the design of

experimental periods is provided in Figure 1. For two days before until 10 days after treatment

began, foals were housed together if weaned, or with their dams, in covered stalls that were

shielded from direct sunlight and provided with wall fans. Foals were treated by mouth for 5

successive days. All study personnel involved in drug administration and data collection were

blinded to treatment. For Experiment 1, treatments were ERY basea (25 mg/kg bwt 3 times

daily), CLAb (7.5 mg/kg bwt twice daily) or AZIc (10 mg/kg bwt once daily). A lactose-based

powder was compounded to be visually indistinguishable from the macrolide preparations. This

placebo was given at times when antibiotics were not scheduled. For Experiment 2, treatments

were ERY, RIF (10 mg/kg twice daily) or ERY/RIF combination. Following completion of

treatment, from days 5 to 10, foals remained in covered stalls during the period of maximal

hyperthermia risk and monitored. On Day 10, foals and dams were moved into pasture

enclosures containing shade structures and kept there until the end of the experimental period

(Day 39). Feed and hay were provided both in shaded and open areas; water was only available in

open areas. Investigators were masked as to treatment assignments. Rectal temperatures were

recorded 3 times daily while foals were stalled (at approximately 0600, 1300, and 2000 h) and

twice daily (0600 and 1700 h) after they were turned out. Respiratory rate, heart rate and faecal

consistency were recorded once daily at 0600 h throughout experiments. In addition to the

scheduled temperature checks, foals kept outside were observed regularly (approximately every 2

h on the hottest days, less frequently on cooler days) during daylight hours for tachypnoea or

other signs of heat stress. When a foal was suspected of heat stress, its rectal temperature was

immediately checked. If the rectal temperature was 39.4°C or above, the foal was brought with

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 its dam into a stall, placed in front of a box fan, and its rectal temperature checked every 2 h
Accepted Article until it was below 38.9°C. If the rectal temperature was 40.0°C or above, the foal was also hosed

with cold water until the rectal temperature was below 38.9°C.

Sweat tests

Quantitative intradermal terbutaline sweat tests (QITST) were performed as previously described

[10] daily on Days -2, -1, and 0 to establish baseline sweating responses for each foal and on

Days 1, 2, 5, 9, 24, and 39 after treatment began. In brief, each foal was restrained without the

use of sedatives and a 5 × 30 cm strip of hair was clipped from one side of the neck parallel to

and 2.5 cm below the dorsal margin of the neck. Starting at the cranial aspect of this strip, 6

intradermal injections of 0.1 mL of serial 10-fold dilutions of the β-adrenergic agent terbutaline

sulphate in 0.9% saline were made through a 25-gauge needle at approximately 2.5 cm intervals

as follows: 0 (control), 0.1, 1, 10, 100 and 1000 mg/L. Preweighed 3 × 6 cm strips of absorbent

padd were taped over each injection site. Thirty minutes later, pads were removed and absorbed

sweat quantified as weight change. Opposite sides of the neck were used alternately for

successive tests.

Data Analysis

All statistical calculations were performed using a commercial software packagee. In order to

meet test assumptions of normal distribution and equal variance, sweat weights were log10-

transformed for analysis. Sweat weights were analysed in two different ways. First, effects of

treatment, terbutaline concentration, and day on sweat weight were explored by 3-factor and 2-

factor within-subjects repeated-measures analysis of variance (ANOVA) general linear model

procedures as previously described [6]. Second, plots of log10 sweat weight vs. log10 terbutaline

concentration for each treatment-day combination were modelled by simple linear regression

and the slopes of the fitted lines were obtained. Effects of treatment and day on terbutaline

slopes were examined by repeated-measures analysis of covariance with treatment and day as

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 within-subjects factors and terbutaline concentration as a covariate. Significant day × terbutaline
Accepted Article and treatment × terbutaline interactions in these analyses indicate that regression lines are not

parallel. Rectal temperature, heart rate, and respiratory rate data were subjected to 2-factor

(treatment, day) repeated-measures ANOVA. When significant treatment or day interactions

were found by any of these analyses, effects were further tested at each level of each factor and,

when significant, post hoc multiple pairwise comparisons were obtained with Bonferroni

confidence interval adjustments. Normality of distribution of studentised residuals and

heteroscedasticity of data were evaluated by the Shapiro-Wilk tests and Mauchly’s tests of

sphericity, respectively. Greenhouse-Geisser corrections were applied to data that violated the

sphericity assumption. Influence of experimental order (period) on sweat weights were evaluated

as effect of replicate on day × terbutaline interactions in 3-factor mixed ANOVAs with one

between-subjects factor (period) and two within-subjects factors (day and terbutaline

concentration). Significance in all analyses was ascribed only to P values <0.05.

Results

Effects of clarithromycin and azithromycin on sweat responses

One foal given four doses of ERY developed severe diarrhoea and was euthanised on day 2 of

treatment after failing to respond to fluid resuscitation and supportive care. Gross necropsy

revealed moderate diffuse acute to subacute colitis with oedema of the colonic wall and tan

discoloration of the colonic mucosa. There was no evidence of R. equi infection. This foal was

replaced in the study. Four other foals developed mild self-limiting diarrhoea (one each in the

ERY and CLA groups and two in the AZI group). On 10 occasions in three foals (seven times in

a single foal), rectal temperatures >39.4°C were recorded while foals were in stalls (two during

the baseline period, seven in the CLA group and one in the ERY group); three of these were

>40°C. During the first 3 days in paddocks, there was a single instance of temperature >39.4°C

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 (ERY group). There was no effect of treatment on rectal temperature, heart rate or respiratory
Accepted Article rate (P>0.05 for treatment × day interactions).

Because sweat responses conducted during the baseline period did not differ (P>0.05 for

each antibiotic), data for the three pretreatment tests (days -2, -1, and 0) were combined and

used for further analyses. Significant 3-factor treatment × day × terbutaline interaction for

terbutaline-induced sweat responses was not found (P = 0.2), indicating that ERY, CLA, and

AZI did not differ in their effects on sweat responses expressed as day × terbutaline interactions

(Fig 2). There were significant effects of day (P<0.0005) for all three antibiotics; the effects of

day were significant (P<0.05) for all terbutaline concentrations above 0.1 mg/L (Fig 2).

Compared to baseline (pretreatment) values, sweat weights were significantly lower over at least

four terbutaline concentrations on post-treatment days 1 to 9 (Fig 2) for ERY and days 2 and 5

for CLA and AZI. There were significant effects of treatment (P<0.05) at all days after baseline

and effects of treatment were significant (P<0.05) for 15 out of 30 post-baseline terbutaline

concentrations. Compared with ERY sweat responses at each of these 15 significant points, 8

and 14 of these were greater in the CLA and AZI groups respectively and 4 were greater in AZI-

than in CLA-treated foals (Fig 2). When slopes of terbutaline plots were analysed (Table 1), there

was a significant treatment × day interaction (P = 0.02). Effects of day were significant for each

antibiotic, extending from day 1 to days 9, 2, and 5 for ERY, CLA, and AZI, respectively.

Effects of treatment were significant on days 1 and 5; CLA and AZI groups were significantly

different from ERY but not from each other.

Experimental period did not influence sweat responses for any treatment (P>0.05 for all

period × day × terbutaline 3-way mixed ANOVA analyses).

Effect of rifampicin on erythromycin-induced suppression of sweating

Oral treatments were generally well tolerated by subject foals. Nine foals developed mild self-

limiting diarrhoea over the course of treatment (four in ERY, two in RIF, three in ERY/RIF

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 group). One foal had a single episode of hyperthermia during the baseline period in stalls. No
Accepted Article instance of hyperthermia was recorded during the first 3 days in paddocks. There was no effect

of treatment on rectal temperature, heart rate, or respiratory rate.

The 3-factor treatment × day × terbutaline repeated-measures interaction was highly

significant (P<0.0005) indicating an effect of antibiotic treatment type on sweat responses as

assessed by day × terbutaline interactions. There were significant effects of day (P<0.0005) on

terbutaline sweat responses for ERY and ERY/RIF treatments, but not for RIF (P = 0.8). The

effects of day were significant (P<0.05) for all concentrations of terbutaline in foals given either

ERY or the ERY/RIF combination. Compared to baseline (pretreatment) values, sweat weights

were significantly lower for at least 2 terbutaline concentrations on each post-treatment day (Fig

3) for ERY and ERY/RIF. There were highly significant effects of treatment (P<0.0005) at all

days after baseline and effects of treatment were significant (P<0.05) for 21 out of 30 post-

baseline terbutaline concentrations. For most (12/21) of these significant treatment effects, both

ERY groups differed significantly from RIF but not from each other. On day 1, however, sweat

amounts were higher at all terbutaline concentrations for ERY/RIF than they were for ERY

alone (Fig 3). When slopes of terbutaline plots were analysed (Table 2), there was a highly

significant treatment × day interaction (P<0.0005). There were significant effects of day for ERY

and ERY/RIF but not for RIF (P = 0.07). Effects of treatment were significant on days 1

through 24. All pairwise comparisons among treatments were significantly different on day 1

whereas sweat responses on days 2 and 5 were greater for RIF than for the two ERY groups.

Consistent with results found for sweat weights (above), RIF significantly reduced the effect of

ERY on day 1.

Experimental period did not influence sweat responses for any treatment.

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 Discussion
Accepted Article Consistent with the results of our previous study [6], ERY treatment suppressed sweating during

and after treatment. Erythromycin-induced anhidrosis is the likely explanation for reports of

hyperthermia in foals treated for R. equi pneumonia with this antibiotic [4]. Because foals in the

current study were protected from direct sunlight for 5 days after treatment regimens were

completed, signs of heat stress attributable to treatment were not seen. In the previous study, six

of ten foals that were given ERY then immediately moved outside developed signs of heat stress

within 3 days whereas only one of 24 ERY-treated foals in the current experiment became

hyperthermic during the first 3 days after turnout [6].

Standard treatment doses of the macrolide antibiotics CLA and AZI also inhibited sweating

albeit with lower potency than did ERY. There is a single previous report of AZI-associated fatal

hyperthermia [11] and the authors are aware of multiple foals that developed signs of heat stress,

fatal in some cases, during treatment with CLA. On the basis of the findings in this and our

previous study, all macrolide antibiotics commonly used for treatment of R. equi pneumonia

suppress sweating. These antibiotics may all pose commensurate risk of hyperthermia when

given to foals kept in hot sunny conditions.

In addition to antimicrobial activity, macrolide antibiotics have broad anti-inflammatory,

prokinetic, and immune-modulating effects [12]. Macrolides with 14- or 15-member lactone

rings suppress secretory responses of respiratory epithelial cells to multiple different

secretagogues [12-15]. These actions likely explain the salutary effect of ERY and AZI in

humans with panbronchiolitis, cystic fibrosis, and other chronic respiratory diseases typified by

mucus hypersecretion [12]. The mechanisms by which secretion is suppressed are not fully

defined but it is likely that macrolides act to limit secretagogue-induced calcium entry into cells

thereby preventing ion flow through calcium-dependent chloride and potassium channels. It is

not known whether or not inhibition of calcium entry and ion channel function is involved in

macrolide-induced anhidrosis of foals but it is worth noting that the necessary cellular machinery

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 exists. Epinephrine- and ATP-induced rises in cytosolic calcium have been demonstrated in
Accepted Article explanted equine sweat gland cells and these cells possess calcium-activated chloride and

potassium channels [16-19].

Rifampicin delayed maximal suppression of sweating by ERY. Sweat responses were

analysed in two different ways, i.e. both as absolute sweat weights and as slopes of sweat weight vs.

terbutaline plots. Both of these analyses when applied to sweat responses on day 1 showed that

combination of ERY with RIF was less suppressive than ERY alone. Notwithstanding this early

post-treatment difference, it is clear from Figure 3 that the addition of RIF had only a minor

overall effect on ERY-induced anhidrosis. This was somewhat surprising in light of reports that

RIF administration reduced the bioavailability of CLA in foals at least 8-fold [9; 20] via induction

of the intestinal efflux transporter P-glycoprotein [20]. Because absorption by foals of the

macrolide tulathromycin was likewise reduced [21], it is reasonable to assume that ERY

absorption is lower when given with RIF. Unfortunately, serum ERY concentrations were not

measured as part of this study. One possible explanation for our findings is that ERY-induced

anhidrosis occurs at doses much lower than those used to treat pneumonia. Efficacy of low-dose

treatment has been demonstrated previously for the immunomodulatory effects of macrolides in

panbronchiolitis treatment [12].

In summary, we have shown that ERY, CLA and AZI used at standard antimicrobial

doses in foals induce sweating dysfunction. Additionally, co-administration of ERY with RIF

also suppressed normal sweat responses whereas RIF alone had no effect. Effects of RIF on

CLA- and AZI-induced anhidrosis were not examined in this study. Foals treated with any of

these antibiotics should be considered at risk for hyperthermia both during treatment and in the

immediate post-treatment period. Future work should be aimed at determining the mechanism

of macrolide-induced anhidrosis.

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 Authors’ declaration of interests
Accepted Article The authors have declared no competing interests.

Ethical animal research

All procedures were approved by the University of Florida’s Institutional Animal Care and Use

Committee.

Sources of funding

This study was supported by funds from the Grayson-Jockey Club Research Foundation and the

Merial Summer Scholars Program.

Authorship

Study design was by A.L. Stieler Stewart and R.J. MacKay. Data analysis was performed by R.J.

MacKay. All authors contributed to data collection, study execution, and manuscript preparation.

Figure Legends

Fig 1: Summary of experimental design. QITST = quantitative intradermal terbutaline sweat test.

Fig 2: Weights (mean ± s.d.) of sweat collected into absorbent pads over intradermal saline or

terbutaline injections in foals treated with either erythromycin, clarithromycin, or azithromycin.

For each terbutaline concentration within a treatment group, days with significantly different

sweat weights are marked with an asterisk; time- and concentration-matched values across the

three treatments that are significantly different do not share a letter. Terbutaline concentrations

shown in the right-hand key to each panel are mg/L.

Fig 3: Weights (mean ± s.d.) of sweat collected into absorbent pads over intradermal saline or

terbutaline injections in foals treated with either erythromycin, rifampicin, or the combination of

erythromycin and rifampicin. Within each treatment points differing significantly from baseline

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 are marked with an asterisk; matched points that are significantly different among treatments do
Accepted Article not share a letter.

Manufacturers’ addresses
a
Erythromycin USP; Westlab Pharmaceuticals, Westlab Pharmacy, Gainesville, Florida, USA.
b
Biaxin®; AbbVie Inc., North Chicago, Illinois, USA.
c
Zithromax®, Pfizer Inc., New York, New York, USA.
d
StayFree Ultrathin Maxi Pads, Energizer Holdings, Inc., St. Louis, Missouri, USA.
e
IBM® SPSS® Statistics 21; IBM North America, New York, New York, USA.

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Table 1. Effect of treatment with erythromycin, clarithromycin, and azithromycin on slopes of

terbutaline plots.

Day Slopes† of Terbutaline Plots P-value¶

Erythromycin Clarithromycin Azithromycin

0 0.195 ± 0.045 0.194 ± 0.045 0.202 ± 0.055 0.9

1 0.070 ± 0.050*,a 0.106 ± 0.079*,b 0.141 ± 0.063*,b 0.03

2 0.049 ± 0.037* 0.101 ± 0.078* 0.11 ± 0.062* 0.07

5 0.069 ± 0.086*,a 0.158 ± 0.06b 0.147 ± 0.077*,b 0.03

9 0.127 ± 0.084* 0.157 ± 0.075 0.171 ± 0.069 0.3

24 0.164 ± 0.058 0.175 ± 0.069 0.194 ± 0.066 0.5

39 0.207 ± 0.068 0.213 ± 0.064 0.187 ± 0.069 0.6

P-value§ <0.0005 <0.0005 0.001

†
Slopes (mean ± s.d.) of lines fitted to plots of log10 sweat weight vs. log10 terbutaline

concentration by simple linear regression

¶
Effect of treatment on day shown in first column of row. Pairs of values with different

superscripted letters are significantly different

§
Effect of day (time). Values significantly different than baseline (day 0) are marked with an

asterisk.

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 Table 2. Effect of treatment with erythromycin, rifampicin, or erythromycin/rifampicin on
Accepted Article slopes of terbutaline plots.

Day Slopes† of Terbutaline Plots P-value¶

Erythromycin/

Erythromycin Rifampicin Rifampicin

0 0.199 ± 0.022 0.208 ± 0.035 0.217 ± 0.050 0.5

1 0.051 ± 0.049*,a 0.217 ± 0.061b 0.108 ± 0.060*,c <0.0005

2 0.059 ± 0.057*,a 0.160 ± 0.091b 0.048 ± 0.044*,a <0.0005

5 0.073 ± 0.082*,a 0.215 ± 0.052b 0.087 ± 0.037*,a <0.0005

9 0.168 ± 0.086,a 0.197 ± 0.080a 0.109 ± 0.059*,b 0.007

24 0.154 ± 0.027*,a 0.202 ± 0.061b 0.181 ± 0.047*,ab 0.03

39 0.222 ± 0.034 0.237 ± 0.038 0.214 ± 0.069 0.7

P-value§ <0.0005 0.07 <0.0005

†
Slopes (mean ± s.d.) of lines fitted to plots of log10 sweat weight vs. log10 terbutaline

concentration by simple linear regression

¶
Effect of treatment on day shown in first column of row. Pairs of values with different

superscripted letters are significantly different

§
Effect of day (time). Values significantly different than baseline (day 0) are marked with an

asterisk.

This article is protected by copyright. All rights reserved.

Accepted Article

This article is protected by copyright. All rights reserved.

Accepted Article

This article is protected by copyright. All rights reserved.