Comparison of the efficacy of four antimicrobial treatment schemes against experimental Ornithobacterium rhinotracheale infection in turkey poults pre-infected with avian pneumovirus.

Abstract

The clinical efficacy of drinking-water administration of enrofloxacin for 3 and 5 days, amoxicillin for 5 days and florfenicol for 5 days for the treatment of respiratory disease induced by an experimental Ornithobacterium rhinotracheale infection in turkeys pre-infected with avian pneumovirus (APV) was assessed based on clinical, bacteriological and histopathological examinations. Experimental groups of 15 susceptible 3-week-old turkeys were each inoculated oculonasally with APV subtype A and 3 days later with susceptible O. rhinotracheale bacteria. Antimicrobial treatment started 1 day after O. rhinotracheale inoculation. After infection, the birds were examined and scored for clinical signs, swabbed daily and weighed at different times. Five birds were euthanized and examined for macroscopic lesions at necropsy at 5 days post bacterial inoculation, and the remainder at 15 days post bacterial inoculation. Samples of the turbinates, trachea, lungs, air sacs, heart and pericardium were collected for bacteriological and/or histological examination. Recovery from respiratory disease caused by an APV/O. rhinotracheale dual infection was most successful after enrofloxacin treatment, irrespective of treatment duration, followed by florfenicol. Amoxicillin treatment was not efficacious. Clinical signs and the number of O. rhinotracheale organisms re-isolated from the trachea and the different respiratory organs were significantly reduced by enrofloxacin treatment for 3 and 5 days. O. rhinotracheale bacteria were not re-isolated from the tracheas of the birds treated with enrofloxacin except for one bird in the 5-day group, as early as 1 day after medication onset. In the group treated with enrofloxacin for 5 days, O. rhinotracheale organisms with a higher minimal inhibitory concentration value (x8) were isolated starting 2 days following treatment onset, initially from a single turkey and subsequently from the other animals.

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Department of Pathology, Bacteriology and Avian Diseases







Comparison of the efficacy of four antimicrobial treatment
schemes against experimental Ornithobacterium
rhinotracheale infection in turkey poults pre-infected with
avian pneumovirus


Journal: Avian Pathology
Manuscript ID: CAVP-2006-0034.R1
Manuscript Type: Original Research Paper
Date Submitted by the
Author:
06-Mar-2006
Complete List of Authors: Marien, Maja; Faculty of Veterinary Medicine, Bacteriology
Nauwynck, Hans; Faculty of Veterinary Medicine, Ghent University,
Laboratory of Virology
Duchateau, Luc; Faculty of Veterinary Medicine, Ghent University,
Department of Physiology and Biometrics
Martel, An; Faculty of Veterinary Medicine, Ghent University,
Department of Pathology, Bacteriology and Avian Diseases
Chiers, Koen; Faculty of Veterinary Medicine, Ghent University,
Department of Pathology, Bacteriology and Avian Diseases
Devriese, Luc; Faculty of Veterinary Medicine, Ghent University,
Department of Pathology, Bacteriology and Avian Diseases
Froyman, Robrecht; Bayer HealthCare AG, Animal Health Division
Decostere, A; Faculty of Veterinary Medicine, Ghent University,
Keywords:
Ornithobacterium rhinotracheale, avian pneumovirus, antibiotic
treatment, turkeys



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Pathology, 34 (3), 204-211.

1
Author’s responses to the comments by the editor and the referees concerning
the paper “Comparison of the efficacy of four antimicrobial treatment schemes
against avian pneumovirus and Ornithobacterium rhinotracheale dual infection in
turkeys”.

As suggested by the editor we revised the manuscript according to the referee’s
comments, and the manuscript was read through by someone with English as first
language and corrected where needed.

Response to comments by Referee 1:
• Although the goal of the experiment was to determine the efficacy of
antimicrobial treatment against ORT, the birds were indeed infected first with
APV. The reason for this is that clinical scores were included as a parameter
for the evaluation of the efficacy of the antimicrobials and it had been
demonstrated in a previously conducted experiment that infection with ORT
alone did not cause clinical signs. Priming with APV, however, resulted in
obvious clinical signs. Also, in the field, infections with ORT are often
preceded by infections with APV. Marien, M., Decostere, A., Martel, A.,
Chiers, K., Froyman, R. & Nauwynck, H. (2005). Synergy between avian
pneumovirus and Ornithobacterium rhinotracheale in turkeys. Avian
• Response to the referee’s comments about the statistical design: Although the
birds were indeed followed up until 15 dpbi, as expected, all clinical scores
returned to zero from day 10 onwards. We intended to perform two pre-
planned comparisons. A first comparison, based on area under the curve
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referee.

2
between 0 and 5 dpbi enabled us to observe the early effects of the infection.
Furthermore, at this time, all animals (15 per group) were still available, which
was no longer the case after that day. A second comparison was aimed at
investigating the evolution over time from 0 to 9 dpbi. This enabled us to
understand how the infection evolves over time, and furthermore to study
whether the treatment groups differ in this period with respect to the clinical
score. We understand that all this may not have been very clear to the reader,
and therefore have clarified this in the text (lines 198-200).
Title
• According to the referee’s suggestion, the title has been adjusted to the
following: “Comparison of the efficacy of four antimicrobial treatment
schemes against experimental Ornithobacterium rhinotracheale infection in
turkey poults pre-infected with avian pneumovirus”.
Materials and methods
• Line 91 (lines 92-97): The in-house serum neutralization test has been
described in the manuscript as requested.
• Line 99-100 (lines 105-107): This sentence has been adjusted according to the
referee’s comment.
• Lines 103-106 (lines 109-116): The method of the preparation of the
inoculation suspension has been described more precisely, as requested by the
• Line 106-108 (line 118): The reference for the MIC values has been specified
as requested.
• Lines 110-113 (lines 120-124): We supplied some extra information about the
drugs used in the study as requested by the referee. It should be noted that,
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euthanised at 5 dpbi. It was randomly determined in advance which animals

3
currently, in Europe, as far as we know, there are no registered antimicrobials
for which ORT specifically is an indication.
• Lines 151-157 (lines 162-168): In preliminary experiments, we determined
that the average weight of mucus absorbed by a swab was 200 µg. This
amount was subjected to minimal variation.
• Lines 158-159 (lines 170-172): This sentence has been adapted according to
the comments of the referee.
• Lines 177-179 (lines 191-195): The requested information is provided in the
text.
• Line 262 (line 185 and line 285): This is adapted to the referee’s comment.
Results and Discussion
• The response to the referee’s comments about the statistical design was
already dealt with above.
• We decided not to show all the data in the figures to maximize clarity. Since
no more clinical signs were observed from 10 dpbi onwards (mentioned in
lines 220-221), and no more ORT organisms could be retrieved from the
tracheal swabs from 11 dpbi onwards (mentioned in lines 238-239), we
decided to leave out the results from after these dates so that the figure could
be clearer on the whole.
• There were no criteria for the selection of the five birds per group that were
were going to be killed. We clarified this in the manuscript in line 154.
Furthermore, we can say that the clinical signs were very similar in terms of
severity in all birds of one group. We added this information in the manuscript
in lines 253-254.
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the plasma or serum is measured. This is performed in the same way in the

4
• In response to the referee’s comments concerning the body weight at the start
of the experiment, we would like to say that the initial body weight was taken
into account in the statistical analysis of the final body weight. A correction
was made for the difference in initial body weight (see lines 209-210). We
therefore do not think it is necessary to provide the actual figures of the initial
body weight, as this would unnecessarily lengthen the manuscript.
• The referee indeed raises a justified question as to whether other bacteria (e.g.,
E. coli) were isolated from the experimental birds rather than ORT. We cannot
exclude the possibility that other bacteria, although undetected, might have
been present. The animals were SPF and kept in separate HEPA-filtered
isolation rooms. In this experiment we isolated the ORT bacteria on a selective
medium containing 5 µg/ml gentamicin en polymyxin to suppress the growth
of contaminating bacteria.
• In line 217 we specifically mentioned that mortality was not observed during
the experiment.
• Basic Pharmacokinetics, 1999, University of Manchester, M. Rowland: “The
apparent volume of distribution at steady state Vd(ss) (equilibrium) is the
pharmacokinetic parameter that relates the amount of drug in the body (A) to
the concentration in the plasma or serum (C): V=A/C.” The Vd(ss) is
determined by intravenous injection of a drug after which the concentration in
different experiments. To support our statements, we added in the publication
the ranges of the Vd(ss) as determined in the different experiments (lines 316-
318). In our publication we tried to explain why the amoxicillin treatment did
not show a significant reduction in the parameters assessed in our study, and
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referee’s comments.

5
the Vd(ss) is one of the pharmacokinetic parameters that has to been taken in
account.
• Line 301 (line 321-322): This reference has been deleted.
• Lines 317-318 (lines 336-341): According to Bush (2003) the antibacterial
spectrum of amoxicillin is identical to that of ampicillin, and there are few
differences in antibacterial activity, especially against Gram-negative bacteria.
Furthermore, according to Otten et al. (1975), the difference between
ampicillin and amoxicillin lies in the pharmacokinetics of amoxicillin after
oral uptake. Therefore, we believe it is permissible to compare ampicillin MIC
values to amoxicillin MIC values. We clarified this in the manuscript in lines
341-343.
Bush, K. (2003). Β-Lactam antibiotics: penicillins. In Antibiotic and
chemotherapy, 8th edition. Finch R.G., Greenwood D., Norrby S.R., and
Whitley R.J. (ed.), Churchill Livingstone, New York.
Otten, H., M. Plempel, and W. Siegenthaler. (1975). Neuere Penicilline. In
Antibiotika – Fibel. Otten H., Plempel M., and Siegenthaler W. (ed.), Thieme,
Stuttgart.

Response to comments by Referee 2:
• As mentioned above, we altered the title of the manuscript according to the
• In response to the referee’s comment that the main finding is that enrofloxacin
treatment for three days is more efficacious than enrofloxacin for five days, we
have to disagree. It is indeed true that the 3-day enrofloxacin treatment
provided overall better results in comparison with the 5-day treatment, but in
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our opinion, this is attributed to the fact that after 2 days of enrofloxacin
treatment ORT isolates were found with a higher MIC value. The fact that this
rise in MIC value appeared as early as 2 dpbi should make it clear that this
also could have taken place in the 3-day treatment group. The observed rise in
MIC is most likely not due to the treatment duration (5 days versus 3 days).
We tried to clarify this in the manuscript in lines 357-359.
• Figure 2: The reason we wanted to show all the individual values instead of
only the mean values was to demonstrate that the rise in mean ORT titres in
the enrofloxacin treated groups could be attributed first to a rise in a single
animal and gradually in more animals. We also feel that the variation within a
specific group is important in the developmental process of an infection
model. Therefore, we prefer to show all the points in the figure.
• The different specific remarks made by the referee have been adjusted
accordingly.
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1
CAVP – 2005-0005
1
Comparison of the efficacy of four antimicrobial treatment schemes against
2
experimental Ornithobacterium rhinotracheale infection in turkey poults pre-
3
infected with avian pneumovirus
4

5
Maja Marien, 1,* Hans Nauwynck,2 Luc Duchateau,3 An Martel,1 Koen Chiers,1 Luc
6
Devriese,1 Robrecht Froyman,4 & Annemie Decostere.1
7

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1Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine,
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Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium, 2Laboratory of Virology,
10
Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke,
11
Belgium, 3Department of Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent
12
University, Salisburylaan 133, B-9820 Merelbeke, Belgium and 4Bayer HealthCare AG,
13
Animal Health Division, D-51368 Leverkusen, Germany.
14
15
Short title: antibiotic treatment APV/ORT infection 16
17
*To whom correspondence should be addressed. Tel: + 32 9 264 74 34. Fax: + 32 9 264 74 18
94. E-mail: maja.marien@UGent.be
19

20

21

22

23

24

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Amoxicillin treatment was not efficacious. Clinical signs and the number of ORT organisms

2
CAVP – 2005-0005 26
Comparison of the efficacy of four antimicrobial treatment schemes against experimental 27
Ornithobacterium rhinotracheale infection in turkey poults pre-infected with avian 28
pneumovirus 29
Abstract
30
31
The clinical efficacy of drinking-water administration of enrofloxacin for 3 and 5 days, 32
amoxicillin for 5 days and florfenicol for 5 days for the treatment of respiratory disease 33
induced by an experimental Ornithobacterium rhinotracheale (ORT) infection in turkeys pre-34
infected with avian pneumovirus (APV) was assessed based on clinical, bacteriological and 35
histopathological examinations. Experimental groups of 15 susceptible 3-week-old turkeys 36
were each inoculated oculonasally with APV subtype A and three days later with susceptible 37
ORT bacteria. Antimicrobial treatment started one day after ORT inoculation. After infection, 38
the birds were examined and scored for clinical signs, swabbed daily and weighed at different 39
times. Five birds were euthanised and examined for macroscopic lesions at necropsy at 5 days 40
post bacterial inoculation (dpbi), and the remainder at 15 dpbi. Samples of the turbinates, 41
trachea, lungs, air sacs, heart and pericardium were collected for bacteriological and/or 42
histological examination. 43
Recovery from respiratory disease caused by an APV/ORT dual infection was most successful 44
after enrofloxacin treatment, irrespective of treatment duration, followed by florfenicol. 45
46
re-isolated from the trachea and the different respiratory organs were significantly reduced by 47
enrofloxacin treatment for 3 and 5 days. ORT bacteria were not re-isolated from the tracheas 48
of the birds treated with enrofloxacin except for one in the 5-day group, as early as one day 49
after medication onset. In the group treated with enrofloxacin for 5 days, ORT organisms with 50
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(Marien et al., 2005). Dual infection with APV and ORT indeed resulted in more severe

3
a higher minimal inhibitory concentration (MIC) value (x 8) were isolated starting two days 51
following treatment onset initially from a single turkey and subsequently from the other 52
animals. 53
54
Introduction
55
56
Viral and bacterial respiratory tract infections frequently occur in diseased turkeys of all ages 57
and may cause considerable financial losses due to reduced growth, an increased mortality 58
rate, high medication costs and a higher number of condemnations at processing (van Empel 59
& Hafez, 1999). 60
Ornithobacterium rhinotracheale (ORT) is an infectious pathogen that has been 61
ascribed an etiological role in the respiratory disease complex in turkeys. This Gram-negative 62
bacterium is mostly regarded as a facultatively pathogenic organism, and in field cases, 63
simultaneous isolation of ORT, respiratory viruses and/or other bacteria is frequently 64
encountered. One of the viruses judged to have a triggering role is avian pneumovirus (genus 65
Metapneumovirus - MPV) (APV) (van Empel et al., 1996). Recently, in a longitudinal study 66
performed by Van Loock et al. (2005), it was shown that both APV and ORT infections often 67
occur between hatch and slaughter on Belgian turkey farms. In a previous study using an 68
optimized experimental in vivo model, it was demonstrated that ORT following APV 69
inoculation in 3-week-old turkeys has a synergistic effect on the course of respiratory disease 70
71
clinical signs, macroscopic and histological findings, and a longer persistence of ORT in the 72
respiratory tract compared with the single infections. 73
Disease caused by ORT may be reduced by preventing predisposing factors including 74
inadequate ventilation, high ammonia levels, too high or too low relative humidity and 75
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respectively. Neutralization was tested on Vero cells in 96-well cell culture microplates using

4
infection with additional pathogenic agents (van Empel & Hafez, 1999). In practice, however, 76
ORT infections are mostly dealt with using different antibiotics such as amoxicillin, 77
ampicillin, doxycycline, tetracycline, trimethoprim/sulphonamide, enrofloxacin and 78
florfenicol. Hitherto, the actual in vivo efficacy of antibiotics for the treatment of ORT 79
infections in poultry has not yet been investigated. This is to a great extent rooted in the fact 80
that, only until very recently (Marien et al., 2005), no suitable infection model was available. 81
The objectives of the present study are to compare the efficacy of enrofloxacin, amoxicillin 82
and florfenicol for treatment of respiratory disease due to experimental ORT infection in 3- 83
week-old turkeys following APV challenge. The efficacy was evaluated on the basis of 84
several parameters, i.e., clinical signs, histopathological findings, re-isolation and titration of 85
the bacterium, and weight gain. 86
87
Materials and methods
88
89
Turkeys. Seventy-five specific pathogen free (SPF) turkeys (AFSSA, Ploufragan, France) 90
were used in this study. The turkeys were hatched in our facilities. The birds were housed on 91
litter in separate isolation rooms with HEPA-filtered air, had free access to food and water 92
and received 16 hours of light per day. At two weeks of age the birds were shown to be free 93
from maternally-derived antibodies to ORT and APV by means of an ELISA available 94
commercially (Biochek, Gouda, the Netherlands) and an in-house serum neutralization test, 95
96
standard procedures. Briefly, serial twofold dilutions of the sera were made and incubated for 97
1 h at 37°C with an equal volume of virus suspension (subtype A), containing 100 TCID50 98
APV. The reading was based on the absence or presence of a cytopathic effect over 7 days. 99
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blood and counting the number of colonies. The minimal inhibitory concentrations (MICs) of

5
The SN-titres were the reciprocal of the highest serum dilution that inhibited the cytopathic 100
effect in 50% of the wells.
101
102
Virus. The APV strain A/T6/96 (subtype A) was used. The strain was isolated during a 103
respiratory outbreak on a Belgian turkey farm (Van de Zande et al., 1998). The virus stock 104
had a titre of 5.0 log10 50% ciliostatic dose (CD50)/ml after the third passage in tracheal organ 105
cultures.
106
107
Bacterium. The ORT type strain LMG 9086T was used, which was originally isolated from a 108
turkey with a respiratory tract infection. The strain was serotyped as type A in an agar gel 109
precipitation test (Hafez & Sting, 1999) performed by Professor H. M. Hafez (Institute of 110
Poultry Diseases, Free University of Berlin, Germany). The strain was stored at -70°C. The 111
organism was cultured for 48 h at 37°C on Columbia agar (Oxoid LTD, Basingstoke, 112
Hampshire, England) with 5% sheep blood in a 5% CO2 atmosphere. The ORT bacteria were 113
transferred into brain heart infusion broth (Oxoid) for 24 h at 37°C with agitation. The 114
bacterial challenge inoculum was prepared by washing the cultured bacteria twice in 115
phosphate-buffered saline (PBS) followed each time by five minutes of centrifugation at 116
3,000 rpm at 4°C. The resulting pellet was re-suspended in PBS to obtain a final 117
concentration of 8.6 log10 colony-forming units (cfu)/ml. Confirmation of the number of 118
cfu/ml was done by inoculating ten-fold dilutions in PBS on Columbia agar with 5% sheep 119
120
amoxicillin, florfenicol and enrofloxacin for this challenge strain were 2 µg/ml, 1 µg/ml and 121
≤0.03 µg/ml, respectively, as determined according to Devriese et al. (2001).
122
123
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(dpbi)). The concentration of each antibiotic to be administered in the water could be

6
Antimicrobial agents. Three antimicrobial agents were used in this study: enrofloxacin 124
(Baytril® 10% oral solution, Bayer, Leverkusen, Germany), amoxicillin (powder form) 125
(Suramox 50®, soluble powder, Virbac S.A., Carros, France) and florfenicol (soluble form) 126
(Nuflor®, Schering-Plough S.A., Xochimilco, Mexico) with manufacturer-recommended 127
doses of 10 mg/kg for enrofloxacin and 20 mg/kg for amoxicillin and florfenicol. 128
129
Experimental design. Seventy-five SPF turkeys were randomly divided into 5 groups of 15 130
birds at one day of age. 131
In all groups, turkeys first received APV, and subsequently received ORT three days later. 132
Each bird was inoculated with APV by the oculonasal route at a dose of 4.4 log10CD50. ORT 133
was likewise administered oculonasally with a dosage of 8 log10 cfu. For inoculation with 134
APV and ORT, a total of 250 µl was divided equally over the nares and eyes.
135
Four groups received antibiotic treatment: enrofloxacin 10 mg/kg for 3 days (group 136
E3), enrofloxacin 10 mg/kg for 5 days (group E5), florfenicol 20 mg/kg for 5 days (group F), 137
and amoxicillin 20 mg/kg for 5 days (group A). Starting at 24 hours post bacterial inoculation, 138
the drinking water was medicated with the appropriate antimicrobial agent. The birds were 139
continuously dosed and received their daily medication over a 24h period. To enable correct 140
dosing, the daily water uptake and mean group body weights were determined. All turkeys 141
were weighed immediately before APV inoculation, before ORT inoculation, at day 5 after 142
ORT inoculation, and finally at the end of the experiment (15 days post bacterial inoculation 143
144
calculated accurately on the basis of the water consumption and body weight data. The fifth 145
group was included as an untreated control group (group C). 146
All birds were clinically examined on a daily basis throughout the experiment. The 147
clinical signs were scored as described in Van de Zande et al. (2001). Briefly, the clinical 148
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7
condition of each bird was assigned a score from 0 (absence of clinical signs) to 7 (nasal 149
exudate with extremely swollen sinuses and frothy eyes, poor general condition and 150
anorexia). The mean clinical score was calculated for each experimental group. 151
Tracheal swabs were collected from the animals in all groups at 3 days post viral 152
inoculation (dpvi) to confirm infection with APV, and daily until 11 dpbi for ORT titration. 153
The tracheal swabs were taken using cotton-tipped aluminium-shafted swabs (Copan 154
Diagnostics Inc., Corona, USA) and placed in 1 ml PBS supplemented with Ca2+ and Mg2+ 155
and in the case of virus titration an additional 10% fetal calf serum (Gibco, Invitrogen 156
Corporation, Merelbeke, Belgium), penicillin (1000 U/ml) (Biopharma, Rome, Italy) and 157
kanamycin (0.5 mg/ml) (Gibco). Processing occurred as described below. 158
Five birds of each group were randomly selected and were sacrificed at 5 dpbi. The 159
remaining 10 birds were sacrificed at 15 dpbi. The birds were necropsied and examined for 160
gross lesions. Samples of the turbinates, trachea, lungs, air sacs, heart and pericardium were 161
collected for bacterial isolation and processed immediately as described below. Finally, 162
samples from the turbinates, trachea and lungs were taken and fixed in 10% neutral buffered 163
formalin for histopathological examination. 164
165
Virological and bacteriological titration of tracheal swabs. The viral titre in log10CD50 per 166
g mucus and the number of cfu of ORT per g mucus were determined. This was done using 167
the procedures described in Marien et al. (2005).
168
169
Bacteriological titration of tissue suspensions and swabs. Samples of the turbinates, 170
trachea and lungs were titrated for ORT from the 25 birds sacrificed at 5 dpbi. The number of 171
cfu of ORT per g mucus was determined as described in Marien et al. (2005). 172
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