The treatment of infectious ovine keratoconjunctivitis in pre-export
feedlot sheep in Western Australia.
Fraser Robert Murdoch
BVMS MANZCVS (Sheep Medicine) FHEA MRCVS
This thesis is presented for the degree of Doctor of Philosophy of
Murdoch University.
2016
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I declare that this thesis is my own account of my research and contains as its main content work which has not been submitted for a degree at any tertiary education institution.
……………………………………………..
Fraser R. Murdoch
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Abstract
Approximately two million sheep are exported from Australia annually, worth in excess of
$170 million to the Australian economy. With the live export industry facing increasing scrutiny it is essential that the industry take steps to optimise the welfare of those animals involved.
Infectious ovine keratoconjunctivitis (IOK) is a significant, infectious eye disease of sheep and is the reason for the rejection of many sheep from the live export chain. Establishing a practical and effective treatment protocol has the potential to reduce economic losses associated with rejected stock and to improve the welfare of those animals presenting with clinical disease.
Injectable oxytetracycline (OTC) has been used as a treatment worldwide and has been shown to be effective. However, in pre-embarkation feedlots the number of animals is so large that such individual treatment is not feasible. This research investigated the clinical efficacy and impact on animal health of OTC given in-feed or in-water.
Oxytetracycline is absorbed from the gastro-intestinal tract following oral administration in feed or water. This research showed that oral administration of OTC results in changes to the rumen microflora population.
Although the change in rumen microflora corresponded with reduced feed intakes, feed intakes returned to normal following cessation of treatment, indicating that any changes to the ruminal microbiome are transient. Oxytetracycline given in-feed for five days results in a significant clinical improvement in IOK.
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In-water OTC caused a persistent decrease in feed and water intake rendering it an unsuitable treatment.
Mild cases of IOK can be successfully treated with in-feed OTC for a five-day period at a dose of 20 mg/kg bodyweight. Sheep with more severe IOK can be successfully treated with two intramuscular injections of OTC at 20 mg/kg bodyweight four days apart.
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Ethics Approval
All experimental work was approved by the Murdoch University Animal Etchics committee under permit numbers: R2159/08, R2409/11, R2460/11 and R 2613/03.
The use of a questionnaire was approved by Murdoch University Human Research Ethics and Integrity Committee under project number 2014/205
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Table of Contents
1 Introduction ...... 22
2 Literature Review ...... 25
2.1 Infectious keratoconjunctivitis in other species ...... 26
2.2 Nomenclature...... 27
2.3 Clinical presentation ...... 27
2.4 Impacts on production ...... 28
2.5 Infectious ovine keratoconjunctivitis and the export of live animals ...... 30
2.6 Risk Factors ...... 31
Season ...... 31
Housing ...... 33
Vectors ...... 33
Breed ...... 34
Age ...... 35
Environmental factors ...... 36
Concurrent disease ...... 36
Carrier animals ...... 37
2.7 Microbiological causative agents ...... 38
Rickettsiae ...... 38
Mycoplasma sp...... 39
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Moraxella sp...... 40
Chlamydia sp...... 40
2.8 Other causes of keratoconjunctivitis ...... 41
2.9 Treatment ...... 42
2.10 Antibiotics...... 45
Topical agents ...... 45
Injectable ...... 48
Oral treatments ...... 50
3 General Aims ...... 52
4 Epidemiology of infectious ovine keratoconjunctivitis at a pre-export feedlot ...... 53
4.1 Introduction...... 53
4.2 Materials and Methods ...... 53
4.3 Results ...... 55
4.4 Discussion ...... 58
4.5 Conclusions...... 62
5 Identification of the microbiological causes and possible treatment of infectious ovine keratoconjunctivitis in a pre-export quarantine feedlot...... 63
5.1 Materials and methods ...... 64
Statistical analysis ...... 66
5.2 Results ...... 66
5.3 Discussion ...... 70
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5.4 Conclusions...... 77
6 Determination of the efficacy of in-water medication in the treatment of ovine infectious keratoconjunctivitis...... 78
6.1 Introduction...... 78
6.2 Hypothesis ...... 80
6.3 Materials and Methods ...... 80
6.4 Results ...... 83
6.5 Discussion ...... 92
6.6 Conclusion ...... 97
7 Maximising intake of in-water oxytetracycline ...... 98
7.1 Introduction...... 98
7.2 Hypotheses ...... 99
7.3 Materials and methods ...... 99
Statistical analysis ...... 100
7.4 Results ...... 101
7.5 Discussion ...... 104
7.6 Conclusions...... 106
8 Determination of oral bioavailability of oxytetracycline in sheep...... 107
8.1 Introduction...... 107
8.2 Materials and Methods ...... 109
Sample analysis ...... 110
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Statistical analysis ...... 110
8.3 Results ...... 112
8.4 Discussion ...... 114
8.5 Conclusions...... 124
9 The impact of oral oxytetracycline on rumen health ...... 125
9.1 Introduction...... 125
9.2 Materials and Methods ...... 131
Treatments ...... 132
Rumen fluid samples ...... 132
Rumen microbial profiling ...... 132
Methodolgy for T-RFLP ...... 133
Methodolgy for Pyrosequencing ...... 134
Blood samples ...... 135
Statistical Analysis ...... 135
9.3 Results ...... 138
Bodyweight and body condition score ...... 140
Feed Intake ...... 140
Water Intake ...... 143
Plasma Beta-Hydroxybutyrate (BHB) concentrations ...... 144
Faecal Score ...... 145
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Rumen pH ...... 146
Plasma OTC concentrations ...... 147
Rumen fluid parameters ...... 148
9.4 Discussion ...... 155
9.5 Conclusions...... 159
10 The use of medicated feed to treat infectious ovine keratoconjunctivitis ...... 160
10.1 Introduction...... 160
10.2 Hypothesis ...... 161
10.3 Materials and Methods ...... 161
Preparation of medicated pellets (in-feed medication)...... 161
Statistical analysis ...... 162
10.4 Results ...... 162
10.5 Discussion ...... 165
11 Clinical efficacy of in-feed OTC ...... 166
11.1 Introduction...... 166
11.2 Hypothesis ...... 166
11.3 Materials and methods ...... 166
Statistical analysis ...... 167
Plasma sample analysis ...... 168
11.4 Results ...... 169
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11.5 Discussion ...... 172
11.6 Conclusion ...... 174
12 The treatment of infectious ovine keratoconjunctivitis with in-feed medication in a pre-embarkation feedlot...... 175
12.1 Introduction...... 175
12.2 Hypotheses ...... 175
12.3 Materials and Methods ...... 176
12.4 Results ...... 177
12.5 Discussion ...... 183
13 General Discussion ...... 184
14 Conclusions ...... 192
15 Appendix 1 ...... 193
16 Appendix 2 ...... 201
17 Appendix 3 ...... 202
18 References ...... 209
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Table of Figures
Figure 5.1 Eye grades over time ...... 69
Figure 6.1 Mean feed intake (kg/head/day) over time. Shaded area represents the duration of Oral treatment. Error bars represent standard errors ...... 85
Figure 6.2 Mean water intake (L/head/day) over time. Shaded area represents the duration of oral treatment. Error bars represent standard errors...... 87
Figure 6.3 Mean clinical eye grade (average both eyes) over time. Shaded area represents the duration of Oral treatment. IM OTC treatment given on day 0 and 4. Error bars represent standard errors...... 89
6.4 Mean plasma OTC concentrations at different time points during the experiment...... 91
Figure 6.5 Mean plasma OTC concentration during treatment period for the Oral and the IM treatment groups ...... 91
Figure 7.1 Change in feed intake (g/head/day) over time. Shaded box represents treatment period. Error bars represent standard errors ...... 102
Figure 7.2 Change in water intake (ml/head/day) over time. Shaded box represents treatment period. Error bars represent standard errors...... 102
Figure 7.3 OTC intakes (based on Experiment 1) for different doses of OTC during treatment period...... 103
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Figure 8.1 Plasma OTC concentrations over time following IV treatment, error bars show standard error ...... 112
Figure 8.2 Plasma OTC concentrations over time following medication with in-feed (IF) OTC with error bars showing standard error...... 113
Figure 8.3 Plasma OTC concentrations over time following medication with in-water (IW)
OTC. Error bars show standard error...... 113
Figure 9.1 Graph of mean feed intake over time for Treatments (11mg/kg and 22mg/kg oral
OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.
...... 141
Figure 9.2 Graph of mean ME intake over time for Treatments (11mg/kg and 22mg/kg oral
OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.
...... 142
Figure 9.3 Graph of mean water intake over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors...... 143
Figure 9.4 Graph of mean plasma BHB concentrations over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors. Black line represents BHB concentration at which sheep are at risk of sub- clinical ketosis...... 144
Figure 9.5 Graph of mean faecal score over time for Treatments (11mg/kg and 22mg/kg oral
OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.
...... 145
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Figure 9.6 Graph of mean rumen pH over time for Treatments (11mg/kg and 22mg/kg oral
OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.
...... 146
Figure 9.7 Graph of mean plasma OTC over time for Treatments (11mg/kg and 22mg/kg oral
OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.
...... 147
Figure 9.8 nMDS of rumen bacterial communities associated with oral dose of OTC taken on day 5. Treatments are: 11mg OTC/kg LW/day () and 22mg OTC/kg LW/day (). Each point in the ordination shows the overall microbial profile of an individual animal. The closer two points are in the ordination the more similar are their profiles...... 150
Figure 9.9 Abundance of rumen bacteria classified to level of phyla for individual control and
OTC treated sheep. (Cont) untreated control sheep, (Low) treated with 11 mg OTC/kg live weight/day and (High) treated with 22 mg OTC/kg live weight/day...... 151
Figure 9.10 Abundance of rumen bacteria classified, where possible, to level of family for individual control and OTC treated sheep. (Cont) control sheep, (Low) 11 mg OTC/kg live weight/day and (High) 22 mg OTC/kg live weight/day...... 153
Figure 10.1 Feed intake (g/head/day) over time. Shaded box represents treatment period.
Error bars represent standard errors...... 163
Figure 10.2 Water intake (kg/head/day) over time. Shaded box represents treatment period.
Error bars represent standard errors...... 164
Figure 11.1 Feed intake (g/hd/day) of Control vs IF treatments over time. Shaded box represents treatment period. Error bars represent standard errors...... 171
Figure 11.2 Water intake (ml/hd/day) of Control vs IF treatments over time. Shaded box represents treatment period. Error bars represent standard errors...... 171
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Figure 11.3 Change in eye grade over time - Control vs IF treatments. Shaded box represents treatment period. Error bars represent standard errors...... 172
Figure 12.1 Adjusted IOK mean scores for each treatment and day...... 179
Figure 12.2 Average eye scores on each day, grouped according to Score on Day 0...... 181
Table of Tables
Table 4.1 Historical shipping data showing numbers and percentages of sheep rejected on arrival at the feedlot due to Imfectious Ovine Keratoconjunctivitis. * depicts significant result...... 56
Table 4.2 Historical shipping data provided by the exporter outlining, by season, the number of sheep rejected due to clinical Infectious Ovine Keratoconjunctivitis. *depicts significant findings...... 57
Table 4.3 Data gatherd by questionnaire showing maximum and minimum percentages of
IOK cases per shipment ...... 57
Table 4.4 Data gatherd by questionnaire showing maximum and minimum percentages of mild IOK cases per shipment in lambs, wethers and ewes...... 58
Table 5.1 Treatments tested for control of Infectious Ovine Keratoconjunctivitis ...... 66
GNR = Gram-negative rod; GPC = gram-positive cocci; GPR = gram-positive rod
Table 5.2 Microbial flora isolated from clinically healthy eyes, Mycoplasma conjunctivae positive is a percentage of total Mycoplasma spp...... 67
Table 6.1 Linear models and P-values for the analysis of water intake, feed intake, clinical eye score and plasma OTC concentration after treatments...... 84
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Table 6.2 Mean feed intake (kg/head/day) for treatments on different days. Within Day, means with different letters differ significantly (P<0.05)...... 86
Table 6.3 Mean water intake (L/head/day) for treatments on different days. Within Day, means with different letters differ significantly (P<0.05)...... 88
Table 6.4 Mean clinical eye grade (average of both eyes) for treatments on different days.
Within Day, means with different letters differ significantly (P<0.05)...... 90
Table 6.5:OTC dose received by the Oral treatment group...... 90
Table 7.1: Treatments in experiment to optimise dose and palatability ...... 100
Table 7.2: Significance levels (P-Values) for terms in model with respect to change in feed or change in water intake...... 101
Table 9.1: Faecal scoring system (Le Jambre et al., 2007) ...... 131
Table 9.2: Significance table for change in measurements between pre- and post- treatment parameters...... 139
Table 9.3 One-way ANOSIM of rumen microbial communities associated with OTC treatment. For each microbial group the influence of oral OTC treatment was investigated.
Where significant differences in rumen microbiota were detected, the pairwise* differences between treatments were investigated further...... 149
Table 10.1 Feed intake between the groups throughout the experiment ...... 163
Table 11.1 Significance levels (P values) for terms used in models to analyse data. Cov
=covariate ...... 170
Table 12.1: Significance levels (P Values) for analysis of pink eye scores on each day post- treatment (control vs in-feed vs injection) ...... 178
Table 12.2 Significance levels (P values) for analysis of change in pink eye score over time
...... 179
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Table 12.3 Change in eye score from day 0 to day 10 ...... 180
Table 12.5: Treatment recommendations (Treat vs Not effective) of different routes of administration (In-feed vs Injection) at different degrees of severity of disease (Score) on day 8 of experiment...... 182
Table of equations
Equation 1: Calculation of bioavailability ...... 111
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Acknowledgments
Throughout the course of this PhD I have been privileged to have the unending support and guidance of a host of dedicated and encouraging people. Firstly, sincere thanks must go to the Australian Postgraduate Authority for providing me with financial support over the course of the PhD and also to Meat and Livestock Australia for funding the project in its entirety.
Many people ask why I decided to undertake a PhD and in truth that is a difficult question with no simple answer. It was under the guidance and tutelage of Helen Chapman that I first became involved in this project and her infectious enthusiasm for sheep medicine inspired me both to continue with the project and to pursue a career in small ruminant medicine and production. In a similar vein, I had many friends who were either presently undertaking or had recently completed PhDs and their positive experiences encouraged me to undertake one myself.
Of course this thesis wouldn’t have come together without the support of my supervisors.
Firstly, Mike Laurence, my principal supervisor, has been a remarkable help throughout. It is hard to express my gratitude to him for his continued support and guidance, and his commitment to the project has kept me going despite falling behind targets at times. Never again shall I dare to put two spaces after a full stop. I feel honored to consider Mike a supervisor, colleague and friend.
Ian Robertson has been an equally supportive supervisor. A man who always has time to help no matter how many other things he has to deal with, Ian has always showed genuine
18 interest in my career. He has already given me great advice which I will continue to benefit from.
As with all projects, this one has very much been a team effort. Huge thanks must go to
Stacey McFarlane for all her technical assistance; no one knows how to keep track of labels better! Equally, thanks must go to John Abbott who always brought a smile to those days in the sheep pens. Without the support and cooperation of the staff at the feedlots we would not have been able to undertake this project. Sincere thanks to our partners at DAFWA,
SARDI, and CSIRO, and to Garth Maker from the Metabolomics department, for all their invaluable assistance and contributions to the analysis of samples generated from the project.
Finally huge thanks to my friends and family. Repeatedly hearing the question “Is your thesis finished yet?” certainly does grate but it provided constant motivation and I know that everybody asked only because they genuinely cared.
Lastly thanks must go to Jill: I couldn’t have done this without you. Thank you for enabling me to go on this journey, and for believing in me. I hope you feel it has all been worth it!
I would like to dedicate this thesis to my close friend Serena Finlayson, who taught me that no matter what life throws at us we must strive to make the most of each and every day.
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Abbreviations
bwt – bodyweight
CSIRO – Commonwealth Scientific and Industrial Research Organisation
CTC – Chlortetracycline
DAFWA – Department of Agriculture and Food, Western Australia
GI – gastro-intestinal
HPLC-MS – High performance liquid chromatography – Mass-Spectrometry
IBK – Infectious bovine keratoconjunctivitis
ID – Identification
IOK – Infectious ovine keratoconjunctivitis
LA – Long acting
LSD – least significant difference
MIC – Minimum inhibitory concentration nMDS – non-metric multidimensional scaling
OTC – Oxytetracycline
OTU – operational taxonomic units
PCR – polymerase chain reaction
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RDP – ribosomal database project
SARDI – South Australian Research and Development Institute
TRF – terminal restriction fragment
T-RFLP – terminal restriction fragment length polymorphism
UK – United Kingdom
VFAs – Volatile Fatty Acids
WA – Western Australia
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1 Introduction
Western Australia started exporting live sheep in 1845 (Georges et al., 1985). Today
Australia remains overall the world’s largest exporter of livestock and the industry’s welfare standards are a global benchmark (Australian Government Department of Agriculture,
2011). Sheep are a major part of this export trade. Although numbers have declined since their peak of around seven million per annum in 2001, there were almost 2.3 million head of sheep exported in 2012. Of these sheep exported, 80% left from Western Australia and the remainder from South Australia and Victoria. The Middle East remains the strongest market for exports with 98% of exports going to this region. Other markets include southern and northern Asia. As wealth and development increase in these countries so too does the demand for protein, and consequently the demand for meat increases.
The live export trade remains a heavily scrutinised and controversial industry. Many countries that import animals are reliant on these imports for food. Supply of processed meat has increased, but trade in live animals from Australia remains dominant because the market for processed meat is curtailed by limited availability of refrigeration and freezers among large proportions of the population in countries of destination. Furthermore, cultural and religious beliefs are such that the fresh meat market continues to dominate. Halal and
Kosher meat have strict requirements for the treatment of animals pre, during and post- slaughter. Although these requirements can be addressed in Australia allowing suitably prepared meat to be exported chilled or frozen, many countries still prefer to slaughter under the supervision of local religious officials to ensure strict adherence to requirements.
All exported animals are subject to the Australian Standards for the Export of Livestock
(ASEL) legislation (Australian Government Department of Agriculture, 2011). These
22 standards are in place to ensure animal health and welfare remain a priority throughout the export chain and that livestock sourced for export are fit and healthy to travel. Any animals failing to meet these standards should be removed from the export chain. These standards help to maintain the health and welfare of the animals and market and public confidence in the industry. ASEL stipulates a number of reasons why an animal would be unfit for travel, ranging from body condition to disease status (Australian Government Department of
Agriculture, 2011). Infectious ovine keratoconjunctivitis (IOK) is identified as one reason for rejection. There is also the requirement that animals have vision in both eyes.
Anecdotal figures from exporters indicate that approximately 0.5-1% of sheep are rejected annually because of the presence of IOK (G.Robinson Per. comms). This figure relates to only those animals permanently removed from the export chain and covers only those animals deemed untreatable or those that fail to respond to treatment. These sheep are deemed to have no commercial value and are typically sold to the pet meat trade. However, this figure excludes those sheep held back for treatment and carried over to subsequent shipments. An independent review of the industry reported that IOK is the most important reason for rejection (Farmer, 2011).
Although sheep will typically recover from IOK with appropriate treatment, there is a cost associated with this treatment in medication, labour hours and additional feeding. In addition to the financial costs associated with the disease, IOK has been recognised as having a detrimental effect on animal welfare. Conjunctivitis is considered to be an irritating condition in humans (Leibowitz, 2000) and corneal ulceration is considered a very painful condition in humans (Wirbelauer, 2006). Assessment of pain and discomfort in a prey animal such as a sheep is notoriously difficult (Murdoch et al., 2013). Considering the
23 findings of Leibowitz (200) and Wirbelauer (2006) it is reasonable to believe that sheep suffering from these conditions are also feeling pain, even though they show little or no signs of discomfort. However, it is essential to act on the assumption that the sheep are in pain, so that animals suffering from IOK receive effective treatment to minimise any possible impact on their welfare and long-term health. This is not only in the obvious interest of the animals but in the interest of the live export industry, whose animal welfare standards must bear rigorous scrutiny.
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2 Literature Review
Infectious Ovine Keratoconjunctivitis (IOK), an infectious eye disease of sheep, has been reported throughout the world. Early reports document its presence in Australia (Symons,
1912; Beveridge, 1942). Outbreaks have also been reported throughout the world: United
Kingdom (Hosie, 1988); Pakistan (Shahzad, 2013); Norway (Akerstedt, 2004); Netherlands
(Ter, 1988); South Africa (Van Halderen et al., 1994); Ivory Coast (Formenty and Domenech,
1992); New Zealand (Cooper, 1967); Switzerland (Janovsky et al., 2001); Spain (Fernandez-
Aguilar et al., 2013); Slovakia (Kovac et al., 2003) and Israel (Lysnyansky et al., 2007; Hadani et al., 2013).
Accurate figures relating to the numbers of outbreaks per year are sparse. In the UK up to
25 outbreaks per annum have been reported (Greig, 1989) whereas in Norway over 4000 individual cases were reported in one year (Akerstedt, 2004). Severe outbreaks can occur up to 10 times per year in the Netherlands with morbidity ranging from a low percentage up to
100% (Konig, 1983a). In all of these instances it is likely that these figures do not reflect the true incidence as many cases will either be treated by farm staff or will resolve spontaneously and go unreported.
Although IOK is sometimes regarded as a relatively mild condition, farmers in New Zealand still considered it the seventh most important disease affecting sheep (Simpson and Wright,
1988). Those farmers considered parasites, lambing related problems, footrot and lameness more important than IOK.
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2.1 Infectious keratoconjunctivitis in other species
Infectious keratoconjunctivitis is seen in species other than sheep, although the aetiological agents vary. Infectious bovine keratoconjunctivitis (IBK) presents with similar clinical signs in cattle (McConnel et al., 2007). Although Moraxella bovis is the most common agent associated with IBK, Moraxella bovoculi (Angelos et al., 2007) and Moraxella ovis have both been implicated (O'Connor et al., 2012). Disease has also been reported in yaks and yak crosses (Bandyopadhyay et al., 2010).
Domestic goats have been reported to be affected by infectious keratoconjunctivitis (Baas et al., 1977; Busch, 1988) and the agents responsible for IOK are also associated with disease in goats (Walridge B.M. , 2002). Wild caprinae: Chamois; Ibex; Mouflon and Tar are also susceptible to infectious keratoconjunctivitis (Tschopp et al., 2005). Although the disease is typically relatively mild in domestic animals, disease in wild populations is more severe and often fatal (Nicolet, 1975; Degiorgis et al., 2000; Belloy et al., 2003). Fatalities were attributed to mis-adventure and increased predation risk associated with impaired vision.
Infectious keratoconjunctivitis has also been found in populations of wild deer (Gortazar et al., 1998). Although the agent responsible for infection wasn’t identified it is postulated that a Chlamydia sp. may have been involved. Several studies highlight the occurrence of infectious keratoconjunctivitis in reindeer (Rehbinder and Nilsson, 1995; Evans et al., 2008).
It is thought that a herpes virus is the causative agent of disease in this species (Tryland et al., 2009).
Keratoconjunctivitis has also been reported in camelids, both old and new world. A case in a llama of keratoconjunctivitis attributed to Moraxella liquefaciens has been described
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(Brightman et al., 1981). Outbreaks due to Moraxella canis (Tejedor-Junco et al., 2010) have been reported in herds of dromedary camels in Lanzarote.
2.2 Nomenclature
Infectious ovine keratoconjunctivitis is sometimes referred to by other names. Pink Eye is the most commonly used alternative (Cooper, 1974; Konig, 1983b; Moore and Whitley,
1984; Baker et al., 2001; Motha, 2003; Dun, 2009), but the terms contagious ovine ophthalmia (Beveridge, 1942), contagious conjunctivo-keratitis (Cooper, 1967), heather blindness (Sinclair, 1955; Cooper, 1974; Townsend, 2007), snow blindness (Hosie, 1989) and inclusion body keratoconjunctivitis (Kovac et al., 2003) have all been used to describe the same disease.
2.3 Clinical presentation
Infectious ovine keratoconjunctivitis is considered to be an irritating (Egwu et al., 1989) and painful condition (Greig, 1989; Akerstedt, 2004) and can occur in one or both eyes (Greig,
1989).
The clinical disease progression has been differentiated into four phases (Egwu, 1991). The initial phase is characterised by inflammation of the conjunctiva with sheep showing signs of blepharospasm (blinking) and increased lachrymation (Egwu et al., 1989). These clinical signs will often be the first that the producer or stock person notices. Increased lachrymation is evidenced by dirt on the fleece of the cheek.
The next phase is an early keratitis. Keratitis is inflammation of the cornea and initially presents as corneal oedema (Grahn B.H. and Peiffer, 2007). Increased lachrymation and
27 blepharospasm due to the increased discomfort associated with keratitis can also be seen at this stage.
Following the initial keratitis a mucopurulent discharge is described, often associated with a superficial corneal ulcer. A corneal ulcer occurs if parts of the epithelial and stromal layer of the cornea are lost, thus exposing the descemet’s membrane layer (Slatter, 2001b). At this stage blindness may be a feature, which is relatively easy to detect by the producer as affected sheep are difficult to move and often walk into objects. Cooper (1967) found that up to 25% of sheep in an outbreak can become blind and the blindness can last for 1-2 weeks.
The final stage involves diffuse ulceration of the cornea and occasionally hypopyon.
Hypopyon is the accumulation of pus in the anterior chamber of the eye (Slatter, 2001a).
Corneal neovascularisation is a feature of more severe cases. When this occurs it typically starts at the limbus and will move towards the centre of the cornea (Walridge B.M. , 2002).
The pain associated with IOK results through damage to the corneal epithelium exposing the trigeminal nerve endings (Maggs et al., 2008).
2.4 Impacts on production
Some consider IOK to be of minor importance to the sheep industry (Axelsen, 1961) but it is associated with a number of issues that can be of economic significance. Studies in Australia have found that compared to healthy sheep, IOK-affected sheep will have reduced weight gain for the duration of the disease (Axelsen, 1961). The same study found that cross-bred ewes lost weight over the period of infection, whereas merino ewes showed weight gain, but that this gain was significantly lower than in uninfected sheep. Weight loss and poor appetite have also been described in an outbreak in Nigeria (Osuagwuh and Akpokodje,
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1979). In this outbreak two lambs died and this was attributed to starvation as a result of blindness associated with corneal oedema. Weight loss has also been attributed to blindness and anorexia (Akerstedt, 2004) in infected sheep.
Carcass quality can be affected by bruising following mis-adventure. Slaughter weights can be reduced as a result of secondary anorexia (Akerstedt, 2004). Outbreaks in lamb finishing feedlots in North America have resulted in weight loss (Hopkins et al., 1973); this outbreak, however, involved both IOK and polyarthritis. Weight losses at pasture can be attributed to reduced feed intakes associated with reduced vision (Radostits, 2007a).
Outbreaks of IOK around joining and lambing can have an economic impact through reduced twinning rates. These have been reported following an outbreak around joining and ewes could be at a higher risk of developing pregnancy toxaemia (Axelsen, 1961). Pregnancy toxaemia is associated with a reducing plane of nutrition in late pregnancy (Radostits,
2007b). This can be a direct sequela of IOK infection. In addition to reduced conception rates, losses can occur around lambing in IOK infected ewes that are associated with the visual deficits suffered by the sheep. These could include squashing lambs or suffocation of lambs with placenta (Hofland et al., 1969). It is not a common occurrence, but blind ewes have been shown sometimes to be capable of rearing lambs (Sinclair, 1955) although success depends on careful monitoring.
Outbreaks of IOK appear to have no impact on wool production (Axelsen, 1961). This is interesting as it is generally considered that any disease leading to periods of inappetance can have an impact on wool production. If prolonged enough, a break in the wool fibre can be seen 2-6 weeks after the insult. However, if the period of inappetance is not prolonged fibres may be reduced in diameter and length but without a break (Groverman, 1992).
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While some authors report little or no negative effect on production (Hosie, 1988), most suggest that the principal economic impact of an outbreak of IOK relates to the costs associated with treatment (Hosie, 1988). These costs comprise both drug costs and the costs of time to treat the animals.
2.5 Infectious ovine keratoconjunctivitis and the export of live animals
Australia is the second largest exporter of live sheep worldwide. In 2012, 2.3 million sheep were exported live from Australia with 80% of these leaving from Western Australia. The
Middle East remains the major export destination accounting for 98% of sheep exports. In
2012 the value of sheep exported was $201 million (MLA, 2013). Exports continued in 2013, although total numbers had dropped to just below 2 million sheep worth $172 million.
Western Australia continued to export 82% of these sheep and Kuwait remained the biggest importer, taking 45% of all sheep exported from Australia (MLA and Livecorp, 2014).
Within the export industry the most significant impact on production associated with IOK is weight loss, but the costs associated with treatment and rejection of animals from the export supply chain must also be taken into consideration. The literature attributes weight loss to decreased feed intake resulting from impaired vision and consequent difficulty in locating feed. Given that IOK is considered a painful condition (Greig, 1989) it could be argued that this pain may also contribute to weight loss. Although difficult to compare directly, studies looking at the pain associated with mulesing have reported weight loss in the week following mulesing (Paull et al., 2008). Cattle with infectious bovine keratoconjunctivitis have poor weaning weights compared to those without IBK (Killinger et al., 1977). The reduced weight gain was attributed to pain and impairment of vision. Across species feed and water intake is consistently modified by painful stimuli, and bodyweight
30 analysis is a useful tool for monitoring effectiveness of analgesia (Stasiak et al., 2003). It is likely that any weight loss associated with IOK is multi-factorial.
Standard policy in the export industry states that any animal showing clinical signs of infectious disease is not fit for export and therefore will be held back (Surman, 1968;
Australian Government Department of Agriculture, 2011), either to be treated before the next shipment or culled (Surman, 1968). Culled animals are deemed to have no commercial value and are humanely euthanased and used in the pet food market. The cost associated with loss of sale in addition to costs associated with treatment, labour and additional feed to hold the animal over for the next shipment all contribute to the exporter’s financial loss attributable to IOK.
2.6 Risk Factors
Infectious ovine keratoconjunctivitis is a multi-factorial disease and the aetiology of IOK outbreaks is often complex. A number of microorganisms have been implicated in outbreaks of IOK. Aside from the infectious agents, several environmental factors are also considered important in the aetiology of IOK.
Season Outbreaks have been reported throughout the year in different parts of the world.
In the United Kingdom outbreaks tend to occur in autumn and winter (Arbuckle and Bonson,
1980; Andrews et al., 1987; Egwu, 1992b). Increased cases in the autumn in Norway are associated with close contact and increased animal supervision. Sheep are grazed at very low stocking densities over the summer months (Akerstedt, 2004) thus reducing close contact and minimizing spread of IOK. However, some cases have been reported in Wales in spring and summer (Sinclair, 1955).
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In North America cases are seen in winter and spring (Riley Jr and Barner, 1953). Cases in
Switzerland occur throughout the grazing season (Nicolet, 1975) in sheep and this coincides with cases seen in wild caprinae in summer and autumn owing to the proximity of domestic animals to the wild population (Belloy et al., 2003).
Outbreaks in Australia are generally reported in the warmer months (Axelsen, 1961;
Surman, 1968). Outbreaks in South Africa, parts of which have a similar climate to Western
Australia, are also seen in the dry and warm months (Van Halderen et al., 1994). Those in
New Zealand typically occur in summer and autumn (Cooper, 1967; Motha et al., 2003), however some atypical outbreaks have been reported in winter (Motha et al., 2003). The reasons for these unseasonal outbreaks were unclear. It is postulated that a novel agent was involved or an outbreak in a naïve flock. The typical outbreaks in New Zealand have been attributed to increased droving and yarding of sheep during these times for routine husbandry coupled with dry and dusty conditions (Cooper, 1967). Like Australia, outbreaks in Croatia are reported in the warmer spring and summer months (Naglic et al., 1999).
Environmental conditions in Israel are similar to some parts of Australia but cases there are reported in the colder months (January – March) (Lysnyansky et al., 2007). In Nigeria, which has a hot a humid climate, cases are reported in October at the end of the rainy season
(Osuagwuh and Akpokodje, 1979). Inclement weather, whether that be cold, wintery weather or hot, dry and dusty summer weather, has been described as a stressor which can predispose to outbreaks (Michael, 1953).
Seasonal patterns of IOK outbreaks appear variable throughout the world. Although outbreaks are generally expected during the warmer months in Australia there is a paucity of literature relating to IOK outbreaks at other times of year in Australia.
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Ultra-violet light can be a contributing factor to infection (Hosie, 1988) which is consistent with outbreaks occurring in the warmer months in Australia. Equally, extreme winter weather is a contributing factor to development of disease (Hosie, 1988).
Housing The housing of sheep is considered to be a pre-disposing factor to the spread of disease.
Disease is spread by direct contact between sheep (Hopkins et al., 1973; Hosie, 1989) and by transmission of infective agents in the lacrimal fluid (Blakemore, 1947). Experimentally, infection can be confined to a group by the use of solid, high partitions between infected sheep and non-infected sheep (Blakemore, 1947). The spread of disease through close transmission goes some way to explaining the seasonal variation of outbreaks in different countries. In countries where sheep are housed in winter to lamb, forced close contact can occur (Akerstedt, 2004). Equally over the autumn and winter where supplementary feeding is required, the use of troughs encourages increased sheep to sheep contact (Cooper, 1967;
Egwu, 1992b; Dun, 2009). High stocking densities will increase sheep to sheep contact and can predispose to an outbreak of IOK (Osuagwuh and Akpokodje, 1979).
Grouping of sheep in feedlots is considered an important factor in aiding rapid spread of infection in an outbreak (Hopkins et al., 1973). Outbreaks in the warmer months are often attributed to dry, hot and dusty conditions (Cooper, 1967). Further to these conditions, grazing tall, seeding grass is considered to be another risk factor (Osuagwuh and Akpokodje,
1979). It is postulated that the grass, contaminated with infected tear secretions, causes trauma to the eyes and an infection is then established (Dun, 2009).
Vectors Vectors can play a significant role in the spread of IOK. Under Australian conditions it has been suggested that flies (Musca domestica) are involved in transmission (Beveridge, 1942).
33
This conclusion was reached when the increased occurrence of disease in the summer months was noted. Flies are more active at this time than in the winter months when there is negligible fly activity. In other countries flies have also been implicated in the spread of disease: in Europe (Konig, 1983a); Nigeria (Osuagwuh and Akpokodje, 1979) and India
(Nanda and Abdussalam, 1943). Strong evidence for their involvement in the Nigerian case was suggested given the disappearance of disease after flies were controlled.
It is thought that flies can directly spread infection between sheep by carrying infected lacrimal fluid (Dun, 2009). Flies are also thought to cause small puncture wounds on the conjunctiva during feeding and the subsequent irritation could allow colonisation by bacteria (Graham-Smith, 1930).
In the UK, on the other hand, where most outbreaks occur in the colder months, it is thought that flies are not always significant vectors, (Egwu, 1992b).
Breed Although IOK can occur in any breed (Radostits, 2007a), some reports exist of variations in infection rates between breeds. In Australia the condition was originally considered to be unique to pure-bred merinos. This was based on an experiment where only pure-bred sheep were experimentally infected, while in cross-bred sheep an infection could not be established (Edgar, 1931). The author of this study postulated that the cross-bred sheep may have an immunity of some form to the infection. Subsequently, the theory that IOK was unique to merinos was disproved following successful experimental infection of cross-bred sheep (Beveridge, 1942).
In the United Kingdom some experimental work has been done to determine if there was any breed predisposition to IOK. These studies looked at Cheviots and Scottish Blackface.
34
Cheviots were initially thought to be more susceptible (Wall, 1982), but a similar study found there to be no difference (Hosie, 1988).
As there is no conclusive evidence of a breed pre-disposition, the variety of breeds that are involved in the live export supply chain are not likely to have an impact on the occurrence of
IOK outbreaks.
Age Sheep of all ages are susceptible to IOK (Beveridge, 1942; Michael, 1953; Jones, 1983), even lambs as young as 5 to 10 days old (Jones et al., 1976). A number of studies have looked at the severity of disease in different age groups. Disease is thought by some to be more severe in adult animals (Greig, 1989; Radostits, 2007a). In contrast some consider IOK to be more severe and outbreaks more frequent in younger sheep (Sinclair, 1955). Beveridge
(1942) found that outbreaks were most common and severe in weaners in Australia. Dun
(2009) found that outbreaks can occur in weaners where frequent handling was a factor;
IOK can be spread through handling of the face and head while animals are being drenched.
Hosie (1988) concurred when frequent outbreaks were described in weaners and this was related to frequent handling. Radostits (2007) reports weaners as being the most severely affected age group.
Hosie (1988) found a seasonal variation in rate of infection depending on age group. Ewes were found most likely to be affected in winter because at that time of year they had been gathered for mating. Increased exposure to ultra-violet light meant that both lambs and ewes could be affected in the summer. Weaned lambs were most likely to be affected in autumn because of increased handling at that time of year for husbandry procedures.
Just as sheep of all breeds enter the live export supply chain, so do sheep of all age
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Environmental factors Environmental, mechanical and climatic factors act together with the causative microorganisms to establish an infection (Lysnyansky et al., 2007). Numerous such factors have been implicated in the establishment of IOK.
Routine husbandry procedures such as lamb marking, worming, weaning and pre-mating checks necessitate forced close contact through yarding. Yarding and high stock densities are recognised risk factors (Edgar, 1931; Beveridge, 1942; Cooper, 1967; Hosie, 1988). In addition, many irritants have been considered to predispose animals to IOK. Dust (Edgar,
1931; Cooper, 1967) and long grasses (Edgar, 1931; Michael, 1953; Cooper, 1967; Dun,
2009) are the most commonly cited irritants.
Exposure to ultra-violet light and heat (Cooper, 1967) are also considered risk factors
(Palgrave, 1910; Hosie, 1988) and exposure to cold and wet weather is likely to predispose to IOK (Palgrave, 1910; Edgar, 1931; Blakemore, 1947).
Concurrent disease Blakemore (1947) considered the presence of another pre-existing or concurrent disease to be a risk factor for IOK. Both joint-ill and parasitism were thought to be of significance.
Michael (1953) reported that dosing infected sheep with an anthelmintic improved recovery from IOK. Deficiencies in Vitamin A have been considered a risk factor in sheep (Hofland et al., 1969). This is similar to a finding with IBK in cattle (el-Sanousi et al., 1978). This theory was, however, disproven in sheep in India where sheep were managed in such a way that
Vitamin A deficiency was unlikely nevertheless became infected with IOK and no effect was seen when infected sheep were treated with Vitamin A supplementation (Patwardhan et al.,
1975).
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Carrier animals Outbreaks of IOK are often associated with the introduction of new animals to a flock. These animals are generally free of clinical disease but act as carriers of the organisms. This was well illustrated during an outbreak of IOK in Croatia (Naglic et al., 2000). No cases of IOK had been reported in Croatia before the importation of sheep from Australia. The imported sheep were quarantined prior to being mixed with native sheep and showed no signs of ocular disease, suggesting that they were asymptomatic carriers of IOK. Outbreaks of IOK occurred in native sheep following introduction of the Australian sheep to the native flock.
Palgrave (1910) identified that introducing new animals with clinical IOK enabled spread of disease. Currently in Western Australia, introducing purchased stock is recognised as a risk factor for IOK (Suijdendorp, 2005). Introduction of carrier rams (Andrews et al., 1987; Hosie,
1988) can partly explain the outbreaks described around joining time. Cooper (1967) found in farmer surveys that mixing of bought-in and home-bred animals was a risk factor in an outbreak of IOK. Radostits (2007) also states that animals bought in, be they clinically infected or apparently healthy carriers, are the source of infection for a flock. Dun (2009) agrees that infection is introduced by carrier animals.
It has been shown that immunity to the organisms considered to be two of the primary pathogens, Mycoplasma conjunctivae and Chlamydophila pecorum, is usually poorly developed and can lead to recurrence of infection in individuals (Baas et al., 1977; Hosie,
1989). Mycoplasma conjunctivae can persist in the eyes and nares for months (Baker et al.,
1965; Hosie, 1989).
It is difficult to avoid many of the risk factors that pre-dispose to outbreaks of IOK when sheep are being exported for most of the year from Australia. The requirement to hold
37 animals in a pre-embarkation feedlot for a quarantine period before shipping serves to exacerbate the risk factors associated with close contact, irritants and forced handling. Pre- embarkation feedlots are arguably a perfect environment for the combination of risk factors to maximise the chances of IOK spread and animal morbidity.
2.7 Microbiological causative agents
Numerous studies have looked with varying success at the microbiological aetiology of IOK.
While the aetiology of IBK is more clearly defined, there is still considerable debate regarding the aetiology of IOK (Lindqvist, 1960; Jones et al., 1976; Egwu, 1991).
Rickettsiae Rickettsiae were first isolated from sheep and considered to be the causative agent of IOK from early reports in South Africa (Coles, 1931). Coles named this organism Rickettsia conjunctivae. An organism that was considered the same was also found in clinical cases of
IOK in Tunisia (Cordier and Menager, 1937), Algeria (Donatien and Lestoquard, 1937),
French Congo (Malbrant, 1939), central France (Lafenetre, 1938) and the French island of
Levant (Pigoury, 1937). Beveridge (1942) described isolating an organism in sheep with IOK in Australia that matched the description given by Coles (1931). However, further studies have concluded that this organism is not a Rickettsia sp.
Blakemore (1947) found similar evidence to suggest that Rickettsia conjunctivae was a causative agent of IOK; however, he considered that the organism could not be classified as a Rickettsia because of the absence of an arthropod vector. In 1948 it was suggested that this organism be renamed Colesiota conjunctivae (Rake, 1948). Although the presence of inclusion bodies in these early studies were considered sufficient to identify the organism, further work has suggested this may not be the case. Inclusion bodies seen by Coles (1931)
38 have since been considered to be goblet cells (Surman, 1973). Surman (1973) puts forward a strong argument to suggest that Mycoplasma sp are in fact the primary organisms involved.
Akerstedt (2004) postulates that the organism found in Coles’ early (1931) study thought to be Rickettsia was in fact a Mycoplasma sp.
Mycoplasma sp. Mycoplasma sp. are considered a major cause of IOK (Baas et al., 1977; Radostits, 2007a).
Early studies have suggested that the Rickettsia organism found by Coles (1931) was in fact a Mycoplasma sp. Surman (1968) isolated Mycoplasma sp. from clinical cases of IOK and was able experimentally to induce clinical infection with the isolates. On the basis of this result it was proposed to classify the organism as Mycoplasma conjunctivae var ovis.
Langford (1971) was also able to isolate a Mycoplasma sp. from clinically infected IOK sheep eyes. Langford (1971) postulated that Mycoplasma sp. may be present in healthy ocular tissue but may become pathogenic following a trauma or stress. This theory was based around an outbreak that occurred in sheep grazing stubble fields where frequent high winds created dusty conditions. It is considered that there were a number of organisms involved and that the primary pathogen was Mycoplasma conjunctivae (Baker et al., 2001).
Cases in Australia, Europe and North America have been attributed to Mycoplasma conjunctivae (Hosie, 1989). It is thought that other bacteria can be involved as they will colonise the primary lesion created by Mycoplasma conjunctivae resulting in more severe clinical signs (Kovac et al., 2003). Hosie (1989) agreed with this finding but stated that this secondary colonization has not been proven experimentally. Infectious keratoconjunctivitis has been successfully induced experimentally by the inoculation of Mycoplasma conjunctivae into the ocular tissue in sheep (Jones et al., 1976) and goats(Baas et al., 1977).
39
In both instances the disease replicated the clinical signs expected in a naturally occurring
IOK infection.
Moraxella sp. Moraxella species have been referred to in the literature as a possible causative agent of
IOK outbreaks. Moraxella sp. have been re-classified from Neisseria to Branhamella and finally to Moraxella (Akerstedt, 2004).
Moraxella sp. have been consistently isolated from clinically affected sheep (Baker et al.,
1965); however, it is thought that Moraxella ovis is not pathogenic even in the presence of corneal damage (Jones et al., 1976). Langford (1971) found that Neiserria ovis (Moraxella ovis) was part of the normal ocular flora and that this may explain its consistent finding by
Baker (1965). Hosie (1989) also found that Branhamella ovis (Moraxella ovis) was frequently isolated from normal and abnormal eyes. In a study of infected sheep, 27% were found to have Mycoplasma conjunctivae and 28% were found to have Moraxella ovis (Akerstedt,
2004) which does highlight its presence in infected eyes.
Chlamydia sp. Chlamydia sp. have been implicated in outbreaks of IOK (Hopkins et al., 1973; Cooper, 1974;
Andrews et al., 1987; Hosie, 1989; Lysnyansky et al., 2007). Hosie (1989) and Lysnyansky
(2007) consider Chlamydia psittaci ovis to be one of the primary pathogens in IOK.
Chlamydial conjunctivitis is thought to be an acute infectious disease of young lambs and goats (Wyman, 1983) and it can be associated with polyarthiritis (Hopkins et al., 1973;
Wyman, 1983). Hopkins (1973) found that clinical signs of chlamydial conjunctivitis can be an early warning sign of subsequent polyarthritis in feedlot lambs. Chlamydia psittaci has been experimentally shown to induce conjunctivitis, although not severe IOK (Wilsmore et
40 al., 1990). Wilsmore (1990) goes on to conclude that C. psittaci reduces the corneal immunity so that secondary organisms, like Moraxella ovis, are able to colonise.
2.8 Other causes of keratoconjunctivitis
Aside from the bacterial causes of keratoconjunctivitis discussed, some less common causes have been described.
Fungal keratoconjunctivitis is considered rare in small ruminants (Moore and Whitley,
1984). Hopkins (1973) isolated Aspergillus spp and Mucor spp from eyes showing clinical signs of keratoconjunctivitis, although these were not considered to be primary pathogens.
Parasites have been cited as a cause of keratoconjunctivitis in sheep. Oestrus ovis, a nasal bot fly, can cause similar clinical signs to IOK (Townsend, 2007). The adult fly lays eggs on the mucus membranes of the face. This typically occurs around the external nares, but it is also possible for the eggs to be directly laid around the eyes. Following hatching, the larvae migrate. This migration can be through the nasolachyrimal system to the eye or directly from the muco-cutaneous junction around the eye to the conjunctiva. In either instance keratoconjunctivitis can develop (Moore and Whitley, 1984). Oestrus ovis is found in
Western Australia. In some areas of South Africa another nasal bot fly, Gedolstia hasleri, can cause similar clinical signs (Moore and Whitley, 1984).
Thelazia spp. can also cause similar clinical signs to IOK. The Musca spp of fly is the intermediate host and they can deposit Thelazia spp. larvae on the conjunctiva while they feed on ocular secretions (Townsend, 2007).
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2.9 Treatment
Infectious ovine keratoconjunctivitis is a condition that ranges in severity. Cooper (1967) found in a survey of farmers that only 23% sought veterinary treatment for outbreaks of
IOK, and those that did tended to do so when they first encountered the disease. Some consider the disease to be self-resolving (Nanda and Abdussalam, 1943; Toop, 1964). Toop
(1964) found that recovery started on the third or fourth day after clinical signs developed and that it took a further seven days for clinical signs to resolve altogether. Akerstedt (2004) found the duration of clinical disease to be longer, with recovery not beginning until one week after clinical signs were first seen and complete recovery not occurring until 3-4 weeks after initial signs.
Under experimental conditions, Baas (1977) found that severe cases could take up to 12 weeks to resolve. Nanda (1943) describes cases recovering without any treatment.
Blakemore (1947) observed that only mild cases would resolve within a week of clinical signs developing, whereas those with more severe clinical signs, such as corneal involvement, had a very prolonged recovery time. In contrast Moore and Whitley (1984) reported that spontaneous recovery occurs even in severe cases within three weeks. Cooper (1967) found little difference in clinical cases between those receiving treatment and those not.
Beveridge (1942) found that in sheep with bilateral IOK where one eye was treated and the other left untreated there was often no difference in recovery rates between the two eyes, although this was not always the case. In contrast to this, Edgar (1931) found a significant difference between treated and non-treated groups. Those treated showed return to normal vision whereas those left untreated showed no progress and continued to have visual deficits. The first reported outbreak in Michigan was found to take almost two
42 months for clinical signs to resolve in severe cases despite various treatments being used
(Riley Jr and Barner, 1953).
Early descriptions of IOK recognized the importance of treating cases early to limit spread throughout a group (Symons, 1912). Symons (1912) goes on to describe the difficulties in treating large groups but highlights the importance of persevering with treatment as success in treating diminishes the more severe the clinical signs. Isolating clinical cases will not only protect clinically healthy sheep from exposure but will allow for easier treatment (Michael,
1953) of the sick animals. Ideally, sheep should be isolated indoors to provide protection from UV light (Nanda and Abdussalam, 1943; Riley Jr and Barner, 1953; Toop, 1964).
Avoiding bright sunlight is thought to reduce the risk of flies and therefore decrease potential spread of disease (Nanda and Abdussalam, 1943). Toop (1964) describes the ease of providing feed and water to sheep with reduced visibility through isolation. Combining isolation of affected sheep and treatment will reduce the length of an outbreak (Cooper,
1967).
Treatments have varied over time. In the early 1900s sheep were treated both systemically and topically. Oral administration of a purgative of epsom salts was recommended (Symons,
1912) to be followed up with hyposulphite of soda and mitre which could be added to the drinking water. In combination with these systemic treatments it was suggested that the eyes should be bathed with a warm strong solution of boracic acid ideally for half an hour at a time at least twice daily. Following bathing a dilute solution of mercuric chloride or salicylic acid should be applied (Symons, 1912). Symons (1912) recommended that in severe cases where ulceration was present a combination of yellow oxide of mercury, boracic acid and Vaseline be applied to the eye.
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Michael (1953) describes a number of home remedies including the application of tobacco juice to the eye. Cutting the lower palpebral vein and angling the head so as to allow the blood to flow across the surface of the eye (Michael, 1953) was also suggested. Shepherds traditionally considered blowing sugar on to the eye to be effective (Sinclair, 1955). Michael
(1953) also suggested sugar as a treatment. More primitive treatments included applying ground up slate to the conjunctiva (Michael, 1953), the theory being that that the fine particles would scrape away the lesions. Topical kerosene has been considered as a treatment. Beveridge (1942), however, found kerosene to have no beneficial effect on clinical signs and in some cases it made the condition worse. It is not surprising that kerosene worsened the clinical signs given that it is considered to be an irritant to eyes
(Chilcott, 2006).
Topical Zinc Sulphate has been used in various concentrations. Beveridge (1942) found that using either 10% or 60% concentration solutions of Zinc Sulphate topically made no difference between treatment and no treatment, however, using Zinc Sulphate in 30% solution gave mixed results: some eyes improved and others did not. In contrast to this
Edgar (1931) found a 2.5% solution applied topically to be effective. A combination of 1%
Zinc Sulphate and 2% Boric acid has also been found to be an effective topical treatment
(Nanda and Abdussalam, 1943). In human cases of conjunctivitis Zinc Sulphate is considered to reduce clinical signs but has no effect on the underlying cause (Wood, 1999).
Topical ethidium bromide has also been used topically to good effect (Cooper, 1961;
Arbuckle and Bonson, 1980). Arbuckle (1980 found a 1% solution to be effective whereas
Copper (1961) found a 0.5% solution to be effective within 48 hours.
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Michaels (1953) studied the effect of oral doses of Riboflavin (Vitamin B2) in the treatment of IOK. A dose of 15mg was given daily for three-five days. Those treated with riboflavin were found to have reduced lacrimal discharge within 48-72 hours compared to those receiving no treatment. Sheep showing more severe signs had a variable response.
2.10 Antibiotics
A number of antibiotics have been used in the treatment of IOK. The choice of antimicrobial should ideally be based on known sensitivity to the organism involved. Given the potential involvement of a variety of organisms, antibiotics that are chosen should have a broad spectrum of activity, including activity against Mycoplasma sp., and should reach sufficient concentrations to exceed the MIC in lacrimal fluid (Regnier et al., 2013).
Topical agents A number of studies have been conducted looking at the efficacy of various topical treatments. Chloramphenicol, Chlortetracycline (Aureomycin Powder), Cloxacillin and OTC are the most commonly used. In general, for topical treatments to be effective they require frequent applications on a daily basis which can make their use in large groups impracticable
(Moore and Whitley, 1984). Normal corneal anatomy plus the constant washing from the tear film prevent high levels of drug penetration of the cornea (Davidson, 2009). As a result the contact time for ophthalmic suspensions is short and only about 1-10% of the drug is absorbed by the corneal stroma therefore frequent applications are required to maintain minimum inhibitory concentrations in the ocular tissue (Davidson, 2009).
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2.10.1.1 Chloramphenicol
Riley (1953) found topical chloramphenicol to be effective in the treatment of IOK. Severe cases required twice daily treatment for 5-10 days whereas milder cases only required once daily treatment for 2-3 days. Sinclair (1955) also found twice daily topical chloramphenicol to be effective, but a 14-day treatment course was required. In contrast to these studies a number of other authors have found Chloramphenicol to be ineffective. When topical chloramphenicol was compared to topical chlortetracycline (CTC), chloramphenicol was found to be no better than giving no treatment and in some cases sheep were clinically worse following treatment (Dickinson and Cooper, 1959). Cooper (1961) also found chloramphenicol to be ineffective. Resistance has been identified to chloramphenicol in
Mycoplasma conjunctivae (Naglic et al., 1999).
Chloramphenicol has been found to cause a severe aplastic anaemia in humans (Page,
1991). This condition can occur indirectly through ingestion of chloramphenicol residues in meat. Given that a minimum residue could not be established and that there was a risk of exposure to chloramphenicol residues, the drug has now been withdrawn from use in food producing animals in Australia (Page, 1991; Australian Government and Australian Pesticides
And Veterinary Medicine Authority, 2014).
2.10.1.2 Chlortetracycline
Topical Chlortetracycline (CTC) commonly comes in a powder form under the trade-name
AureomycinTM. A number of authors have studied the efficacy of CTC in the treatment of
IOK. Dickinson (1959) treated IOK cases twice daily until clinical signs disappeared. A good clinical response was typically seen within 24-48 hours. Relapses following cessation of
46 treatment were common. The same author compared the efficacy of CTC against chloramphenicol and against no treatment, and found that CTC was more effective than both chloramphenicol and no treatment (Dickinson and Cooper, 1959). Cooper (1961) and
Toop (1964) found topical CTC to be an effective treatment. Mycoplasma conjunctivae can be eliminated following a three day course of CTC applied topically twice daily (Egwu et al.,
1989). Mild cases can be treated with a single application of topical CTC (Hosie, 1988), although this is likely to be effective only in extensively managed flocks. Intensively managed flocks would require frequently repeated applications of CTC (Hosie, 1989). Hosie
(1988) found that a single application of CTC was not effective prophylactically. Dun (2009) also considered a single topical CTC application effective in mild cases.
2.10.1.3 Cloxacillin
Cloxacillin is reported to be effective in treating ocular disease in cattle and sheep. It’s efficacy in the treatment of IBK has been well documented (Buswell and Hewett, 1983;
Daigneault and George, 1990). Cloxacillin is used in an ointment formulation which provides an increased contact time with the ocular tissue because of the greater viscosity of the preparation and the slow release of the drug from droplets which have been found to settle in the inferior cul-de-sac (McConnel et al., 2007). This allows for less frequent applications while still maintaining adequate drug concentrations. Although Cloxacillin is an effective treatment of IBK, where only Moraxalla sp. are thought to be involved, the involvement of
Mycoplasma sp. in IOK will potentially limit the use of cloxacillin due to its mechanism of action.
A single application of cloxacillin was found to be more effective than no treatment or treating with a powder containing neomycin/sulphacetamide (Webber et al., 1988).
47
Cloxacillin was found to reduce clinical signs but signs reappeared following cessation of treatment (Lysnyansky et al., 2007). However, when compared to either CTC (Hosie, 1988) or OTC (Greig, 1989) both were found to be more effective than cloxacillin.
2.10.1.4 Oxytetracycline
Topical oxytetracycline (OTC) preparations are available in powder and aerosol forms in
Australia and are registered for the treatment of IOK.
Topical OTC given 3-4 times daily has been found to be effective in shortening the course of disease (Wyman, 1983; Moore and Whitley, 1984; Townsend, 2010). If uveitis or ulceration is present then the addition of 1% atropine topically three times a day is beneficial (Wyman,
1983). Greig (1989) considers topical treatment with OTC applied at least once daily for 5-6 days to be effective if treatment is initiated in early/mild cases. Naglic (2000) found that topical OTC was only effective in mild cases when it was applied 2-3 times per day for one week, however clinical signs usually returned following cessation of treatment.
Injectable
2.10.1.5 Systemic injection
Konig (1983b) conducted a study of various injectable drugs used in the treatment of IOK.
Spiramycin was found to be an effective treatment, but following cessation of treatment there were a number of relapses. A high number of relapses followed treatment with tiamullin, which was found to be less effective than spiramycin or OTC even though it achieved high concentrations in the lacrimal fluid.
Injectable OTC was found to be an effective treatment. Naglic (1999) found that tetracyclines, tiamulin, tylosin and enrofloxacin were effective systemic treatments for IOK.
48
In order to limit the emergence of resistance in the fluroquinolone (e.g. enrofloxacin) group of antibiotics, their use in food producing animals has been prohibited in Australia (Cheng et al., 2012).
Hosie (1995) found OTC injection effective in rapidly reducing the clinical signs of IOK in all stages of infection. This was also found by Kovac (2003), although this study found that in severe cases it took four days for clinical signs to resolve. Despite reduction in clinical signs of IOK, Mycoplasma conjunctivae is not eliminated and the sheep therefore remain carriers
(Kovac et al., 2003). Andrews (1987) and Townsend (2010) also found injectable OTC to be effective in severely affected sheep. Systemic long acting OTC can be useful in controlling an outbreak of IOK (Greig, 1989). Dun (2009) considers that systemic long acting OTC can be used to halt the progress of an outbreak.
Injecting Florfenicol at a dose of 40 mg/kg intra-muscular results in adequate concentrations in the lacrimal fluid to exceed the minimum inhibitory concentration (MIC) of Mycoplamsa conjunctivae and therefore makes it a suitable choice for the treatment of IOK (Regnier et al., 2013).
Injectable cephalosporin was found to be less effective than injectable OTC in the treatment of IOK (Greig, 1989).
2.10.1.6 Sub-conjunctival injections
Sub-conjunctival injections have been used to treat infections of the cornea and anterior tissues of the orbit (Regnier, 2007). Injections should be placed in the bulbar conjunctiva
(Whitley and Moore, 1984; Regnier, 2007) with a maximum volume of 0.5 mL per eye in
49 sheep (Whitley and Moore, 1984). The benefit of this method lies in providing sustained drug concentrations at the site of infection and can be used in place of frequent topical drug application. Injections of water soluble substances like short acting OTC are likely to provide therapeutic levels for 8-12 hours (Regnier, 2007). Dun (2009) found sub-conjunctival injections of OTC to be effective. Sub-conjunctival injection of a penicillin-streptomycin combination drug was found to be less effective than a systemic injection of OTC (Greig,
1989).
Aside from the risk of intraocular injections (Whitley and Moore, 1984), subconjunctival injections are difficult to do and time consuming, so that this method of treatment is less practicable in large groups of sheep (Dun, 2009).
2.10.1.7 Combinations: topical and systemic
Hosie (1988) found that for severe cases of IOK a combination of a single CTC topical treatment with a systemic OTC injection was the most effective treatment. Konig (1983) also found combinations to be effective for severe cases. Naglic (2000) considers the optimum treatment for IOK to be once daily topical OTC in combination with LA OTC injections given every three days for three consecutive treatments.
Oral treatments There is limited information in the literature about oral medications for the treatment of ocular infections. Because of the difficulty of administering frequent topical treatments to young children oral antibiotics have been shown to be an effective alternative in the treatment of conjunctivitis in humans (Wald, 1997). Oral erythromycin was found to be more effective than topical erythromycin at eliminating Chlamydia sp. in ocular infections of
50 children (Patamasucon et al., 1982). There is some evidence that oral doxycycline can be of use in the treatment of ocular infections in horses (Matthews, 2009). A combination of injectable OTC and in feed OTC in the treatment of IBK in cattle suggested that this combination was more effective than procaine penicillin administered sub-conjunctivally
(Eastman et al., 1998). This experiment wasn’t able to conclude whether the oral OTC medication was effective or not.
Although numerous studies have been done on both treatment and pre-disposing factors of
IOK, there is a paucity of information regarding the unique environment of a pre-export feedlot. This thesis aims to establish a suitable treatment protocol that is both effective and practical to use in the feedlot environment where up to 80,000 sheep are held prior to being exported.
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3 General Aims
The review of the current literature reveals a paucity of information relating to the epidemiology and control of IOK in pre-export feedlots. For this reason the following were considered as general aims for this thesis:
to record data on IOK cases entering a feedlot;
to document whether any of the risk factors identified in the literature are present
in live-export pre-export feedlots and to determine whether anything can be done
to mitigate these factors
considering that much research has been published on microbiological causative
agents of IOK, to determine if these agents are present in feedlot conditions and
associated with clinical disease
to test the efficacy and impact on the animals of various common treatments for
these organisms
to measure bioavailability, clinical efficacy and the impact on the rumen
microbiome of either in-feed or in-water oxytetracycline
to identify practical and effective treatments for IOK in pre-export feedlots that
house up to 80,000 sheep
given that IOK is a progressive disease, to determine if a severity of disease
treatment cut-off can be established so that a decision can be made on which
treatment, if any, would be most appropriate at different stages of the disease
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4 Epidemiology of infectious ovine keratoconjunctivitis at a pre-
export feedlot
4.1 Introduction
With increasing scrutiny of the live export trade in Australia, it is essential that the industry address areas of concern throughout the chain. Infectious ovine keratoconjunctivitis (IOK) has been cited as a problem throughout the export chain and to date only anecdotal evidence exists as to the gravity of the issue. It has been estimated by exporters that 0.5-1% of sheep are rejected from a shipment because of IOK (G. Robinson and M. Curnick pers comms) at departure from the pre-export feedlot. In real terms this equates to approximately 10,000-20,000 sheep per year, based on 2013 totals. However, this figure does not include those animals that are held over for treatment until the following shipment.
The aims of the experiment described in this chapter were to:
1. Establish whether accurate data could be obtained of cases of IOK on arrival and
departure from a pre-export feedlot
2. Determine the prevalence of IOK on arrival and on departure from a pre-export
feedlot
3. Determine seasonal patterns in the prevalence of IOK at a pre-export feedlot
4.2 Materials and Methods
Data were received from an exporter of live sheep who recorded cases of IOK on arrival at the feedlot. The feedlot is located approximately 50km south of metropolitan Perth in
53
Western Australia and is a designated pre-export quarantine facility with capacity to accommodate more than 80,000 sheep. All sheep were inspected on arrival at the feedlot. It is at this point that the exporter decided which animals were not fit for entry to the feedlot.
Rejected animals were deemed to have no commercial value and the producer received no payment for them.
Sheep were rejected for a number of reasons. These included severe lameness, IOK, emaciation/weakness and wounds. Stock inspectors were employed at the feedlot to carry out these inspections. Only sheep with advanced IOK, whereby vision was impaired and/or, in the opinion of the inspector, treatment was unlikely to be successful, were rejected.
Sheep showing more mild clinical signs of IOK were not rejected but were treated.
Sheep arriving at the feedlot will have come from a number of different properties from around the state of Western Australia. Esperance was the furthest recorded origin at 690km from the feedlot. Most sheep originated from 150-300km from the feedlot.
On arrival at the feedlot, sheep were grouped into types based on the commercial market they were destined for. Each batch of sheep are classified as a line. This could involve mixing of different lines of sheep. Once grouped, sheep were moved into the sheds where they were housed for the duration of the quarantine period pertaining to their countries of ultimate destination.
Odds ratios and their 95% confidence intervals (95% CI) were calculated to assess the risk of
IOK in different months and to identify a seasonal pattern.
Feedlot inspectors were invited to complete a questionnaire (Appendix 1). The questionnaire was designed to get an overall impression of the percentage of animals
54 presenting at the feedlot with IOK of varying severity and was approved for use by the
Murdoch University Human Ethics Committee. Data from the exporter represented those animals with severe IOK only; the questionnaire was designed to capture information about the animals that had milder IOK.
4.3 Results
Historical data were obtained for a twelve-month period from an exporter (Table 4.1).
Within that period 1,168,372 sheep were exported from that pre-export feedlot. Of those sheep exported, 681 (0.06%) were rejected on account of IOK.
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Shipment Total Percentage of Date number Number shipment Lower 95% Upper 95% sheep in rejected rejected with Odds Confidence confidence shipment with IOK IOK Ratios interval interval 7/08/2012 74272 7 0.01 1.03 0.36 2.94 12/08/2012 19421 5 0.03 2.82 0.90 8.89 1/10/2012 73541 13 0.02 1.94 0.77 4.85 10/11/2012 69641 100 0.14 15.75* 7.32 33.90 10/12/2012 67303 62 0.09 10.10* 4.62 22.07 7/01/2013 73456 47 0.06 7.01* 3.17 15.52 1/02/2013 29450 40 0.14 14.90* 6.67 33.26 26/02/2013 28224 25 0.09 9.71* 4.20 22.46 8/03/2013 75170 60 0.08 8.75* 4.00 19.14 6/04/2013 70510 76 0.11 11.82* 5.45 25.64 20/04/2013 76602 18 0.02 2.57* 1.08 6.16 15/05/2013 76164 46 0.06 6.62* 2.99 14.66 1/06/2013 23034 3 0.01 1.43 0.37 5.52 23/06/2013 77627 23 0.03 3.25* 1.39 7.57 14/07/2013 70548 9 0.01 1.40 0.52 3.75 21/08/2013 68319 22 0.03 3.53* 1.51 8.26 22/09/2013 76685 7 0.01 1.00 4/10/2013 72029 26 0.04 3.96* 1.72 9.11 7/11/2013 46376 92 0.20 21.77* 10.10 46.96 Table 4.1 Historical shipping data showing numbers and percentages of sheep rejected on arrival at the feedlot due to Imfectious Ovine Keratoconjunctivitis. * depicts significant result.
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Sheep being shipped in Spring, Summer or Autumn were more likely to have IOK than those shipped in Winter (Table 4.2). Sheep being shipped in either Spring and Summer were 1.8
(1.55-2.10) times more likely to have IOK than those shipped in Autumn and Winter.
Total Number Percentage Lower 95% Upper 95% Season number rejected rejected with Odds Confidence confidence sheep with IOK IOK Ratio interval interval Spring 338272 238 0.07 3.40* 2.60 4.40 Summer 198433 174 0.88 4.24* 3.21 5.60 Autumn 298446 200 0.67 3.24* 2.46 4.26 Winter 333221 69 0.02 1.00 Spring and Summer 536705 412 0.08 1.80* 1.55 2.10 Autumn and Winter 631667 289 0.04 1.00 Table 4.2 Historical shipping data provided by the exporter outlining, by season, the number of sheep rejected due to clinical Infectious Ovine Keratoconjunctivitis. *depicts significant findings.
Questionnaires were sent to five inspectors and three responded (60% response rate). The three responses were combined and are shown in Table 4.3.
Mild IOK (%) Severe IOK (%) Minimum Maximum Minimum Maximum Spring October 15 25 1 5 November 20 35 5 10 Summer January 35 50 10 20 February 30 40 10 20 Autumn April 25 35 5 15 May 15 25 2 15 Winter July 5 15 2 5 August 5 15 2 5 Table 4.3 Data gatherd by questionnaire showing maximum and minimum percentages of IOK cases per shipment
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In Table 4.4 the breakdown of mild cases in lambs, wethers and ewes respectively are summarised. The respondents failed to give data for severe cases in different ages of animal. The seasonal pattern highlighted by the rejection data from the exporter was confirmed by the exporters with higher percentages of both mild and severe cases seen during the warmer months.
Lambs Wethers Ewes Minimum Maximum Minimum Maximum Minimum Maximum (%) (%) (%) (%) (%) (%) Spring October 15 30 5 10 1 15 November 20 35 10 15 5 10 Summer January 35 50 15 20 5 10 February 30 50 15 20 5 10 Autumn April 30 50 15 20 5 10 May 25 35 10 15 1 5 Winter July 20 20 5 10 1 5 August 15 20 5 10 1 5 Table 4.4 Data gatherd by questionnaire showing maximum and minimum percentages of mild IOK cases per shipment in lambs, wethers and ewes.
The inspectors reported that, for entire male animals, only ram lambs were exported so no figure could be given for rams individually. 95% of sheep inspected were Merinos. The inspectors commented that the proportion of cross-bred sheep with IOK was much lower than in merinos, although they were unable to enumerate this.
4.4 Discussion
The Australian Standards for the Export of Livestock (ASEL) lists the reasons why sheep should be rejected from a consignment for shipment. These standards state that animals with IOK must be rejected (Australian Government Department of Agriculture, 2011). Sheep
58 must also have vision in both eyes. These data received from the exporter were limited in that only those animals deemed too severely affected with IOK for treatment were recorded.
The figure of 0.06% rejections represented the point prevalence of severe IOK at entry to the pre-export feedlot. This data did not capture the mild cases that would be treated during the quarantine period so that they were fit to be shipped. To conform to current legislation, no severe cases of IOK should be identified on arrival at the feedlot as they would not be deemed fit to have travelled from the farm (Moir, 2010). These cases are deemed to be of no commercial value to the exporter and the producer is not paid for them.
It is likely that this discourages producers from sending inappropriate sheep to the pre- export feedlots.
Anecdotally IOK is seen more commonly in lambs and in the spring and summer months on arrival at the pre-export feedlot. This pattern was confirmed by the inspectors’ responses to the questionnaire. They highlighted that most cases were seen in lambs, followed by wethers with the fewest cases in ewes. Although IOK has been reported in animals of any age (Hosie, 1989), infection in lambs is considered to be mild and more transient (Egwu et al., 1989) compared to more severe infections in adults (Hosie, 1989). Although there is a paucity of data relating to the situation in Australia, one contradicting report highlights that outbreaks in weaners are most common and when they do occur are severe (Beveridge,
1942). Although the responses from the inspectors did not give definitive figures, they did suggest that disease was seen most commonly in younger animals. The responses did not allow differentiation of severity of disease between the different age groups.
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The data confirmed a significant seasonal pattern to the prevalence of IOK on entry to the pre-export feedlot. Axelsen (1961) and Surman (1968) reported that cases of IOK in
Australia were most commonly seen in the spring and summer months. As spring and summer in Western Australia are typically hot and dry months, sheep are likely to be exposed to a number of the recognised risk factors for IOK. Hosie (1988) considered exposure to ultra-violet light a risk factor for developing IOK. Cooper (1967) also identified heat as a risk factor. Yarding is recognised as a time for the spread of disease (Edgar, 1931;
Michael, 1953; Cooper, 1967; Dun, 2009) and in Western Australia yarding in the dry months will lead to increased exposure to dust, which Edgar (1931) and Cooper (1967) identified as another risk factor for IOK. Given the high degree of exposure to a number of significant risk factors it is not surprising that cases of IOK were seen in lines of sheep on arrival at the feedlot.
In addition to the sheep identified as suffering from severe IOK on arrival at the pre-export feedlot, a number of sheep arrived with milder IOK. These sheep typically presented with tear staining of the wool on the cheek and conjunctivitis. Sheep with milder IOK are considered treatable and would usually be separated into a batch for treatment. The feedlot management considered that many of these milder IOK cases had resulted from irritation to the eyes during the truck journey. Although this hypothesis could not be confirmed it is possible that sheep would have been exposed to irritants in the preparation for loading and during the transport itself. The questionnaire to the inspectors aimed to capture an impression of the percentage of mild cases presenting at the pre-export feedlot. Their responses highlighted the high numbers of sheep arriving at the feedlot with clinical signs of
60 mild IOK. Although these animals were not rejected outright they did require treatment to enable them to be fit for export.
Attempts were made to validate this data by independently analysing a shipment. At the pre-export feedlot sheep typically arrive over a 2-day period for a shipment which can contain up to 80000 sheep. There are six loading banks for trucks, so 12 people would be required, two at each loading bank, to carry out the inspection. Two people would be required at each loading bank so that both eyes of the sheep could be inspected. Feedlot management were concerned that the additional staff would slow down the procedure and, despite lengthy discussions, the experiment could not proceed. The experiment was designed to be two-pronged: the attempt to analyse a shipment on arrival, and an attempt to track cohorts of sheep through the quarantine period to identify if there were certain groups that were more likely to develop IOK. After unloading, however, groups of sheep were sorted into batches according to sheep type, and with this mixing it was not possible to follow farm cohorts through the quarantine period.
Mixing sheep poses two potential risks with reference to IOK. Firstly, introducing animals from different farms will create the risk that naïve sheep will be exposed to carriers. This phenomenon was highlighted by Naglic (2000) and cited as the explanation for how IOK was introduced to native sheep in Croatia in 1995 following contact with imported Australian sheep. Secondly, sheep are held in pens during the sorting process which allows for close contact. Hopkins (1973), Hosie (1989) and Akerstedt (2004) described IOK being spread by close contact between animals. Once housed, sheep are fed an ad-lib pellet ration. Trough feeding allows for further close contact and this has also been linked to the spread of IOK
(Cooper, 1967; Egwu, 1992b; Dun, 2009). Once infection is identified, Hopkins (1973) found
61 that grouping of sheep in a feedlot was an important factor in aiding the spread of an outbreak of IOK.
Data were not available from the exporter for rejections on leaving the pre-export feedlot.
Currently at Fremantle Port, final inspections are carried out at the wharf by official AQIS inspectors rather than during loading for outward transport from the feedlot. This process is currently under review as concerns have been raised about unsuitable sheep being presented at the wharf (Farmer, 2011). Any cases of IOK that have been unresponsive to treatment during the quarantine period will be re-assessed at the feedlot. These cases will either be held back for the next shipment or culled depending on the assessment of the feedlot management regarding their likely response to treatment. Guidelines have been generated as part of this project to aid their decision making with these cases.
4.5 Conclusions
It was not possible to validate the data obtained from the exporter for rejection rates of IOK on arrival at the feedlot or data for rejection rates on leaving the feedlot. The historical data obtained shows a prevalence of severe IOK on arrival at the feedlot of 0.06% over the year.
Sheep were more likely to have IOK in spring and summer than in autumn and winter. Mild cases of IOK are frequently identified in sheep arriving at the pre-export feedlot; this can affect as many as 50% of sheep during summer. Mild cases are more commonly seen in lambs; there are very few cases among ewes.
With high numbers of sheep arriving at the feedlot with clinical signs of mild IOK it is essential that an appropriate treatment strategy is identified to minimize the impact of an outbreak in the pre-export feedlot and to enable these animals to be exported following the quarantine period.
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5 Identification of the microbiological causes and possible
treatment of infectious ovine keratoconjunctivitis in a pre-export
quarantine feedlot.
Infectious ovine keratoconjunctivitis continues to be a concern within the sheep live-export chain and in particular at the pre-export feedlot. The work described in Chapter 4 has highlighted that although outright rejections represent a small percentage of sheep arriving at the feedlot, there is a high percentage of animals presenting with mild IOK that require treatment. The cost of both the rejections and the treatment of the disease represents a significant loss to the industry.
A number of treatments are used throughout the world to try and alleviate the clinical signs of IOK. Although some work has been done to assess treatments in other countries (Konig,
1983b; Hosie, 1995) there is a paucity of literature evaluating treatments used in Australia, and in particular in the unique environment of a pre-export feedlot.
The aims of this experiment were to:
1. describe normal ocular flora in sheep in a pre-export feedlot
2. assess the efficacy of a number of commonly used treatments at reducing the clinical
appearance of IOK in naturally infected sheep
3. assess the impact of those treatments on the bacterial population in the ocular
environment.
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5.1 Materials and methods
Fifty-one merino and merino cross sheep with no clinical signs of ocular disease were selected from the pre-export feedlot. These sheep had been at the feedlot for five days and were randomly selected from a group with no clinical signs of IOK. Both eyes were swabbed using a cotton-tipped, wooden-shafted swab which was inserted into the fornix, between the conjunctiva and the globe. The swab was gently rotated for ten seconds and then removed. Swabs were plated onto horse blood agar plates in a manner designed to obtain isolated colonies (Harrigan and McCance, 2014) and the shaft was then cut and the swab tip placed in a Mycoplasma broth. New gloves were worn for each sheep to minimize the risk of contamination between animals.
Eighty merino and merino cross sheep with naturally occurring IOK infections were selected from those sheep held back from a shipment owing to the presence of clinical eye disease.
Sheep were selected on the basis of the clinical grade of infection. The grading system
(Appendix 2) was adapted from existing systems (Konig, 1983b) making the grades numerical. Only sheep with grade 2 (conjunctivitis) or grade 3 (corneal oedema) in both eyes were selected for this experiment. Affected sheep showed no other clinical signs of systemic disease. All sheep were transported to the Murdoch University on-campus farm (Perth,
Western Australia) where they were randomly assigned on passing through the race into 8 groups (n=10) and housed in group pens in an open sided shearing shed. This allowed for ease of monitoring and for treatments to be easily administered.
Both eyes were photographed and graded on day zero and a swab was taken for bacterial culture in the same manner described for those without clinical disease. All eyes were graded by the same two people on each occasion using the grading system in Appendix 2.
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All eyes were graded and photographed on days 0, 4, 6, 8 and 10. The left and right eye scores were added to give group scores for both eyes and the change from day 0 to the end of the trial. Swabs were repeated on day 10 as previously described.
The 8 groups were subdivided, as shown in Table 5.1, into a control group and 7 treatment groups. The control group was monitored until day 20 and eyes were graded and photographed on days 0, 4, 6, 8, 10, 12, 18 and 20.
Group Treatment given
Oxytetracycline injectable (Alamycin LA 300, Norbrook Lab. Aust. Pty Limited) 1 20mg/kg given as a single intramuscular injection into neck muscle. Dose calculated based on accurate sheep weights.
Cloxacillin 500mg/3g (Orbenin Eye Ointment, Zoetis) administered at a dose of 2 125mg per eye (one quarter of a tube applied as a streak to each eye, repated after 2 days if still clinical signs).
3 No treatment, control group
Oxytetracycline powder 200mg/g administered orally in the drinking water for 5 4 days. 2g of powder (400mg drug)/head/day based on each sheep drinking 4L water/day.
Oxytetracycline 20mg/g, (Terramycin Pink Eye Powder, Pfizer Animal Health), 5 applied topically twice daily to eyes for 5 days.
Oxytetracycline 20mg/g, (Terramycin Pink Eye Powder, Pfizer Animal Health). Applied topically to eyes twice daily for 5 days PLUS 6 Oxytetracycline injectable 20mg/kg (Alamycin LA 300, Norbrook Lab. Aust. Pty Limited) given as single intramuscular injection into the neck muscle. Dose calculated based on accurate sheep weights.
7 Oxytetracycline 2.0mg/g, (Terramycin Pink Eye Aerosol, Pfizer Animal Health). Applied topically to the eyes twice daily for 5 days.
8 Oxytetracycline (Alamycin 300 LA Norbrook Lab. Aust. Pty) at a dose of 20mg/kg on day 0 and day 4 by intramuscular injection into the neck muscle. Dose
65
calculated based on accurate sheep weights.
Table 5.1 Treatments tested for control of Infectious Ovine Keratoconjunctivitis
Statistical analysis Statistical analyses were performed using statistical package SPSS version 17. An ANOVA with Bonferroni corrections were used for normally distributed data. The Kruskal-Wallis
ANOVA was used for continuous data that were not normally distributed. Paired tests were used to measure the changes in individual sheep between the start and the end of the trial.
5.2 Results
A total of 20 different organisms were cultured from the 102 control eyes. Of these organisms, Bacillus species were the most commonly isolated with 87.3% positive.
Moraxella ovis was the second most commonly isolated organism with 38.2% followed by
Staphylococcus chromogens with 33.3%. Only 4.9% of control animals were positive for
Mycoplasma species, only one of which was positively identified as Mycoplasma conjunctivae (Table 5.2).
66
Organism Positive N (%) Moraxella ovis 39 (38.2) Mycoplasma species 5 (4.9) M.conjunctivae positive 1 (25.0) M.ovis + Mycoplasma sp. 3 (15) M.ovis + M.conjunctivae 1 (0.9) Staphylococcus epidermis 20 (19.6) Bacillus species 89 (87.3) Bacillus sp. Dry yellow 12 (11.8) Staphylococcus chromogens 34 (33.3) Streptococcus pyogenes 0 (0) Arcanobacterium pyogenes 0 (0) Mannheima haemolytica 2 (2) Non-haemolyticMoraxella sp. 2 (2) Gram-negative cocco-bacilli 0 (0) Acinetobacter sp. 8 (7.8) Unidentified GPR 0 (0) Shewanella sp. 0 (0) Pasteurella multocida 0 (0) Unidentified 0 (0) Corynebacterium sp. 0 (0) Staphylococcus haemolyticus 38 (37.3) Gram-negative diplococcic 0 (0) Unidentified GNR 0 (0) Staphylococcus aureus 3 (2.9) Neisseria sp. 2 (2.0) Proteus sp. 0 (0) Unidentified GPC 0 (0) Staphylococcus intermedius 0 (0) Coliform 2 (2) Pseudomonas aeruginosa 0 (0) E.coli 0 (0) Staphylococcus hyicus 0 (0) Streptococcus Group G 1 (1) Streptococcus species 4 (3.9) Pseudomonas sp. 0 (0) Aeorcoccus viridians 5 (4.9) Micrococcus sp. 2 (2) Kocuria varians 1 (1) Enterococcus faecalis 0 (0) Brevundimonas dimuta 13 (12.7)
GNR = Gram-negative rod; GPC = gram-positive cocci; GPR = gram-positive rod Table 5.2 Microbial flora isolated from clinically healthy eyes, Mycoplasma conjunctivae positive is a percentage of total Mycoplasma spp.
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All sheep continued to eat and drink normally throughout the course of the experiment. An estimation of water intake was done for animals in group 4 based on residual water volumes.
The group scores for eyes and the change from day 0 to the end of the experiment is shown in Figure 5.1. A one-way ANOVA applied to the difference between the total score at day 0 and the total scores at day 10 for the different groups showed a significant difference overall between the groups (p < 0.001). The order of improvement, ranging from the greatest reduction in clinical score to the least reduction, was 8, 6, 1, 4, 2, 3, 7 and 5.
It was found that two injections of long acting Oxytetracycline (OTC) treatment (Group 8) and Oxytetracycline single injection combined with topical oxytetracycline powder (Group 6) were the most effective treatments. Both topical OTC preparations, powder (Group 5) and aerosol (Group 7), were less effective than giving no treatment at all. Group 8 was found to be significantly better than Groups 2, 3, 4, 5 and 7, and Group 6 was significantly better than
Groups 3, 5, and 7. Therefore the study showed that Group 3, the control group, was significantly worse than Groups 6 and 8. No significant difference was noted between
Groups 8 and 6.
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Figure 5.1 Eye grades over time
Eye grades over time Group 1 Group 2 Group 3 60 Group 4 Group 5 Group 6 Group 7 Group 8 50
40
30 Total Eye Grades
20
10
0 0 1 2 3 4 5 6 7 8 9 10 11 12 Days post-treatment
Group 1: Single Alamycin LA 300™ injection 20mg/kg. Group 2: Orbenin Eye Ointment™. Group 3: No treatment. Group 4: Oxytetracycline powder in the water. Group 5: Terramycin Pink Eye Powder™. Group 6: Terramycin Pink Eye Powder™ plus Alamycin LA 300™ 20mg/kg single injection. Group 7: Terramycin Pink Eye Spray™. Group 8: Two injections Alamycin LA 300™ 20mg/kg 4 days apart.
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All groups showed a significant reduction in Moraxella ovis autoagglutinating (p<0.05) and
Mycoplasma spp. (p<0.05) indicating that regardless of treatment chosen there was a reduction in the growth of M. ovis and Mycoplasma spp. over the course of the experiment.
For Mycoplasma spp the change is less in the control group than in group 7 (topical OTC aerosol) (p<0.033). This indicates that giving no treatment is more effective at reducing
Mycoplasma spp. growth than treating with topical OTC aerosol.
Moraxella ovis and Mycoplasma spp. were reduced in all treatment groups and in the control group.
The use of the topical powder treatment gave a better result than no treatment for Moraxella ovis.
The use of the topical powder treatment gave a poorer result than no treatment for Mycoplasma spp.
5.3 Discussion
Despite the fact that the sheep were showing no clinical signs of IOK, a number of organisms were cultured in this current experiment. There is limited literature describing the normal
70 ocular flora of sheep eyes. One study found that Micrococcus spp., Streptococcus spp.,
Achromobacter spp., Corynebacterium spp., Bacillus spp., and Moraxella spp. were found in the ovine conjunctival sac of a healthy sheep showing no clinical signs of ocular disease
(Spradbrow, 1968). Bacillus spp. were the bacteria most frequently isolated from healthy eyes in this experiment. It is likely that this frequent occurrence was due to the presence of
Bacillus spp. in dust (Lee et al., 2009). Pre-embarkation feedlots are particularly dusty environments. Dust is stirred up by the constant movement of sheep through the yards at the feedlot.
Staphylococcus chromogenes was also frequently isolated from healthy eyes. S. chromogenes has been associated with sub-clinical mastitis in sheep (Fthenakis, 1994) and cattle (Devriese et al., 2002), but it has not been reported as an ocular pathogen. It is postulated that this bacteria is another environmental organism that contaminated eyes in dusty conditions.
The presence of both Moraxella ovis and, to a lesser extent, Mycoplasma spp. in ocular tissue of healthy sheep highlights the potential for sheep to remain carriers of the organisms responsible for IOK. Infectious ovine keratoconjunctivitis is thought to have been introduced into the native sheep population of Croatia through the introduction of asymptomatic carrier sheep from Australia (Naglic et al., 2000). Naglic (2000) found that Mycoplasma conjunctivae was always identified in sheep flocks where IOK was present even in animals without clinical disease, and this matches the findings of the current experiment.
The presence of IOK-causing organisms in ocular tissue alone is not always enough to result in clinical disease. Spradbrow (1968) considered that the organisms worked synergistically with environmental factors to cause clinical disease.
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Numerous treatments have been recommended for IOK, many of which are no longer used.
Treatments suggested by stockmen to the researchers as being effective for IOK included kerosene and cod liver oil. Kerosene is frequently used in the developing world as an emergency treatment for ocular injuries prior to attending hospitals (Ajite and Fadamiro,
2013) despite its being irritant to the eye (Pe'er and BenEzra, 1988). Cod liver oil has also been reported as an effective treatment for external infections of the eye in humans
(Stevenson, 1936; Ajite and Fadamiro, 2013). Neither treatment was considered suitable for testing in this experiment.
The results of this experiment highlighted the variation in clinical response seen following treatment of IOK with different compounds. Previous work in the United Kingdom demonstrated that a combination of injectable OTC and topical OTC was an effective treatment for IOK (Hosie, 1988). This combination was ranked as one of the most effective treatment in this experiment and it was found that utilising topical OTC in conjunction with injectable OTC resulted in a more effective treatment than a single injection of OTC alone.
However, due to the increased labour requirements required for topical treatment it was considered practically to be less effective than 2 injections of injectable OTC.
Considering the pharmokinetics of topical OTC, this finding was unexpected. Aqueous topical treatments have been shown to have a half-life of a few minutes in lacrimal fluid
(Ward, 1991), therefore any additional benefit of the topical preparation would be minimal.
When used alone, in Group 5, OTC topical powder was found to be the least effective treatment, with the sheep showing a worsening of clinical signs following cessation of treatment.
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Animals in Group 7 and Group 5 received topical aerosol OTC spray and OTC powder respectively, and neither group demonstrated a significant clinical improvement following treatment. On the contrary, topical OTC treatments (both powder and aerosol) were found to reduce clinical signs during application of the treatment, but clinical signs worsened following cessation of treatment and in many individuals, the clinical signs became worse than on initial presentation. Given the limited contact time of aqueous topical treatments it is likely that therapeutic concentrations of drugs would not be maintained in the ocular tissues.
The tear half-life of aqueous topical preparations is short (McConnel et al., 2007). McConnel et al (2007) also found that any antimicrobial preparations sprayed onto the eye were likely to be washed away by the tear film within a few minutes. The topical application of powders and solutions containing dyes have been found to be irritant, resulting in increased tear production (Brown et al., 1998) and therefore decreased contact times. Only about 1-10% of drug solutions and suspension applied topically will be absorbed by the corneal stroma
(Davidson, 2009). As a result of these pharmacokinetic principles, frequent applications are required to maintain minimum inhibitory concentrations in the ocular tissue.
It was found that sheep showed aversion to topical treatments, in particular the topical OTC spray. Sheep were reluctant to move into the race for treatments or examination following commencement of treatment with the aerosol. The results of the experiment showed that those animals that received no treatment showed a significantly greater clinical improvement than those treated with topical oxytetracyclines.
As a treatment protocol, both powder and aerosol topical treatments are labour intensive and therefore impractical for use on a large number of animals, aside from the issues of
73 efficacy and aversion to application seen in this trial. This agreed with Davidson’s (2009) findings that drugs manufactured in powder form and those containing dyes, such as the topical OTC spray used, are irritating and have low drug bioavailability and are not recommended for application to eyes.
Cloxacillin was also investigated as a topical agent (Treatment group 2). Unlike the aqueous
OTC topical medications, Cloxacillin is in an ointment base and this formulation allows for increased contact time with the ocular tissue because of the increased viscosity of the preparation and the slow release of drug from droplets which have been found to settle in the inferior cul-de-sac (McConnel et al., 2007). Cloxacillin has been advocated as a treatment for infectious keratoconjunctivitis in both cattle and sheep. In cattle the clinical effect of cloxacillin treatment has been well documented (Buswell and Hewett, 1983;
Daigneault and George, 1990) and the drug has been shown to be effective. However,
Infectious Bovine Keratoconjunctivitis (IBK) is known to be caused by Moraxella spp. only, organisms which have good sensitivity to cloxacillin. In IOK the involvement of Mycoplasma spp. will limit the efficacy of cloxacillin. Cloxacillin has limited to no effect against
Mycoplasma spp. owing to the absence of a cell wall. Penicillins like cloxacillin act on receptors in the cell wall of bacteria to cause bacterial death (Rang et al., 2000).
Hosie (1988) found that a single dose of Aureomycin Powder ™ (Chlortetracycline) administered topically was more effective than cloxacillin ointment at treating clinical IOK in hill sheep in the United Kingdom. Sheep in Group 2 received treatment with cloxacillin ointment. Of those animals treated with topical preparations, these sheep showed the greatest clinical improvement. Overall, however, animals in Group 2 showed only the fifth greatest clinical improvement. Given the increased labour effort required in applying the
74 treatment and lower levels of drug bioavailability weighed against that of more effective treatments, cloxacillin ointment could not be recommended as a treatment of choice.
In-water medications would appear to be the least labour-intensive and therefore the most suitable treatment protocol for use on large numbers of animals. The use of in-water or in- feed medications makes control of dosing difficult; some animals are likely to be under- dosed and potentially some will be over-dosed. It was found during this treatment experiment that, on average, the sheep were drinking two litres of medicated water per head per day, which would deliver a sub-therapeutic dose, according to the manufacturer’s guidelines.
Davidson (2009) recommended a dose of 22mg/kg once daily for five days for oral administration of tetracycline – this is more than double the dose rate used in the current experiment (880mg for a 40kg sheep versus the 400mg active ingredient used in the current experiment). Normal levels of water intake for sheep are known to be around four litres per day. This can vary widely as a result of a number of factors including weather, diet, water quality and access to water. Although a clinical improvement was seen in the sheep in
Group 4 (OTC in the water), this improvement was less than seen with other treatment protocols. Animals in Group 4 (OTC in the water) showed the 4th greatest clinical improvement. These animals improved more than those in the control group and those receiving topical medications. Although the use of in-water antibiotic medication shows promise, further work is required to establish individual intakes and to assess any impact on rumen health.
The use of systemic antibiotic therapy for IOK has been studied throughout the world.
Systemic antibiotic therapy is usually given by way of an intramuscular injection of a long-
75 acting antibiotic preparation. This limits the labour requirements for treatment and has been found to be effective (Hosie, 1995). Hosie et al (1995) found that a single injection of long-acting oxytetracycline at a dose of 20mg/kg body weight halted signs and produced a rapid clinical cure of severe conjunctivitis. Konig (1983) also found a single injection of long- acting oxytetracycline to be effective in treating clinical cases. The findings of this experiment align with the work by Hosie (1995) and Konig (1983), and demonstrate that general principles of treating IOK can be applied to treatment of the disease in a feedlot.
Axelson (1961) reported that feed intakes can be reduced and animals will lose weight as a result of infection with IOK. It would be expected that sheep with severe clinical signs would have limited vision and would be likely to be disorientated and might struggle to locate feed and water troughs, and that this might be a limiting factor with the use of in-feed or in- water medications.
However, the sheep in the trial showed only mild signs of IOK, and it was noted that all animals in the experiment continued to eat and drink as normal.
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5.4 Conclusions
Up to 20 different species/genera of organisms were isolated from the eyes of healthy sheep including Moraxella ovis and Mycoplasma conjunctivae. This result highlights the potential carrier status of healthy sheep that can act as a source of infection to naïve animals.
This experiment has highlighted the limited value of using topical treatments for IOK in sheep. The use of topical OTC products has been found to worsen the clinical signs of IOK and giving no treatment is more effective that using either of these products. Injectable OTC was shown to be the most effective treatment, and this result is similar to studies carried out under more extensive situations in other countries. Although not the most effective treatment tested, in-water OTC was more effective than giving no treatment. Given the relative ease of administration of medication in water to large numbers of animals, this treatment warrants further investigation. Establishing that injectable OTC is the most effective enables this treatment to be used as a useful standard with which to compare any treatments used in subsequent experiments.
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6 Determination of the efficacy of in-water medication in the
treatment of ovine infectious keratoconjunctivitis
6.1 Introduction
The experimental work of the pilot study described in Chapter 5 highlighted the potential for the use of in-water OTC as a treatment for IOK. Although this treatment wasn’t the most effective of those tested, its ease of use in a large feedlot scenario suggested that it warranted further investigation. It is likely that high numbers of sheep will require treatment for IOK in pre-export feedlots. Logistically, injecting thousands of sheep requires a high number of work hours and could divert labour away from other jobs. High standards of hygiene and good injection technique, which can be difficult to maintain when treating large numbers of animals, are essential in order to minimize the risk of carcass damage resulting from intra-muscular injections.
As described in Chapter 5, a dose of 400 mg Oxytetracycline powder was administered per head in the water as per the manufacturer’s recommendations. This rate of administration resulted in a 50kg sheep drinking 4 L of water per day consuming a dose of 8 mg/kg OTC.
There is no evidence in the literature of successfully treating ocular infections in sheep with oral medication. In cattle, IBK has been treated with orally administered OTC at a dose of 22 mg/kg bodyweight for five days (Davidson, 2009). Research undertaken to establish an effective oral dose of tetracycline in calves demonstrated that a dose of 25 mg/kg was effective in achieving plasma concentrations of OTC greater than 1µg/mL (Luthman et al.,
1989). The effect of adding OTC to water on palatability and intakes in ruminants hasn’t been quantified. Luthman (1989) reported that the addition of OTC at doses of 400 mg/L or
78
800 mg/L water had no negative effect on water intake in pigs, however ruminants may respond differently.
Given that the clinical improvement seen in the oral OTC group in the pilot study was greater than in those receiving no treatment, it is hypothesised that adequate plasma concentrations of OTC are achieved to result in a clinical effect when the OTC is given as a solution. Tetracyclines are known to be absorbed following oral administration in many species (Pijpers et al., 1991; Levison and Levison, 2009). However, there is a paucity of literature relating to the absorption of OTC following oral administration in sheep and its use in the treatment of disease. The pharmacokinetics of doxycycline (Castro et al., 2009) and enrofloxacin following oral administration (Bermingham, 2002) have been documented in sheep. Existing literature on the oral administration of OTC in water to sheep is limited to a study that considered the impact on rumen microbial population. (Munch-Petersen and
Armstrong, 1958). In that study no effect of oral OTC administration on the appetite of the sheep was found, but this was not quantified and no comment was made on the effect, if any, on water intake.
Given the subjective nature of assessment of feed and water intake in the pilot study and a lack of published evidence of any impact of in-water OTC on feed and water intake, it is important to undertake further work to quantify any change following OTC administration.
This forms the basis for the study outlined in this chapter.
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6.2 Hypothesis
1. Addition of OTC in the drinking water such that a 50kg sheep drinking 4L of water will get a 22 mg/kg dose for 5 days will have no adverse effect on feed and water intake.
2. Inclusion of OTC in the drinking water such that a 50kg sheep drinking 4L of water will get a dose of 22 mg/kg for 5 days is an effective treatment for IOK.
3. OTC is absorbed into the blood stream at detectable levels following administration by both intra-muscular and in-water routes.
6.3 Materials and Methods
Twenty seven Merino and Merino cross, mixed age and mixed sex sheep were selected from a pre-export feedlot in the South West of Western Australia. All sheep selected had naturally occurring clinical signs of IOK and were recent arrivals at the feedlot. None of the sheep had undergone any antibiotic treatment for IOK or any other bacterial disease.
Selected sheep were in apparent good health aside from the presence of IOK.
At selection all sheep’s eyes were examined and graded. Only those with a grade 2 or grade
3 in both eyes were selected for the experiment. The grading scale was as used in Chapter 5:
Grade 2 being conjunctivitis and epiphora and Grade 3 additionally having corneal oedema.
All identification (ID) tags were recorded and the sheep were transported to Murdoch
University Animal House.
Sheep were housed in purpose built, individual raised pens with visual contact with other sheep on three sides. Individual housing was chosen to allow close monitoring and to enable individual measurements to be taken daily. The housing was a new environment for the
80 sheep so an acclimatisation period of two days was allowed before the start of the experiment. All sheep were weighed on entering the animal house and again at the end of the experiment. Throughout the experiment sheep were fed the standard export pellet ration that is fed in the feedlot of origin. Feed was delivered in a trough and water was provided in a bucket. Sheep were randomly assigned to one of three Treatment groups:
Group 1: control animals, which received no treatment. (Control)
Group 2: treated with OTC (Alamycin LA 300, Norbrook laboratories, Australia) given
by intra-muscular injection at a dose of 20 mg/kg on day 0 and day 4. (IM)
Group 3: treated with water soluble OTC (CCD OTC, CCD Animal Health, Australia)
administered in the drinking water for 5 days. Water concentration was calculated so
that a sheep that drank 10 % of its live-weight per day received a dose of 22 mg/kg
OTC.
Ocular swabs were taken from all sheep on days 0, 5 and 10. A sterile, cotton-tipped wooden-shafted swab was placed into the fornix, the area between the eyeball and the eyelid, and held there for 15 seconds during which time the swab was gently rotated. Swabs were plated onto horse blood agar plates in a manner for achieving a single culture (Bonner and Hargreaves, 2011). The swab tip was then placed in Mycoplasma broth. Samples were submitted to the Animal Health Laboratory at the Department of Agriculture and Food WA,
South Perth. Here they were incubated at 37oC for 24 hours before the plates were read and colonies identified.
Individual feed and water intakes were calculated daily by measuring feed and water provided and quantity of residuals. Faecal scoring was performed daily to crudely assess any
81 impact on rumen function. Faecal scoring was done using a 1-5 scale with 1 being firm and 5 being diarrhoea (Greeff and Karlsson, 1997).
All eyes were graded on every second day during the experiment to assess the clinical eye grade. The grading system used is described in the pilot study (Chapter 4).
To determine some of the pharmacological properties of OTC following administration by the two different routes, IM and Oral, samples of blood and lacrimal fluid were taken from all sheep in Groups 2 and 3. To collect lacrimal fluid an un-stained schirmer tear test strip
(Haag-Streit, UK) (STT) was placed in the fornix of the eye and held in place for 15 seconds.
Strips were placed individually in a plain 5 mL tube and then frozen at -80oC prior to analysis. Lacrimal fluid samples were taken on days 0, 1, 3, 5, 7 and 10.
Blood samples were taken at the same time points as lacrimal fluid samples from all sheep in Groups 2 and 3. A 3 mL sample was taken from the jugular vein using a 20 gauge, 1 inch needle collecting into a sterile EDTA vacutainer tube. Following collection, samples were centrifuged for 3 minutes at 4000 rpm and the plasma was transferred into a sterile cryovial tube and stored at -80 oC until analysis.
6.3.1.1 Sample Analysis
Tear strips were extracted in methanol, while plasma samples were cleaned using solid phase extraction (SPE) prior to analysis. Samples were analysed using a specially developed liquid chromatography-tandem mass spectrometry method (LC-MS/MS). High-performance liquid chromatography-mass spectrometry (HPLC-MS) is well suited to analysis of OTC concentrations (Oka et al., 1997). This method was developed using OTC solution to
82 determine the appropriate LC conditions and MS/MS transitions. In total five transitions were monitored to reduce the chance of matrix interference.
6.3.1.2 Statistical Analysis
Linear mixed models (LMM), which included treatment effects, covariates and appropriate random effects, were used to analyse the data. Covariance structures were defined for random terms as required and simplified where likelihood ratio tests indicated that this was possible. Hierarchical tests (Type I sums of squares) and a 5% level of significance were used to assess whether treatment and covariate effects were significant. When covariates were fitted after treatment effects they explained within treatment variance; when they were fitted before treatment effects, treatment effects were adjusted for covariance.
Predicted treatment means and standard errors (SE) were corrected to mean and covariate values where appropriate.
6.4 Results
In Table 6.1 the structure of the models used for the analysis of water and feed intake, and the clinical eye score, are summarised.
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Linear models and P-values
Water Plasma OTC Intake Feed Intake Pink eye score
Fixed term P-Value P-Value P-Value P-Value
Liveweight <0.001 <0.001 0.095 <0.001
Treatment 0.008 0.162 0.136 <0.001
OTC Dose 0.001 <0.001 0.062
Day <0.001 <0.001 0.006 0.077
Day.Treatment 0.002 <0.001 <0.001 0.275
Table 6.1 Linear models and P-values for the analysis of water intake, feed intake, clinical eye score and plasma OTC concentration after treatments.
The liveweight range was 30.5 kg to 57.5 kg (mean 44.15 kg) at the beginning of the experiment and 33.5 kg to 67.5 kg (mean 46.25 kg) at the end of the experiment.
Feed intake was significantly affected by liveweight (p value <0.001). Data were corrected for liveweight and a significant difference in feed intake was seen between the treatments on different days (Table 6.1 and Figure 6.1).
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Figure 6.1 Mean feed intake (kg/head/day) over time. Shaded area represents the duration of Oral treatment. Error bars represent standard errors
85
Feed intake – kg/head/day
Day Control IM Oral
0 1.442 1.37 1.429
1 1.458a 1.285 1.185b
2 1.361a 1.241a 0.579b
3 1.572a 1.882a 0.903b
4 1.295a 1.25a 0.746b
5 1.896a 1.659a 1.268b
6 1.632a 1.414 1.033b
7 1.812a 1.697 1.387b
8 1.48 1.33 1.081
9 1.815 1.695 1.627
10 1.728a 1.49 1.12b
Table 6.2 Mean feed intake (kg/head/day) for treatments on different days. Within Day, means with different letters differ significantly (P<0.05).
Feed intake decreased in the Oral treatment group during the treatment period but returned to maintenance levels following cessation of treatment.
Feed intake was significantly lower in the Oral treatment compared to the Control treatment on days 1, 2, 3, 4, 5, 6, 7 and 10 (Table 6.2). Feed intake was significantly lower in the Oral treatment compared to the IM treatment on days 2, 3, 4 and 5 (Table 6.2). There was no significant difference in feed intakes between the Control and the IM treatments on any of the days (Table 6.2).
86
Water intake was significantly affected by liveweight (P < 0.001). There was a significant difference in water intake between the treatments and on different days (p =0.002) (Figure
6.2).
Figure 6.2 Mean water intake (L/head/day) over time. Shaded area represents the duration of oral treatment. Error bars represent standard errors.
6
5
4
L/head/day
- 3 Control IM 2 Oral
Mean waterintake Mean 1
0 0 1 2 3 4 5 6 7 8 9 10 Day
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Water intake – L/head/day
Day Control IM Oral
0
1 3.273 3.623 3.981
2 2.586a 4.289b 2.203a
3 3.336a 3.845a 2.092b
4 2.577 3.158a 1.481b
5 4.053a 4.567a 3.537b
6 3.836 3.475 2.814
7 4.046 3.678 3.204
8 3.461a 3.123 2.37b
9 4.324 4.126 3.645
10 3.308 2.845 2.703
Table 6.3 Mean water intake (L/head/day) for treatments on different days. Within Day, means with different letters differ significantly (P<0.05).
Water intake in the Oral treatment group was significantly lower than in the Control or IM treatment groups during the in-water treatment period. Following cessation of in-water treatment, water intake in the Oral treatment group returned to pre-treatment levels following cessation of treatment. Day 8 was the exception, where intake of the Oral treatment group reduced for one day before returning to similar levels to the other groups for the remainder of the monitoring period.
Sheep in the Oral treatment group drank significantly less than sheep in the IM treatment on days 2, 3, 4 and 5 and drank significantly more than the Control treatment on day 2
(Table 6.3). The Oral treatment group drank significantly less water than the Control treatment group on days 3, 5 and 8 (Table 6.3).
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Clinical eye grades changed over the course of the experiment (Figure 6.3). A significant difference was seen between the treatment groups on different days throughout the experiment (p<0.001) (Table 6.4).
Figure 6.3 Mean clinical eye grade (average both eyes) over time. Shaded area represents the duration of Oral treatment. IM OTC treatment given on day 0 and 4. Error bars represent standard errors.
3
2.5
2
1.5 Control IM
Clinical eye Clinical grades 1 Oral
0.5
0 0 1 2 3 4 5 6 7 8 9 10 Day
Clinical eye grades did not differ significantly at the end compared to the start for both the
Control and the Oral treatment groups. After initially worsening, eye grades of the Oral treatment group made a marked improvement on day 5. This improvement, however, was not sustained and clinical eye grades in this group returned to levels similar to those at the start of treatment (Figure 6.3).
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Clinical Eye Grades
Day Control IM Oral
0 2.303 2.238 2.104
3 1.865a 1.849a 2.382b
5 1.99a 1.46 1.271b
7 1.803 1.626a 2.106b
10 1.928 1.46a 2.104b
Table 6.4 Mean clinical eye grade (average of both eyes) for treatments on different days. Within Day, means with different letters differ significantly (P<0.05).
Animals in the IM treatment group received a dose of 20 mg/kg of OTC on days 0 and 4.
Those in the Oral treatment group received a variable dose over time as a result of variable water intake (Table 6.5).
Day 1 Day 2 Day 3 Day 4
Mean mg OTC 662.4 372.0 353.9 254.1 consumed
Average dose 14.7 8.2 7.8 5.6 mg/kg
Table 6.5:OTC dose received by the Oral treatment group.
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Plasma OTC concentrations measured on day 1 and day 5 of treatment differed between the
Oral and IM treatment groups. Serial measurements taken between these two points over the treatment period were not significantly different between the groups (Figure 6.4).
6.4 Mean plasma OTC concentrations at different time points during the experiment.
1 0.9 0.8 0.7
0.6 0.5 oral (µg/mL) 0.4 IM 0.3 0.2
Oxytetracycline Plasma concentration 0.1 0 0 1 3 5 7 10 Days
For mean plasma OTC concentrations over the treatment period, the IM treatment group was significantly higher than the Oral treatment group (Figure 6.5).
Figure 6.5 Mean plasma OTC concentration during treatment period for the Oral and the IM treatment groups
1.2
1
0.8
0.6
µg/mL) ( 0.4
0.2 Mean plasma OTC concentration OTC plasma Mean 0 IM Oral
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Analysis of the lacrimal fluid proved to be unsuccesful. Following extraction of the samples from the STTs they were run through the liquid chromatography-tandem mass spectrometer. Unfortunately the results proved difficult to interpret. All samples, including those in the control group, showed a spike reaction at the site where an OTC spike was anticipated.
6.5 Discussion
Feed intake decreased during the in-water treatment period in the Oral treatment group.
This coincided with a decrease in water intake in the same animals during this time. It is hypothesised that the decrease in feed intake was a consequence of the reduced water intake. All animals were fed a dry, pellet-based ration. As the water intake decreased in these animals the hydration state would also decrease resulting in a decreased palatability of a dry feed and a decreased appetite. Despite the decreased water intake, no animals appeared clinically dehydrated. There were no signs of skin tenting or sunken eyes on clincal examination. Water intake can be linked to dry matter intake in sheep. Within two days of reduced water intake, feed intake will be noticeably reduced (Forbes, 2007b). Forbes
(2007b) attributed this to the requirement of water to enable passage of feed through the digestive tract. Frequency of drinking has also been shown to decrease feed intake (Squires and Wilson, 1971) whereby decreasing the number of drinks per day will also decrease feed intake. Animals will typically drink twice the weight of dry matter intake (Forbes, 2007a).
Despite alterations in feed and water intake this is a pattern that can be seen in the results of this experiment.
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It is also hypothesised that the decrease in water intake during the in-water treatment period in this oral treatment was an effect of the palatability of the OTC dissolved in water.
Both feed and water intakes were decreased following the commencement of the Oral treatment. Palatability effects are typically seen immediately after a change occurs (Arnold et al., 1980). This is possibly associated with an unpleasant taste in the water caused by the addition of OTC powder, and following cessation of treatment, feed and water intakes did increase. Although post-treatment measurements fluctuated over the monitoring period, the trend was towards pre-treatment levels. The return to normal intake levels following cessation of treatment coupled with the dramatic drop in intake on day one of treatment is sufficient to conclude that OTC powder dissolved in the water was unpalatable to the sheep.
During the treatment period intakes appeared to remain constant. It is not known if extending the treatment period would allow the sheep to adapt to the new taste, possibly leading to improved intake over time.
Although sheep are not expected to gain weight during the live export process, it is important that they maintain their weight. A number of studies have looked at risk factors associated with deaths within the export process (Norris et al., 1989a; Norris et al., 1989b;
Richards et al., 1989; Richards et al., 1991). Inanition and salmonellosis have been highlighted as major causes of death during the export process (Richards et al., 1989).
Richards et al. (1989) highlighted the importance of maintaining regular feed intake to minimise the risk of either of the major causes of death. Although feed intake decreased during the Oral treatment it did not drop to zero intake. Despite maintaining some intake it
93 would be preferable to avoid a decrease in feed intake, and therefore methods to reduce the impact of the Oral treatment on feed intake should be explored.
An improvement in the clinical eye score was seen only in the IM treatment group. In contrast to Chapman et al. (2010), who found a clinical improvement in the oral treatment group, there was no overall improvement despite some initial improvement. It is suggested that the unpalatable taste of the OTC dissolved in water at a concentration that would deliver a dose of 22mg/kg if an animal drank 10% of its liveweight, caused the animals to drink too little water to deliver an effective dose of OTC. Intake of such a low dose of OTC is unlikely to allow concentrations in the plasma and ocular tissues to be sufficiently high to exceed the MIC of the pathogenic organisms. Further work needs to be done to determine the concentration of OTC available following oral administration.The benefits of giving IM medication over medication in the water is that a known concentration is given. In-water doses are very variable depending on intake. Intake itself is also dependant on a number of factors: climatic, availability of clean water, and adequate trough space. However, if it were possible to optimise the in-water concentration such that animals received an effective dose, this treatment would be suitable for a feedlot and would be less labour intensive than giving multiple IM injections. Additionally, blanket treatment of a group with a high number of clincal cases would allow treatment of those animals with early clincal disease, whose clinical signs might not have been seen by feedlot staff.
Changes in clinical appearance between the Oral and IM treatment groups can be attributed to the difference in the OTC dose. In the Oral treatment group the highest average dose was
14 mg/kg OTC on a single day. It is difficult to compare this directly with the intramuscular dose of 20 mg/kg OTC (Alamycin LA 300, Norbrook laboratories, Australia) which is a long
94 acting preparation. Shorter acting preparations, those designed to last 24 hours, are given daily at a dose of 8 mg/kg (Engemycin Intervet/Schering-Plough Animal Health Australia).
Comparing daily mg/kg doses alone, sheep in the Oral treatment group had a potentially effective oral dose on days 1 and 2 only, and the remainder of the course was therefore at sub-therapeutic levels. Comparing given doses alone does not take into account the concentration of drug available. This highlights the requirement for further work to establish the bioavailability of OTC through various methods of administration.
This experiment indicates that OTC can be detected in plasma following both IM and oral routes of administration. Although plasma concentrations are higher following IM injection, the experiment shows that this difference is not dose related as dose was not a significant covariate in the model. Therefore, the hypothesis is that the difference was due to a different bioavailability of the drug when given by different routes of administration.
Failure of lacrimal fluid analysis has been attributed to a substance that shows a similar structure to OTC through the liquid chromatography tandom mass-spectrometer, a phenomenon known as matrix effects (Trufelli et al., 2011). Although HPLC-MS is considered to be highly selective it is still possible for endogenous impurities to exist which intereferes with the reliabilty of the results (Trufelli et al., 2011). The presence of these matrix effects makes interpretation of the data impossible. To confirm that the substance could not be attributed to the STT strips and not to the lacrimal fluid collected plain STT strips were prepared as described and the fluid generated run through the liquid chromatography tandem mass-spectrometer. The plain STT strip showed no reponse at the OTC site. It was considered that STT strips may not be suitable for such analysis, however they have been
95 used to harvest tears for analysis and been successfully used in elephant seals for the analysis of doxycycline (Freeman et al., 2013).
Ocular tissue is protected by a tear film which has many functions including the provision of white blood cells to the cornea and conjunctiva and protection via specific and non-specific antibacterial substances (Davidson and Kuonen, 2004). These substances can act directly on the bacteria themselves or can act to modulate the host’s immune system. It is possible that such substances could themselves provide matrix effects.
Alternative methods of sample collection were considered including cathetirisation of the naso-lacrimal gland. The use of this technique in studies in calves has shown that OTC concentrates in lacrimal fluid and ocular tissue (George, 1985). Studies in sheep where a bioassay technique was used have also shown that OTC will concentrate in the ocular tissues
(Nouws and Konig, 1983), albeit at low concentrations. Both these studies were conducted using intra-muscular injection only. Given the higher bioavailability of OTC following intra- muscular injection compared to oral dosing, it is possible that drug concentrations in the lacrimal fluid are below detectable levels following oral administration of the drug. Further exploration of lacrimal fluid analysis was ruled out. Nouws and Konig (1983) discuss the passive diffusion of the non-protein bound fraction of drugs into lacrimal fluid. Given the low proportion of protein binding of OTC (Ziv and Sulman, 1972) it is feasible for the drug to concentrate in lacrimal fluid. Although concentration of OTC in the lacrimal fluid has not been confirmed in this study, previous written reports of this and the clincal improvement seen in the groups treated with OTC compared to the control group in this experiment indicate that OTC must reach sufficient concentrations in the ocular tissue/fluid to result in a clinical response. Following systemic administration of OTC in cattle, OTC has been shown to
96 concentrate in the corneal tissues (Davidson and Pickett, 2009). If this is true in sheep this may go some way to explaining any clinical effect.
6.6 Conclusion
This experiment showed that OTC can be detected in plasma following both IM injection and oral routes of administration. Although significant clinical response to treatment in the
Oral treatment group was not seen, this lack of response can potentially be attributed to the low intakes seen following commencment of medication in the water. This observation, combined with the promising findings in the pilot study (Chapter 5), led the researchers to believe that it may be possible to alter the dose such that optimal intakes are achieved. The goal would be to alter the concentration of OTC in the water in such a way and to such an extent that water intake was not adversely affected, resulting in greater concentrations of
OTC available for absorption from the GI tract. This aim formed the basis for the experiment summarised in the following chapter.
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7 Maximising intake of in-water oxytetracycline
7.1 Introduction
The previous experiment highlighted the negative impact on water and feed intake associated with a high concentration of OTC in the drinking water. In order to maximise water intake and mitigate some of the palatability issues it was hypothesised that reducing the dose to the point where intake wasn’t affected might enable therapeutic concentrations of OTC to be achieved in plasma. It was also hypothesised that mitigating the adverse taste by adding dextrose to the water might be effective in reducing the negative impact on water intake.
It is generally considered that most domestic animals are attracted to sweet tastes
(Goatcher and Church, 1970b). Sugars are often added to medicated feedstuffs and water to increase palatability (Pers. comm. K. Nairn). Fowl demonstrate a preference for sucrose- flavoured water (Kare et al., 1957). However, studies have found that sheep can remain indifferent to sweet flavours (Goatcher and Church, 1970b). Goatcher (1970) found that sheep showed minimal preference for any sugar added to water, but once concentrations exceeded 20% sheep began to show a dislike for additional sugars in their water.
Concentrations of glucose above 5% in water have been shown to decrease the sheep’s intake of water; concentrations lower than 5% have a minimal impact on water intake
(Arnold et al., 1980). In contrast, it is thought that increased dry matter intake of pasture species with high water soluble carbohydrate (sucrose) concentrations result because of increased palatability (O'Donovan et al., 2011).
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Although there is evidence in other species of a predilection for sweet tastes, the existing evidence surrounding taste preferences in sheep is inconclusive. The trend is such that sheep are not averse to sweet tastes at low concentrations in water. Increasing the concentration of OTC in-water between the experiments in Chapters 5 and 6 resulted in a reduction in water intakes and consequently in OTC intakes. If the concentration of OTC in the water were reduced, but this reduction were more than off-set by the consequent increase in water intake, it could mean that higher concentrations of OTC were available for absorption.
7.2 Hypotheses
1. A lower concentration of OTC in the water will increase palatability and lead to a subsequent increase in water intake. The result would be an effective increase in the dose of OTC.
2. The addition of dextrose to water medicated with OTC will not improve the palatability of water medicated with OTC.
7.3 Materials and methods
Thirty adult Merino cross ewes were randomly selected from the Murdoch University teaching flock. Animals were housed in a purpose built animal house in individual raised pens with sheep contact on at least one side.
As in previous experiments, sheep were randomly assigned to five treatments (n=6, see
Table 7.1). All sheep were weighed on entry to the animal house and again on leaving the animal house. A two-day acclimatisation period was allowed prior to commencing
99 measurements to allow the sheep to adapt to the pellet ration and the individual pens. All sheep were fed a maintenance pellet ration during this period from a trough and water was given in a bucket.
The concentrations of OTC in the water were calculated such that a 50kg sheep drinking four litres per day would consume the treatment dose. Treatments started after two days of baseline measurements and lasted for five days. Following this there was a further five days of monitoring. Measurements of feed and water intake were taken daily.
Treatment Dose rate and water additive
1 2% dextrose (Control) 2 2% dextrose and 11 mg/kg OTC 3 2% dextrose and 16.5 mg/kg OTC 4 11mg/kg OTC 5 16.5 mg/kg OTC
Table 7.1: Treatments in experiment to optimise dose and palatability Statistical analysis For each animal all pre-treatment feed and water intakes were averaged over the acclimation period and change from the pre-treatment average was calculated for each post-treatment date. A linear mixed model was fitted to the calculated changes on all post- treatment dates. The model included fixed effects for pre-treatment liveweight, treatment, post-treatment date and treatment by date interaction; and random effects for animal and the animal by date interaction. An autoregressive model allowed for correlations between measurements made on the same animal on different dates and different residual variance on each date. 5% LSD’s were calculated to compare treatment means to zero, i.e. whether treatment caused a change from the pre-treatment mean, and to compare treatments.
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7.4 Results
The addition of dextrose made no significant difference to either feed or water intake, with or without the addition of OTC (feed intake P = 0.099, water intake P = 0.154, see Table 7.2).
Adding OTC to water at a dose rate of 16 mg/kg and 11mg/kg caused both feed and water intake to fall significantly after treatment began (feed intake P = 0.003, water intake P =
0.007, see Table 7.2 and Figure 7.1). Water intake fell more at the 16 mg/kg dose rate compared to the 11mg/kg dose rate (P = 0.051, see Table 7.2 and Figure 7.2).
Over time, particularly after the OTC treatment ended, feed and water intakes recovered to near the levels of the no OTC treatment. Recovery of intakes commenced from day 2 of treatment.
Total OTC quantities consumed per day were no different for the 11mg/kg treatment compared to the 16 mg/kg dose rates (see Figure 7.3).
Term Change in feed Change in water intake intake
Weight 0.846 0.323
OTC (with or without OTC) 0.003 0.007
OTC.dextrose (OTC with or without 0.099 0.154 dextrose)
OTC dose (11 or 16mg/kg) 0.951 0.051
OTC.dextrose.dose (11 or 16mg/kg 0.626 0.437 dextrose)
Time <0.001 <0.001 time.OTC 0.004 0.005
Table 7.2: Significance levels (P-Values) for terms in model with respect to change in feed or change in water intake.
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Figure 7.1 Change in feed intake (g/head/day) over time. Shaded box represents treatment period. Error bars represent standard errors
Figure 7.2 Change in water intake (ml/head/day) over time. Shaded box represents treatment period. Error bars represent standard errors.
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Figure 7.3 OTC intakes (based on Experiment 1) for different doses of OTC during treatment period.
mg OTC consumed mgOTC
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7.5 Discussion
Following results from the experiment described in Chapter 6, the current experiment was designed to try and mitigate the assumed negative palatability effects of including OTC in water. This pilot trial was designed using knowledge from previous experiments where feed and water intakes were reduced following the addition of OTC to plain water. As such no control group was included of just plain water. This limited our ability to draw conlusions on the effect of adding dextrose to plain water alone. Dextrose was chosen as an additive because anecdotal evidence shows that it has been used in the poultry industry as a supplement in medicated water to improve water intake. Additions of 1 and 2% glucose to drinking water for dairy cows has shown to have no negative impact on intakes (Osborne et al., 2002). However, sheep appear to be less attracted to sweet tastes compared to other domestic species (Goatcher and Church, 1970b). Despite this there are no reports in the literature of a negative impact on water or feed intake by sheep associated with the inclusion of low concentrations of sugars. Increasing concentrations of sucrose in water had no impact on intake for sheep until an inclusion rate of 20% was reached which resulted in a slight decrease in intake (Goatcher and Church, 1970a). Although a preference for sweet tastes is typical of some animals like cattle and goats, the same is not consistently true with sheep where significant variation exists between animals (Goatcher and Church, 1970b).
Dextrose is readily soluble in water and has a sweet taste. Given the lack of strong evidence that this taste would be attractive to sheep it was unknown whether adding dextrose to the water would counteract any negative effects of OTC on palatability. The previous experiment looked at the effects of using 22mg/kg OTC dissolved in water to treat IOK.
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Lower OTC doses were selected in this experiment with the hypothesis that if sheep tolerated the taste they would consume more water and increase the therapeutic dose.
The lack of effect of adding dextrose to water in order to mitigate taste mirrors the results of the previous experiment with regard to decreased intakes. This correlates well with work done in pigs where sweetening the water was also found to be ineffective in increasing water intakes (Maenz et al., 1993). The addition of tetracycline to the drinking water of pigs, however, had no impact on water intake (Luthman et al., 1989) which may further highlight the peculiar palate of the sheep compared to other domestic animals.
The addition of in-water OTC reduced feed and water intake to different levels when added at different concentrations. Total consumption of OTC data from the previous experiment was used to extrapolate daily consumption of OTC in this experiment. This showed that there was no difference in OTC intakes at differing dose rates of OTC (Figure 7.3). Despite lowering the concentration of OTC in the water, intake was still reduced. It would appear that the higher the concentration in water, the less the sheep will drink and consequently the lower the amount of OTC consumed.
Although growth is not required in sheep within the live export chain, it is important that feed and water intakes are maintained. Any factors which result in a reduction of feed or water intake should be minimized to reduce the risk of deaths associated with inanition and salmonellosis (Richards et al., 1989). Given that the administration of in-water OTC is associated with decreased feed and water intakes it is prudent to address the significance of this before contemplating this as a suitable treatment for IOK.
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7.6 Conclusions
The results of the experiment have disproved the hypothesis that lowering the concentration of OTC in the water will result in increased water intakes. Adding dextrose in an attempt to improve the palatability of the water had no effect in mitigating the negative effect of OTC on daily water consumption.
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8 Determination of oral bioavailability of oxytetracycline in sheep.
8.1 Introduction
Although it has been demonstrated that OTC is absorbed at detectable concentrations following oral administration, it is important to determine how bioavailable OTC is following oral administration in sheep. Once this has been established the dose of OTC could be modified with a view to maximizing water intake without decreasing available concentrations of OTC in the blood.
Only when a drug is administered intravenously can it be assumed that all of the drug will be active; administration by any other route is subject to the bioavailability of that drug
(Baggot, 2004). The bioavailability of any drug can be defined as the concentration at which a drug enters the circulation without having undergone any change (Baggot and Giguere,
2013). It is not the dose of the drug given that determines the therapeutic effect, rather the concentration of the drug in the bloodstream following absorption (Neuschl, 2000), and ultimately the concentration at the target site. It is important when a drug is used therapeutically to treat or prevent disease that it is absorbed sufficiently to achieve adequate concentrations within the body fluids and tissues (Luthman et al., 1989).
The addition of OTC to water results in a solution that is typically readily absorbed from the gastro-intestinal tract. While the rumen is considered to have good absorptive ability, dilution of drug in the large volume of rumen fluid can slow absorption (Droumev et al.,
1992). In addition to the large volumes of fluid the presence of microorganisms in the rumen and a pH of 5.5-6.6 can result in denaturing of some antibiotics (Baggot and Giguere,
2013). Rumen microflora can inactivate drugs through metabolic or chemical reactions
(Toutain et al., 2010). An example of this is Chloramphenicol which is known to be
107 deactivated by reduction of the nitro group in the rumen of goats by rumen microbes
(Theodorides et al., 1968).
Absorption across the mucosal wall relies on the drug being soluble in lipids. Oxytetracycline is lipid soluble but newer generation tetracyclines, like doxycycline, are more lipid soluble and therefore more readily absorbed (Castro et al., 2009). Doxycycline is 5-10 times more lipid soluble than OTC (Abd El-Aty et al., 2004). Absorption across cell membranes is optimal between pH 4-7 (del Castillo, 2013) which suits the environment within the rumen.
Tetracyclines have extended half-lives of 6-10 hours because of entero-hepatic circulation whereby drug excreted in the bile is reabsorbed in the intestine (del Castillo, 2013). If adequate drug concentrations are maintained, entero-hepatic circulation will prolong the pharmacological effects of the drug (Gibson and Skett, 2001b).
In order to achieve as high a circulating concentration of OTC as possible, it is important to understand the oral bioavailability of the drug. If a concentration of OTC can be delivered through the drinking water or through medicated feed such that an effective plasma concentration is achieved then the use of oral medications in the feedlots would be a viable option. An effective drug concentration is one that exceeds the Minimum Inhibitory
Concentration (MIC) of the target organism where the MIC is the minimum concentration of a drug that will inhibit growth of an organism in vitro (Boothe, 2001b).
This experiment was modelled on a similar study investigating the pharmacokinetics of enrofloxacin given to sheep orally and by IV injection (Bermingham, 2002) and was designed to determine the bioavailability of OTC following oral administration in both water and feed.
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8.2 Materials and Methods
Six adult Merino cross ewes were selected at random for the experiment. A three-way cross over study design was used with a 10-day rest period between study periods to ensure adequate clearance of drug. The sheep were randomly assigned into three groups of two animals per group. They remained in that assigned group for all three study periods.
Sheep were housed for 36 hours prior to the start of the experiment to allow them to become accustomed to the housing and feed. All animals were offered a diet of oaten chaff and commercial maintenance pellets.
The three treatments were:
IV: 8mg/kg OTC (Engemycin, MSD Animal Health, Bendigo, VIC, Australia) given by intravenous injection once
IW: 22mg/kg OTC (CCD OTC, CCD Animal Health, Geelong VIC, Australia) given orally in- water once.
IF: 20 mg/kg OTC (Terramycin 200, Phibro Animal Health, Girraween, NSW, Australia) given orally in-feed once.
The two sheep in each group were rotated for each experiment period meaning that all sheep were used in each treatment group once. All animals were weighed and housed in individual, raised pens with sheep contact on at least one side. Fleece was clipped over the neck to facilitate catheter placement. Indwelling catheters (16 gauge, 2 ¾”) were placed in the jugular vein of each sheep and an extension set was attached to facilitate easier sampling. Catheters were sutured in place using nylon sutures and a net bandage was used to provide additional protection over the neck area.
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The two sheep in the IF treatment had feed withheld for 12 hours prior to commencement of the experiment to improve feed intake over a short period of time. Sheep in the IW treatment were given their dose by a drench using a Magrath feeder tube (Springer Magrath
Co. MN, USA). Using a Magrath feeder ensured the medicated fluid was placed in the oesophagus to gaurantee that the required dose was given.
Blood samples were taken via the catheter. On each occasion the first 2 mL of blood was discarded as this removed any blood or flush fluid that remained in the extension set. A blood sample was taken from all the sheep five minutes prior to administration of any treatment. This sample provided a base-line value. Serial blood samples were then taken at
1, 2, 3, 5, 10, 15, 20, 30, 40, 60 minutes and 2, 4, 6, 8, 12 and 24 hours following treatment.
The catheter and extension set were flushed using heparinised saline following each sample.
Blood was transferred to a plain vacutainer and then centrifuged at 2000rpm for 15 minutes. Serum was decanted off and stored in cryovials at -80 C until analysis.
Sample analysis All serum samples were analysed using High Performance liquid Chromatography with
Tandem Mass Spectrometry as described in Chapter 6.
Statistical analysis Plasma OTC concentrations over a 24 hour period for animals in each treatment were examined graphically. Average OTC values for each treatment at each time of measurement were estimated using split plot analysis of variance and these values were used to calculate the total plasma OTC over a 24-hour period (the area under the concentration vs. time curve). OTC bioavailability for IF and IW treatments were calculated by comparing their total
110 plasma OTC over a 24 hour period to the same value for the IV treatment, Equation 1 describes this calculation.
Equation 1: Calculation of bioavailability
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8.3 Results
Oxytetracycline was detected in the plasma of sheep in all three treatment groups. Plasma concentrations peaked at a mean of 415.57 (+/- 51.3) µg/mL at 1 minute post-injection following IV administration. Following this peak, plasma concentrations steadily declined
(Figure 8.1). In both the IF and IW treatments, plasma concentrations were not detected within the first 30 minutes of monitoring. In the IF group OTC was first detected in the plasma at 120 minutes, followed by peak concentration of 4.12 (+/- 0.03) µg/mL at 6 hours
(Figure 8.2). In the IW group OTC was first detected in plasma at 30 minutes post-treatment.
Peak plasma concentrations of 5.34 (+/-0.04) µg/mL OTC were seen in the IW group at 12 hours (Figure 8.3).
Figure 8.1 Plasma OTC concentrations over time following IV treatment, error bars show standard error
700.00
600.00
500.00
400.00
300.00
200.00
100.00
Plasma OTC concentrations (µg/mL) concentrations OTC Plasma 0.00 -5 1 2 3 5 10 15 20 30 40 60 120 240 360 480 720 1440
Time (mins)
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Figure 8.2 Plasma OTC concentrations over time following medication with in-feed (IF) OTC with error bars showing standard error.
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Plasma OTC OTC Plasma concentration(µg/mL) 0.00 -5 1 2 3 5 10 15 20 30 40 60 120 240 360 480 720 1440 Time (mins)
Figure 8.3 Plasma OTC concentrations over time following medication with in-water (IW) OTC. Error bars show standard error.
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00 Plasma OTC concentration (µg/mL) concentration OTC Plasma
0.00 -5 1 2 3 5 10 15 20 30 40 60 120 240 360 480 720 1440
Time (mins)
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The half-life of OTC for the IV group was 3.96 hours, IF group 279.9 hours and IW group 51.5 hours.
The oral bioavailability of OTC in the IF treatment was 18% and the bioavailability of OTC in the IW treatment 27%.
8.4 Discussion
Oral bioavailability is defined as the fraction of the drug that reaches the systemic system unchanged following oral administration (Rang et al., 2008). This measurement is considered to be of greater clinical importance than the rate of drug absorption (Baggot and
Giguere, 2013). Oxytetracycline was shown to be absorbed and detectable in plasma following administration IV and both IF and IW. IW medication was more bioavailable than
IF (27% vs 18%).
Boluses of drug given IV are instantly available in the body and require no absorptive process. This results in the rapid achievement of maximum plasma concentrations. Al-
Nazawi (2003) found that a peak plasma concentration of OTC of 850 µg/mL was reached at two minutes post-injection of 5 mg/kg OTC. Similar results were found in this experiment where a dose of 8 mg/kg was given IV. A dose of 20 mg/kg tetracycline given IV has been shown to achieve a peak plasma concentration of 200 µg/mL (Rajaian, 2007). Despite being a comparison between OTC and tetracycline, there is a considerable difference in the peak plasma concentrations reported by Rajaian (2007) and Al-Nazawi (2003). Rajaian (2007) analysed samples by spectrofluorimetric analysis whereas Al-Nazawi (2003) analysed samples by fluorescence. Spectrofluorimetric analysis is considered to be less sensitive in detection of OTC or tetracycline than fluorescence (Hayes Jr and DuBuy, 1964). This
114 difference in sensitivity would, most likely, explain the difference in peak plasma concentrations.
A number of factors affect the amount of active drug in the plasma following oral administration. In humans approximately 60-80% of OTC is absorbed following oral administration (Chambers, 2006; Rang et al., 2007a). In humans OTC is absorbed primarily from the stomach and the proximal small intestine (Chambers, 2006). Any alteration in gut motility will impair absorption of the drug (Rang et al., 2007a). Oxytetracycline is known to be chelated by a number of ions that can be found in the feed. Calcium ions are the most common chelating agents (Rang et al., 2007a), but magnesium, aluminium, iron or zinc can also bind OTC (Chambers, 2006). Once chelated, OTC is no longer available for immediate absorption and therefore the rate of absorption will be prolonged. It is recommended that tetracyclines are given orally to calves or lambs in milk. Given the high levels of calcium in milk it is not surprising that this leads to a poorer absorption than if they were given in water (Neuschl, 2000). Neuschl (2000) conducted a study comparing chlortetracycline (CTC) administered in milk and in water. The oral bioavailability of CTC was 33% lower in those sheep dosed with milk than with water, although the author did not state the exact bioavailability. In the same study, the maximum concentrations of CTC in plasma were lower in medicated milk-fed animals compared to those that received medicated water, 1.72
µg/mL and 2.2 µg/mL respectively. The half-life of CTC was also longer in the medicated milk group compared to the medicated water group, 8.8 hours and 8.6 hours respectively. This longer half-life implies that there is a delay in absorption from the gut (Neuschl, 2000) caused by the chelation of CTC with milk calcium.
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Chlortetracycline is considered to be the most poorly and inconsistently absorbed of the tetracycline group (Neuschl, 2000). By comparing the maximum plasma concentrations obtained in this experiment, it is concluded that a larger concentration of OTC is absorbed compared to CTC. Additionally the concentrations of OTC in plasma after IF and IW administration remained above the MIC for sensitive organisms, reported as 0.5 µg/mL
(Neuschl, 2000). Oral dosing with tetracycline in milk can achieve effective plasma concentrations (Luthman et al., 1989). Luthman et al. (1989) found that the maximum concentration was reached in plasma four hours after administration and that levels were maintained above 1µg/mL with a dose of 25 mg/kg.
Drugs are absorbed from the gastro-intestinal tract through the mucosa. Following absorption they are taken via the circulation to the liver and then distributed throughout the body. Even after absorption there is the risk that the drug can be inactivated by enzymatic activity in the wall of the gastrointestinal mucosa or liver (Rowland and Tozer,
1995a; Rang et al., 2007a) making less active drug available. In mono-gastrics, splanchnic blood flow increases after eating, and this improves circulation between the gut and the liver, which in turn can increase drug availability (Rang et al., 2007a). In order to achieve maximal absorption, it is best for the drug to be in an area with a large area of permeable mucosal surface (Rowland and Tozer, 1995a). This is found in the small intestine. To achieve maximal absorption it is desirable for the drug to reach this area as quickly as possible. In humans this occurs within 2-4 hours. If maximal absorption does not occur within this time then there is a greater chance of drug de-activation and therefore less active drug available for absorption (Rowland and Tozer, 1995a).
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In ruminants, any oral dose must pass through the reticulum, rumen, omasum and finally abomasum before reaching the small intestine. Transit times in the ruminant are far greater than in the mono-gastric owing to the retention of material in the rumen. For feeds to pass through the entire gastro-intestinal tract in a ruminant the time taken can vary from 30-50 hours for highly digestible feeds, such as concentrates, and up to 50-80 hours for more fibrous feeds (Fuller, 2004). Fluid, however, will pass through quicker than any particulate matter. The reported liquid half-life in a ruminant varies in the literature ranging from 15-20 hours (Herdt, 2002; Toutain et al., 2010). Water will only leave the rumen as it is replaced.
Replacement fluid comes only via the oesophagus from either saliva, drinking water or succulent feeds (Herdt, 2002). As concentrate feeds are not succulent and do little to stimulate the rumen, leading to a reduction in saliva production, there is potential for prolonged liquid transit times in the rumen as fluid is not being replaced (Herdt, 2002). The same is true with animals that have reduced feed intakes, whether that be due to shy feeding, concurrent disease or other reasons. If the drug becomes bound to fibrous material with high cellulose contents, the time taken to pass through the gastro-intestinal tract can be 50-60 hours, this being adequate time to break down the cellulose (Toutain et al., 2010).
Although absorption is considered to occur only within the abomasum and the small intestine, the results of this experiment indicate some absorption must occur across the rumen mucosa. Oxytetracycline was first detected in plasma at 30 minutes for the IW group and two hours for the IF group. Given that the transit time for liquid through the rumen is considered to be from 6-20 hours it is unlikely that these plasma concentrations would be the result of absorption from more caudal parts of the gastro-intestinal tract. The rumen is lined by a keratinised stratified squamous epithelium providing a small surface area for
117 absorption (McKellar, 1994). Absorption of OTC occurs by passive transfer across the mucosal membrane (Rang et al., 2007a) until an equilibrium is reached.
The reticulo-rumen volume can be estimated by the following equation (National Research
Council, 2007)
Volume of reticulo-rumen (L) = 0.77 x bwt (kg)0.57-3.49
As an example, for a 50kg sheep the reticulo-rumen is likely to have a volume of 4.5 L. The volume of the reticulo-rumen in sheep is generally within the range of 3-15 L (Dehority,
2004). Given this large volume it is likely that a low concentration gradient will be present.
This results in slow absorption of OTC across the rumen mucosa (McKellar, 1994).
Lipid soluble drugs will easily cross the rumen mucosa (McKellar, 1994). Tetracyclines have varying lipophicity with Doxycycline being the most lipophillic, and OTC slightly less lipophilic
(Ziv and Sulman, 1974; Boothe, 2001a). Ionisation of a drug also affects its ability to cross the gastrointestinal mucosa. Tetracyclines are ionised at all pHs so they cannot easily cross this membrane (Ziv and Sulman, 1974). It has been suggested that ionised drugs will form bonds with a material in the gastro-intestinal tract to reduce their polarity (Ziv and Sulman,
1974). Ziv (1974) also postulated that ionised lipophillic drugs could be absorbed by passing through a small number of large pores within the mucosal wall.
This experiment supports the hypothesis that OTC is absorbed through the rumen mucosa, despite being of relatively low lipophillicity and being ionised.
The bioavailability of a drug depends on the characteristics of the preparation. For example oxytetracycline hydrochloride is more readily absorbed than oxytetracycline dihydrate
(Baggot, 2008). Bioavailability also depends on the individual animal. Between animals there
118 will be a variation in enzyme activity, intestinal motility and pH of the gastrointestinal tract
(Rang et al., 2007a) all of which can have an influence on bioavailability. Therefore, oral bioavailability can vary between animals. This is particularly important in animals that have gastrointestinal disease or conditions affecting the circulatory system (Rang et al., 2007a).
Disease status of the animal can also impact on the oral bioavailability. In pigs, ill animals were found to have a higher oral bioavailability of tetracyclines (Pijpers et al., 1991). It is thought that this is due to a decreased gastric function which increased the overall time available for absorption of the drug (Pijpers et al., 1991). The decreased gastric function resulted in the time to reach maximum plasma concentrations to be increased from 1.7 hours in healthy pigs to 7 hours in ill pigs.
Droumev (1992) studied the oral bioavailability of tetracycline in sheep using a slow release intra-ruminal bolus. The oral bioavailability was found to be 12-13%. This value is lower than the values calculated for either IF or IW in this experiment. Droumev (1992) attributed the low bioavailability to the design of the bolus, which ensured that it remained in the reticulo- rumen, and this caused the released drug to be greatly diluted. In addition to the anatomical location it was considered that additional gastro-intestinal factors had an influence: attachment of the drug to food particles; chelation; and disintegration of the drug in the rumen microflora (Droumev et al., 1992).
In this experiment, incorporating OTC in water or in rapidly digestible pellets allowed the drug to be available for absorption more quickly than through a slow release mechanism.
This minimized the risk of denaturing the OTC and increased its bioavailablity.
The time of peak plasma concentration varied between the IV group and the IF and IW groups. When a drug is given by an extra-vascular route it has to rely on absorption into the
119 vascular system which accounts for the delay in achieving plasma concentrations (Rowland and Tozer, 1995b). The peak plasma concentration represents the point at which elimination of the drug exceeds absorption resulting in decreasing concentrations of the drug (Rowland and Tozer, 1995b). Given the number of factors which can impact absorption of OTC following oral administration it is expected that the peak plasma concentration will be lower than following IV administration. In contrast, the plasma concentration of oral OTC was found to remain higher than that of IV OTC over time. This relates to the slow absorption of drug from the gastrointestinal tract over time which offsets the rate of elimination to a degree (Rowland and Tozer, 1995b). The same is true for any dosage regime where the drug is given extra-vascularly, e.g. intra-muscular injection. Despite a delay in reaching peak plasma concentrations, both the IF and IW groups maintained plasma concentrations above the MIC for 22 hours.
With some drugs the peak plasma concentration can be increased by increasing the dose of the drug, but this would be unlikely to affect the time to peak concentration. However, tetracyclines are incompletely absorbed from the gastrointestinal tract and the remaining drug is eliminated unchanged (Chambers, 2006). This remaining active drug has the potential to be active against gastrointestinal microflora. In humans this can result in overgrowths of yeasts, Pseudomonas spp. and Enterococci spp. This is typically only seen following long-term use of the drugs (Chambers, 2006). By increasing the dose, it is likely that more active drug will be left in the gastrointestinal tract resulting in a higher risk of negative effects on the microflora. Droumev (1992) found that giving 60-70 mg/kg of tetracycline orally resulted in a maximum plasma concentration of 6.6 µg/mL. This dose is three times higher than what was used in this experiment, yet the maximum concentration
120 is only marginally higher than the IF and IW plasma concentration. It would appear that significantly increasing the dose administered of oral OTC has little effect on the maximum plasma concentration and given the risk to the gastro-intestinal microflora it would not be advisable.
The degree of distribution throughout the body that follows absorption impacts on the efficacy of an antimicrobial (Al-Nazawi, 2003). The tetracyclines are generally widely distributed (Chambers, 2006; Papich and Riviere, 2009). The volume of distribution of a drug is defined as the volume of fluid required to hold the amount of drug within the body at the plasma concentration (Rang et al., 2008) Al-Nazawi (2003) found that the volume of distribution in sheep was 13.4 L/kg. This is greater than the volume of the sheep’s body water which is an indication of excellent penetration of the drug throughout all the body tissues (Al-Nazawi, 2003). Volumes of distribution greater than 1 L/kg show that the drug is distributed outside of the extracellular fluid. This indicates penetration into intracellular spaces or binding to tissue sites (Papich and Riviere, 2009). A wide volume of distribution will maximise the potential of OTC reaching the ocular tissue where it is required in IOK cases. There is reported to be an age-related change in the volume of distribution. The volume of distribution is higher in young animals and decreases with age because of decreasing extracellular and total body fluid and the increased volume of the gastrointestinal tract (Nouws et al., 1983). Given the wide age range amongst sheep at the feedlot, there may be a varying efficacy of OTC treatment.
All tetracyclines are protein bound within plasma. Protein binding of drugs limits the availability of active forms in the plasma (Papich and Riviere, 2009) as it is the unbound form which is active (Rang et al., 2007a). Oxytetracycline is the least protein bound of the
121 tetracyclines (Ziv and Sulman, 1974). In sheep 21-25% of OTC is protein bound (Papich and
Riviere, 2009).
To ensure that a steady state of plasma concentration is maintained it is important to have an understanding of the elimination characteristics of the drug. The tetracyclines are eliminated from the body primarily through glomerular filtration in the kidneys but also through the liver in the bile to the gastrointestinal tract (Short, 1994; Chambers, 2006). In the liver OTC is conjugated with glucoronides which makes them hydrophilic (Rang et al.,
2007a). This conjugated OTC is excreted back into the intestines via the bile ducts. The bacteria in the small intestine produce a high concentration of ß-glucuronidase enzymes which can break down these glucoronide-OTC conjugates, thus releasing OTC (Gibson and
Skett, 2001b). The hydrolysed OTC is then available to be re-absorbed back into the circulation whereas the OTC which is not hydrolysed in this way is excreted from the body in the faeces (Gibson and Skett, 2001b). This reabsorption is known as enterohepatic circulation (Chambers, 2006) and can increase the duration of action of the drug (Gibson and Skett, 2001b) because more drug becomes available for absorption following the initial absorption.
The half-life of the drug is the determining factor in the duration of action. The half-life is defined as the time taken for the concentration of drug in plasma to reduce by 50% (Rang et al., 2007b). The longer the half life, the longer the duration of action of a drug (Rang et al.,
2007b). In humans the half-life of tetracyclines varies between 6-12 hours (Chambers,
2006). In sheep following intra-venous administration of OTC the half-life is 3.2 hours (Al-
Nazawi, 2003), which is similar to the 3.96 hours found in this experiment. Half-life determines the dosing frequency of a drug. If the half life is long, then to maintain steady
122 plasma concentrations, the frequency of administration can be reduced (Gibson and Skett,
2001a). With antimicrobial therapy it is important to maintain a steady therapeutic level to minimise the risk of under-dosing, which can lead to development of resistance. To maintain this the dosage frequency should be equal to or less than the half-life (Gibson and Skett,
2001a). However, other factors can impact on the plasma concentration achieved, mainly the absorption of the drug and then the subsequent distribution of the drug. As expected because of the slower absorption of the drug, half-life for OTC following oral administration is much longer. Available data from this experiment showed that the half-life of the IW group was 51.5 hours and of the IF group 279.94 hrs. These values cannot be guaranteed given the limited data available; further samples beyond 24 hours would be required to confirm these values. Equally the length of half-life is only part of the requirements for the drug to be effective, time above the Minimum Inhibitary Concentration is a great importance also.
The oral bioavailability varied between the IF and the IW group. Many of the factors described can be used to explain this. Medication incorporated into a feed pellet requires breakdown of that pellet before it can be available for absorption. This is in contrast to the
IW group where the drug is dissolved in the water and immediately available for absorption.
Increasing the time within the gastro-intestinal tract results in less drug being available for absorption, and therefore a lower oral bioavailablity.
One limitation of the experiment is that in both the IF and IW groups the drug was administered over a short period of time which would not reflect what would happen in sheep fed a medicated pellet on an ad lob ration or medicated water where small quantities would be consumed throughout the day altering the drug pharmacokinetics.
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8.5 Conclusions
Oxytetracycline was detectable in plasma following both intravenous and oral administration. Plasma concentrations were relatively low following oral administration.
This may limit their use in a clinical context if the concentrations achieved are lower than the MIC of the target bacteria. Despite this concern, initial impressions from the work described in Chapters 5 and 6 indicate a degree of clinical effect following dosing with OTC in the drinking water. Further work is required to establish a concentration that is well enough tolerated in water for adequate water intake to be maintained during treatment.
Given the percentage of drug that is not absorbed and therefore theoretically remaining active within the gastrointestinal tract, it is important to determine what, if any, effect this active drug would have on the rumen microflora. Since palatability of medicated water was considered a limiting factor in intake, this could be a limiting factor in delivering an effective dose via the oral route in sheep, given the pharmacokinetic data discussed in this chapter.
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9 The impact of oral oxytetracycline on rumen health
9.1 Introduction
As with all herbivorous animals, ruminants rely on micro-organisms for their general health
(Doetsch and Robinson, 1953) and for the digestion of plant constituents (Bell et al., 1951;
Hobson, 1969; Cunha et al., 2011). The rumen houses a complex microbial environment.
The ruminant and the microbial population within the rumen are reciprocally beneficial
(Hungate, 1966a). The rumen continually mixes incoming feed with micro-organisms and saliva secreted by the host through frequent rumen contractions. In addition to mixing, the contractions enable movement of organisms and digesta from the rumen into the omasum and abomasum (Hobson, 1969). The ruminant supplies feed which the microbial population break down into Volatile Fatty Acids (VFAs) and gases. The VFAs are absorbed and used by the ruminant to provide energy (Hungate, 1966a; Kamra, 2005). In forage fed ruminants, saliva serves to maintain a neutral pH within the rumen, thus maintaining a stable microbial population (Hobson, 1969). Gases produced, mainly carbon dioxide and methane, sit in the gas layer in the dorsal rumen prior to being excreted (Hobson, 1969).
Early research highlighted the role of bacteria in the fermentation of plant material
(Pasteur, 1863) revealing that this bacterial digestion could generate products that could be used by the ruminant host (Zuntz, 1879). Given that ruminants typically consume a diet high in plant material it is important that they can digest the cellulose in the plant matter (Lodge et al., 1956). Mammals do not produce the cellulase enzyme required for the breakdown of cellulose (Doetsch and Robinson, 1953). It has long been known that microbes in the rumen can digest cellulose (Hofmeister, 1881) through the production of cellulase (Doetsch and
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Robinson, 1953). Since these early researchers, many others have looked at the complex microbial population within the rumen and what effects diet can have.
Most of the bacteria within the rumen microbial population are obligate anaerobes
(Hungate, 1966b; Flint, 1997; Kamra, 2005). The rumen maintains an anaerobic environment to support these organisms (Hungate, 1966b; Dehority, 2004). For optimal function of the rumen microbes the pH should be maintained between 6.0-7.0 (Hungate, 1966b; Dehority,
2004; Kamra, 2005) and at a temperature of 39-40 oC (Hungate, 1966b; Dehority, 2004;
Kamra, 2005).
The rumen microbial ecosystem exists in a fairly constant state maintained through the frequent addition of food material and removal of products of digestion (Hungate, 1966b).
The ecosystem is made up of 1010-1011 cells/mL of bacteria, 104-106 cells/mL ciliate protozoa, 103-105 zoospores/mL of anaerobic fungi and 108-109 cells/mL bacteriophages
(Kamra, 2005). The different groups work together, both synergistically and antagonistically.
Some microbes depend on others for the supply of nutrients (Hobson, 1969) whereas some are antagonised by compounds released by others (Kamra, 2005). This complexity of microbes in the rumen is required to enable digestion of the wide variety of components of the diet (Hungate, 1966b). A variety of microbes also allows for maximum biocatalytic capacity within the rumen as multiple bacteria will provide multiple pathways for breakdown of feedstuffs, thus maximising the amount of energy generated (Hungate,
1966b).
The fungi found in the rumen are obligate anaerobes and are involved in the breakdown of fibre (Kamra, 2005). It is estimated that fungi make up 10% of the rumen micro-organism
126 population (Chaucheyras-Durand and Ossa, 2014). Given the low fibre content, lower pH and higher transit times seen with pelleted diets, growth of fungi is limited (Kamra, 2005).
Bacteriophages are present in the rumen and are specific to certain bacteria (Kamra, 2005).
They are responsible for turnover of bacteria; lysis of bacteria releases protein which can be useful to the ruminant (Kamra, 2005).
More than 200 species of bacteria have been identified using culture-based techniques
(Chaucheyras-Durand and Ossa, 2014). With the use of PCR techniques many more organisms have been identified (Chaucheyras-Durand and Ossa, 2014). Using 16s ribosomal rRNA sequences approximately 7000 bacterial species have been identified within the rumen micro-organism population (Chaucheyras-Durand and Ossa, 2014). Most bacteria in the rumen are cocci and short rods measuring 0.4-1 µ in diameter and 1-3 µ in length
(Hungate, 1966b). The nature of the bacteria varies depending on the diet given. Hungate
(1966b) found that in ruminants on a high fibre diet of hay and forage the bacteria were mainly gram negative, whereas those in ruminants on a low fibre diet high in concentrates were mainly gram positive. High concentrate diets lead to an increased proportion of
Lactobacilli sp. which are better suited to the more acidic conditions in the rumen associated with concentrate feeding (Hungate, 1966b). The diversity of rumen micro- organisms is greater in animals fed forage based diets than in those on concentrate-based diets (Fernando et al., 2010). The most common phyla of bacteria are Bacteroidetes,
Firmicutes (Cunha et al., 2011; Castro-Carrera et al., 2014) and Proteobacteria
(Chaucheyras-Durand and Ossa, 2014). The primary functions of bacteria within the rumen are the breakdown of cellulose, production of fatty acids and the synthesis of proteins and vitamins (Doetsch and Robinson, 1953).
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The microbial population of the rumen is susceptible to daily fluctuations in the environment, micro-environment of the rumen and the physiology of the animal (Ziemer et al., 2000; Chaucheyras-Durand and Ossa, 2014). Diet is a key factor in changing the micro- environment of the rumen (Hungate, 1966b). Many of the organisms within the rumen are sensitive to pH, particularly a reduction in pH (Flint, 1997). Concentrate diets are known to reduce pH (Flint, 1997) and potentially create an unstable microbial population (Lee et al.,
1982). Lee (1982) found that the instability of the microbial environment was due to increased bacterial activity resulting in an accumulation of acid. Feeding of roughage with the concentrate can help to stabilise the micro-environment (Lee et al., 1982). Ziemer
(2000) considered that although diet changes will alter the microbial population there is little evidence to show that the whole microbial community is affected.
Although there is a wide variety in microbial population it is difficult to quantify the exact role played by any of the groups of microbes (Doetsch and Robinson, 1953; Kamra, 2005).
The earliest recorded attempts at culturing rumen bacteria were in 1884 (Tappeiner, 1884).
No successful attempts to isolate rumen bacteria occurred until the late 1940s (Hobson,
1969). The earliest success at culturing rumen bacteria was in 1947 when some were distinguished but full characterisation of the bacteria was not completed (Gall et al., 1947).
The difficulty in culturing rumen bacteria lies with provision of suitable conditions mimicking the rumen environment and the supply of primary nutrients normally provided by other bacteria within the rumen (Hobson, 1969). Only a small percentage of the rumen microbial population can be identified by culture (Tajima et al., 1999), estimated to be 20%
(Chaucheyras-Durand and Ossa, 2014). Reliance on culture-based techniques alone will severely underestimate microbial diversity within the rumen (Fernando et al., 2010). Most
128 rumen bacteria are similar in size and shape and therefore indistinguishable under a microscope (Hungate, 1966b). Prevotella spp. and Butyrivibrio spp. appear to be the most common rumen bacteria to be cultured (Flint, 1997).
Advances in molecular techniques have allowed a greater understanding of the genetic diversity of the microbial population (Tajima et al., 1999). Most of the rumen microbes are found in the fibrous layer of the rumen (Brosh et al., 1983) and are therefore difficult to identify by culture. Molecular techniques enable identification of organisms attached to plant material within the fibrous layer as their nucleic acid can still be recovered (Ziemer et al., 2000). The use of rRNA-based techniques is essential to advance knowledge and understanding of the rumen microbial population (Flint, 1997). Using such techniques allows for a direct comparison to be made of the rumen microbial population before and after an alteration to either the rumen environment or diet (Ziemer et al., 2000).
Given the importance of a stable rumen microbial population to the general health and productivity of ruminants, it is essential to fully determine the effect of oral antibiotic medication on the rumen microbial population. Much has been done to determine the suitability of antibiotics for inclusion in rations for the purposes of growth promotion
(Bartley et al., 1951; Neumann et al., 1951; Munch-Petersen and Armstrong, 1958;
Klopfenstein et al., 1964). Research has also been done to assess the impact of antibiotics given orally on milk production (Rusoff et al., 1952). Most work to date has been done looking at the use of aureomycin (chlortetracycline). Klopfenstein (1964) found that including aureomycin resulted in increased weight gains and faster growth. Neumann
(1951), however, found no difference in growth with the inclusion of aureomycin.
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Aureomycin has been found to alter the rumen microflora when given orally (Bartley et al.,
1951; Neumann et al., 1951; Horn et al., 1955; Munch-Petersen and Armstrong, 1958;
Klopfenstein et al., 1964; Purser et al., 1965). Neumann (1951) and Purser (1965) found that total bacterial counts were unchanged by inclusion of antibiotics, but the diversity of bacteria was different. In contrast Bartley (1951) found no difference in direct microscopic examination of the rumen microflora between those treated and those untreated. The addition of OTC has been found to change the microbial population, but as no cultures were obtained the true nature of the change could not be identified (Munch-Petersen and
Armstrong, 1958). Early research focused more on the effects of antibiotics on digestibility as opposed to directly on the organisms (Bell et al., 1951; Horn et al., 1955).
This experiment was designed to determine the effects of OTC on the rumen microflora.
Given the paucity of reports in the literature relating to the effects of OTC it was an important experiment to carry out. As ruminants rely heavily on a stable and functional microbial population for the digestion of feedstuffs and maintenance of general health, it is important to assess any effects on this associated with OTC before recommending its use.
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9.2 Materials and Methods
Twelve mixed-age Merino-cross wethers with permanent rumen fistulas in place were used in this experiment. The wethers were of varying ages and sizes, so attempts were made to put equal numbers of old and young animals in each group to minimise overall differences in weight. The sheep had previously been fistulated using a standard two-stage process
(Dougherty, 1955; Saeed et al., 2007). The fistula was located in the left paralumbar fossa and enabled direct access to the rumen. All sheep were housed in a purpose-built animal house at CSIRO Floreat, WA. Sheep were housed in individual pens and contact between sheep through pens was allowed. An acclimatisation period of one week was allowed prior to commencing the experiment for sheep to adapt to the housing and pellet ration. Sheep were weighed and body conditions were measured (Russel et al., 1969) by the same operator three times: on entry to the animal house, once during the experiment and again at the end of the experiment. Sheep were split into two treatment groups and to balance the treatments each group consisted of three older and three younger wethers. Individual feed and water intake and faecal scores were measured on a daily basis throughout the experiment. Faecal scores were measured as a visual assessment of faecal consistency using a 1-5 scale, with 1 being firm pellets and 5 being diarrhoea (Le Jambre et al., 2007), Table
9.1, and were conducted by the same person each time.
Faecal Score Description 1 Firm, well formed pellet 2 Soft, pellet form 3 Soft faeces, loss of pellet structure 4 Paste like faeces 5 Watery, liquid diarrhoea Table 9.1: Faecal scoring system (Le Jambre et al., 2007)
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Alternative feedstuffs (chaff) were offered to sheep to mitigate the decrease in feed intake when it occurred during the experiment.
Treatments Both treatment groups were treated for five days with water soluble Oxytetracycline (OTC)
(CCD OTC, CCD Animal Health, Geelong VIC, Australia). Treatment group 1 was given
22mg/kg liveweight of OTC and Treatment group 2 was given 11mg/kg liveweight OTC. OTC powder was diluted in water and administered directly into the rumen via the rumen fistula each day for 5 consecutive days.
Rumen fluid samples Rumen fluid samples were taken on days -5, -3, -1, 0, 1, 2, 3, 4, 5, 6, 7, 9, 11, 14, 16 and 18.
On day 0 a rumen fluid sample was taken immediately prior to administration of medication and a follow up sample was taken 1 hour after medication. Rumen fluid samples were taken by inserting a rigid plastic tube through the fistula and removing fluid by capillary action.
Samples were placed on ice after collection. Samples taken prior to commencement of treatment were used as controls.
Rumen pH of the samples was measured immediately on collection using an electronic pH meter, which was calibrated daily. Rumen fluid samples were frozen (-20 oC)and analysed later for Volatile Fatty Acid (VFA) and Ammonia concentrations. Separate samples were taken immediately prior to drug administration on day 0 and on day 5. These were frozen at
-80oC and sent for microbial profiling.
Rumen microbial profiling The microbial profiling methodology used to investigate rumen bacterial, archaeal, fungal and protozoan communities was based on the terminal restriction fragment length polymorphism (T-RFLP) technique. T-RFLP is a culture-independent technique for profiling
132 microbial communities based on differences at the nucleic acid or genome level (Torok et al., 2008). This tool has been used to investigate changes in gut bacterial communities associated with dietary modification, such as addition of feed enzymes, prebiotics, organic acids and antimicrobials in the poultry industry (Geier et al., 2009; Geier et al., 2010; Torok et al., 2011). The advantage of the technique is that it is high-throughput, high resolution and capable of providing a “snap shot” of the entire microbial community at any particular time. Hence, it is an ideal initial screening tool that requires no prior knowledge of the actual microorganisms present within the community. This method was deemed appropriate for the purposes of this experiment, namely to assess the impact of oral OTC on rumen microflora.
Where significant treatment differences are detected other molecular techniques can be used to identify organisms involved. In this experiment, the organisms identified as being affected by OTC treatment were further characterised by pyrosequencing technology.
Pyrosequencing is a method of DNA sequencing based on the sequencing by synthesis principle. Pyrosequencing can be used to identify the microbial genome sequence and assign phylogenetic information to the population. In this experiment taxonomic assignment was done using the ribosomal database project (RDP), which assigned taxonomy to family level.
Methodolgy for T-RFLP Total nucleic acid was extracted from the 24 freeze dried samples, representing the pre and post treatment sample from both Treatment groups, using a South Australian Research and
Development Institute (SARDI) proprietary method (Torok et al., 2008). The bacterial ribosomal DNA from the extracted material was amplified with universal 16S bacterial
133 primers, one of which was labelled with fluorescent dye. Archael, fungal and protozoan communities were also amplified using universal group specific primers. The resulting amplicons were restricted with a specific recognition sequence restriction enzymes and electrophoretically separated on a capillary DNA sequencer (ABI 3730, Applied Biosystems).
Data were analysed using GeneMapper (Applied Biosystems) to determine positions of terminal restriction fragments (TRF).
Methodolgy for Pyrosequencing Bacterial 454 pyrosequencing was done with primers 27F and 534R (Nossa et al., 2010). This primer pair amplified a 598 bp product from the most variable region of the 16S rRNA (V2-
V3). Bacterial DNA was amplified from total nucleic acid using the FastStart HiFi PCR system
(Roche Diagnostics). Each primer included sequences to facilitate the sequencing of products in the Roche/454 system. The forward PCR primer consisted of a related set of primers with different “barcode” sequences, which enabled the identification of individual samples. PCR amplicons were analysed for specificity by electrophoresis on a 2% agarose gel. PCR amplicons were purified using Agencourt® AMPure® XP (Beckman Coulter) and quantified according to the “Amplicon Library Preparation Methods Manual GS Junior
Titanium Series” protocol (Roche Diagnostics). Pooled samples (n=12 (controls)) were sequenced using the Roche/454 GS Junior Genome Sequencer and Titanium chemistry according to the manufacturer’s instructions (Roche Diagnostics).
The rumen bacterial communities from the control (n=12), Treatment group 1 (22 mg/kg)
(n=6) and Treatment group 2 (11 mg/kg) (n=6) were analysed. 16S rRNA sequences were assigned to operational taxonomic units (OTU) with 97 % within group similarity, a level approximately equivalent to a species designation. OTU tables were produced in Qiime v
1.4.0 (Caporaso et al., 2010) with standard defaults and taxonomic assignment determined
134 using the greengenes RDP classifier database with a cut-off threshold of 80%. OTU tables were filtered to remove the possibility of sequencing errors and chimeras, by eliminating
OTU with less than five sequences and present in less than two samples BLASTn with nucleotide collection (nr/nt) databases and the Megablast algorithim (National Center for
Biotechnology Information [NCBI]) were also used to identify similarity of OTU of interest to sequences available in public genome sequence databases (Altschul et al., 1990).
Blood samples Blood samples were collected from the jugular vein using a 20 gauge, 1” needle and a vacutainer tube, plain tube for OTC concentrations and lithium heparin for BHB analysis.
Blood samples were centrifuged at 2000 rpm for 15 minutes and the plasma titrated off.
Plasma was frozen and analysed at a later date. Blood samples collected on days -3, 0, 1, 2,
3, 4, 5, 7, 9, 11, 14, 16 and 18 were analysed for BHB concentrations. Blood samples were also collected on days 0, 1, 2, 3, 4, 5, 7, 9 and 11 for measurement of plasma OTC concentrations.
Statistical Analysis For all data except those associated with rumen microbe profiling the following analyses were carried out. For each animal all pre-treatment values were averaged and change from the pre-treatment average was calculated for each post-treatment date. A linear mixed model was fitted to the calculated changes on all post-treatment dates. The model included fixed effects for Treatment (11 vs 22), post-treatment date and their interaction; and random effects for animal and the animal by date interaction. An autoregressive model allowed for correlations between measurements made on the same animal on different dates. 5% LSD’s were calculated to compare Treatment means to zero, i.e. whether treatment caused a change from the pre-treatment mean, and to compare the two
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Treatments. For pellet and total ME intake the analysis has also been performed with water intake included in the fixed model, i.e. as a covariate.
For the rumen profiling analysis, data points from GeneMapper analysis were validated and outputs generated for statistical analysis using queries within a custom built database
(Torok et al., 2008). Queries in the database were used to compare duplicate T-RFLP profiles and identify synonymous fragment sizes (±2 bp). The resulting fragments were treated as
OTU, representing particular microbial species or taxonomically related groups. Operational taxonomic units (OTU) obtained from the 24 rumen samples were analysed using multivariate statistical techniques (PRIMER 6, PRIMER-E Ltd., Plymouth, UK). These analyses were used to examine similarities in sheep rumen microbial communities and to identify
OTU accounting for the differences observed in microbial communities (Torok et al., 2008).
Bray-Curtis measures of similarity (Bray and Curtis, 1957) were calculated to examine similarities between rumen microbial communities from the T-RFLP generated (OTU) data matrices, following standardization and fourth root transformation. The Bray-Curtis similarity co-efficient (Bray and Curtis, 1957) is a reliable measure for biological data on community structure and is not affected by joint absences that are commonly found in microbial data (Clarke, 1993). Analysis of similarity (ANOSIM) (Clarke, 1993) was used to test if rumen microbial communities were significantly different between treatments. This is a non-parametric method of ranked siddimilarities. The R-statistic value describes the extent of similarity between each pair in the ANOSIM analysis, with values close to unity indicating that the two groups are entirely separate and a zero value indicating that there is no difference between the groups.
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Similarity percentages (SIMPER) (Clarke, 1993) analyses were done to determine which OTU contributed most to the dissimilarity between treatments. The overall average dissimilarity
( ) between microbial communities of ruminants on two treatments shown to significantly differ were calculated and the average contribution of the ith OTU ( i ) to the overall dissimilarity determined. Average abundance ( y ) of important OTU in each of the groups was determined. OTU contributing significantly to the dissimilarity between treatments were calculated ( i/SD(δi)>1). Percent contribution of individual OTU ( i%) and cumulative percent contribution (Σ i%) to the top 65% of average dissimilarities were also calculated.
Unconstrained ordinations were done to graphically illustrate relationships between treatments using non-metric multidimensional scaling (nMDS) (Shepard, 1962a; Kruskal,
1964). nMDS ordinations attempt to place all samples in an arbitrary two-dimensional space such that their relative distances apart match the corresponding pair-wise similarities.
Hence, the closer two samples are in the ordination the more similar are their overall microbial communities. “Stress” values (Kruskal’s formula 1) reflect difficulty involved in compressing the sample relationship into the 2-D ordination.
For pyrosequencing analysis OTU obtained from the rumen of 24 samples were analysed using multivariate statistical techniques (PRIMER 6, PRIMER-E Ltd, Plymouth, UK). These analyses were used to examine similarities in sheep rumen bacterial communities and to identify OTU accounting for differences observed in bacterial community structure. Bray-
Curtis measures of similarity (Bray and Curtis, 1957) were calculated to examine similarities between ruminal bacterial communities from the pyrosequencing generated (OTU) data matrices, following standardization and fourth root transformation. Similarity percentages
(SIMPER) (Clarke, 1993) analyses were done to determine which OTU contributed most to
137 the dissimilarity between treatments. Bacteria OTU contributing significantly to the dissimilarity between treatments were calculated (Diss/SD>1). Unconstrained ordinations were done to graphically illustrate relationships between treatments using non-metric multi-dimensional scaling (nMDS) (Shepard, 1962b, a; Kruskal, 1964). nMDS ordinations were done to place all samples in an arbitrary two-dimensional space such that their relative distances apart match the corresponding pair-wise similarities. Hence, the closer two samples are in the ordination the more similar are their overall bacterial communities.
“Stress” values (Kruskal’s formula 1) reflect difficulty involved in compressing the sample relationship into the 2-D ordination.
9.3 Results
In all cases for each animal all pre-treatment values were averaged and change from the pre-treatment average was calculated for each post-treatment date. In Table 9.2 the level of significance for all the measurements taken is summarised.
Intake - kilo’s of Intake - Water Faecal rumen Plasma Plasma feed MJ ME intake score pH BHB OTC
P- P- P- P- Term P-value P-value value value P-value value value
Time <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Water <0.001 <0.001
Treatment 0.646 0.745 0.670 0.948 0.184 0.265 0.154
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Time.Treatment 0.455 0.164 0.019 0.858 0.140 0.122 0.258
Table 9.2: Significance table for change in measurements between pre- and post- treatment parameters.
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Bodyweight and body condition score The mean bodyweight of animals in the Treatment 1 group (11mg/kg OTC) was 72.7 kg prior to commencement of treatment compared to 65.3kg for animals in the Treatment 2 group
(22 mg/kg OTC). In both groups, bodyweight decreased up to day 3 before increasing again.
The mean weight loss in both Treatments was 2.8 kg. There was no significant difference between the Treatment groups.
Feed Intake Feed intake reduced over time following commencement of the treatment, although it returned to pre-treatment levels following cessation of treatment (Figure 9.1). There was no significant difference between the two Treatment groups. When water was fitted as a co- variant it was highly significant (p<0.001) suggesting a significant association between feed and water intake.
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Figure 9.1 Graph of mean feed intake over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.
1400
1200
1000
800
600 11 mg/kg OTC 400
22 mg/kg 200 OTC
mean grams mean (g)pellets of consumed 0 -10 -5 0 5 10 15 20 -200 days
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Metabolisable energy
The total mega-joules (MJ) of metabolisable energy (ME) were calculated (Figure 9.2) to account for the addition of chaff when intake dropped significantly. As with feed intake, energy intake decreased following the start of treatment and recovered after treatment finished. It appeared that sheep preferentially ate chaff over pellets during the treatment period. As with pellet intake, there was no significant difference between the Treatments over time.
Figure 9.2 Graph of mean ME intake over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.
20.00
18.00
16.00
14.00
12.00
10.00 11 mg/kg 8.00 OTC
6.00 Mean MJ ME consumed MEMJ Mean 4.00 22 mg/kg OTC 2.00
0.00 -10 -5 0 5 10 15 20
Days
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Water Intake Mean water intake fell significantly following the start of treatment (Figure 9.3). There was a significant difference in mean water intake over time and also a significant difference between the Treatments on days 4, 10, 14 and 15.
Figure 9.3 Graph of mean water intake over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.
4500
4000
3500
3000
2500
2000 11 mg/kg OTC 1500 22 mg/kg
1000 OTC mean litres of water consumed water of litres mean 500
0 -10 -5 0 5 10 15 20 Days
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Plasma Beta-Hydroxybutyrate (BHB) concentrations Mean BHB concentrations rose significantly following commencement of treatment.
Concentrations peaked at day 8 and returned to pre-treatment concentrations on approximately day 13 (Figure 9.4). There was no significant difference between Treatments over time.
Figure 9.4 Graph of mean plasma BHB concentrations over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors. Black line represents BHB concentration at which sheep are at risk of sub-clinical ketosis.
1.200
1.000
0.800
0.600 11 mg/kg OTC
0.400 22 mg/kg OTC
0.200 mean plasma plasma mean BHB concentrations (mmol/L
0.000 -5 0 5 10 15 20 days
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Faecal Score Faecal score changed significantly over time and increased significantly after the start of
OTC treatment (Figure 9.5). There was no significant difference between the Treatments over time.
Figure 9.5 Graph of mean faecal score over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.
5
4
3 11 mg/kg
Faecal score Faecal OTC
2
1 -10 -5 0 5 10 15 20 Days
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Rumen pH Rumen pH changed significantly over time and increased significantly after the start of OTC treatment (Figure 9.6). There was no difference between the Treatments over time.
Figure 9.6 Graph of mean rumen pH over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.
7.80
7.60
7.40
7.20
7.00 11 mg/kg 6.80 OTC 22 mg/kg
mean rumen pH rumen mean 6.60 OTC
6.40
6.20
6.00 -10 -5 0 5 10 15 20 days
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Plasma OTC concentrations OTC was detected in the blood of all animals during the OTC treatment period.
Concentrations peaked at day 5 and low levels were still detected on day 9, 4 days after treatment ceased (Figure 9.7). There was no significant difference in the concentration of
OTC in the plasma in the two treatment groups over time.
Figure 9.7 Graph of mean plasma OTC over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.
0.700
0.600
0.500
0.400
0.300 11mg/kg OTC
0.200 22 mg/kg OTC 0.100
mean plasma OTC concentration (µg/mL)concentration OTC plasma mean 0.000 0 2 4 6 8 10 days
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Rumen fluid parameters There was a significant effect of OTC treatment on rumen bacterial, archael and fungal populations, but no effect was seen on protozoal populations. Despite this inter-animal variability, a subjective judgement can be made for the bacterial, archaeal and fungal communities in the rumen communities of animals pre-treatment compared to after the start of OTC treatment. This difference relates to both presence/absence of unique OTU, as well as shifts in abundance of common OTU. The number of OTU shared among the animals post-treatment was reduced compared with the number observed pre-treatment, indicating a reduced biodiversity in micro-organisms within the rumen after OTC treatment commenced.
Multivariate statistical analyses were used to investigate differences in rumen bacterial, archaeal, fungal and protozoan communities associated with OTC treatment. Significant differences (P=0.001) associated with OTC treatment were detected in the rumen bacterial, archaeal and fungal communities (Table 9.3). OTC treatment did not influence the rumen protozoan communities (Table 9.3). Both Treatments altered the rumen bacterial, archaeal and fungal communities when compared with pre-treatment populations. However, it was only within the bacterial communities that there was also a significant difference associated with the OTC dose (Table 9.3). The influence of OTC dose on rumen bacterial communities is summarised in Figure 9.8.
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Bacteria (R=0.703, P=0.001) pre-treatment 11 OTCa 22 OTCb pre-treatment 0.774 0.816 11 OTC 0.001 0.141 22 OTC 0.001 0.050 Archaea (R=0.448, P=0.001) pre-treatment 11 OTC 22 OTC pre-treatment 0.618 0.616 11 OTC 0.001 0.009 22 OTC 0.001 0.366 Fungi (R=0.489, P=0.001) pre-treatment 11 OTC 22 OTC pre-treatment 0.434 0.689 11 OTC 0.004 0.083 22 OTC 0.002 0.177 Protozoa (R=0.063, P=0.203) * For each pairwise comparison the R value (Grubb et al.) and P value (italics) are indicated.
P < 0.05 is significant.a 11 OTC = Daily oral dose of 11 mg OTC per kg liveweightb 22 OTC =
Daily oral dose of 22 mg OTC per kg liveweight
Table 9.3 One-way ANOSIM of rumen microbial communities associated with OTC treatment. For each microbial group the influence of oral OTC treatment was investigated. Where significant differences in rumen microbiota were detected, the pairwise* differences between treatments were investigated further.
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Figure 9.8 nMDS of rumen bacterial communities associated with oral dose of OTC taken on day 5. Treatments are: 11mg OTC/kg LW/day () and 22mg OTC/kg LW/day (). Each point in the ordination shows the overall microbial profile of an individual animal. The closer two points are in the ordination the more similar are their profiles.
Pyrosequencing of bacterial communities generated 90396 reads with an average of 3700 reads per sample. 1631 OTU were detected once data had been filtered. Fifteen bacterial phyla as well as unclassified bacteria were identified in the rumen of the sheep (Figure 9.9).
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Figure 9.9 Abundance of rumen bacteria classified to level of phyla for individual control and OTC treated sheep. (Cont) untreated control sheep, (Low) treated with 11 mg OTC/kg live weight/day and (High) treated with 22 mg OTC/kg live weight/day.
151
The majority of sequences identified belonged to the Bacteroides (23.1%), Firmicutes
(41.3%), Proteobacteria (14.9%), Fibrobacteres (8.2%) and unclassified bacteria (8.5%).
Rumen bacteria could be further classified into 45 groups based on class, order or family
(Figure 9.10).
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Figure 9.10 Abundance of rumen bacteria classified, where possible, to level of family for individual control and OTC treated sheep. (Cont) control sheep, (Low) 11 mg OTC/kg live weight/day and (High) 22 mg OTC/kg live weight/day.
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Control samples had a more diverse rumen microbiota (6-14 bacterial phyla detected per sheep with an average of 11.1 phyla) compared to the post-treatment samples (3-7 bacterial phyla per sheep with an average of 4.7 phyla). Bacteroidetes were more abundant in the rumen of control sheep (46.1%) compared to treated sheep (0.1%). The differences in
Bacteroidetes were due to greater abundance of unclassified Bacteroidales (10.6%) and
Prevotellaceae (32.2%) in the control sheep than in the treated sheep (less than 0.1% of each). Control sheep also contained Fibrobacteres (16.3%) and Spirochaetes (2.6%), while these phyla contributed <0.01% to the rumen bacterial population in OTC treated sheep.
Control sheep contained 16.3% Fibrobacteraceae and 2.5% Spirochaetaceae, while the OTC treated sheep contained less than 0.1% each of these families. There was a greater abundance in OTC-treated sheep of Firmicutes (65.1%), Proteobacteria (19.4%) and unknown bacteria (14.9%) than in the control sheep (17.6%, 10.3% and 2.1% respectively).
The difference in Firmicutes was predominantly due to the Lachnospiraceae, which were more abundant in the treated sheep (55.1%) than in the control sheep (7.2%). The increased
Proteobacteria detected in treated sheep was due to increased abundance of
Enterobacteriaceae (19.0%) which accounted for <0.1% in control sheep. Although
Proteobacteria were less abundant in the controls, Aeromondales (8.6%), were more abundant in the controls which were absent from the treated sheep. The percentage contribution of bacterial taxa to overall community structure for individual sheep is shown in Appendix 3.
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9.4 Discussion
The two dosage regimes used in this experiment were chosen to assess the recommended dose (22 mg/kg) and half the recommended dose of OTC. The dose of 11 mg/kg OTC was used to determine the impact of a lower dose such that if a severe impact was seen with the higher dose recommendations could potentially be based on the lower dose.
Experimental work in Chapter 6 highlighted a decrease in both water and feed intake following inclusion of OTC medication in water. The results of this experiment also demonstrated negative effects of OTC on feed and water intakes. In Chapter 6 the reduced feed intake was attributed to a concurrent decrease in water consumption. In this experiment sheep were offered non-medicated water and the OTC was administered directly into the rumen via the fistula. This was done to ensure each sheep received the correct dose of OTC to allow a true assessment of the effect of the two doses on intake and rumen health.
It is postulated that the decrease in feed intake is directly related to the effects of the OTC on the rumen microflora. Analysis of the data from the rumen fluid showed that the
Treatments significantly changed the populations of rumen microbes. What is evident from these depictions is that the rumen microbiota were variable among animals on the same treatment and that there was a need for robust statistical analysis when investigating treatment differences. The two Treatments caused the populations to change in different ways such that the rumen microbe profile at the end of the experiment differed between
Treatments. This, however, did not lead to a difference between Treatments in any of the other variables, namely feed intake, energy intake, faecal score, BHB concentrations or
155 rumen pH. Pyrosequencing was done to speciate the microflora populations that were affected and the results are included as Appendix 3.
Bacteroidetes spp., Firmicutes spp. and Fibrobacter spp. are the most common genera of the rumen bacterial population (Fernando et al., 2010; Castro-Carrera et al., 2014) and these were the main genera impacted by the treatment administered.
Bacteroidetes spp. were reduced following treatment. Prevotellacae are the most common species of the phylum Bacteroidetes (Stewart et al., 1997). Prevotellacae are gram negative, anaerobic rods or coccibacilli which are found in the rumen in association with a wide variety of feedstuffs (Stewart et al., 1997). They have no action against cellulose but will degrade and utilise starch and are important in the degradation of protein (Stewart et al.,
1997). Fermentation products of Prevotellacae include proprionate, which is an important energy precursor in the ruminant. Members of the Prevotellacae genera have been shown to develop plasmids conferring resistance to tetracyclines (Flint and Stewart, 1987); however, given the reduction in population seen in this experiment the significance of this is unclear. Longer term monitoring of the rumen microflora population would be required to assess if this had the potential to be a significant concern.
Fibrobacter spp. were seen to reduce following treatment. Fibrobacter succinogenes is a common bacteria found in the rumen microflora and its main function is the breakdown of cellulose (Stewart et al., 1997). Interestingly F. succinogenes is considered to be resistant to many in-feed antibiotics such as Avoparcin (Stewart et al., 1997). Given that OTC is also effective against gram positive organisms it would be reasonable to expect no change in the population following treatment; however, the decrease would indicate that F. succinogenes is not resistant to OTC. Given the importance of Fibrobacter spp. in the initial breakdown of
156 cellulose, a reduction in their activity will limit further breakdown of cellulose by other organisms (Mackie et al., 2002) and consequently minimize effective digestion of feed.
Animals in both Treatment groups altered their body weight following the introduction of
OTC. This weight loss is probably attributable to loss of rumen mass as opposed to loss of muscle mass. This can be confirmed by only a mild increase in BHB concentrations that indicates a relatively low level of fat catabolism. None of the animals entered a clinically significant state of negative energy balance which is evident when the BHB level exceeds 1.5 mmol/L. Beta-hydroxy butyrate is produced in a number of ways including from the rumen microbes and through fat catabolism in the liver. Although BHB concentrations increased during treatment, they only briefly exceeded 0.8 mmol/L which is considered a safe value for the estimation of the nutritional status of a flock (O'Doherty and Crosby, 1998).
O’Doherty and Crosby (1998) found that ewes consuming half their ME requirements had
BHB concentrations of 1.18-1.25 mmol/L, which was in excess of values found in this experiment.
Not all bacterial genera were reduced following treatment. Firmicutes spp. increased.
Lachnospiraecae, of which Lachnospira multiparus is a member, were responsible for this increase. These bacteria further breakdown cellulose following initial digestion (Mackie et al., 2002). Proteobacteria populations also increased following treatment. There is some debate surrounding the importance of Proteobacteria in the rumen microflora (Kang et al.,
2013): originally they were thought to be of limited importance because of their relatively low numbers, but recently their metabolic importance has been considered more important than previously thought when judged on their numbers alone. (Kang et al., 2013).
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Despite the observed changes within the rumen microflora it is important to recognize the limitations of the pyrosequencing technique in identifying all changes present. The rumen functions through the interactions of bacteria, fungi and protozoa (Mackie et al., 2002).
Given that there were no significant changes seen in fungal or protozoal populations, and that the animals returned to normal feed intakes following cessation of treatment it is likely that any changes to bacterial populations were transient and rumen microbiota returned to normal in a relatively short time post-OTC treatment.
The decrease in feed intake appeared to be offset to a degree by the introduction of chaff.
Chaff was mixed through the pellet ration and sheep appeared to preferentially eat the chaff during the treatment period and for a number of days following treatment. Further work would be required to definitively confirm that feeding chaff during the treatment period would maintain feed and energy intake. The decrease in feed intake was only temporary, and intake levels returned to levels similar to those pre-treatment, 1 week post- treatment.
Rumen pH increased over time but the mean pH did not exceed that of a healthy sheep, which should be in the range of 6.5-7.5. Prior to introduction of OTC the pH was lower than
6.5. This is to be expected in sheep on a diet consisting of only pelleted feed (Briggs et al.,
1957; Mould and Orskov, 1983).
It is encouraging that OTC was absorbed following administration directly into the rumen.
Plasma concentrations of OTC were comparable to those in the previous experiment, even though the OTC dose was administered directly into the rumen as opposed to relying on water intake.
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There were no significant differences between the two different doses of OTC given in this experiment and it appears that the responses to soluble OTC were not dose-dependent.
There was a cumulative dose effect whereby peak plasma OTC concentrations were achieved after four days in the 22 mg/kg Treatment and after five days in the 11 mg/kg
Treatment. Plasma concentrations were lower than those found following intra-muscular injections of OTC, namely 0.94 µg/mL, (Chapter 6) which is to be expected given the lower bioavailability seen following oral administration.
9.5 Conclusions
Although some changes in rumen microflora were found in the Treatment groups, these changes were restricted to the bacterial populations with no significant changes seen in either the fungal or protozoal populations. As the rumen microflora work symbiotically it is likely that any change will be transient and the population will modify such that function is maintained. Despite changes in microflora, all other parameters indicative of rumen health showed no significant difference.
Feed and water intake were again reduced in this experiment, and this has the potential to be problematic within the live export chain. To date in-water medication has been assessed.
Work described in Chapter 8 indicated that OTC is absorbed following administration in feed, however the impact of administering OTC in-feed on feed and water intakes is unknown.
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10 The use of medicated feed to treat infectious ovine
keratoconjunctivitis
10.1 Introduction
Previous chapters have described several negative effects associated with in-water medication on the physiology and behaviour of the sheep. However, the positive clinical response of IOK to OTC treatment encouraged the researchers to consider alternative options. It was shown in Chapter 8 that OTC was absorbed when administered in feed.
A number of early studies have considered the impact of medicated feed on rumen function
(Bell et al., 1951; Neumann et al., 1951; Perry et al., 1954; Munch-Petersen and Armstrong,
1958). Both Neumann et al (1951) and Perry et al (1954) found that feed intake was transiently reduced for 2-4 days following introduction of feed medicated with aureomycin.
Bell et al (1951) reported a marked anorexia and severe diarrhoea associated with aureomycin medicated feed: it was postulated that this was not a palatability issue but rather was related to digestive disturbance. In contrast to these reports Munch-Petersen and Armstrong (1958) found that no change in appetite was seen following the introduction of medicated feed. Given the conflicting reports in the literature and the paucity of studies looking at OTC inclusion it was important to determine if there were any negative effects of in-feed medication with OTC.
With the negative effects on feed and water intakes seen with in-water OTC medication, a palatability experiment was conducted first to determine the effect of in-feed medication on feed intake.
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10.2 Hypothesis
1. The addition of oxytetracycline to feed will have no impact on feed intakes
10.3 Materials and Methods
Thirty adult Merino cross ewes were sourced from the Murdoch University teaching flock.
All sheep selected were in good health and had received no treatments in the month preceding the experiment. Sheep were housed in individual pens in a purpose built animal house ensuring they had sheep contact on at least one side. All sheep were weighed on entry to the animal house and a 2-days acclimatisation period was observed prior to commencement of the experiment.
The sheep were randomly assigned as they came through the race to two treatment groups of fifteen sheep.
IF. This treatment received non-medicated pellets for the first 2 days followed by 5 days of medicated pellets and then 5 days of non-medicated pellets.
Control. This treatment received only non-medicated pellets throughout the study period.
Feed and water measurements were recorded daily at the same time each day for all animals. All sheep were weighed at the end of the experiment.
Preparation of medicated pellets (in-feed medication). Medicated pellets were manufactured so that a 50 kg sheep eating 2% of its bodyweight in pellets per day would consume a dose of 20mg/kg OTC. Pellets were manufactured by
Wellard Rural Exports at their feedmill. The pellets consisted of a mixture of lupins, grain
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(Triticale) and chopped straw. The OTC powder (Terramycin, Phibro Animal Health Pty Ltd,
Girraween, Australia) was mixed in with the cereal and 15 minutes was allowed in the mixer to ensure adequate distribution of the powder throughout the mixture. Following this, chopped straw was added and a further 10 minutes of mixing occurred. The mixture was then heated to approximately 70oC for 2 minutes. The heat was generated by application of steam, which moistened the mixture to enable better compaction into the pellet shape.
Statistical analysis For each animal all pre-treatment feed and water intakes were averaged. A linear mixed model was fitted to the feed and water intakes on all post-treatment dates. The model included fixed effects for pre-treatment liveweight and intakes, treatment, post-treatment date and treatment by date interaction; and random effects for animal and the animal by date interaction. An autoregressive model allowed for correlations between measurements made on the same animal on different dates and different residual variance on each date.
5% Least Significant Differences (LSD) were calculated to compare the two treatments.
10.4 Results
There was no significant difference in the weight of sheep between the groups: sheep in the
IF group had a mean weight of 40.5 (+/- 2.88) kg and those in the control group 42.2 (+/-
4.21) kg pre-treatment and 41.33 (+/- 2.92 kg) and 42.2 (+/-4.54)kg post-treatment.
Both feed and water intakes were significantly impacted by the introduction of medicated feed to the sheep in the IF treatment when compared to those in the control group
(P<0.001, see Figure 10.1 and 10.2). In Table 10.1 the mean feed intake values are summarised.
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Feed intake fell after the introduction of medicated feed and recovered to near the levels of the control animals by the end of the experiment. Similarly, water intake fell after the introduction of pellets, recovered and then fell slightly again before the end of the experiment.
IF group Control group Mean intake (g) during 820.33 (+/- 330.6) 1173.00 (+/- 45.2) treatment Mean intake (g) following 1002.57 (+/- 159.7) 1240.29 (+/- 22.1) treatment Mean intake (g) over whole 993.75 (+/- 256.7) 1308.75 (+/- 38.7) experiment Table 10.1 Feed intake between the groups throughout the experiment
Figure 10.1 Feed intake (g/head/day) over time. Shaded box represents treatment period. Error bars represent standard errors.
1600
1400
1200
1000
800 Control OTC
600 feed intakefeedg/head/day 400
200
0 1 2 3 4 5 6 7 8 9 10 11 Day
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Figure 10.2 Water intake (kg/head/day) over time. Shaded box represents treatment period. Error bars represent standard errors.
4000
3500 3000 2500 2000 Control 1500 OTC 1000
water intakemL/head/day water 500 0 0 1 2 3 4 5 6 7 8 9 10 Day
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10.5 Discussion
Feed intake in the IF group dropped sharply in the first two days following the introduction of the medicated feed. Following this drop, intake slowly increased to levels closer to those prior to introduction of medicated feed. Water intake also dropped sharply in the early treatment period before slowly returning to levels comparable with those of the control group following cessation of treatment.
All sheep were offered a pelleted ration at a standard weight of 1.5 kg daily. The pellet provides 11 MJ/kgDM of metabolisable energy and was calculated to contain 90% dry matter (Caporaso et al.). This would equate to a kilogram of feed providing 9.9 MJ of metabolisable energy as fed. The maintenance energy requirements of sheep vary with age, type and the environment in which they are kept. As a guide, adult sheep require 0.185 MJ
ME kg -1 (Ball et al., 1998) per day for maintenance, equivalent to 9.25 MJ ME for a 50kg sheep. The IF group showed a reduction in intake during the treatment period which resulted in their mean energy intake reducing to 8.1 MJ ME which was above their maintenance energy requirements according to Ball (1998) of 7.2 MJ ME. Given that maintenance, and not growth, is the aim for sheep during pre-embarkation, and given the recovery of intake to previous levels following cessation of treatment, this reduction in intake may be an acceptable consequence of treatment.
As with feed intake, water intake was seen to drop but still remained within acceptable volumes for maintenance.
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11 Clinical efficacy of in-feed OTC
11.1 Introduction
The previous experiment has demonstrated that, although feed intake was reduced, feed intakes remained sufficient for maintenance despite the addition of medication to the pellets. In Chapter 5 it was concluded that OTC was absorbed into the blood at detectable levels following administration in the feed. On the basis of these findings it was judged important to assess the clinical efficacy of administering OTC by this route to animals with naturally occurring clinical IOK.
11.2 Hypothesis
1. Administration of pellets medicated with OTC will be effective in the clinical
improvement of IOK.
11.3 Materials and methods
Thirty mixed age and mixed breed sheep with clinical IOK were selected from a pre-export feedlot. Sheep were selected with either grade 2 (conjunctivitis) or grade 3 (conjunctivitis and corneal oedema) infection in both eyes. No treatment had been given to the sheep prior to selection for this experiment. The sheep were transported to a purpose built animal house facility at Murdoch University where they were housed in individual pens with sheep contact on at least one side.
All sheep were weighed on arrival at the animal house and on leaving. Sheep were accustomed to the pelleted ration prior to selection, therefore a 24-hour period was allowed for acclimatisation to the change in housing.
The sheep were randomly assigned into 2 treatment groups of fifteen sheep each:
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Control. These sheep received non-medicated pellets throughout the duration.
IF. These sheep received medicated pellets for 5 days followed by 5 days of un-medicated pellets.
Medicated pellets were as described in the in-feed palatability trial, section 10.2.
Swabs for bacterial culture were taken from all sheep on arrival at the animal house and again on leaving. Swabs were taken by placing a sterile cotton-tipped wooden-shafted swab in the fornix of the eye between the third eyelid and the conjunctiva; these swabs were then plated on sheep blood agar in the standard manner to obtain a single culture before the tip was broken off into a Mycoplasma broth. Plates and Mycoplasma broths were submitted to the Department of Agriculture microbiology laboratory in South Perth for culture growth and analysis.
Feed and water intake were recorded for individual sheep daily. Eye grades were assessed and recorded in all sheep on days 0, 1, 3, 5, 7 and 10.
Statistical analysis For each animal all pre-treatment feed and water intakes were averaged. A linear mixed model was fitted to the feed and water intakes on all post-treatment dates. The model included fixed effects for pre-treatment liveweight and intakes, treatment, post-treatment date and treatment by date interaction; and random effects for animal and the animal by date interaction. An autoregressive model allowed for correlations between measurements made on the same animal on different dates and different residual variance on each date.
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For each eye on each animal all pre-treatment eye grades were averaged. A linear mixed model was fitted to the eye grade data on all post-treatment dates (days 0, 2, 4 and 7). The model included fixed effects for pre-treatment eye grade, treatment, post-treatment date and treatment by date interaction; and random effects for animal, the animal by date interaction and eyes within dates and animals. An autoregressive model allowed for correlations between measurements made on the same animal and eyes on different dates and different residual variance on each date.
A split plot analysis of covariance was used to analyse bacterial scores from each eye of each animal made at the end of the experiment. Pre-treatment score for the appropriate bacteria was used as a covariate measurement and eye within animal was used as the split factor.
5% LSD’s were calculated to compare the two treatments.
Plasma sample analysis Plasma samples were analysed for concentrations of OTC. The method used for sample analysis is as described in Chapter 6.
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11.4 Results
The mean weight at the start of the experiment was 39.3 kg for the control group and 38.5 kg for the IF group. There was no significant difference in weight between the animals in the groups and no significant change in their weight over the experimental period.
Feed intakes reduced significantly during the treatment period in the IF treatment but recovered after treatment ended (P=0.049, see Table 11.1 and Figure 11.1)
Water intake was not significantly reduced and there was no significant reduction in water intake associated with the drop in feed intake in the IF group (Figure 11.2).
Clinically, sheep in the IF treatment showed a significant improvement in eye grade during treatment compared to those in the control group (P=0.007, see Table 11.1 and Figure
11.3). Following cessation of treatment there was a degree of worsening of eye grades in the treatment group but they still remained significantly lower than those in the control group.
Sheep in the treatment group showed a reduction in bacterial load for Moraxella ovis only following IF treatment compared to the control group (P=0.004, see Table 11.1). There was no difference seen between the treatments in Mycoplasma conjunctivae load.
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Term Feed intake Water intake M.ovis Mycoplasma Eye grade eye grade (cov) 0.035 feed intake (cov) 0.056 0.130 water intake (cov) 0.361 0.034 weight 0.826 0.914 day (time) <0.001 <0.001 <0.001 treatment (cont v IF) 0.049 0.285 0.004 0.231 0.007 day.treatment <0.001 0.054 0.008
Table 11.1 Significance levels (P values) for terms used in models to analyse data. Cov =covariate
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Figure 11.1 Feed intake (g/hd/day) of Control vs IF treatments over time. Shaded box represents treatment period. Error bars represent standard errors.
1600
1400
1200
1000
800 Control 600 IF
feed intakefeedg/head/day 400
200
0 0 1 2 3 4 5 6 7 8 9 10 Day
Figure 11.2 Water intake (ml/hd/day) of Control vs IF treatments over time. Shaded box represents treatment period. Error bars represent standard errors.
6000
5000
4000
3000 Control IF
2000 Water intakeWatermL/head/day 1000
0 0 1 2 3 4 5 6 7 8 9 10 Day
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Figure 11.3 Change in eye grade over time - Control vs IF treatments. Shaded box represents treatment period. Error bars represent standard errors.
4
3
2 control
IF Mean eye eye Meangrade 1
0 0 2 4 7 Day
Despite the reduction in intake of medicated pellets, a significant improvement in clinical eye grade within the IF treatment group was seen in this experiment. In line with the results in Chapter 10, the sheep in the treatment group exceeded the minimum intake of 7.1 MJ
ME required for maintenance. During the treatment period the mean intake was 9.1 MJ ME and throughout the whole experiment period the mean intake was 10.8 MJ ME for those in the IF group.
11.5 Discussion
This experiment was designed to determine if in-feed (IF) medication had any effect on clinical IOK. It has been shown that there was a reduction in feed intake following introduction of medicated pellets (Section 10.1). Despite the reduction in intake of medicated pellets, a significant improvement in clinical eye grade within the IF treatment group was seen in this experiment. In line with the results in Chapter 10, the sheep in the treatment group exceeded the minimum intake of 7.1 MJ ME required for maintenance.
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During the treatment period the mean intake was 9.1 MJ ME and throughout the whole experiment period the mean intake was 10.8 MJ ME for those in the IF group.
In contrast to the results when OTC was included in water, the results of this experiment show the degree of feed and water intake reduction was lower when OTC is added to the pelleted ration. It is likely that the difference relates to the difference in palatability between in-feed and in-water medications, with OTC in-feed being more palatable than OTC in-water.
Bacteriology samples taken before and after treatment indicated that effective concentrations of OTC were reaching the ocular tissue resulting in the reduced bacterial load seen post-treatment. This is supported by experimental work in Chapter 8 that showed that following administration of OTC in feed, detectable concentrations were achieved in the plasma. For an antimicrobial drug to be effective it must exceed the Minimum Inhibitory
Concentration (MIC) of the target bacteria. The MIC of Mycoplasma conjunctivae for OTC is reported to range from 0.78-6.2 µg/mL depending on the strain (Egwu, 1992a). The MIC for
Moraxella ovis for OTC is 0.5 µg/mL (Catry et al., 2007). Experimental work reported in
Chapter 8 indicated that peak plasma concentrations above the MIC for both bacteria were achieved.
Oxytetracycline is a bacteriostatic antibiotic (Neu, 1978) and as such only inhibits the growth of the bacteria through interference with protein synthesis, and does not kill the bacteria.
Most bacteriostatic antimicrobials will also kill a large proportion of bacteria, 90-99%, but not a sufficient proportion to be classed as bacteriocidal (Pankey and Sabath, 2004). The inhibition of the bacteria by OTC then relies on the host immune system to remove the organism (Taylor et al., 2002).
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Notably, although there was a decrease in Moraxella ovis colonies following IF treatment, there appeared to be very few other organisms grown from the final sample and in some cases a pure culture of M. ovis was present following IF treatment. This could potentially contribute to the slight deterioration in clinical eye grades following cessation of treatment.
The positive results that followed five days of in-feed medication suggest that extending the duration of treatment might improve the overall result. Sheep are typically in the feedlot for seven days, therefore an extended course would be feasible. Further studies would be required to fully assess the effect of altering the treatment course in this way.
11.6 Conclusion
Treating sheep with OTC medicted pellets for 5 days effectively reduces clinical eye grades and bacterial load. Feed and water intakes are reduced, however adequate levels are consumed throughout treatment for maintenance.
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12 The treatment of infectious ovine keratoconjunctivitis with in-
feed medication in a pre-embarkation feedlot.
12.1 Introduction
It was important to test whether the positive responses seen clinically in sheep treated with
OTC medicated feed in controlled animal house environments were replicated in an actual pre-embarkation feedlot. Up until this point all experiments had focused on small numbers of animals with frequent and close individual monitoring. The experiment reported here mimicked the feedlot environment. This experiment was designed to validate the previous results in an industry context so that the efficacy of in-feed treatment of IOK could be demonstrated as a practical, affordable and realistic option for exporters. Both productivity and animal welfare were hypothesised to improve should the results concur with previous research.
A range of severity of infection was considered in this research, based on selection of sheep with eye grades between 1 and 6. The experiment aimed to assess the efficacy against varying grades of IOK of in-feed OTC medication compared with two injections of OTC, which has been consistently found to be the optimum treatment.
12.2 Hypotheses
Oxytetracycline-medicated feed will be effective in treating mild, up to and including
grade 3, IOK
Two injections of OTC will be effective against all grades of IOK
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12.3 Materials and Methods
Two hundred and seven Merino cross, mixed age sheep with naturally occurring clinical IOK were selected from those rejected at a pre-export feedlot. No sheep had received treatment prior to selection.
Sheep eyes were graded using the grading system previously described (Appendix 1). All sheep had clinical IOK in both eyes, not necessarily of equal severity. Following grading, sheep were drafted into two groups, one group with clinical eye grades 2-4 and the other with clinical eye grades 5-6. From these groups, sheep were randomly drafted into 3 treatment groups:
Group 1 - control group receiving no treatment (n=69 with 40 grade 2-3 and 29 grade 4-5)
Group 2 - intra-muscular injection OTC (Alamycin 300 LA, Nobrook Laboratories Australia
PTY Ltd, Tullamarine, VIC, Australia) (2 doses of 20mg/kg 4 days apart) (n=70 with 45 grade
2-3 and 25 grade 4-5)
Group 3 – in-feed OTC (Terramycin 200, Phibro Animal Health, Girraween, NSW, Australia)
(n= 68 with 36 grade 2-3 and 32 grade 4-5).
Sheep were housed in three separate raised pens in a standard feedlot. Free access to water was given to all sheep. Those in groups 1 and 2 had ad lib access to pelleted feed as would be typical in a feedlot. Sheep in group 3 received medicated feed, as described in Chapter
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10, for 5 days. Accurate measurements of feed intake were not taken, but subjective assessment of residual feed was made; residual feed was removed on a daily basis. Accurate measurement of residuals was not possible owing to contamination of feed and crumbling of pellet diet. Ad lib access to non-medicated pellets was given following cessation of medicated pellets.
Eye grades were recorded every second day from day 0 up to day 10 using the same grading system as before, Appendix 2.
For each eye on each animal all pre-treatment eye grades were averaged. A linear mixed model was fitted to the eye grade data on all post-treatment dates. The model included fixed effects for pre-treatment eye grade, treatment, post-treatment date and treatment by date interaction; and random effects for animal, the animal by date interaction and eyes within dates and animals. An autoregressive model allowed for correlations between measurements made on the same animal and eyes on different dates and different residual variance on each date.
12.4 Results
Eye score on Day 0 was significantly associated with eye scores post treatment (P <0.001).
When treatment means were corrected for score on Day 0 there was a significant effect of
Treatment (P<0.001) on daily eye score but this effect interacted with day-post-treatment such that the effect of treatment on eye score was different on different days post treatment (P<0.001) (Table 12.1). Daily treatment eye score means (corrected for score on
Day 0) are shown in Figure 12.1
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At the end of the experiment animals treated with injections had lower eye scores than those treated with in-feed medication. Both treatments had lower mean eye scores than the control animals.
Fixed Term P Value
Day <0.001
Score on Day 0 <0.001
Treatment <0.001
Day.Treatment <0.001
Score on Day 0.Treatment <0.001
Table 12.1: Significance levels (P Values) for analysis of pink eye scores on each day post-treatment (control vs in-feed vs injection)
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Figure 12.1 Adjusted IOK mean scores for each treatment and day.
The eye score on Day 0 significantly affected the degree of change of eye score over time
(P<0.001) (Table 12.2).
Fixed Term P value
Score on day 0 <0.001
Treatment <0.001
Score on day 0.Treatment 0.048
Table 12.2 Significance levels (P values) for analysis of change in pink eye score over time
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Treatment Control In feed Injection
-0.898 -1.691 -2.318
Standard errors 0.1316 0.1322 0.1304
Table 12.3 Change in eye score from day 0 to day 10
At an average Score on day 0 value of 3.1, the decrease in eye score from Day 0 to Day 10 for the In-feed group was 0.793 (±0.186) (-0.898 vs -1.691 for the In-Feed and Control groups respectively). The decrease in eye score from Day 0 to Day 10 for the Injection group was 1.4206 (±0.186) more than for the Control group (-0.898 vs -2.318). The Injection group had a significantly higher decrease in eye score than the In-Feed group (Table 12.3).
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Figure 12.2 Average eye scores on each day, grouped according to Score on Day 0.
Treatment Control Treatment In feed
5
4
3
2
1
0
Treatment Injection 0 3 6 9
5
4
3
2
1
0 0 3 6 9
Score v Day (1) Score v Day (2) Score v Day (3) Score v Day (4) Score v Day (5)
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The eye score means according to the starting eye score are depicted in Figure 12.2. In-feed
OTC for 5 days was found to be an effective treatment for sheep with IOK up to and including grade 3, whereas injectable OTC was effective in treating IOK up to and including grade 5 (Figure 12.2, Table 12.5).
Score In-feed Injection
1 Treat Treat
2 Treat Treat
3 Treat Treat
4 Not effective Treat
5 Not effective Treat
Table 12.4: Treatment recommendations (Treat vs Not effective) of different routes of administration (In- feed vs Injection) at different degrees of severity of disease (Score) on day 8 of experiment.
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12.5 Discussion
The results of the feedlot experiment are consistent with results seen in the previous experiments. Although, subjectively, feed intake in the medicated feed group did decrease, the amounts of residual feed remaining were small. In-feed OTC is considered effective in treating sheep with eye grades up to and including 3. The hypothesis was that in-feed OTC would be effective up to grade 4; however, the experiment demonstrated that those sheep with grades 4 and 5 need to be treated with intra-muscular OTC to have the best chance of recovery to the point where they could be shipped.
The use of in-feed medication is significantly less labour intensive than injecting individual animals which greatly reduces the cost of the treatment. Within a cohort of sheep at the pre-export feedlot there is likely to be variation in severity of clinical signs of IOK. Survey results from inspectors outlined in Chapter 4 indicated that up to 50% of a shipment could have mild IOK during the high-risk summer period. Given the numbers of animals involved and the labour required to inject those with mild disease it is encouraging that in-feed medication is an effective treatment. Given that IOK is considered a painful condition (Greig,
1989; Akerstedt, 2004) having an effective treatment available that requires minimal labour could contribute to improved welfare of animals in pre-export feedlots.
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13 General Discussion
Infectious Ovine Keratoconjunctivitis continues to affect the live export industry, both in pre-export feedlots and onboard voyages. The live export trade attracts some of the most consistent attention from the public in relation to animal welfare (O'Flynn and O'Dea, 1996).
With ever-increasing scrutiny on the industry it is important to address issues that may compromise animal welfare and give the public reason for concern. IOK is a multifactorial disease with several aetiological agents and is associated with numerous risk factors. These, along with the potential for the development of a carrier state make treating disease and keeping the sheep free from disease within the export chain a significant challenge.
Infectious ovine keratoconjunctivitis is a common disease of sheep worldwide. Although numerous authors have investigated causes of the disease (Livingston.C. W et al., 1965;
Cooper, 1967; Surman, 1973; Cooper, 1974; Hosie, 1988; Egwu et al., 1989; Naglic et al.,
2000) there still remains some debate as to the exact agent responsible. To date much of the published research has investigated outbreaks in the northern hemisphere with few reports of outbreaks in Australia and no investigations of the disease within pre-export feedlots.
The unique environment of a pre-export feedlot poses many challenges to the control of
IOK. In the days leading up to a shipment the feedlot will receive up to 80000 sheep from a number of different sources, some travelling a considerable distance. As discussed in
Chapter 4 this mixing of sheep poses a great risk to the spread of IOK through the exposure
184 of naïve sheep to carriers. Additionally the stress of mixing coupled with a dusty and often hot environment further adds to the risk of developing clinical IOK.
The feedlots try to address some of the risk factors. Many of the holding pens have concrete bases and all areas leading up to the unloading bays are concreted. This minimizes dust, but with that number of sheep moving over a relatively short period of time dust will be unavoidable. All holding and sorting pens are covered to provide shade, which reduces the effect of UV light. Upon arrival sheep are sorted into ‘lines’ whereby they are grouped according to their age and/or type. Keeping age groups together may theoretically be beneficial in reducing the risk of exposure to carrier animals; however, the literature and the responses to questionnaires in the current study highlight that there is no age group that is specifically affected more than any other.
Although IOK is not typically associated with high mortality and therefore, superficially, could be considered less important than other diseases, e.g. Salmonellosis, it is associated with high morbidity. As discussed in Chapter 1 IOK is considered a painful condition and does result in a reduction in feed intake and growth, and this coupled with the rejection from a shipment leads to significant economic losses to the exporter.
Despite efforts by the feedlot management to mitigate some of the risk factors IOK still remains an issue, therefore sourcing an effective treatment is of paramount importance for addressing the welfare concerns raised by the presence of the disease, and for addressing the economic concerns as well.
The initial pilot work of Chapter 5 concluded that injectable OTC was the most effective treatment. This is in agreement with many studies from around the world (Andrews et al.,
185
1987; Greig, 1989; Hosie, 1995; Naglic et al., 1999; Kovac et al., 2003; Dun, 2009; Townsend,
2010). This initial pilot work was also useful in concluding that using topical OTC treatments is no better than giving no treatment. Despite highlighting that injectable OTC was the most effective treatment, there still remained the serious logistical problem of treating all the large numbers of affected sheep in this way. If the feedlot were to rely solely on injectable
OTC it is likely that many of the milder cases would be left untreated, therefore controlling outbreaks would be difficult and welfare issues associated with the disease would not be addressed across all animals.
In-water medication initially showed promise in the pilot work described in Chapter 5. The appeal of a mass medication modality is understandable because it means that it is not necessary to handle animals individually, which is an obvious advantage given the numbers of animals involved. Further experiments with in-water treatment have, however, highlighted limitations. The addition of medication appeared to have a deleterious effect on the palatability of water resulting in low intakes during the treatment period. This leads to very low concentrations of OTC being consumed resulting in a poor clinical response.
Oxytetracycline appears to be more palatable when mixed in-feed, with a reduction in feed intake observed but not to levels below those required for maintenance. Consistently, in- feed medication has resulted in a good clinical response throughout the experiments. The reduction in feed intakes observed appears to be temporary, with sheep returning to intakes comparable with those pre-treatment following cessation of treatment. To date experimental work has focused on a 5-day treatment period and it has been noted that following initial improvement in clinical signs, clinical signs can begin to worsen in the 5-day period following treatment. The researchers would recommend testing a longer period of
186 offering medicated feed – possibly seven days. Sheep are typically in the pre-export feedlot for around 7 days and it may be possible to extend the treatment to this time. In addition to extending the treatment period it would be beneficial to extend the post-treatment monitoring period to assess whether treatment improves recovery rates over a 3 weeks period compared to no treatment. Mimicing the risk factors that the sheep will be subject to following leaving the feedlot and going to the ship would be difficult in an experimental environment therefore longer term monitoring is unlikely to reflect true recovery rates within the live export chain.
Although IOK is a significant cause of rejection from the export chain, there are a variety of other issues present within the chain that must be considered when implementing a treatment strategy. Inanition is one of the most important causes of death in sheep within the live export chain alongside Salmonellosis (Norris et al., 1989a; Barnes et al., 2008).
Failure to eat pellets during the latter part of the quarantine feedlot period has been recognized as a risk factor for deaths during the subsequent voyage (Norris et al., 1989a).
Non-feeders are at highest risk of dying from inanition and salmonellosis and one of the keys to reducing this risk is to ensure sheep continue to eat consistently throughout the live export chain process (Norris et al., 1989a). There are other factors to consider in relation to onboard deaths, including the season of shipment and fatness of the animals (Higgs et al.,
1999). Links between inanition and the development of Salmonellosis have been well documented in the literature (Higgs et al., 1993). Stressed sheep that do not eat are at risk of developing clinical Salmonellosis as a result of environmental contamination (Higgs et al.,
1993). Although the sheep treated with in-feed OTC had a reduced feed intake, intake was still maintained throughout the treatment period and returned rapidly to levels comparable
187 with pre-treatment levels soon after cessation of treatment. If mild IOK lesions were allowed to progress it is likely that this would have a more significant impact on feeding than treating with in-feed OTC.
Advocating the use of antibiotics in-feed, or indeed in-water, could be considered irresponsible use of antibiotics. The development and presence of antimicrobial resistance occurs naturally; however, the inappropriate use of antibiotics is known to be a factor in accelerating the development of antimicrobial resistance (WHO, 2015). The Australian
Government has published a national strategy to minimize antibiotic misuse and development of resistance (Australian Government, 2015). Although the Government recognizes that the use of antibiotics in food animals is comparatively low in Australia compared to worldwide figures, it is still essential to ensure that those that are used are used appropriately.
Antimicrobial resistance develops through antimicrobial use which selects for resistant organisms coupled with a genetic change in the organism (Levy and Marshall, 2004). The genetic change confers resistance by altering the organisms’ ability to defend itself (Witte,
1998). This can be through altering the drug target site, altering metabolism to circumvent affected pathways or through detoxifying the drug (Witte, 1998). These alterations can result in resistance to multiple drugs. Tetracyclines work by inhibiting the protein synthesis of the bacteria. This is the same mode of action as aminoglycosides, macrolides and lincosamides (Levy and Marshall, 2004), therefore resistance to one can infer resistance to drugs in the other groups.
Levy and Marshall (2004) refer to a study in chickens which were fed OTC in feed for growth promotion which led to rapid development of tetracycline resistant E.coli within days but
188 after 2 weeks of use the E.coli was multi-resistant. Antibiotics used in feed for the purposes of growth promotion are given at sub-therapeutic concentrations for prolonged periods of time (Wegener, 2003), typically less than 200g per tonne and for greater than 2 weeks
(McEwen and Fedorka-Cray, 2002). Long term use of an antibiotic, for greater than 10 days, is known to lead to selection of resistant bacteria, not just to that antibiotic but potentially to others too (Levy and Marshall, 2004). In the 1960s the Swann Committee in the UK identified the risk of using drugs which are important in human medicine as growth promoters in animal feed (Witte, 1998). It is recognized that the use of antibiotics in feed at sub-therapeutic concentrations has led to a reservoir of resistant bacteria, and this prompted the European Union to ban the use of a number of medically important drugs for use as growth promoters (Wegener, 2003). Wegener (2003) goes on to discuss the decrease in prevalence of resistant bacteria found in food, food-producing animals and humans since this ban was implemented. Although a risk, the contribution of antibiotic use in animal health to the development of antibiotic resistance affecting humans is considered small and in general is confined to enteric organisms (Levy and Marshall, 2004).
In contrast to the long-term use of in-feed antibiotics, short term use of antibiotics at treatment concentrations for treatment of disease is considered responsible use. The use of antibiotics can always be justified for limited treatment periods in response to disease, or as a prophylactic against disease (McEwen and Fedorka-Cray, 2002). Tetracyclines are considered to be of low importance in human medicine (Scott et al., 2012a) making their choice a responsible one. A study in Canada found no significant link between tetracycline administration to sheep in food or water and the development of resistant Campylobacter
189 sp. (Scott et al., 2012b), although an association was seen with the development of resistant faecal E.coli spp.
Advocating the treatment of sheep at a pre-export feedlot with in-feed OTC is defensible on a number of fronts. Firstly the evidence in the literature points to low risk of development of resistant bacteria following short courses of in-feed antibiotics when given at therapeutic doses in feed. The dose advocated is a therapeutic one and the evidence presented in
Chapters 7 and 8 highlight the clinical response to treatment. Secondly, relying on individual treatment with injectable OTC will result in many sheep not being treated and therefore compromising their welfare. Aside from the high numbers of animals involved, it can be difficult to identify the early clinical signs of IOK in individual sheep when they are grouped together as in a feedlot. The data presented in Chapter 4 highlights the high percentage of mild cases that arrive at the feedlots; blanket treatment of groups with in-feed medication is the most effective way of ensuring that all these cases are treated. The work in Chapter
11 has highlighted the limitations of in-feed medication for the treatment of those sheep with more severe IOK, and given this evidence it would be inappropriate to treat these with in-feed medication. The numbers affected with severe IOK are much lower and they are potentially easier to spot making them more suitable candidates for intramuscular treatment. In-feed OTC medication is not superior to injectable OTC in clinical effect, but it is cheaper and easier to administer to large groups.
The stability of OTC in in-feed medication is unknown. In all the experiments the feed was made up within a week of it being used. It is known that OTC is sensitive to degradation by
UV light (Christiano et al., 2010), therefore storage of medicated feed could be problematic.
Although the in-feed medication used in these experiments is registered for use in sheep in
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Australia, it does not have a label claim for the treatment of IOK. Additionally no export slaughter interval has been established to the researchers’ knowledge. These issues would need to be considered prior to the widespread adoption of this treatment strategy.
Through a series of experiments it has been shown that IOK up to grade 5 is treatable. This information should be useful for feedlot managers to enable them to instigate the most effective treatment at the earliest possible opportunity. This should result in higher recovery rates and fewer culls owing to IOK. This improvement in treatment outcomes will improve animal welfare, which remains a key aim of those involved in the live animal export industry.
Ultimately the aim would be to prevent occurrence of IOK completely. However, as this is a multi-factorial disease with a number of organisms and risk factors involved that is very much easier said than done. In cattle, where the causative agent is more clearly understood, vaccines have been developed to prevent disease. These vaccines are manufactured based on the pili of Moraxella bovis which is important in the stimulation of immunity. As there are a number of different pili the current vaccine provides little, if indeed any, protection against clinical disease (Brown et al., 1998). Given the relative complexity of IOK it is unlikely that a vaccine could be developed to provide adequate protection against outbreaks occurring in the feedlots.
This work has identified a sound treatment protocol for use within the pre-export feedlots in Western Australia. However, anecdotal evidence suggests that outbreaks of IOK continue to occur on board ships. Tracing sheep through the live export chain beyond the feedlot was outside the scope of this study. Further work is required to determine what, if any, difference implementing this treatment protocol in the pre-export feedlot has on subsequent outbreaks of IOK onboard the ships.
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14 Conclusions
Infectious ovine keratoconjunctivitis is a disease that continues to challenge not only the live export industry but also the farming community worldwide. The multitude of factors known to contribute to the occurrence and severity of an outbreak continue to frustrate veterinary practitioners and producers alike. Given the presence of a number of key risk factors within the pre-export feedlots, IOK outbreaks would appear to be unavoidable. It is for this reason that effective and realistic treatment strategies must be in place to ensure treatment of the disease, optimization of animal welfare, reduction in economic losses and an assurance to the general public that the industry is focused on the health and welfare of the animals it exports.
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15 Appendix 1
Determination of patterns in numbers of animals presenting with eye disease
at pre-export feedlots.
Controlling eye disease in sheep within the export industry represents an ongoing challenge.
Infectious ovine keratoconjunctivitis (IOK), Pink Eye, is a common cause for animals to be rejected from shipments due to its infectious nature. Current estimates state that 0.5-1.0% of all sheep are rejected due to Pink Eye infections. This figure only represents those animals which are deemed to have severe Pink Eye and therefore have no commercial value. Accurate figures representing the numbers of mild cases presenting at the feedlots which will be treated do not exist.
The aim of this study is to get an estimation of numbers of animals seen at feedlot entry inspections with mild or severe Pink Eye. This project forms part of a bigger project addressing treatment and control options for Pink Eye within pre-export feedlots. The project has been funded by Meat and Livestock Australia.
Completion of this study is entirely voluntary. The information gathered will remain confidential, all data will be analysed and incorporated into a report for Meat and Livestock
Australia in addition to my PhD thesis.
My supervisor and I are happy to discuss with you any concerns you may have about this study.
Sincerely
193
Dr Fraser Murdoch Dr. Michael Laurence
PhD Candidate Senior Lecturer Production Animal Health [email protected] [email protected]
This study has been approved by the Murdoch University Human Research Ethics Committee (Approval 2014/205). If you have any reservation or complaint about the ethical conduct of this research, and wish to talk with an independent person, you may contact Murdoch University’s Research Ethics Office (Tel. 08 9360 6677 (for overseas studies, +61 8 9360 6677) or e-mail [email protected]). Any issues you raise will be treated in confidence and investigated fully, and you will be informed of the outcome.
194
Infectious ovine keratoconjunctivitis (IOK), or Pink Eye, is a common problem within the pre- export feedlots. Data gathered to date quantifies the numbers of rejections at entry to the feedlot due to IOK. It would be useful to expand on this data and confirm anecdotal evidence of seasonal and age related patterns. The following survey is designed to determine an accurate estimate of numbers of sheep presenting with clinical signs of IOK at the feedlots.
Definitions:
Mild IOK – sheep have reddening of the conjunctiva and discharge from the eyes but no evidence of decreased vision (not walking into things)
Severe IOK – sheep will likely have clouding of the surface of the eye and do show decreased vision.
1. For shipments in the following months please state what you consider to be the
minimum and maximum number of mild cases of IOK as a percentage:
Minimum Maximum
Spring: October ______
November ______
Summer: January ______
February ______
Autumn: April ______
195
May ______
Winter: July ______
August ______
2. For shipments in the following months please state what you consider to be the
minimum and maximum number of cases of severe IOK as a percentage:
Minimum Maximum
Spring: October ______
November ______
Summer: January ______
February ______
Autumn: April ______
May ______
Winter: July ______
August ______
3. For shipments in the following months please state what you consider to be minimum and maximum numbers of sheep affected with IOK (separate percentage for mild and severe) in each class as a percentage of the total shipment:
Minimum Lambs Maximum Lambs
Mild/Severe Mild/Severe
196
Spring: October ______
November ______
Summer: January ______
February ______
Autumn: April ______
May ______
Winter: July ______
August ______
Minimum Wethers Maximum Wethers
Mild/Severe Mild/Severe
Spring: October ______
November ______
Summer: January ______
February ______
Autumn: April ______
May ______
Winter: July ______
August ______
Minimum Ewes Maximum Ewes
197
Mild/Severe Mild/Severe
Spring: October ______
November ______
Summer: January ______
February ______
Autumn: April ______
May ______
Winter: July ______
August ______
Minimum Rams Maximum Rams
Mild/Severe Mild/Severe
Spring: October ______
November ______
Summer: January ______
February ______
Autumn: April ______
May ______
Winter: July ______
August ______
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4. For shipments in the following months please state what you consider to be the
minimum and maximum numbers of sheep affected with IOK (separate percentage
for mild and severe) in each breed as a percentage of the total shipment:
Minimum Merino Maximum Merino
Mild/Severe Mild/Severe
Spring: October ______
November ______
Summer: January ______
February ______
Autumn: April ______
May ______
Winter: July ______
August ______
Minimum Cross breed Maximum Cross Breed
Mild/Severe Mild/Severe
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Spring: October ______
November ______
Summer: January ______
February ______
Autumn: April ______
May ______
Winter: July ______
August ______
We thank you in advance for your time in completing this survey. All data is anonymous and will be used solely for analysis as part of a PhD thesis. Please return completed surveys to:
Dr. Michael Laurence
School of Veterinary and Life Sciences
College of Veterinary Medicine
Murdoch University
Murdoch, WA 6150
or email to:
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16 Appendix 2
Grade 0 – normal eye Grade 1 – epiphora (weeping eye)
Grade 2 – conjunctivitis Grade 3 – corneal oedema (clouding of the cornea)
Grade 4 – corneal ulceration
Grade 5 - corneal neovascularisation
Grade 6 – chronic eye damage
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17 Appendix 3
Percentage contribution* Taxon
(phyla, class, order C C C family) C 1 10 11 12 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 H16 H20 H21 H22 H23 H24 L13 L14 L15 L17 L18 L19
20. 11. 37. 15. 27. 14. 17. 18. Other; 1.5 2.4 1.9 2.0 2.3 0.6 1.3 3.6 3.6 2.9 1.2 1.5 0 9.5 6 1 6 8 6 0.5 8 4.8 1.2 1
Actinobacteria; Actinobacteria; Bifidobacteriales; 0.0 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.2 0.0 0.4 0.5 0.2 0.4 0.1 0.0 0.1 0.0 0.0 0.5
Bacteroidetes; Bacteroidia; Bacteroidales; Other 0.1 1.7 1.4 0.8 0.7 0.1 1.9 2.1 0.8 2.3 0.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Bacteroidetes; Bacteroidia; 15. 15. 13. 12. 18. 13. 12. 10. 10. Bacteroidales; 1.9 5 5 7 1 5.1 1 0 0 7 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0
Bacteroidetes; Bacteroidia; Bacteroidales; Porphyromonadacea e 0.5 2.6 9.1 3.0 0.9 1.4 3.2 0.2 1.4 3.3 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.4 0.0
Bacteroidetes; Bacteroidia; Bacteroidales; 30. 28. 12. 24. 33. 29. 23. 22. 33. 25. 83. Prevotellaceae 37.8 3 0 9 9 8 9 7 5 7 0 6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 0.0
202
Percentage contribution* Taxon
(phyla, class, order C C C family) C 1 10 11 12 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 H16 H20 H21 H22 H23 H24 L13 L14 L15 L17 L18 L19
Bacteroidetes; Flavobacteria; Flavobacteriales; Flavobacteriaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0
Cyanobacteria; 4C0d-2; YS2; 0.0 0.1 0.0 0.4 0.0 0.0 0.0 0.0 0.8 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Elusimicrobia; Endomicrobia 0.0 0.6 0.0 0.2 0.1 0.1 0.1 0.0 0.3 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Fibrobacteres; Fibrobacteres; Fibrobacterales; 18. 17. 10. 31. 11. 24. 28. 16. 22. Fibrobacteraceae 8.3 5 2 9 1 5.8 9 9 8 1 4 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Firmicutes; Bacilli; Lactobacillales; Enterococcaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.1 0.5 1.1 0.3 5.4 0.5 0.0 0.0 0.4 0.4 1.3 1.3
Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.9 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Firmicutes; Bacilli; Lactobacillales; Streptococcaceae 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.3 0.0 0.0 0.0 0.0 0.0 0.1 0.0
Firmicutes; 0.3 1.8 2.2 2.3 0.4 0.4 4.4 0.7 1.3 2.3 0.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.3 0.8 0.0 0.0 0.0 0.0 Clostridia; 203
Percentage contribution* Taxon
(phyla, class, order C C C family) C 1 10 11 12 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 H16 H20 H21 H22 H23 H24 L13 L14 L15 L17 L18 L19
Clostridiales; Other
Firmicutes; Clostridia; Clostridiales; 1.3 4.7 4.0 3.8 3.5 3.8 6.7 3.3 6.3 4.1 6.4 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0
Firmicutes; Clostridia; Clostridiales; Catabacteriaceae 0.1 0.0 0.0 0.2 0.3 0.2 0.2 0.0 0.1 0.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Firmicutes; Clostridia; Clostridiales; Eubacteriaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.3
Firmicutes; Clostridia; Clostridiales; 11. 12. 41. 80. 65. 55. 56. 68. 34. 75. 79. 26. 71. Lachnospiraceae 7.4 6.9 2.9 7.8 5.4 2.1 7.2 8 7.5 5.7 5 8.9 4 8 6 8 6.5 5 6 1 2 0 3 6
Firmicutes; Clostridia; Clostridiales; 28. 13. 60. Ruminococcaceae 1.4 1.6 1.1 3.0 1.2 2.0 4.3 2.6 1.6 4.8 3.8 0.0 4 0.0 0.0 0.0 0.1 0.0 7 5 0.0 0.2 0.1 0.0
Firmicutes; Clostridia; 0.8 2.1 1.9 2.6 2.3 3.9 1.9 2.9 1.8 3.4 2.1 2.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Clostridiales;
204
Percentage contribution* Taxon
(phyla, class, order C C C family) C 1 10 11 12 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 H16 H20 H21 H22 H23 H24 L13 L14 L15 L17 L18 L19
Veillonellaceae
Fusobacteria; Fusobacteria; Fusobacteriales; Fusobacteriaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0
Lentisphaerae; Lentisphaerae; Victivallales; Victivallaceae 0.0 1.5 4.6 0.8 1.0 0.3 1.0 0.3 0.1 4.2 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Lentisphaerae; Lentisphaerae; Z20; 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.2 0.1 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Planctomycetes; Planctomycea; Pirellulales; 0.0 0.3 0.0 0.3 0.3 0.0 0.2 0.5 1.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Proteobacteria; Alphaproteobacteria ; Other; Other 0.0 2.4 0.0 0.6 1.6 0.1 0.9 0.9 0.9 0.1 1.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Proteobacteria; Alphaproteobacteria 0.0 0.5 0.1 1.7 0.3 0.8 0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Proteobacteria; Betaproteobacteria; 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 Neisseriales;
205
Percentage contribution* Taxon
(phyla, class, order C C C family) C 1 10 11 12 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 H16 H20 H21 H22 H23 H24 L13 L14 L15 L17 L18 L19
Neisseriaceae
Proteobacteria; Gammaproteobacter ia; Other; Other 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.4 0.0
Proteobacteria; Gammaproteobacter 25. 37. ia; Aeromonadales; 35.2 0.2 2.4 7 0.2 6 0.1 1.0 0.1 0.0 0.4 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Proteobacteria; Gammaproteobacter ia; Aeromonadales; Succinivibrionaceae 0.9 0.3 0.1 2.4 0.6 0.0 0.1 1.7 0.9 0.1 0.4 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Proteobacteria; Gammaproteobacter ia; Enterobacteriales; Other 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.1 0.0 0.0 0.0 0.1 0.0 0.1
Proteobacteria; Gammaproteobacter ia; Enterobacteriales; 20. 71. 14. 15. 63. Enterobacteriaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 7.7 9.1 7 6.2 7 6 1.4 3.3 5.9 5 8 8.1
Proteobacteria; Gammaproteobacter ia; Pasteurellales; Pasteurellaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.0
206
Percentage contribution* Taxon
(phyla, class, order C C C family) C 1 10 11 12 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 H16 H20 H21 H22 H23 H24 L13 L14 L15 L17 L18 L19
Proteobacteria; Gammaproteobacter ia; Pseudomonadales; Moraxellaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.1 0.0 3.1 0.0
SR1; 0.0 0.0 0.0 0.4 0.0 0.3 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Spirochaetes; Other; Other; Other 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Spirochaetes; Spirochaetes; Other; Other 0.1 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Spirochaetes; Spirochaetes; Spirochaetales; Spirochaetaceae 1.3 2.2 0.6 2.0 8.3 1.1 2.3 4.7 3.4 2.1 2.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0
Synergistetes; Synergistia; Synergistales; Dethiosulfovibrionac eae 0.0 0.0 0.1 0.4 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
TM7; TM7-3; CW040; F16 0.0 0.1 0.0 0.2 1.3 0.1 0.1 0.1 0.7 0.6 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
207
Percentage contribution* Taxon
(phyla, class, order C C C family) C 1 10 11 12 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 H16 H20 H21 H22 H23 H24 L13 L14 L15 L17 L18 L19
Tenericutes; Other; Other; Other 0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.2 0.3 0.0 0.1 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tenericutes; Erysipelotrichi; Erysipelotrichales; Erysipelotrichaceae 0.0 0.1 0.2 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.2 0.0 0.2 0.0 0.0 0.0 0.2 0.0 0.5 0.0 1.2 0.1
Tenericutes; Erysipelotrichi; Erysipelotrichales; vadinHA31 1.2 2.7 6.2 1.4 0.9 0.3 3.2 1.1 2.6 0.5 4.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tenericutes; Mollicutes; Anaeroplasmatales; Anaeroplasmataceae 0.0 0.3 0.2 0.2 0.0 0.0 0.4 0.1 0.9 0.6 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tenericutes; Mollicutes; Mycoplasmatales; Mycoplasmataceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0
Tenericutes; Mollicutes; RF39; 0.0 0.4 0.0 0.2 0.0 0.0 0.2 0.0 0.1 0.2 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
208
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