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Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis. This is the most common tick-borne disease of equidae in southern Africa  and piroplasmosis has been detected worldwide, including in recent studies in Europe , the United States , South America , Jordan  and Asia . Transmission of the parasites via the infected tick may lead to haemolytic anaemia, anorexia, weight loss, transplacental infection and even death in naive individuals. Imidocarb dipropionate, a carbamate, is the drug of choice for the treatment of equine piroplasmosis [7, 8] but severe colic and diarrhoea may occur due to cholinesterase inhibition [9, 10]. Concurrent administration of atropine  or glycopyrrolate  has been advocated to minimise or prevent the side effects observed with imidocarb treatment.
Imidocarb administration causes a significant increase in frequency of defaecation, total faecal output and faecal water content in horses , effects ameliorated by premedication with glycopyrrolate or atropine. However, semi-quantitative assessment of gastrointestinal motility using transabdominal ultrasonography did not reveal significant differences in either small or large intestinal motility in individuals administered imidocarb . Thus, neither the specific impact of imidocarb on equine orocaecal transit time (OCTT) nor its interaction with glycopyrrolate and atropine have been quantified.
The induced lactose 13C-ureide breath test has been validated against gastroenterocolonic scintigraphy for the measurement of OCTT in people  and, more recently, has been validated in vitro for OCTT measurement in horses . The underlying principle of the lactose 13C-ureide breath test is that enzymatic splitting of the glycosylureide moiety of the stable isotope is performed only by intestinal microbes in the large bowel. Subsequent hydrolysis of the released 13C-urea component results in rapid liberation of stable 13CO2 and its appearance in the bicarbonate pool. As glycosylureide cleavage is the rate-limiting step in this process, mass spectrometric analysis of the 13C:12C ratio in expiratory breath after ingestion of the labelled test meal provides an indirect measurement of OCTT .
Using the lactose 13C-ureide breath test to measure OCTT, one aim of this study was to improve our knowledge of the specific effects of imidocarb treatment on equine small intestinal transit and its interaction with the anti-cholinergic compounds atropine and glycopyrrolate. The goal of quantifying the effects of these drugs was to validate an imidocarb protocol for treatment of equine piroplasmosis that resulted in minimal short-term disruption to gastrointestinal motility and function.
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In this prospective randomised study, 4 of 6 healthy horses developed signs of colic together with tachypnoea, tachycardia, increased intestinal borborygmi and an increase in total faecal output when administered a standard i.m. dose of imidocarb accompanied by saline. The symptoms started approximately 30 min after drug administration and lasted up to 5 h, also coinciding with the presence of projectile diarrhoea. The occurrence of both colic  and diarrhoea  after imidocarb administration has been observed previously and was reported to be of similar duration . This duration of systemic side effects observed following i.m. administration of imidocarb correlates to its peak plasma concentration, with a decline measured after 2–4 h . The significant increase in borborygmi noted in the imidocarb/saline group also was in agreement with the ‘violent peristalsis’  or hypermotility of the gastrointestinal tract  previously associated with the cholinergic properties of imidocarb.
Given the clinical evidence that imidocarb caused increased large intestinal motility, it was anticipated that its administration also would cause a significant reduction in OCTT. Cholinergic agents increase jejunal contractility in vitro by increasing both circular and longitudinal smooth muscle activity . However, there was no significant difference in OCTT between the imidocarb/saline or saline control groups in this study, suggesting that there was minimal effect of imidocarb (2.4 mg/kg bwt) on gastric emptying and subsequent small intestinal transit of the labelled test meal. This selective difference in the action of imidocarb on the equine small and large intestinal tract has not been reported previously.
In contrast to combining glycopyrrolate with imidocarb, the combination of atropine at 0.035 mg/kg bwt and imidocarb resulted in variable and often extreme prolongation of OCTT and caecal t1/2, with significant reduction in orocaecal movement of the test bolus when compared with all other test groups. Intestinal borborygmi were reduced significantly in both the imidocarb/glycopyrrolate and imidocarb/atropine groups when compared with the saline control, but this effect was most marked after atropine administration, both in terms of borborygmi score and duration of effect. Individual animal response to atropine also was more variable than to glycopyrrolate. Atropine at the dose used in this study has been shown previously to cause a profound reduction in the rate of solid phase gastric emptying , which also is likely to result in prolongation of OCTT.
Both glycopyrrolate (0.0025 mg/kg bwt) and atropine (0.035 mg/kg bwt) when given i.v. prevented the immediate signs of colic associated with concurrent imidocarb administration in this study. This was in contrast to previously reported findings  that atropine at 0.02 mg/kg bwt did not prevent imidocarb-related colic. The difference in the results of the 2 studies is likely to be due to dose-dependent effects of atropine on equine large intestinal motility. Atropine given at 0.044 mg/kg bwt has been shown to cause a significant reduction in large intestinal motility and development of impactions . Thus, the 0.035 mg/kg bwt dose of atropine used in this study may be the optimum dose to inhibit the action of imidocarb on large intestinal motility while minimising potential side effects. However, as found in this study and previously reported , the 0.035 mg/kg bwt atropine dose will have a deleterious effect on gastric emptying rate and potentially small intestinal motility and may be linked to subsequent colonic impaction .
In the present study, glycopyrrolate (0.0025 mg/kg bwt) successfully prevented the development of diarrhoea and colic associated with imidocarb administration, without causing significant prolongation of OCTT. A small, but significant, reduction in large intestinal borborygmi scores was detected in the imidocarb/glycopyrrolate group compared with the imidocarb/saline group but this was less marked than recorded in the imidocarb/atropine group. This same dose of glycopyrrolate has been previously reported to be sufficient to reduce the cardiovascular effects of xylazine without causing clinical detriment to large intestinal motility [21, 28]. The results of the present study suggest that glycopyrrolate blocks the inhibition of cholinesterase induced by imidocarb  without affecting gastric emptying rate or small intestinal motility. This is in contrast to atropine, which counters the clinical effects of imidocarb administration but at the cost of significant detriment to both small and large intestinal transit. The potential difference found in this study between the effects of the 2 parasympatholytic compounds on equine intestinal motility has not been previously reported and is worthy of further investigation.
The administration of imidocarb/atropine led to a tachycardia of up to 3 h duration, in agreement with effects of atropine described elsewhere , whereas subjects were tachycardic for a maximum of 1 h following imidocarb/glycopyrrolate treatment. This may indicate that atropine 0.035 mg/kg bwt is of greater systemic duration than glycopyrrolate 0.0025 mg/kg bwt in horses.
The lactose 13C-ureide breath test proved sufficiently sensitive in this study to detect significant differences in OCTT caused by drug administration and was a useful diagnostic tool that was simple to perform. Although several digestive processes might contribute to the OCTT, the test results were well described by a one-curve model and a two-curve model of fit was not required .
The lactose 13C-ureide breath test has been validated using in vitro studies for the measurement of OCTT in horses  but has not been reported previously in applied pharmacological studies. Limitations of the stable isotope breath tests in horses might include natural fluctuations in basal 13CO2 production during the test period . This was minimised by removing dietary variations during the study period and by avoidance of changes in exercise level . However, the habitual consumption of C4-rich kikuyu grass by subjects between test days meant that the δ 13C value of the test meal was lower than that of the standard diet. A preliminary study in the test subjects revealed a tendency for the expiratory 13C:12C ratio to fall with time under test conditions after consumption of the unlabelled test meal. This occurred in all subjects with minimal variation and may have been caused by metabolism of the test meal. A progressive decline in basal 13CO2 production could have resulted in uniform overestimation of transit times by the lactose 13C-ureide breath test and values for OCTT in this study were higher than those found in a previous study . Increased enrichment of the test meal with the 13C-isotope would have further reduced any inaccuracies in parameter measurement but was prohibitively expensive for this study. Although direct comparison of drug effect on intestinal transit parameters was not considered to be affected by this technical issue, use of a larger number of subjects would have enhanced the statistical power of the study.
The study was performed during the drought season in the north of South Africa, during which a peak in impaction colics is common in equine practice. However, the randomisation of drug therapies with time was sufficient to remove this potential confounding factor from the interpretation of test results.
It is concluded from this study that imidocarb has deleterious effects on large bowel motility with production of diarrhoea and colic in a majority of animals. As imidocarb/saline did not cause a significant reduction in OCTT, these side effects of imidocarb likely result principally from induction of large intestinal hypermotility rather than from modulation of small intestinal motility. Atropine and glycopyrrolate each prevented these side effects of imidocarb in this study but atropine was associated both with prolongation of OCTT and a significant reduction in large intestinal motility that was profound in certain subjects. Glycopyrrolate in contrast prevented the extreme side effects of imidocarb at therapeutic doses without causing significant changes to OCTT as compared with control individuals. Therefore, based on the drugs evaluated in this study, an effective protocol to ameliorate the gastrointestinal side effects observed with imidocarb is to administer a concurrent dose of glycopyrrolate at 0.0025 mg/kg bwt.