Trial design, compliance and ethics approval
This trial was designed according the guidelines of evidence-based medicine  and reported utilizing the Consolidated Standards of Reporting Trials (CONSORT) -. The study design is a prospective, single-blinded, two-arm parallel clinical dosage determination field trial. The study complied with the European Economic Community Guideline on the demonstration of efficacy for veterinary medicinal products containing antimicrobial substances. Both the Ethics Committee for Animal Experiments of the University of Helsinki in Finland and the National Animal Experiment Board in Finland approved the study protocol. The owners provided their written informed consent in which they agreed to participate with their dog.
Study population, inclusion and exclusion criteria
This study was conducted at the Small Animal Teaching Hospital, Faculty of Veterinary Medicine, University of Helsinki, Finland. The study population was recruited from the patient material of a placebo-controlled, randomized, double-blinded, prospective clinical trial on the effectiveness of oral tylosin in treating recurrent diarrhea in dogs, that was conducted just prior to this current dosage determination trial . Dogs were considered eligible for the oral tylosin dosage determination trial if they responded to a tylosin treatment at a dosage of 25 mg/kg once daily for seven days in the aforementioned trial. Inclusion criteria were that the dogs were at least six months old and had been suffering from recurrent diarrhea responding to tylosin treatment. They were not allowed to have received systemic corticosteroids, nonsteroidal anti-inflammatory drugs, or antibiotics other than tylosin in the 90 days preceding the tylosin dosage determination trial. Lactating bitches were excluded, as were dogs with evidence of systemic or organ-related disease that could secondarily cause diarrhea.
The dogs underwent an initial examination prior to enrollment in the tylosin effectiveness trial to ensure that they met the selection criteria. In brief, the initial examination covered the dogs’ clinical history and physical examination, blood samples for a complete blood count and serum biochemical profile, urinalysis, fecal analysis for endoparasites and enteropathogenic bacteria, and gastroduodenoscopy with mucosal biopsies from the duodenum. The protocol and the results of the initial examination have been published previously .
The primary objective of the current trial was to assess whether daily doses of 5 mg/kg or 15 mg/kg tylosin at a once daily dosing interval for seven days would have similar effects on the fecal consistency in diarrhea relapses to that of an initial dose of 25 mg/kg tylosin once daily for seven days in these dogs, a dosage that has proved effective in controlling canine TRD . Further to assess whether the tylosin dose 5 mg/kg was as effective as the 15 mg/kg dose. To evaluate this, the elimination of diarrhea served as the main criterion. The elimination of diarrhea was fulfilled when noting that on three consecutive days the fecal score for each of the dog’s feces was and remained three or less. A mean fecal consistency score assigned during the last three days of the seven-day treatment period served as the primary outcome measure. To determine the mean fecal consistency score, the owners evaluated and recorded throughout the study the consistency of each of their dog’s stool according to a standardized fecal scoring system previously established by Moxham . This kind of fecal scoring system has proved useful in evaluating treatment success in diarrhea patients ,,-. In brief, the fecal scoring system is based on a nine-point scale, consisting of scores from one to five, with half-point intervals. To ensure uniformity in the fecal consistency scoring, it comprises both a verbal description and a visual picture for each of the fecal consistency scores. Scores 1 and 1.5 represented feces of a hard and dry consistency. Scores 2 and 2.5 indicated well-formed feces that left no mark when picked up. A score of 3 represented feces of a slightly moist consistency, whereas score 3.5 represented feces of a moist consistency, but which still had some definite form. Scores 4 through 5 described feces of poor consistency.
To evaluate whether the tylosin dosage was effective, the investigator calculated a mean fecal consistency score of the scores assigned for each of the dog’s feces during the last three days of the seven-day treatment period based on the owner’s records. A responder was defined as having a mean fecal consistency score of three or less; a non-responder had a score of more than three. The owners were unaware of these definitions.
Secondary outcome measures were defined in order to evaluate the recurrence rate and the time to recurrence of diarrhea at a 25 mg/kg dosage of tylosin. The recurrence of diarrhea was defined as when the dog’s feces had a consistency score of at least four on the scale for at least two consecutive defecations. Further secondary outcome measures were defined to assess the influence of the three different tylosin dosages (5, 15 and 25 mg/kg) on the time of ceasing of diarrhea. The day on which the diarrhea ceased was defined as the day of the treatment period on which the fecal score for each of the dog’s feces was and remained three or less.
When assessing the dogs’ eligibility for this present dosage determination trial all dogs had diarrhea and began their tylosin treatment at a dose of 25 mg/kg once daily for seven days. The dog owners evaluated and recorded the consistency of each of their dogs’ feces. On day seven of the treatment period each dog underwent a physical examination and its fecal consistency records were collected. The investigator recorded the day on which the dog’s diarrhea ceased and calculated a mean fecal consistency score for each dog over the last three days of the treatment period at a tylosin dosage of 25 mg/kg; the dogs defined as responders were deemed eligible to participate in the dosage determination trial. Treatment at 25 mg/kg tylosin was then discontinued for the participating dogs and a follow-up period of up to two months served to determine whether the diarrhea recurred. No additional changes were permitted in the dog’s feeding management or medication. Upon meeting the criteria for the recurrence of diarrhea (fecal score ≥ 4 for at least two consecutive defecations) each dog underwent a physical examination, during which the investigator also assessed and confirmed the fecal consistency by rectal palpation. The dogs then received tylosin (Tylosin tartrate 120 mg and 240 mg tablets, University Pharmacy, Helsinki, Finland; equivalent to 100 mg and 200 mg tylosin) once daily for seven days in doses of either 5 mg/kg or 15 mg/kg.
During the seven-day treatment period, the owners continued evaluating each of their dog’s stools and recorded its fecal consistency according to the guidelines. On day three, the investigator phoned the owner to assure whether the clinical condition of the dog met the criteria of the humane endpoint of the study and whether the dog had to be released from the study and treated accordingly. A humane endpoint was defined as the point at which the dog’s clinical condition was poor and it suffered from excessive diarrhea. On day seven, the dog underwent a second physical examination, including rectal palpation and an evaluation of the dog’s fecal consistency. The fecal consistency records were collected, and a mean fecal consistency score was calculated for each dog based on the last three days of the treatment period at the 5 mg/kg or 15 mg/kg dosages.
Allocation to treatment groups
After the onset of diarrhea within the two-month follow-up period the dogs received an ascending study number denoting the order in which the diarrhea randomly recurred. The investigator then assigned the dogs in a 1:1 ratio to two different dosage groups - either 5 mg/kg or 15 mg/kg - receiving tylosin orally once daily for seven days. All dogs with odd study numbers were assigned to the 5 mg/kg dosage group, and those with even study numbers to the 15 mg/kg dosage group.
The study was single-blinded and thus open to the investigator but blinded for the dog owner. The blinding procedure was carried out by labeling the 120 mg tylosin tartrate tablet bottles as “Tylosin tart. S” and the 240 mg tylosin tartrate tablet bottles as “Tylosin tart. V”. The tablets were visually identical.
Descriptive statistics were used to summarize the data. The Shapiro-Wilk test served to determine whether the data were normally distributed; non-parametric data were expressed as median and range. The Wilcoxon signed rank test was used for non-parametric data to compare the mean fecal consistency scores of the last three days of the treatment period at the 25 mg/kg tylosin dosage to the scores at the 15 mg/kg and 5 mg/kg tylosin dosages. The Mann–Whitney U test was used to examine differences between the mean fecal consistency scores in the 5 mg/kg and 15 mg/kg tylosin dosage groups. To evaluate the effect of each dosage on the time until the diarrhea ceased, the Wilcoxon signed rank test and the Mann–Whitney U test were used. The level of statistical significance was set at P < 0.05; all statistical analyses were performed with a commercially available statistical software system (SPSS 18.0 for Windows).