Tylosin responsive chronic diarrhea in dogs westermarck et al 2005 journal of veterinary internal me

J Vet Intern Med 2005;19:177–186

Tylosin-Responsive Chronic Diarrhea in Dogs Elias Westermarck, Teresa Skrzypczak, Jaana Harmoinen, Jo¨rg M. Steiner, Craig G. Ruaux, David A. Williams, Erkki Eerola, Pernilla Sundba¨ck, and Minna Rinkinen Fourteen dogs had shown chronic or intermittent diarrhea for more than 1 year. Diarrhea had been successfully treated with tylosin for at least 6 months but recurred when treatment was withdrawn on at least 2 occasions. Tylosin-responsive diarrhea (TRD) affects typically middle-aged, large-breed dogs and clinical signs indicate that TRD affects both the small and large intestine. Treatment with tylosin eliminated diarrhea in all dogs within 3 days and in most dogs within 24 hours. Tylosin administration controlled diarrhea in all dogs, but after it was discontinued, diarrhea reappeared in 12 (85.7%) of 14 dogs within 30 days. Prednisone given for 3 days did not completely resolve diarrhea. Probiotic Lactobacillus rhamnosus GG did not prevent the relapse of diarrhea in any of 9 dogs so treated. The etiology of TRD, a likely form of antibiotic-responsive diarrhea (ARD) is unclear. The following reasons for chronic diarrhea were excluded or found to be unlikely: parasites, exocrine pancreatic insufficiency, inflammatory bowel disease, small intestinal bacterial overgrowth, enteropathogenic bacteria (Salmonella spp., Campylobacter spp., Yersinia spp., or Lawsoni intracellularis), and Clostridium perfringens enterotoxin and Clostridium difficile A toxin. A possible etiologic factor is a specific enteropathogenic organism that is a common resident in the canine gastrointestinal tract and is sensitive to tylosin but difficult to eradicate. Additional studies are required to identify the specific cause of TRD. Key words: Clostridium perfringens enterotoxin; Fecal alpha-1-proteinase inhibitor; Prednisone; Probiotic Lactobacillus rhamnosus GG; Serum unconjugated bile acid.

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hronic enteropathies in dogs are treated with a variety of antibiotics (eg, tetracycline, metronidazole, ampicillin, and tylosin), and therefore a new term for diarrheal disorders responding to antibiotic therapy, antibiotic-responsive diarrhea (ARD), has been coined.1 This disorder has been reported to be most typical of German Shepherd Dogs with small-bowel diarrhea.2 Controversy exists over the term ARD and the term idiopathic small intestinal bacterial overgrowth (SIBO); whether these terms are interchangeable or not is not clear. In Finland, tylosin has been used almost exclusively to treat this chronic or intermittent diarrhea, and therefore the disorder has been called tylosinresponsive diarrhea (TRD). Tylosin is a macrolide antimicrobial widely used as a feed additive in food animal production. Its antimicrobial activity is targeted against facultative and obligate anaerobic gram-positive bacteria. In addition, some gram-negative bacteria are sensitive to tylosin.

However, the gram-negative bacteria Eschericia coli and Salmonella spp. are intrinsically tylosin-resistant.3 Tylosin is used in powder form for pigs and poultry. In Finland and some other countries, tylosin also was available in tablet From the Department of Clinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland (Westermarck, Harmoinen, Rinkinen); the National Veterinary and Food Research Institute, Helsinki, Finland (Skrzypczak); the Gastrointestinal Laboratory, Texas A&M University, College Station, TX (Steiner, Ruaux, Williams); the Department of Medical Microbiology, University of Turku, Turku, Finland (Eerola); and the Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland (Sundba¨ck). The study was presented as an oral abstract at the 13th ECVIM-CA Congress in Uppsala, Sweden, 2003.

Reprint requests: Elias Westermarck, PhD, Department of Clinical Veterinary Sciences, Faculty of Veterinary Medicine, P.O. Box 57, 00014 University of Helsinki, Helsinki, Finland; e-mail: elias.westermarck@helsinki.fi.

Submitted June 30, 2004; Revised September 10, October 6, 2004; Accepted November 6, 2004.

Copyright q 2005 by the American College of Veterinary Internal Medicine 0891-6640/05/1902-0005/$3.00/0

form. However, the manufacturer ceased production of tablets several years ago. Helsinki University Pharmacy and other compounding pharmacies subsequently have started making capsules containing tylosin powder. The primary aims of this study were to objectively assess the characteristic clinical and clinicopathologic findings in dogs with TRD and to evaluate the effectiveness of tylosin in treating diarrhea. Special attention was given to determining whether the findings justify the speculation that TRD is an independent disease entity with a common etiology.

Certain enteropathogenic bacteria, such as Clostridium perfringens and campylobacters, have been suggested to play a role in the etiopathogenesis of chronic or intermittent diarrhea, and these bacteria are sensitive to tylosin.4 A lessrecognized enteric pathogen is Lawsonia intracellularis, an obligate intracellular bacterium that causes proliferative enteropathy (PE) and is known to infect a wide variety of host animals. PE most often has been described in pigs, but it is increasingly also diagnosed in horses and occasionally in dogs.5–7 Tylosin is reported to be an effective drug against L intracellularis.8 Because microbial resistance to antibiotics is an expanding problem, efforts have been made to reduce the use of antibiotics, both in veterinary and human medicine. Certain probiotic lactic acid bacteria (LAB) have been shown to be effective in the prevention and treatment of a variety of diarrheal disorders in humans and in experimental mouse models.9,10 Another aim of the present study thus was to evaluate whether probiotic LAB (Lactobacillus rhamnosus GG [LGG]) could be used instead of tylosin to treat or prevent the relapse of chronic diarrhea in dogs with TRD. Antibiotics such as metronidazole and ciprofloxacin are documented to have beneficial immunomodulatory effects on gut mucosa, and to alleviate chronic inflammation in inflammatory bowel disease (IBD).11,12 Many macrolide antibiotics also have immunomodulatory effects.13 Tylosin has been reported to have a positive effect in the treatment of chronic canine enteropathies resembling IBD. However, its exact mode of action in IBD is unknown.14 In addition to having antibacterial properties, tylosin may have anti-inflammatory effects that contribute to its effectiveness in

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treating diarrhea in dogs. If the putative anti-inflammatory or immunomodulatory properties play an important role in treating dogs with TRD, corticosteroids also should have a beneficial effect in dogs with TRD. To test this hypothesis, the dogs in this study also were treated with prednisone and the effect of treatment was evaluated.

To date, no extensive studies concerning the use of tylosin in the treatment of chronic or intermittent diarrhea in dogs have been published. Anecdotal reports by veterinarians and dog owners suggest that tylosin is effective in the treatment of diarrhea, but relapses are common.

Materials and Methods Dogs Fourteen pet dogs were selected for the study. All of the dogs were from a geographical area close to the Faculty of Veterinary Medicine of the University of Helsinki. The dogs were of 12 breeds (3 German Shepherd Dogs and 1 each of Black Newfoundland, White Newfoundland, Doberman Pinscher, Red Irish Setter, Australian Terrier, Siberian Husky, Giant Schnauzer, Shetland Sheepdog, Golden Retriever, Cocker Spaniel, Whippet, and a mixed-breed dog). The dogs all were adults (ranging in age from 2 to 12 years, median 8.5 years). The dogs included 9 males and 5 females. Dogs weighed from 7 to 58 kg (median, 34 kg).

Each dog’s diet during the trial was considered by the owner to be the most suitable for the individual dog and the diets remained unchanged throughout the study.

The dogs fulfilled the following selection criteria. Each dog had chronic or intermittent diarrhea for a minimum of 1 year, which was successfully treated with tylosin for at least 6 months. When tylosin treatment was discontinued, dogs developed diarrhea, which again responded well to tylosin treatment. When the study commenced, all dogs had been on tylosin for at least 1 month and were considered otherwise healthy.

Medication The dosage of tylosina varied between 6 and 16 mg/kg, with a mean dosage of 11.7 mg/kg PO q24h. The dosage of tylosin was not changed during the study from the dogs’ individual pretrial dosage. The dosage of prednisoneb was 1.5 mg/kg PO q24h and that of probiotic LGGc was 1 capsule PO q12h, with each capsule containing 5 3 109 colonyforming units (CFU) of LGG.

Study Protocol All dogs were treated with tylosin and were asymptomatic at the time of enrollment. For the 1st part of the study, tylosin therapy was continued for a period of 2 weeks (tylosin period I; Fig 1). Thereafter, tylosin was discontinued and the dogs were monitored for a period of up to 1 month to determine whether signs of diarrhea would reappear, as suggested by the clinical history. Dogs that did not develop diarrhea during this time were released from the study. Dogs that did develop diarrhea for a period of 3 days (diarrheal period I) moved on to the 2nd part of the study, in which another 2-week tylosin course was initiated (tylosin period II). Dogs were released from the study if they failed to respond to tylosin. After tylosin was discontinued, probiotic therapy was administered to assess its effectiveness in preventing the reappearance of diarrhea. Dogs were followed for a period of up to 1 month. Dogs that did not develop diarrhea during this period were released from the study. Dogs that developed diarrhea for a period of 3 days (diarrheal period II) moved on to the 3rd part of the study, in which prednisone was administered for 3 days (prednisone period). If diarrhea resolved during prednisone treatment, the dogs were released from the study. If diarrhea persisted for 3 days, prednisone treatment

was discontinued and tylosin treatment was reinitiated. After 2 weeks of successful tylosin treatment, the study was completed.

Examinations Performed during the Study Assessment of Clinical Status. During the study, dog owners assessed the severity of the following clinical signs daily: alertness (normal or reduced), appetite (normal, reduced, or considerably reduced), drinking (normal, reduced, or increased), borborygmus (no, occasionally, or often), flatulence (no, occasionally, or often), and vomiting (no, occasionally, or often). Fecal consistency during each defecation was scored on a 9-point scale, from 1 (hard and crumbly) to 5 (watery diarrhea), with half-point increments (Table 1). Instructions on how to evaluate consistency were provided to dog owners both in photo and written form. Grades 4.5 and 5 were classed as unacceptable. Grade4 feces were of poor quality; they were moist and poorly formed with a consistency of putty or porridge. Grade 3 represented feces of good quality that were slightly moist. Ideal feces were those that could be easily picked up and did not leave any mark (grades 2 and 2.5).

Fecal Examination. Dog owners collected a fecal sample every other day for the entire duration of the study. During periods of diarrhea, owners collected a fecal sample on a daily basis. Samples were immediately frozen at 2208C after collection.

Examination for Parasites. Three fecal samples were examined by flotation method and were evaluated for the presence of Giardia antigen by using an enzyme-linked immunosorbent assay (ELISA) kit.d Measurement of Fecal Alpha-1-Proteinase Inhibitor Concentration. Fecal samples from the last 3 days of tylosin treatment periods and 3 consecutive defecations during the diarrheal periods and the prednisone treatment period were collected. The samples were collected into preweighed tubes and immediately frozen at 2208C. The samples were shipped on dry ice to the Gastrointestinal Laboratory at Texas A&M University for measurement by using an in-house, species-specific ELISA.15 Evaluation of Fecal Bacterial Flora. Changes in fecal bacterial flora were studied by computerized gas-liquid chromatography (GLC) of bacterial fatty acids as described earlier.16 In brief, bacterial fatty acids were extracted from the stool samples and measured by GLC. The fatty acid profiles obtained by GLC were evaluated by computer analysis, thus reflecting the associated microbiota.17,18 From 4 dogs, 20–26 fecal samples/dog were collected during the study, and fecal fatty acid profiles were analyzed. For each of these dogs, the fecal fatty acid profiles were compared with those from samples collected on day 1, and results were expressed as similarity percentages. In addition, the fecal fatty acid profiles during tylosin treatment (9–12 samples/dog) and during the days with no tylosin treatment (10–16 samples/dog) were compared.

Examination for Enteropathogenic Bacteria. From the 9 dogs that completed the whole study, fecal samples during tylosin therapy and after 3 days of diarrhea were collected manually from the rectum and transferred to special bacterial transport media.e The samples then were transported to the laboratory and processed within 2 hours after collection. Each sample was cultured for Salmonella spp., Campylobacter spp., Yersinia spp., and C perfringens by using specialized culture media.

For detection of Salmonella, 1 g of feces was inoculated into 1% Rappaport-Vassiliadis-soyapeptonef for enrichment and incubated for ¨ no¨z agarg and 24 hours at 378C. Incubated samples were cultured on O xylose-lysine-decarboxylase agarh and incubated for 24–48 hours at 378C. Typical colonies were subcultured on biochemical media according to the identification schema used by our diagnostic laboratory.

For detection of Campylobacter, samples were cultured on a campylobacter blood-free selective medium (modified CCDA–Preston)i and incubated microaerophilically at 378C. Plates were examined for growth at 48–96 hours. Suspected colonies were subcultured on 5% fastidious anaerobe agar.j Tests for catalase, oxidase, nitrate, hippurate hydrolysis, sensitivity to 30-mg disks of cephalothink and nalidixin,l

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Fig 1. Study protocol.

and growth in a microaerophilic atmosphere at 258C and 428C were performed. Isolates were identified as Campylobacter jejuni or Campylobacter spp. organisms only. For detection of Yersinia, samples were cultured on cefsulodin-irgasan-novobiocin agarm and incubated at 308C for 2 days. Typical colonies were subcultured on biochemical media according to the identification schema used by our diagnostic laboratory. For detection of C perfringens, samples were cultured on trypticase soy agar (TSA) supplemented with 5% defibrinated bovine bloodn and incubated anaerobically at 378C for 48 hours. Presumptive identification of C perfringens was based on the presence of a double zone of hemolysis, lecithinase production, positive reverse Christie, Atkins, Munch-Petersen (CAMP) test, and large non–spore-forming ‘‘boxcar’’-shaped gram-positive bacilli. Typical colonies were cultured

on egg yolk agar plateso for lecithinase production and TSA with Streptococcus agalactiae for the CAMP test, and incubated anaerobically at 378C for 2 days. CAMP-positive colonies, which produced lecithinase, were identified as C perfringens.

During tylosin therapy and after diarrhea for a 3-day period, 1–3 fecal samples/dog were examined for the presence of C perfringens enterotoxin (CPE) and C difficile toxin A. The presence of these toxins was measured by using ELISA methods.p,q The ELISA test for measurement of CPE also was evaluated in a semiquantitative fashion by measuring optical density (OD) by using spectrophotometry at a wavelength of 450 nm (OD450).

For 4 dogs, fecal samples during diarrheal periods were examined for detection of L intracellularis DNA by polymerase chain reaction

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Table 1. Feces scoring system.

Grade 1 1.5 2 2.5

3 3.5 4 4.5 5

Description Hard, dry, and crumbly; ‘‘bullet like’’ Hard and dry Well formed; does not leave a mark when picked up; ‘‘kickable’’ Well formed with slightly moist surface, which leaves a mark when picked up; ‘‘almost sticky to touch’’ Moist, beginning to lose form, leaving a definite mark when picked up Very moist, but still has some definite form The majority, if not all the form is lost, poor consistency, viscous Diarrhea, with some areas of consistency Watery diarrhea

(PCR), by using primers as described elsewhere.19 Serum samples from these dogs were studied by using an indirect fluorescent antibody test (IFAT) to detect serum immunoglobulin G directed against L intracellularis.6

same procedure was followed when testing for differences in SUBA between tylosin treatment periods and diarrheal periods. The Wilcoxon signed rank test also was used when evaluating the differences in the time interval to the appearance of diarrheal signs when tylosin treatment ceased compared with when dogs were treated with probiotic therapy. The difference in the proportion of positive CPE samples between tylosin and diarrheal periods was tested by using the chisquare test. The Mann-Whitney test was used to test for differences in CPE concentrations and in bacterial diversity between samples taken during tylosin treatment and during diarrheal periods. Results are expressed as medians. P-values less than .05 were considered significant.

Results Of the 14 dogs enrolled, 9 completed the study. Two dogs were excluded after the 1st part of the study because no diarrhea developed during the 1-month follow-up. After the 1st diarrheal period and before the probiotic treatment was started, 3 dog owners withdrew their dogs for personal reasons (eg, divorce or moving). The clinical history data results include information on all 14 dogs enrolled. All other data include information on only the 9 dogs that completed the study.

Blood Samples Blood samples were collected during tylosin therapy (tylosin periods I and II) and after a 3-day diarrheal period (diarrheal periods I and II).

Clinical Pathology. CBC and serum alanine transaminase and alkaline phosphatase (ALP) activity; urea, creatinine, glucose, total protein, and albumin concentrations; and serum sodium and potassium concentrations were determined.

Serum Concentrations of Vitamins and Trypsinlike Immunoreactivity (cTLI). Serum concentrations of cobalamin and folate were measured by competitive immunoassay methods.r,s cTLI was measured by using a species-specific ELISA method.20 C-Reactive Protein (CRP). Serum concentrations of CRP were measured at the Gastrointestinal Laboratory at Texas A&M University by using a species-specific ELISA.t The minimal detection limit of the assay was determined to be 7 mg/mL. Serum Unconjugated Bile Acid (SUBA) Concentrations. SUBA concentrations were measured at the Gastrointestinal Laboratory at Texas A&M University by using gas chromatography–mass spectrometry after isolation by solid-phase extraction and anion-exchange chromatography.21

Endoscopic and Postmortem Examination Gastrointestinal (GI) endoscopy was performed in all dogs at the end of the study and endoscopic biopsies from stomach and duodenum were evaluated histopathologically.

Two dogs were euthanized approximately 1 year after the trial period ended. The dogs had continued the tylosin treatment after the trial had ended, and no diarrhea had been reported. The reason for euthanasia was a prostatic abscess in 1 dog and painful hip dysplasia in the other. The dogs were subjected to postmortem examination immediately after euthanasia, and full-thickness biopsies were taken from the duodenum, jejunum, ileocecal junction, and colon.

Statistical Methods For all 9 dogs completing the entire trial, the median alpha-1-proteinase concentrations during the tylosin treatment periods and during the diarrheal periods were recorded. Differences between these periods then were tested by the Wilcoxon signed rank test. The same method was used when comparing tylosin periods and prednisone period. The

Clinical History The clinical signs based on patient history of the dogs are listed in Figure 2. Abnormal loose fecal consistency was the predominant clinical sign, and the dogs showed a mixed pattern of small and large intestinal signs. The majority (9/ 14, 64%) of the owners described their dogs’ feces as watery, mucoid, bloody, or some combination of these. If the consistency of the feces varied during the diarrheal episodes, the owners were allowed to choose more than 1 alternative to describe the consistency. Increased defecation frequency and decreased fecal volume was noted in almost all dogs (11/14, 71%). Typical signs also included borborygmus (8/14, 57%) and flatulence (9/14, 64%). Less than 50% of the dogs vomited during the diarrheal outbreaks (6/ 14, 43%).

Typical age (10/14, 71%) at onset of diarrhea was 3–4 years (range 1–6 years). All of the dogs had suffered from diarrhea for a period of more than 1 year, most (9/14, 64%) for 3 years. The dogs had responded well and quickly to tylosin treatment. Most (13/14, 92%) showed marked improvement within 2 days of initiation of tylosin treatment.

If tylosin treatment was discontinued, diarrhea reappeared in most dogs (12/14, 85%) within 3 weeks and in the other dogs (2/14, 14%) after several months. In all dogs, tylosin treatment had been discontinued at least 2 times and in 7 dogs (50%) of 14 more than 4 times. The dosage of tylosin for long-term use had been tapered to the lowest possible dosage to control clinical signs. Of the 14 dogs included in the study, 6 were fed solely with commercial dog food, 5 were fed a homemade diet exclusively, and 3 were fed commercial dog food mixed with a homemade diet. When the dogs were not receiving tylosin, most (10/14, 71%) showed GI signs if the diet was changed. Conversely, only some dogs (4/14, 29%) showed GI disturbance due to dietary change when on tylosin treatment.

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Results of Fecal Examination

Fig 2. Clinical signs based on patient history of the dogs (n 5 14).

Clinical Status During the Trial During both tylosin treatment periods and at the end of the study, feces were firm (median score, 2.5; Fig 3). In the 2 diarrheal periods, feces often were watery, with median scores of 4.75 and 4.50. During prednisone treatment, the severity of diarrhea decreased, but the median fecal score was 3.25, indicating that fecal consistency was abnormally soft. When the tylosin treatment was started after 3 days of diarrhea, the diarrhea stopped in most dogs (6/9, 67%) in 1 day and in all dogs in 3 days. When tylosin was discontinued at the end of the 1st tylosin treatment period, diarrhea reappeared after 2–30 days (median 7 days; Fig 4).

The 2nd time that tylosin was discontinued and probiotic LGG was started, diarrhea reappeared in all 9 dogs after 3–26 days (median 7 days). Thus, probiotic LGG failed to prolong the asymptomatic period.

Changes in other clinical signs recorded by the dog owners occurred frequently but usually were mild. During the diarrheal periods, many dogs (5/9, 55%) appeared tired, had reduced appetite, and vomited occasionally. Flatulence was noted in 6 dogs (67%) of 9.

Parasites. No parasites were found by flotation, nor was Giardia detected by ELISA in any of the dogs at any time.

Fecal Alpha-1-Proteinase Inhibitor Concentration. The median alpha-1-proteinase inhibitor concentration was 1.35 mg/g during the 1st tylosin period and 14.70 mg/g during the 1st diarrheal period (Fig 5). In the 2nd periods, the medians were 1.57 mg/g and 7.73 mg/g, respectively. The difference between tylosin and diarrheal periods was statistically significant (P 5 .008). During the prednisone period, the median alpha-1-proteinase concentration was 3.92 mg/ g, which was significantly different (P 5 .028) from the median value during tylosin periods.

Changes in Bacterial Flora. Bacterial flora fluctuated during the trial in all dogs, with the median variation in fatty acid profiles describing fecal bacterial flora being 15% among samples taken from 4 dogs during tylosin treatment and 17% among samples taken on days with no tylosin treatment. No significant difference in fatty acid profiles was present between samples taken with and without tylosin treatment (P 5 .173) (Fig 6).

Enteropathogenic Bacteria. C jejuni was isolated from fecal samples of 2 dogs during diarrheal period I. C perfringens was isolated from 5 dogs during both diarrheal periods, and in 1 of these dogs, C perfringens also was isolated while the dog was being treated with tylosin. Neither Salmonella nor Yersinia spp. were present in any samples. No evidence of L intracellularis was found in fecal or serum samples from any of the dogs at any time point.

CPE was detected in 54% (7/13) and 64% (9/14) of the samples during tylosin periods and in 59% (10/17) and 44% (8/18) during diarrheal periods. The difference between these periods was not significant (P 5 .539).

The median CPE concentration (OD450) was 0.137 among samples taken during the 1st tylosin period and 0.134 among samples taken during diarrheal period I. During the 2nd tylosin and the 2nd diarrheal periods, the medians were 0.192 and 0.088, respectively. The difference between the tylosin and diarrheal periods was not statistically significant (P 5 .798; Fig 7). C difficile toxin A was found only in 1 diarrheal fecal sample. All samples from tylosin-treated dogs were negative for this pathogen toxin.

Results from Blood Samples Routine Clinical Pathology Parameters. Results of both hematologic and serum biochemistry tests measured throughout the study were within reference ranges in all dogs except 1 with abnormally high serum ALP activity (.2,000 U/mL) in all 4 samples. The reason for the high ALP activity in this dog was not clarified, because the dog was clinically healthy other than manifesting TRD signs during diarrheal periods. All dogs had total protein and albumin concentrations within the reference ranges for all samples.

Serum Concentrations of Vitamins and cTLI. Three dogs had abnormally low serum cobalamin concentrations.

Two of these dogs also had serum folate concentrations below the reference range, and the 3rd dog had an abnormally high serum folate concentration. These results were detected in the 1st evaluation, and remained unchanged for

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Fig 3. Median (maximum, minimum) grade of fecal consistency of 9 dogs during the different periods of the trial.

all the dogs for the duration of the trial. All the dogs had cTLI concentrations within the reference range.

CRP Concentrations. Serum CRP concentrations were above the detection limit in 3 (33%) of 9 during the 1st diarrheal period and in 1 (11%) of 9 samples during the 2nd diarrheal period. In all serum samples taken during

tylosin treatment, serum CRP concentrations were below the detection limit.

SUBA Concentrations. Median SUBA concentration was 209.6 nM during the 1st tylosin period and 94.6 nM during the 1st diarrheal period (Fig 8). During the 2nd periods, the medians were 139.7 nM and 107.0 nM, respec-

Fig 4. Cumulative number of dogs (n 5 9) with diarrheal signs after tylosin treatment was discontinued.

Fig 5. Fecal alpha-1-proteinase inhibitor concentration of dogs (n 5 9) during the 5 periods. Median values are connected by a line.

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Fig 6. Proportional changes in fecal bacterial flora compared with day 1. Daily values are for dog 2.

tively. The difference between tylosin and diarrheal periods was not significant (P 5 .441).

Endoscopic Examination and Postmortem Examination Findings Gastroduodenoscopy at the end of the study identified only mild changes for the duration of the study. In 3 dogs (3/9, 33%), duodenal biopsies indicated moderate lymphocytic-plasmacytic infiltration and mild distension of lymphtic vessels.

In both dogs that underwent postmortem examination, full-thickness biopsies of the intestine identified only minimal changes. No intracellular organisms were identified.

Discussion Tylosin, tetracycline, or metronidazole is recommended for the treatment of dogs suffering from chronic enteropathies.14 In the present study, the dogs were not treated with any antibiotic other than tylosin. It is unknown how the dogs would have responded to treatment with other antibiotics and consequently we prefer to use the name TRD instead of ARD. TRD may be considered a form of ARD. However, more studies are needed to assess similarities and differences between TRD and ARD. Tylosin is the most common drug used to treat chronic diarrhea of dogs in Finland, and the response to treatment has been very good anecdotally. Consequently, we studied the effects of tylosin in dogs with chronic diarrhea by using both indirect and direct methods.

Based on clinical histories and clinical findings, several characteristics of TRD were identified. TRD typically affects middle-aged, large-breed dogs. The clinical signs and serum folate and cobalamin concentrations indicate that both large and small intestine are involved. The response to tylosin was very rapid, and in 7 of the 9 dogs that completed the entire trial, diarrheal signs resolved within 24 hours. When dogs were on tylosin diarrhea ceased, but after tylosin treatment was discontinued, diarrhea reappeared within a median of 7 days.

To further characterize TRD and clarify the etiology of the disease, a number of different tests were performed. The

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Fig 7. Clostridium perfringens enterotoxin concentration in fecal samples (n 5 63) during 4 periods. Medians are shown by columns with upper and lower quartiles indicated by bars.

time lag between the discontinuation of medication and the relapse of signs suggests a bacterial etiology for TRD. Most of the common enteropathogenic bacteria were excluded as a causative agents for TRD based on negative culture results and results of ELISA, PCR, and IFAT tests. C jejuni was cultured in fecal samples from 2 dogs during 1 of the diarrheal periods. However, we consider this finding nonspecific because no Campylobacter spp could be isolated in fecal samples from the same dogs during the second diarrheal period. In addition, Campylobacter spp are commonly isolated in the feces of healthy dogs.22 C perfringens was cultured from fecal samples of 5 dogs during 1 period of diarrhea. However, C perfringens apparently can readily be cultured in .80% of dogs with or without diarrhea. Thus, isolation of this organism is not diagnostic of a cause-and-effect relationship.22,23 Detection of CPE is the most widely used diagnostic tool for diarrhea due to C perfringens in dogs because it is the only test that has been associated with the disease in dogs. On the other hand, asymptomatic dogs also have been documented to

Fig 8. Serum unconjugated bile acid (SUBA) concentrations of dogs (n 5 9) during 4 periods. Median values are connected by a line.

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have positive CPE assay results.22 In this study, no statistical difference was observed in the prevalence of CPE between tylosin treatment and diarrheal periods. Thus, CPE can be concluded not to be the cause of diarrhea in dogs with TRD. However, it is noteworthy that the ELISA used in our study is not validated for dogs, and its sensitivity and specificity are uncertain. Also, the measurement of CPE with optical density with spectrophotometry at a wavelength of 450 nm has not been validated as a semiquantitative method. Nevertheless, this immunoassay is considered the most reliable assay available for CPE diagnosis.23 The fecal samples also were examined for C difficile toxins, but the test results were positive only in 1 sample.

Only 40% of intestinal bacteria in humans are estimated to be culturable.24 Therefore, TRD may be caused by a bacterium not yet isolated or characterized. We also utilized molecular biological methods in our attempt to identify the causative agent of TRD. L intracellularis was considered a potential microbial cause of TRD because it is an obligate intracellular organism that has not been successfully grown on conventional cell-free media. It affects enterocytes in the ileum and colon and causes acute or chronic diarrhea in pigs, horses, and sometimes dogs.5,6 Because of the difficulty in isolating L intracellularis, it may be underdiagnosed as a cause of canine enteropathies. However, no L intracellularis organisms were found in the present study in PCR and IFAT tests or in the histologic examination of dogs subjected to postmortem examination.

In humans, measurement of SUBA concentrations has been shown to be a sensitive and specific diagnostic test for the diagnosis of SIBO.25 SUBA concentrations also are reported to be a suitable indirect test for SIBO in dogs.

SUBA concentrations are described as increasing 10- to 20fold in some dogs with SIBO.21 However, it recently has been documented that no significant differences occurred when duodenal bacteriology and SUBA concentrations were assessed before and during antibiotic therapy.26 In the present study, no increase occurred in SUBA concentrations during the times when the dogs had diarrhea. This observation does not preclude the presence of abnormal numbers of bacterial species that do not deconjugate bile acids. In addition, neither clinical signs nor colabalamin and folate assays suggested similarities between SIBO and TRD. Orally administered antibiotics have been documented to change the gut microbiota, but these changes are not permanent and normal gut microbiota soon is reestablished.27 No causative bacterial agent was detected in our study, which may reflect very subtle changes in the gut microbiota. Such changes may go undetected with traditional methods of culture. GLC analysis of fecal fatty acid profiles is reported to be a sensitive method for evaluating bacterial changes in feces.17 We observed no significant changes when tylosin-treated and untreated dogs were compared.

Tylosin is reported to affect bacterial adhesion to gut mucosa.13 Hence, the rapid and good response to tylosin may be due to the drug’s ability to interfere with pathogen adhesion, which does not alter the fecal fatty acid profile.

Bacterial adhesion is important for the pathogen to at least transiently colonize the host and cause clinical illness. Interference with bacterial adhesion properties may therefore reduce pathogenicity. The history of the dogs participating

in our study indicates that even when tylosin treatment is continued for many years, its effect on controlling TRD does not appear to diminish. The effective dosage of tylosin was on average lower than the dosage proposed in the literature.2 This observation may further support the hypothesis that the antidiarrheal effect of tylosin is based on impeded pathogen adhesion, because subtherapeutic doses of antimicrobials have been shown to affect bacterial adhesion.28 Additional studies are warranted to evaluate the potential effects of tylosin on bacterial adhesion.

Immunomodulatory effects of tylosin on gut mucosa also could explain the favorable effects of this drug in the treatment of TRD. IBD is a common cause of chronic and intermittent diarrhea in dogs.29 This disease is believed to result from an altered mucosal response to bacterial and dietary antigens in the gut, which leads to chronic inflammation.14 In the present study, however, only mild histopathologic inflammatory changes were observed in the intestinal biopsies taken during endoscopy or at postmortem examination. Nonetheless, histopathologic samples were collected when the dogs were on tylosin and therefore asymptomatic. Anti-inflammatory treatment with prednisone or another immunomodulator is one of the cornerstones of IBD therapy. In addition, certain antibiotics, such as metronidazole and fluoroquinolones, may possess immunomodulatory properties.30 We evaluated the ability of prednisone to control TRD signs. During the 3-day prednisone treatment, diarrhea did not resolve completely in any dog. Thus, prednisone was not as effective as tylosin in treating diarrheal signs. This finding implies that either TRD signs are not caused by IBD or the mechanism of tylosin action on the gut mucosa does not resemble that of prednisone. However, the fecal score improved somewhat during the 3-day prednisone period. This observation suggests that tylosin may have either direct or indirect (ie, bacteria-mediated) immunologic properties. The dogs were treated with a smaller dosage of prednisone than that generally recommended for large-bowel IBD in dogs.31 In addition, prednisone was only administered for 3 days, which may have been too short a treatment period for a successful outcome. This approach was mainly due to the fear of unwanted adverse effects related to prednisone, and dog owners were reluctant to give their pets doses of prednisone that could cause polydipsia and polyuria. Thus, a larger dosage of prednisone or a longer treatment period might have produced better control of TRD signs.

Evidence of an inflammatory response in TRD dogs was seen in view of serum CRP concentrations increasing in 3 dogs during diarrheal periods, but being undetectable during tylosin treatment. Although serum CRP cannot be used as a diagnostic test for IBD because it can be increased in other inflammatory conditions, it is useful in differentiating dogs with infectious or inflammatory diseases from healthy dogs.32 Some evidence of changes in the permeability of intestinal mucosa caused by TRD was observed because fecal alpha-1-proteinase inhibitor concentrations were significantly increased in dogs during diarrhea as compared with during tylosin treatment. This finding indicates that TRD is associated with increased permeability and GI protein loss, although serum protein and albumin concentrations were

Tylosin-Responsive Diarrhea

within the reference ranges for all dogs throughout the study.

Whether the cause for reduced permeability is due to inflammatory or microbial changes triggered by tylosin is a subject for future studies. Fecal alpha-1-proteinase concentration is reported to be a very sensitive measurement for GI protein loss.33,34 Feces contaminated with blood also can have increased alpha-1-proteinase concentration and thus if hematochezia is present, the alpha-1-proteinase results cannot be used to suggest increased GI protein loss.

Although no tylosin-associated adverse effects were recognized in any of the dogs during this trial, the use of antibiotic agents for extended periods should be discouraged. Microbial resistance to antibiotics is a growing problem. In humans, interest has emerged in the potential therapeutic use of probiotics in patients with IBD and SIBO. Certain probiotic LAB have been documented to down-regulate inflammatory response and increase antimicrobial activity in humans.35 In dogs, probiotic Enterococcus faecium has been reported to reduce fecal numbers of C perfringens36 and stimulate immune functions.37,38 However, because E faecium may be associated with some risks, it was not chosen for this study.39 Instead, we investigated the potential beneficial effects of LGG, a probiotic used in human patients.35 Unfortunately, LGG did not prevent the relapse of diarrhea in any of the dogs. Whether other probiotics or canine-derived LAB would have a beneficial effect in dogs with TRD remains to be determined.

In conclusion, no specific etiologic factor for TRD was identified. A likely factor is an enteropathogenic organism that is a common resident of the canine GI tract. It must be sensitive to tylosin, although in most cases it cannot be completely eradicated. Another explanation for the favorable response may lie in tylosin’s ability to interfere with bacterial adhesion to gut mucosa, thus preventing an as yet unidentified pathogen from colonizing the mucosa and causing diarrhea. In addition, tylosin may possess some immunomodulatory properties based on the notion that prednisone had a positive effect on the fecal consistency, despite a rather low dosage and short treatment period. Additional studies will be required to identify the definite etiology of TRD.

Footnotes Tylosin tartrate 240 mg capsules (equivalent to tylosin 200 mg), Yliopiston apteekki, Helsinki, Finland b Prednison 5 mg tablets, Orion Pharma Inc, Espoo, Finland c Gefilus capsules (5 3 109 CFU), Valio Inc, Helsinki, Finland d ProSpect Giardia Microplate Assay, Alexon-Trend, Ramsey, MN e Probact Transport Swab, Technical Service, Heywood, UK f Rappaport Vassiliadis medium, Lab, Lancashire, UK g ¨ Ono¨z agar, Merck, Darmstadt, Germany h Xylose-lysine-decarboxylase agar, Lab, Lancashire, UK i Modified CCDA–Preston medium, Lab, Lancashire, UK j 5% fastidious anaerobe agar, Lab, Lancashire, UK k Cephalothin, Oxoid, Hampshire, England l Nalidixin, Oxoid, Hampshire, England m Cefsulodin-irgasan-novobiocin, Lab, Lancashire, UK n Trypticase soy agar supplemented with 5% defibrinated bovine blood, BBL, Cockeysville, MD a

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Egg yolk agar prepared according to Summanen P, Baron EJ, Citron DM, et al. Appendix C: Preparation of media and reagents. In: Wadsworth Anaerobic Bacteriology Manual, 5th ed. Belmont, CA: Star Publishing;1993:168. p Techlab Clostridium perfringens Enterotoxin Test, Blacksburg, VA q Premier Clostridium difficile toxin A, Meridian Diagnostic Inc, Cincinnati, OH r IMMULITE Folic Acid, Diagnostic Products Corporation, Los Angeles, CA s IMMULITE Vitamin B12, Diagnostic Products Corporation, Los Angeles, CA t Canine C-Reactive Protein Assay, Tridelta Development Limited, Bray, Ireland o

Acknowledgments The study was supported by Hills’ Pet Nutrition, Inc. We thank veterinary students Eeva-Liisa Ahonen and Kaisa Lassila, who did their pregraduate work based on this project. Their contribution of keeping in touch with pet owners and collecting samples from dogs was invaluable. Dr Marjukka Anttila is acknowledged for examining fecal and serum samples for Lawsonia intracellularis. The work was done at the Department of Clinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, Finland.

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