Comparison of a chlorambucil-prednisolone combination with an azathioprine-prednisolone combination for treatment of chronic enteropathy with concurrent protein-losing enteropathy in dogs: 27 cases (2007–2010)

Julien R. S. Dandrieux School of Veterinary Science, University of Liverpool, Neston CH64 7TE, England.

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 Dr med vet, DACVIM
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Peter-John M. Noble School of Veterinary Science, University of Liverpool, Neston CH64 7TE, England.

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 BVM&S, PhD
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Timothy J. Scase Bridge Pathology Ltd, PO Box 2877, Bristol, BS8 9FH, England.

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Peter J. Cripps School of Veterinary Science, University of Liverpool, Neston CH64 7TE, England.

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Alexander J. German School of Veterinary Science, University of Liverpool, Neston CH64 7TE, England.

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Abstract

Objective—To compare treatment protocols for chronic enteropathy and concurrent protein-losing enteropathy that used prednisolone in conjunction with either azathioprine or chlorambucil in dogs.

Design—Retrospective case series.

Animals—27 dogs.

Procedures—All dogs had hypoalbuminemia (serum albumin concentration, < 18.0 g/L) and chronic enteropathy as diagnosed via complete gastrointestinal tract investigations including intestinal biopsy. Dogs received either an azathioprine-prednisolone combination (group A; n = 13) or a chlorambucil-prednisolone combination (group C; 14). Response to treatment was assessed by evaluation of body weight gain, serum albumin concentration, and duration of primary treatment.

Results—No significant pretreatment differences were detected between groups for any baseline variable (signalment and weight), clinicopathologic variable (albumin, cobalamin, and folate concentrations), or histopathologic findings. After treatment, serum albumin concentration and weight gain were significantly greater in group C. Median survival time for group A dogs was 30 days (95% confidence interval, 15 to 45 days) and was not reached for group C dogs. Duration of primary treatment was positively associated with the histopathologic presence of mild lacteal dilatation and use of a chlorambucil-prednisolone combination.

Conclusions and Clinical Relevance—Results suggested that a chlorambucil-prednisolone protocol is more efficacious for treatment of chronic enteropathy and concurrent protein-losing enteropathy, compared with an azathioprine-prednisolone combination. Given these findings, a prospective randomized clinical trial is warranted.

Abstract

Objective—To compare treatment protocols for chronic enteropathy and concurrent protein-losing enteropathy that used prednisolone in conjunction with either azathioprine or chlorambucil in dogs.

Design—Retrospective case series.

Animals—27 dogs.

Procedures—All dogs had hypoalbuminemia (serum albumin concentration, < 18.0 g/L) and chronic enteropathy as diagnosed via complete gastrointestinal tract investigations including intestinal biopsy. Dogs received either an azathioprine-prednisolone combination (group A; n = 13) or a chlorambucil-prednisolone combination (group C; 14). Response to treatment was assessed by evaluation of body weight gain, serum albumin concentration, and duration of primary treatment.

Results—No significant pretreatment differences were detected between groups for any baseline variable (signalment and weight), clinicopathologic variable (albumin, cobalamin, and folate concentrations), or histopathologic findings. After treatment, serum albumin concentration and weight gain were significantly greater in group C. Median survival time for group A dogs was 30 days (95% confidence interval, 15 to 45 days) and was not reached for group C dogs. Duration of primary treatment was positively associated with the histopathologic presence of mild lacteal dilatation and use of a chlorambucil-prednisolone combination.

Conclusions and Clinical Relevance—Results suggested that a chlorambucil-prednisolone protocol is more efficacious for treatment of chronic enteropathy and concurrent protein-losing enteropathy, compared with an azathioprine-prednisolone combination. Given these findings, a prospective randomized clinical trial is warranted.

Protein-losing enteropathy is a syndrome characterized by loss of albumin through the intestinal wall and has been reported in many breeds including Basenji, German Shepherd Dog, Rottweiler, Soft-Coated Wheaten Terrier, and Yorkshire Terrier.1 Various intestinal lesions can be responsible for the protein loss; primary and secondary lymphangiectesia have both been described as well as CEs characterized by inflammation of the intestines, infectious or parasitic diseases, and neoplastic diseases.1

When PLE arises secondary to CE, it is usually considered to have a guarded prognosis because of the unpredictable response to treatment and risk of relapse.1 Survival is usually poor with published reports estimating a 66% death rate within 5 months in 1 study on hypercoagulability in PLE-affected dogs2 and a 47% 1-year survival rate in Rottweilers with PLE.3 Recent research abstracts also report either a 32% mortality rate within 2 yearsa or up to a 58% risk of deathb,c in such cases. Two abstracts have reported mean survival times for dogs with PLE of 90 days (range, 2 to 2,544 days)d and 701 days.e However, specific details regarding the exact treatments used in those 2 studies were not reported.

Given that hypoalbuminemia is a negative prognostic indicator in cases of CE,4,5 most clinicians use aggressive immunosuppressive combination treatments in such cases. Although the efficacy of immunosuppressive treatment has been assessed in other diseases, including immune-mediated hemolytic anemia,6–9 atopic dermatitis,10 and anal furunculosis,11,12 information regarding efficacy of immune-suppressive treatment for CE is limited. Use of prednisolone alone, the combination of prednisolone and azathioprine,4,13 cyclosporine,14 and methotrexate15 have all been described, but none have been compared in an objective study. Treatment success in dogs with PLE secondary to CE can be short-lived with a combination of prednisolone and azathioprine or lead to unacceptable adverse effects (among which bone marrow suppression16 and pancreatitis17 are reported). Adverse effects have also been described with use of cyclosporine, including gastrointestinal tract signs and gingival hyperplasia,14,18 and cost can be prohibitive.

The use of azathioprine is not recommended in cats, given that this species has low activity of thiopurine methyltransferase, the enzyme that metabolizes azathioprine, which increases the risk of adverse effects.19 Instead, the alkylating agent chlorambucil has been widely used as an alternative in this species. This drug is usually well tolerated, and a favorable outcome has been reported in cases of intestinal small cell lymphoma.20,21 Despite the fact that this drug is used routinely for some dermatological22 and oncological23 conditions in dogs, the use of chlorambucil has not yet been reported for treatment of gastrointestinal tract diseases in this species.

For this reason, the purpose of the study reported here was to compare the use of a combination of prednisolone and chlorambucil with a combination of azathioprine and prednisolone for treatment of dogs with CE and concurrent PLE. The null hypothesis was that there would be no difference in the response of dogs to these 2 immunosuppressive protocols.

Materials and Methods

Case selection—Dogs referred to the internal medicine service of the Small Animal Teaching Hospital, University of Liverpool, for investigation of chronic gastrointestinal tract disease in a 3-year period between September 2007 and September 2010 were retrospectively reviewed. The aim was to include dogs with the syndrome of CE with concurrent PLE. Accordingly, eligibility criteria included having clinical signs consistent with small intestinal disease, identification of hypoalbuminemia (< 18 g/L), and complete diagnostic investigation (including endoscopy) to eliminate other causes of both gastrointestinal tract disease (eg, systemic disorders, infectious diarrhea, and physical gastrointestinal tract diseases such as partial obstructions of the intestinal tract) and hypoalbuminemia (eg, hepatic disease, renal disease, and blood loss). Additionally, dogs could not have received corticosteroids in the 3 weeks before treatment initiation. Finally, to minimize variation in management, all dogs were examined by one of the authors (JRSD, PJMN, or AJG) at all visits.

Medical records review—For all cases, the diagnostic approach was similar and the medical records were required to include the following information: complete history and physical examination, detailed laboratory investigations (CBC and serum biochemical profile [eg, concentrations of urea, creatinine, total bilirubin, calcium, phosphate, total protein, and albumin and serum activities of alkaline phosphatase and alanine aminotransferase]), a bile acid stimulation test, urinalysis including urine protein-to-creatinine ratio, fecal parasitology for Giardia intestinalis cysts (via zinc sulfate centrifugal flotation) and nematode parasites (via flotation with sugar solution), and fecal bacteriologic culture (for Salmonella spp and Campylobacter spp). Serum trypsin-like immunoreactivity and folate and cobalamin concentrations were measured in 23 dogs (11 dogs in group A and 12 in group C). In the remaining 4 dogs, the referring veterinarian had undertaken such tests and they were not repeated in the present study. All dogs underwent survey abdominal radiography and abdominal ultrasonography, and intestinal biopsies were performed. In 1 dog, full-thickness biopsy specimens (from the stomach, duodenum, jejunum, and ileum) were collected at exploratory coeliotomy, whereas in the remaining dogs, biopsy specimens were collected during gastrointestinal endoscopy. Endoscopy of the proximal portion of the gastrointestinal tract was performed in all dogs, and colonoscopy was performed in 1 dog in group A and 8 dogs in group C. Dogs underwent endoscopy of the distal portion of the gastrointestinal tract when biopsy of the ileum was deemed necessary because of ultrasonographic changes or hypocobalaminemia. During endoscopy, multiple mucosal specimens were collected from the stomach (corpus and fundus) and proximal portion of the duodenum and from the ileum in cases with colonoscopy. All specimens were submitted for histologic analysis. Trained animal technicians cared for all dogs during their hospitalization for clinical procedures.

Study design—The study was a retrospective case series assessing treatment outcomes in a cohort of dogs with CE and concurrent PLE. It is reported according to the Strengthening and Reporting of Observational Studies in Epidemiology (STROBE) statement guidelines.24

Concurrent medications and treatment—Previous treatments in both groups prior to referral included various anthelmintics, antimicrobials (mainly potentiated amoxicillin and metronidazole), and probiotics. There was no difference between groups in treatments used. The type of diet used varied among dogs and was decided on a case-by-case basis, depending on the recommendation of the primary clinician, owner preference, and palatability.

Initial dosages of immunosuppressive medication—For dogs of group A, the initial dosage of prednisolone was 2 mg/kg (0.9 mg/lb), PO, every 24 hours or divided every 12 hours, and the initial dosage of azathioprinef was 2 mg/kg, PO, every 24 hours.25 For group C, the initial dose of prednisolone was 1 to 2 mg/kg (0.5 to 0.9 mg/lb), PO, every 24 hours or divided every 12 hours, and the initial dosage of chlorambucilg was determined according to body surface area at 4 to 6 mg/m2, PO, every 24 hours for the first 7 to 21 days, before considering a dose reduction.26–28 For both groups, doses were rounded up or down to the nearest whole tablet (or half tablet for prednisolone). For both azathioprine and chlorambucil, for which the smallest tablet size exceeded the computed daily dose, medication was pulsed (ie, given at an interval that would ensure that the overall dose was equivalent to the calculated daily dose).

Given the retrospective nature of the study, the choice of treatment was not randomized. During the first part of the study period, most dogs were treated with the azathioprine-prednisolone combination, and chlorambucil-prednisolone was used in the second part. This change in primary treatment was, in part, due to the perception that many dogs that received azathioprine-prednisolone did not respond well, such that either additional immune-suppressive drugs were needed or dogs were euthanized because of lack of improvement in their clinical signs.

Study follow-up and dosage alterations—All study dogs were returned to the clinic for an initial reassessment 7 to 14 days after the initiation of treatment. Thereafter, follow-up investigations were conducted at approximately 2- to 4-week intervals. At each visit, dogs were weighed and body condition score judged by a 9-integer unit scale,29 a physical examination was performed, and blood was taken for hematologic and serum biochemical analysis. In addition to the official reevaluation visits, contact was maintained with clients by e-mail and, if necessary, follow-up telephone calls. The amount of contact required varied depending on the individual case. All dogs were followed-up until they died, were euthanized, or were lost to follow-up. All dogs that were lost to follow-up had been rechecked for at least 4 months. The reason for loss to follow-up was not recorded in all cases but included dogs that responded well and returned to the care of the referring veterinarian after the first 4 months of treatment.

The initial administered dosages of immunosuppressive medication were tapered gradually (prednisolone first, with chlorambucil or azathioprine tapered thereafter) depending on response, as judged by the clinician at follow-up. Prednisolone dosage was tapered by 25% to 33% every 2 to 4 weeks, whereas the chlorambucil or azathioprine dosages were both tapered by decreasing dosage, administration frequency, or both (eg, q 24 h to q 48 h to q 72 h). The decision to change drug dosages was based on both objective (eg, increases in body weight and albumin concentration) and subjective (eg, improvement in clinical signs) measures. In cases that achieved remission, dosages were slowly tapered and attempts were made to stop administration of medication altogether (prednisolone first, then azathioprine or chlorambucil).

If dogs did not have improvement in their diarrhea within the first 2 weeks, rescue treatment with cyclosporine was discussed with the owner. When dogs initially responded and subsequently relapsed after reductions in dose of the primary immunosuppressive treatment, drug dosages were increased to the last effective dosage. If there was still no response or adverse effects were too important, cyclosporine treatment was then offered. Ultimately, treatment for 3 dogs in group A and 1 dog in group C was changed to cyclosporine. To avoid any possible confounding effect of cyclosporine treatment, duration of primary treatment was the outcome measure used in survival analyses.

Outcome measures—Response to treatment was assessed by changes in body weight, serum albumin concentration, improvement in clinical signs, and duration of primary treatment. The latter was determined as the number of days from commencement of immunosuppressive treatment until either the time cyclosporine was added to the treatment to control the signs, the time of death or euthanasia, or the time that the dog was lost to follow-up (censored). To increase objectivity, the primary outcome measure chosen was a change in serum albumin concentration, whereas secondary outcome measures included a change in body weight, duration of primary treatment, and time to death or euthanasia. Improvement in clinical signs was reported in both groups but given the subjectivity of assessment (eg, an activity index had not been consistently performed), these were not compared between groups.

Time to death was defined as the time (in days) until the dog was euthanized or died. The reasons for death were recorded in both groups. All deaths were considered to be related to the primary disease and were treated as such for survival analysis.

Histologic assessment—Tissue samples collected during endoscopy were fixed in neutral-buffered 10% formalin, routinely dehydrated, and embedded in paraffin by use of standard procedures. Slides (4 μm) were stained with H&E. Results of the histologic assessment performed at the time (by a number of pathologists) were used in case management by excluding cases that were not consistent with CE (eg, lymphoma or primary lymphangiectasia). All cases were reviewed, on a single occasion, by a European board-certified pathologist (TS) unaware of group assignments, and assessed and graded using recently published internationally accepted criteria.30

Statistical analysis—Given that some of the variables were not normally distributed (as determined via the Shapiro-Wilk test) and other datasets contained categorical data, nonparametric analyses were used. For data reported in proportions, the Fisher exact test was used. For changes in body weight and albumin concentration within treatment groups, the Wilcoxon signed rank test was used. For comparisons of body weight and albumin between treatment groups, the Mann-Whitney test was used. Kendall rank correlation was used to examine whether associations existed between duration of primary treatment and either corticosteroid dose or absolute serum cobalamin concentration.

Odds ratios (with 95% CIs) were used to compare failure of primary treatment and failure of all treatment (including any rescue treatment) between groups. Survival curves were generated by the Kaplan-Meier method, and survival plots were compared by use of the log-rank test. For this analysis, any dog that died or was euthanized was classified as dead, and any dog still alive at the time it was lost to follow-up was censored. To determine which variables affected duration of primary immunosuppressive treatment, multivariable Cox regression was performed. The outcome of interest was duration of primary treatment, and the predictors tested included age, sex, pretreatment body weight, pretreatment albumin concentration, treatment group, diet type (fat-restricted vs non-fat-restricted), prednisolone dosage, ascites, crypt dilation, and lacteal dilation (defined as normal, mild, moderate-marked [given only 2 dogs had marked lacteal dilation]).30 Continuous explanatory variables were entered into the model both as continuous and in a categorical form, with their quartiles or other biologically sensible values as cutoffs between categories (eg, albumin concentration, < 15 g/L). In the final model, the choice of competing linear versus categorical formats was based on whether the effect of the variable appeared linear, whether there were changes in the deviance residual, or whether there were changes in the Akaike and Bayesian Information Criteria. Given that data were incomplete for cobalamin, a separate Cox regression model was created for the 23 dogs with complete data, which included cobalamin status (classified as either normal [0] or decreased [1]), treatment group, albumin concentration, ascites, lacteal dilation, and body weight. For all models tested, continuous variables in the proportional hazards regression models were linear in the log hazard plot.

Statistical analyses were performed with a computer software program,h and descriptive statistics were used to report baseline data (either median and range, or mean ± SD). The level of significance was set at P < 0.05 for 2-sided analyses.

Results

Signalment and baseline data—Three hundred seventy-two dogs were referred with gastrointestinal tract signs during the time frame of the study, 39 of which had hypoalbuminemia and had had a complete set of diagnostic investigations performed. After exclusion of other causes and elimination of dogs that had recently received corticosteroid treatment, 27 dogs met the study inclusion criteria.

Thirteen dogs were treated with a combination of azathioprine and prednisolone (group A) and 14 dogs with a combination of chlorambucil and prednisolone (group C). There were no significant differences in any of the baseline variables between groups (Table 1).

Table 1—

Median (range) or numeric values of baseline measurements of variables in dogs with CE and concurrent PLE treated with azathioprine-prednisolone (group A) or chlorambucil-prednisolone (group C).

VariableGroup A (n = 13)Group C (n = 14)P value
Age (y)7.8 (2.8–12.3)6.5 (0.8–10.0)0.34
Sex4 M, 3 NM, 0 F, 6 NF1 M, 5 NM, 1 F, 7 NF0.45
BreedBorder Collie (n = 2), Cardigan Corgi (1), CKCS (1), Cocker Spaniel (1), Dalmatian (2), English Springer Spaniel (1), Golden Retriever (1), Jack Russell Terrier (1), mixed breed (2), Staffordshire Bull Terrier (1) 
Weight (kg)16.9 (8.3–30.0)9.8 (3.4–63.6)0.076
BCS3 (2–5)4 (3–5)0.13
Duration (mo)1.8 (0.7–12.0)2.0 (1.0–8.0)0.54
Ascites8/137/140.57

BCS = Body condition score. CKCS = Cavalier King Charles Spaniel. F = Sexually intact female. M = Sexually intact male. NF = Neutered Female. NM = Neutered male. WHWT = West Highland White Terrier. — = Not applicable.

Baseline clinicopathologic assessments—There were no significant differences in most pretreatment serum biochemical variables between groups, including values for albumin, folate, and cobalamin concentrations; trypsin-like immunoreactivity; and urine protein-to-creatinine ratio (Table 2). The only significantly (P = 0.002) different variable was food-withheld bile acids concentration, which was greater in group A (4.7 μmol/L [0.4 to 18.3 μmol/L]), compared with group C (1.0 μmol/L [0.0 to 18.0 μmol/L]). However, when the proportion of abnormal results was compared (2/13 vs 1/14), there was no difference between groups (P = 0.53), and postprandial bile acid concentrations did not differ significantly (P = 0.19).

Table 2—

Median (range) values of clinicopathologic variables in the same dogs as in Table 1.

VariableReference intervalGroup AGroup CP value
Albumin (g/L)23.0–31.014.0 (12.0–17.1); 13/1315.4 (11.0–17.7); 14/140.64
Cobalamin (ng/L)> 200636 (99–2001); 4/11169 (62–724); 8/120.26
Folate (μg/L)3.5–8.57.7 (2.4–14.6); 1/119.0 (3.5–19.5); 0/120.37
TLI (μg/L)> 6.08.3 (3.9–11.2); 1/1110.6 (7.8–26.6); 0/120.071
Bile acids concentrations (μmol/L)
 Food withheld0.0–15.04.7 (0.4–18.3); 2/131.0 (0.0–18.0); 1/140.002
 Postprandial0.0–25.06.7 (4.0–34.5); 1/134.2 (0.4–18.0); 0/140.19
UPCR< 0.500.10 (0.01–0.50); 0/130.16 (0.02–0.88); 2/140.45

TLI = Trypsin-like immunoreactivity. UPCR = Urine protein-to-creatinine ratio.

Proportion of dogs with abnormal values in each group is given for each variable. Results for TLI, folate, and cobalamin were not included for 4 dogs (2 in group A and 2 in group C) because those measurements had previously been determined at different laboratories.

Dosages of immunosuppressive treatment administered—In group A, given rounding of dosages, the median (range) dosages for prednisolone and azathioprine were 2.0 mg/kg (1.3 to 3.8 mg/kg/d [0.6 to 1.9 mg/lb/d]) and 1.6 mg/kg (0.7 mg/lb/d; 0.8 to 2.3 mg/kg/d [0.4 to 1.0 mg/lb/d]), respectively. For group C, the median (range) dosages for prednisolone and chlorambucil were 1.7 mg/kg (0.8 mg/lb; 1.0 to 2.4 mg/kg/d [0.5 to 1.1 mg/lb/d]) and 4.4 mg/m2/d (2.1 to 5.8 mg/m2/d), respectively. The median dosage of prednisolone administered in group A was greater than the median dosage administered in group C (P = 0.022).

Histopathologic findings—There were no significant differences between groups for any histopathologic gastric variable (P > 0.13 for all). Further, no differences were evident between groups for small intestinal histologic assessments (Table 3). Lymphoplasmacytic infiltration was scored as 0 for 5 dogs in group A and 4 dogs in group C, as 1 for 6 dogs in group A and 5 dogs in group C, and as 2 for 2 dogs in group A and 5 dogs in group C. No dogs had a score of 3.

Table 3—

Median (range) values of histopathologic findings in the duodenum in the same dogs as in Table 1.

Histopathologic findingGroup AGroup CP value
Villous stunting0 (0–1)0 (0–1)0.41
Epithelial injury0 (0–1)0 (0–3)0.94
Crypt dilation0 (0–3)1 (0–3)0.14
Lacteal dilation1 (0–3)1 (0–3)0.17
Fibrosis0 (0–1)0 (0–2)0.96
Intraepithelial lymphocytes0 (0–2)0 (0–2)0.47
Lymphocytes and plasma cells0 (0–1)1 (0–2)0.07
Eosinophils1 (1–2)1 (0–2)0.26
Neutrophils1 (0–2)1 (0–2)0.48

Serum albumin concentration—At the 2-week recheck (Figure 1), serum albumin concentration had increased significantly in both groups (increase in group A, 36% [range, −9% to 76%; P = 0.004]; increase in group C, 56% [range, 18% to 145%; P = 0.003]), but the magnitude of increase was greater for group C than for group A (P = 0.021). At this stage, 11 of 12 (group A) and 5 of 13 (group C) dogs reexamined still had low albumin concentrations. When the surviving dogs were reassessed 16 weeks after commencing treatment, serum albumin concentration was 22.5 g/L (range, 15.9 to 28.0 g/L) in group A and 29.4 g/L (range, 21.0 to 38.0 g/L) in group C, with 2 of 4 (group A) and 2 of 11 (group C) dogs still having low albumin concentrations at this stage. Statistical analysis of these results was not conducted given the small numbers of remaining group A dogs.

Figure 1—
Figure 1—

Box-and-whisker plots of serum albumin concentrations over time in dogs with CE and concurrent PLE treated with azathioprine-prednisolone (A) or chlorambucil-prednisolone (C). Each box indicates the interquartile range, the horizontal line indicates the median, the whiskers indicate the 10th and 90th percentiles, and circles indicate outliers. Whiskers and outliers are not depicted for 1 group that included < 6 dogs.

Citation: Journal of the American Veterinary Medical Association 242, 12; 10.2460/javma.242.12.1705

Clinical signs and body weight—Clinical signs (gastrointestinal, ascites, or both) improved in 6 of 13 dogs in group A and 12 of 14 dogs in group C during the first 2 weeks of treatment (P = 0.046). At this stage, body weight had decreased in group A (median change, −14% [range, −28% to +2%]; P = 0.004) but had not changed in group C (median change −1% [range, −13% to +21%]; P = 0.42), with a significant (P = 0.003) difference between groups.

Additional treatments—Other treatments were used as deemed to be necessary by the attending clinician. Metronidazole was used concurrently in 5 of 13 (38%) and 6 of 14 (43%) group A and group C dogs, respectively (P = 0.83). Additional immunosuppressive treatment was subsequently added if response was thought to be suboptimal, again based on a decision of the attending clinician. Cyclosporine was added in 3 of 13 (23%) group A dogs and in 1 of 14 (7%) group C dogs (P = 0.31).

Various diets were used as adjunctive treatment for dogs in both groups. These included a fat-restricted dieti (5 dogs in group A and 2 dogs in group B), a fat-restricted hydrolyzed soy dietj (3 dogs in group C), a non–fat-restricted hydrolyzed soy dietk (3 dogs in group A and 4 dogs in group C), a non–fat-restricted hydrolyzed chicken dietl (3 dogs in group C), a single-source protein dietm (1 dog in group A), a home-prepared exclusion diet (cooked chicken and rice; 2 dogs in group A), nutritional support through a gastrostomy tube with a liquid critical care dietn (2 dogs in group A), or nutritional support through an esophagostomy tube either with a liquid critical care dietn (1 dog in group C) or a blended hydrolyzed chicken dietl (1 dog in group C). There was no significant (P = 1.0) difference in type of diet (fat-restricted vs non–fat-restricted) between treatment groups.

Survival—All study dogs had follow-up until the time of primary treatment failure (as judged by the attending clinician), death (including euthanasia) or censoring. At the time of assessment, primary treatment was deemed to have failed in 12 of 13 group A dogs, whereas primary treatment was considered to have failed in fewer group C dogs (4/14); relative to group C, the OR for primary treatment failure was 30 (95% CI, 3 to 730; P < 0.001). Median duration of primary treatment was significantly (P = 0.02) shorter in group A (30 days [range, 2 to 599 days]) than in group C (253 days [range, 5 to 494 days]).

Cyclosporine was added in the treatment of 3 dogs in group A and 1 dog in group C because clinical signs were not controlled with the primary treatment. Of these, only 1 dog (from group A) became a long-term responder (over 140 days after treatment change).

At the end of the study, 2 of 13 dogs were still alive in group A, and 10 of 14 were still alive in group C (P = 0.01; Figure 2); relative to group C, the OR for treatment failure was 12 (95% CI, 2 to 163; P < 0.001). Median survival time for group A dogs was 30 days (95% CI, 8 to 40 days), whereas median survival time was not reached in group C.

Figure 2—
Figure 2—

Kaplan-Meier survival curve comparing dogs treated with azathioprine-prednisolone (solid line) and those treated with chlorambucil-prednisolone (dashed line).

Citation: Journal of the American Veterinary Medical Association 242, 12; 10.2460/javma.242.12.1705

In group A, both dogs needed continuing treatment to control the clinical signs. In group C, treatment was completed for 3 dogs and 7 needed further treatment or were in the process of having treatment tapered off. Specifically, 1 dog successfully completed treatment but relapsed 2 months later. This dog responded again after treatment was restarted.

Two dogs (1 from each group) died suddenly after the onset of acute respiratory distress 2 and 5 days after treatment initiation (pulmonary thromboembolism was suspected, although a necropsy was not performed); the remaining deaths were the result of elective euthanasia. Reasons for death in group A dogs were lack of response (n = 8, based on persistence of clinical signs and no increase in serum albumin concentration, 2 of which were started on cyclosporine but were no longer responding) and development of additional clinical signs (2; diagnoses included suspected disseminated intravascular coagulation and thoracic effusion). In group C, reasons for euthanasia included lack of response (1 dog that was started on cyclosporine with limited improvement for 132 days at which time euthanasia was performed) and the development of complications (2; diagnoses included suspected disseminated intravascular coagulation and pancreatitis). The 2 dogs that were suspected to have developed disseminated intravascular coagulation were admitted with marked lethargy, ascites, and hypoalbuminemia. Response to supportive and immune-suppressive treatment was minimal. In both instances, owners elected euthanasia after 8 days because of the lack of improvement. One of these dogs had developed ecchymoses and thrombocytopenia at the time of clinical deterioration; the other dog developed thrombocytopenia, high APTT, and high circulating fibrinogen degradation products. When all censored dogs (2 dogs in group A and 10 dogs in group C) were taken into account as treatment failure (ie, deceased because of their gastrointestinal tract disease), 3-month survival rate was 4 of 13 dogs and 12 of 14 dogs for groups A and C, respectively; 6-month survival rate was 2 of 13 dogs and 11 of 14 dogs, for groups A and C, respectively.

Factors associated with duration of primary immunosuppressive treatment—Cox regression analysis was used to assess the factors associated with duration of primary treatment (Table 4). The following factors were found to be significantly associated: treatment group, albumin concentration in quartiles, body weight in quartiles, presence of ascites, and lacteal dilation.

Table 4—

Hazard ratios from a multivariable Cox regression for duration of primary treatment in the same dogs as in Table 1.

VariableHazard ratio95% CI*P value
Age (y)1.560.88–2.750.13
Sex
 MaleReferent
 Female16.520.60–458.890.098
Body weight (kg; in quartiles)
 ≤ 9.60Referent
 9.61–16.500.00010.00–0.060.004
 16.51–24.700.0040.00–0.270.001
 > 24.700.040.00–1.720.092
Albumin (g/L; in quartiles)  
 ≤ 13.0Referent 
 13.1–14.21.380.19–9.970.75
 14.3–16.0482.141.32–176, 722.710.040
 > 16.05.470.05–647.670.48
Treatment group
 AReferent
 C0.00010.00–0.060.004
Prednisolone dosage (mg/kg)1.560.29–8.540.61
Diet group
 Not fat restrictedReferent
 Fat restricted0.530.01–35.340.76
Ascites
 AbsentReferent  
 Present0.020.00–0.450.013
Crypt dilation
 NoneReferent
 Present4.830.16–150.250.37
Lacteal dilation
 NoneReferent
 Mild0.010.00–0.680.032
 Moderate-marked1.590.04–60.970.80

Age, body weight, and albumin concentrations are pretreatment values.

For treatment group, duration of primary treatment was positively associated with dogs receiving the chlorambucil-prednisolone combination (hazard ratio for group C vs group A, 0.0001 [95% CI, 0.00 to 0.06]; P = 0.004). Dogs with an albumin concentration between 14.3 g/L and 16.0 g/L had a significantly shorter time receiving primary treatment than dogs with albumin concentration < 13.0 g/L (hazard ratio, 482.14 [95% CI, 1.32 to 176,722.71]; P = 0.040), but no significant difference was found for dogs with albumin concentration < 14.3 or > 16.0 g/L. Dogs weighing between 9.61 and 24.70 kg had a significantly longer duration of primary treatment than dogs that weighed < 9.61 kg (hazard ratio for body weight between 9.61 and 16.50 kg vs body weight ≤ 9.60 kg, 0.001 [95% CI, 0.00 to 0.06]; P = 0.04; hazard ratio for body weight between 16.51 and 24.70 kg vs ≤ 9.60 kg, 0.004 [95% CI, 0.00 to 0.2]; P = 0.001). Dogs with ascites at initial evaluation had a significantly longer time of primary treatment than dog without ascites (hazard ratio for presence of ascites, 0.02 [95% CI, 0.00 to 0.45]; P = 0.013). Dogs with mild lacteal dilation (hazard ratio, 0.01 [95% CI, 0.00 to 0.68]; P = 0.028) had a significantly longer time of primary treatment, compared with dogs without lacteal dilation. However, the effect of corticosteroid dose was not significant (hazard ratio/mg of prednisolone/kg, 1.56 [95% CI, 0.29 to 8.54]; P = 0.61), and there was also no significant association between corticosteroid dose and duration of primary treatment, when tested in isolation (P = 0.25). No significant association between duration of primary treatment and sex, diet group, or crypt dilation was found.

A further multivariable Cox regression model was tested with only the data from the 23 dogs in which cobalamin results were available from the in-house laboratory. Given the low number of dogs involved, only the factors previously found to be significant were included in addition to serum cobalamin, namely treatment group, albumin concentration, body weight, ascites, and lacteal dilation. Treatment group (hazard ratio for group C vs group A, 0.10 [95% CI, 0.01 to 0.75]; P = 0.025) remained significant, but there was no effect for cobalamin status (hazard ratio, 3.39 [95% CI, 0.56 to 20.81]; P = 0.19), albumin concentration (hazard ratio, 0.78 [95% CI, 0.49 to 1.23]; P = 0.828), body weight (hazard ratio, 0.99 [95% CI, 0.88 to 1.11]; P = 0.87), ascites (hazard ratio, 0.21 [95% CI, 0.04 to 1.11]; P = 0.067), or lacteal dilation (hazard ratio for mild dilation, 0.34 [95% CI, 0.07 to 1.74]; P = 0.20). Further, there was no significant association between serum cobalamin concentration and duration of primary treatment (P = 0.60).

Discussion

Results of this retrospective study suggested that a chlorambucil-prednisolone drug combination may be more effective than azathioprine-prednisolone in the management of canine CE with concurrent PLE. In this respect, there were greater increases in albumin concentration and body weight with treatment, longer duration of primary treatment, and longer survival. Given that the study was retrospective, conclusions must be made cautiously and more objective clinical trials are now needed to confirm or refute the findings.

The basis for use of chlorambucil as an alternative to azathioprine warrants some discussion. Historically, the usual treatment protocol for CE and concurrent PLE in our hospital was a combination of prednisolone and azathioprine, as suggested by current expert opinion.26 However, given a perceived lack of efficacy, a decision was made to look for alternatives in the hope that outcomes might improve. A chlorambucil-prednisolone combination was used because these drugs were commonly used in cats for both refractory CE and low-grade lymphoma with success. Chlorambucil also has the advantage of being more affordable than cyclosporine.

The reason for the apparently improved outcome for dogs treated with chlorambucil in the present study is not known, and there are various possibilities. First, onset of action of azathioprine is considered to be slow and it is generally accepted that a period of 11 days is required before the onset of clinical effects.31 This could be a limitation when treating severely affected animals and, indeed, 4 of the group A dogs were euthanized within this time frame versus 2 of the group C dogs. Thus, the improved response rate with chlorambucil might, in part, be the result of a shorter time to reach therapeutic effect. However, to our knowledge, no precise data are available about the duration of onset for chlorambucil in dogs to support this possibility, although it is usually considered to be slower in onset of action than other alkylating agents.32 Adverse effects were uncommon in both groups, and bone marrow suppression was not observed at any stage; therefore, a difference in adverse effects was not likely the reason for the group differences noted.

Most of the baseline data, including signalment, clinicopathologic findings, and histopathologic patterns, were similar between the groups, suggesting that, despite the lack of randomization, the groups were similar. Thus, the results obtained were not likely the result of known risk factors for poor prognosis in dogs, including hypoalbuminemia and hypocobalaminemia.4,5 Increased specific pancreatic lipase has been described as a risk factor,33 but this was not measured consistently in all dogs and for this reason was not analyzed further. The only significant group difference was a greater median preprandial bile acids concentration in group A. However, none of those dogs had a preprandial bile acids concentration > 20 μmoI/L, which has been suggested as cutoff value for histopathologic abnormalities of the hepatobiliary system.34 Therefore, the difference in preprandial bile acids concentration was not likely clinically important. There was no significant difference in the postprandial bile acids concentration between groups. Finally, 1 dog in group A had trypsin-like immunoreactivity of 3.9 μg/L, which was less than the reference limit (> 6 μg/L; a value < 3.0 μg/L is consistent with exocrine pancreatic insufficiency). This result was not considered to be consistent with clinical exocrine pancreatic insufficiency, but retesting after a month is usually recommended as was the intention for this dog. However, this dog survived only 7 days after initiation of treatment, at which time it was euthanized after developing thoracic and abdominal effusion, consistent with worsening of PLE.

Activity indices have been described for assessing disease severity on the basis of clinical and laboratory findings and can also be used as prognostic markers.5,35 Activity indices were not consistently used for every visit; this was a limitation of the study, and as a result, we cannot be certain that group differences in disease severity did not exist. However, only cases evaluated by 3 study authors were included. Furthermore, objective assessments for the primary outcome measures were chosen, namely albumin concentration, weight change, and duration of primary immune-suppressive treatment. Thus, whereas the absence of activity index data is a notable limitation, the outcomes should nonetheless still be valid.

Several other limitations also need to be mentioned. First, dietary management varied among study dogs. In this respect, some dogs were fed hydrolyzed protein diets, some were fed low-fat diets, nutritional support with tube feeding was occasionally required, and others were fed a home-prepared diet. Dietary management can be highly effective in the management of canine CE,5 and hydrolyzed protein diet is beneficial as the sole treatment in many cases,36 whereas a diet based on reduced fat content is suggested for dogs with lymphangiectasia.1 However, although not a primary outcome measure, there was no significant difference between groups in the number of dogs fed a fat-restricted diet and fat restriction was not found to be significant for duration of primary treatment in the multivariable Cox regression model, making the differences in diet unlikely to be responsible for a better outcome in dogs from group C.

A second limitation was the fact that the median prednisolone dosage was significantly less in dogs treated with chlorambucil-prednisolone (1.7 mg/kg), compared with those treated with azathioprine-prednisolone (2.0 mg/kg). Although prednisolone is used primarily for its immune-modulating properties, several adverse effects from the drug37 could account for an apparent worsening in clinical status. Prednisolone has a catabolic effect, which can be detrimental in dogs in a negative energy balance. Furthermore, it may worsen the hypercoagulable state that often exists2 and may cause signs such as muscle weakness and lethargy. Prednisolone dosage did not have a significant independent effect when included in the multivariable Cox regression analysis, and the overall effect of treatment group remained. No association was detected between the dosage of prednisolone and duration of primary immunosuppressive treatment. However, given the retrospective nature of the study, an independent effect of prednisolone dosage could not be excluded completely. That said, we believe this to be less likely because the majority of dogs (approximately 90%) received between 1 and 2.3 mg/kg/d, and it is unclear whether such dosage differences would have such a profound effect on response. Equivalent daily dosages of prednisone (approx 2 mg/kg/d) lead to improvement in clinical signs of IBD in a recent randomized controlled trial in dogs.38 Further, in that study, approximately 30% of dogs with concurrent hypoproteinemia did not respond well.

Nonetheless, given the uncertainty regarding the possible confounding effect of prednisolone dosage, prospective trials are recommended to verify the present study findings. For instance, a prospective randomized controlled trial could include 4 groups, in which both azathioprine and chlorambucil are paired with either high- or low-dose prednisolone. Two prednisolone-only groups (ie, low-dose and high-dose) could also be considered to determine what effect could be attributable to prednisolone alone and at what dosage, although this would complicate the study design and increase the number of dogs required. Further, results of several studies indicate that hypoalbuminemia is a negative prognostic factor in CE4,5,38 and aggressive treatment with drug combinations is usually recommended from the outset.26 Therefore, ethically, a prednisolone-only group would be difficult to justify in dogs with marked hypoalbuminemia.

A third limitation was that there was a difference in the time frame for case recruitment, with most dogs in group A being enrolled during the first half of the study and most dogs in group C being recruited during the second half. However, 3 of the authors provided primary management of all dogs in the study, and other variables were deemed unlikely to have had an important effect.

A fourth potential limitation was the fact that more censored dogs were in group C than in group A, but those dogs were censored because they were still alive at the time of analysis, not because they had died. Censoring did not occur because of poor outcome and loss to follow-up but rather because of good response to treatment and longer duration of primary treatment.

A final limitation was the fact that discrepancies in histologic assessment of intestinal biopsy specimens have been recognized and emphasize the difficulty of obtaining a final diagnosis.39,40 Most notably, it can be difficult to differentiate cases of CE from alimentary lymphoma.41 If some of the dogs in the present study actually had alimentary lymphoma, this could account for a better response to chlorambucil. A combination of chlorambucil and prednisolone is the treatment of choice in cats with small cell lymphoma, with good outcome.20,21

Dogs with mild lacteal dilation were more likely to have longer duration of the primary treatment than those without lacteal dilation. The reason for this difference was not clear, but this difference might suggest that mild lacteal dilation is secondary to lesions responsive to immunosuppressive treatment (eg, inflammatory disease), whereas different etiologies are present when lacteal dilation is marked (eg, primary lymphangiectasia) or absent. However, this result should be interpreted cautiously because assessing the prognostic importance of histologic examination was not an aim of the study. Similarly, some body weight and albumin values were also associated with longer duration of primary treatment. Although this separation allowed a better fit of the multivariable Cox regression, its clinical importance was unclear. Dogs with ascites at first evaluation had a significantly longer duration of primary treatment than dogs without ascites, but the importance of this finding was unclear. The number of study dogs was small, and multiple variables were included in the model.

Despite the limitations associated with a retrospective study, the 2 groups were equivalent in signalment, baseline data, baseline clinicopathologic assessments, and histopathologic findings. Also, known negative prognostic factors, such as hypoalbuminemia and hypocobalaminemia, were similar between groups. Although there was a difference in the initial prednisolone dosage between groups, prednisolone dosage was not associated with duration of primary treatment. No difference in additional treatments (antimicrobials or fat-restricted diet) was found between the 2 groups. The group treated with chlorambucil improved in all criteria examined while receiving primary treatment. Dogs with CEs with secondary PLE might benefit from treatment with chlorambucil-prednisolone instead of azathioprine-prednisolone.

ABBREVIATIONS

CE

Chronic enteropathy

CI

Confidence interval

PLE

Protein-losing enteropathy

a.

Bota D, Hernandez J. Protein-losing enteropathy in Yorkshire Terriers—retrospective study of 19 dogs (abstr), in Proceedings. 21st Eur Conf Vet Intern Med-CA Cong 2011;237.

b.

Simmerson SM, Wunschmann A, Crews L, et al. Description of protein-losing enteropathy in Yorkshire Terrier dogs using the W.S.A.V.A gastrointestinal classification system (abstr). J Vet Intern Med 2009,23:732.

c.

Craven M, Duhamel GE, Sutter NB, et al. Absence of a bacterial association in Yorkshire Terriers with protein-losing enteropathy and cystic intestinal crypts (abstr). J Vet Intern Med 2009,2:757.

d.

Equilino M, Theodoloz V, Doherr M, et al. Biochemical markers and survival in dogs with protein-losing enteropathy (abstr). J Vet Intern Med 2011;25:691.

e.

Owens SL, Parnell NK, Moore GE, et al. Canine protein-losing enteropathy: a retrospective analysis and survival study in 68 dogs (abstr). J Vet Intern Med 2011;25:692.

f.

Immuran, Aspen, Dublin, Ireland.

g.

Leukeran, Alkopharma Sarl, Luxembourg, Luxembourg.

h.

Stats Direct, version 2.6.2, Stats Direct Ltd, Altrincham, Cheshire, England.

i.

Digestive low fat wet/gastrointestinal low fat wet, Royal Canin, Aimargues, France.

j.

Hypoallergenic moderate calorie dry, Royal Canin, Aimargues, France.

k.

Hypoallergenic dry, Royal Canin, Aimargues, France.

l.

z/d wet, Hill's Pet Nutrition, Topeka, Kan.

m.

Sensitivity Control chicken and rice wet, Royal Canin, Aimargues, France.

n.

Enteral care, PetAg, Hampshire, Ill.

References

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  • Figure 1—

    Box-and-whisker plots of serum albumin concentrations over time in dogs with CE and concurrent PLE treated with azathioprine-prednisolone (A) or chlorambucil-prednisolone (C). Each box indicates the interquartile range, the horizontal line indicates the median, the whiskers indicate the 10th and 90th percentiles, and circles indicate outliers. Whiskers and outliers are not depicted for 1 group that included < 6 dogs.

  • Figure 2—

    Kaplan-Meier survival curve comparing dogs treated with azathioprine-prednisolone (solid line) and those treated with chlorambucil-prednisolone (dashed line).

  • 1. Dossin O, Lavoue R. Protein-losing enteropathies in dogs. Vet Clin North Am Small Anim Pract 2011; 41: 399418.

  • 2. Goodwin LV, Goggs R, Chan DL, et al. Hypercoagulability in dogs with protein-losing enteropathy. J Vet Intern Med 2011; 25: 273277.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Dijkstra M, Graus JS, Bosje JT, et al. Protein-losing enteropathy in Rottweilers. Tijdschr Diergeneeskd 2010; 135: 406412.

  • 4. Craven M, Simpson JW, Ridyard AE, et al. Canine inflammatory bowel disease: retrospective analysis of diagnosis and outcome in 80 cases (1995–2002). J Small Anim Pract 2004; 45: 336342.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Allenspach K, Wieland B, Groene A, et al. Chronic enteropathies in dogs: evaluation of risk factors for negative outcome. J Vet Intern Med 2007; 21: 700708.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Weinkle TK, Center SA, Randolph JF, et al. Evaluation of prognostic factors, survival rates, and treatment protocols for immune-mediated hemolytic anemia in dogs: 151 cases (1993–2002). J Am Vet Med Assoc 2005; 226: 18691880.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Piek CJ, Junius G, Dekker A, et al. Idiopathic immune-mediated haemolytic anemia: treatment outcome and prognostic factors in 149 dogs. J Vet Intern Med 2008; 22: 366373.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Mason N, Duval D, Shofer FS, et al. Cyclophosphamide exerts no beneficial effect over prednisone alone in the initial treatment of acute immune-mediated haemolytic anemia in dogs: a randomized controlled clinical trial. J Vet Intern Med 2003; 17: 206212.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Piek CJ, van Spil WE, Junius G, et al. Lack of evidence of a beneficial effect of azathioprine in dogs treated with prednisolone for idiopathic immune-mediated hemolytic anemia: a retrospective cohort study. BMC Vet Res 2011; 7: 15.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Olivry T, DeBoer DJ, Favrot C, et al. Treatment of canine atopic dermatitis: 2010 clinical practice guidelines from the International Task Force on Canine Atopic Dermatitis. Vet Dermatol 2010; 21: 233248.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. O'Neill T, Edwards GA, Holloway S. Efficacy of combined cyclosporine A and ketoconazole treatment of anal furunculosis. J Small Anim Pract 2004; 45: 238243.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Hardie RJ, Gregory SP, Tomlin J, et al. Cyclosporine treatment of anal furunculosis in 26 dogs. J Small Anim Pract 2005; 46: 39.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Willard MD, Helman G, Fradkin JM, et al. Intestinal crypt lesions associated with protein-losing enteropathy in the dog. J Vet Intern Med 2000; 14: 298307.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Allenspach K, Ruefenacht S, Sauter S, et al. Pharmacokinetics and clinical efficacy of cyclosporine treatment of dogs with steroid-refractory inflammatory bowel disease. J Vet Intern Med 2006; 20: 239244.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Yuki M, Sugimoto N, Takahshi K, et al. A case of protein-losing enteropathy treated with methotrexate in a dog. J Vet Med Sci 2006; 68: 397399.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Rinkardt NE, Kruth SA. Azathioprine-induced bone marrow toxicity in four dogs. Can Vet J 1996; 37: 612613.

  • 17. Moriello KA, Bowen D, Meyer DJ. Acute pancreatitis in two dogs given azathioprine and prednisone. J Am Vet Med Assoc 1987; 191: 695696.

    • Search Google Scholar
    • Export Citation
  • 18. Steffan J, Favrot C, Mueller R. A systematic review and meta-analysis of the efficacy and safety of cyclosporine for the treatment of atopic dermatitis in dogs. Vet Dermatol 2006; 17: 316.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Foster AP, Shaw SE, Duley JA, et al. Demonstration of thiopurine methyltransferase activity in the erythrocytes of cats. J Vet Intern Med 2000; 14: 552554.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Lingard AE, Briscoe K, Beatty JA, et al. Low-grade alimentary lymphoma: clinicopathological findings and response to treatment in 17 cases. J Feline Med Surg 2009; 11: 692700.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Stein TJ, Pellin M, Steinberg H. Treatment of feline gastrointestinal small-cell lymphoma with chlorambucil and glucocorticoids. J Am Anim Hosp Assoc 2010; 46: 413417.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Rosenkrantz WS. Pemphigus: current therapy. Vet Dermatol 2004; 15: 9098.

  • 23. Taylor F, Gear R, Hoather T, et al. Chlorambucil and prednisolone chemotherapy for dogs with inoperable mast cell tumours: 21 cases. J Small Anim Pract 2009; 50: 284289.

    • Crossref
    • Search Google Scholar
    • Export Citation
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