Detection of Mycobacterium avium subspecies paratuberculosis-specific DNA by PCR in intestinal biopsies of dogs

Background: Mycobacterium avium subspecies paratuberculosis (MAP) is the cause of paratuberculosis. MAP infections have not been reliably detected in dogs, but a reemerging debate about the link between MAP and Crohn's disease has renewed interest about the occurrence of MAP in pets.
Hypothesis: This study was undertaken to examine canine intestinal biopsies for the presence of MAP-specific DNA.
Animals: Forty-two dogs with chronic vomiting, diarrhea, or both; and 14 dogs with no gastrointestinal disease.
Methods: All dogs with signs of gastrointestinal disease had a standard work-up for chronic gastrointestinal disease. Endoscopically obtained intestinal biopsies were submitted for histopathologic and molecular investigations. Biopsies were screened for MAP-specific DNA by 3 polymerase chain reaction (PCR) methods (nested, seminested, and triplex real-time PCR). Samples from control dogs were obtained during necropsy.
Results: Histopathology of the biopsies was indicative of inflammatory bowel disease (IBD) in 17 and neoplasia in 6 dogs. Six dogs showing nonspecific changes responded to diet and were classified as having food-responsive enteropathy. In 13 dogs a final diagnosis was not established. MAP-specific DNA was detected and confirmed by sequencing in 8 dogs (19%). These dogs were diagnosed with food-responsive enteropathy (n = 3), IBD (n = 2), and open diagnosis (n = 3). MAP-specific DNA was not detected in dogs with no gastrointestinal disease.
Conclusions and clinical importance: MAP-specific DNA was detected in approximately one fifth of dogs with chronic gastrointestinal disease and might play a role as a pathogenic agent. Apart from animal welfare, the zoonotic aspect warrants further studies addressing the viability of MAP organism in canine intestinal biopsies by culture.     

Phase II, open-label trial of single-agent CCNU in dogs with previously untreated histiocytic sarcoma

Background: Histiocytic sarcoma (HS) is an aggressive neoplasm in dogs, and in most instances, the disease is localized, but not amenable to surgical removal, or is disseminated. Affected patients usually die within 6 months. There have been no prospective studies to determine efficacy of single‐agent chemotherapy in dogs with HS. Hypothesis: Single‐agent CCNU [1‐(2‐chloroethyl)3‐cyclohexyl‐1‐nitrosourea; lomustine] has antitumor activity against HS in dogs. Animals: Twenty‐one dogs with histologically confirmed, nonresectable localized or disseminated HS. Methods: Prospective, open‐label phase II clinical trial in which dogs with previously untreated HS were uniformly treated with CCNU as a single oral dosage of 90 mg/m² every 4 weeks. The primary outcome measure was reduction in tumor size. Results: Fourteen dogs with disseminated HS and 7 with localized HS were enrolled between 1999 and 2008. Overall response rate was 29% (95% confidence interval [CI], 14–50%) for a median of 96 days (95% CI, 55–137 days). Three dogs (1 disseminated, 2 localized) had complete responses lasting for 54–269 days and 3 dogs (2 disseminated, 1 localized) had partial responses lasting for 78–112 days. Conclusions and Clinical Importance: CCNU, when used as a single agent, has activity against HS in dogs. Evaluation of CCNU postoperatively for dogs with resectable localized HS and as part of combination therapy for tumors that are nonresectable or disseminated should be considered.     

Comparison of direct and indirect tests for small intestinal bacterial overgrowth and antibiotic-responsive diarrhea in dogs

Controversy exists over the diagnosis of idiopathic small intestinal bacterial overgrowth (SIBO) in dogs and some clinicians use the term antibiotic-responsive diarrhea (ARD) in preference. However, whether such terms are interchangeable is not clear. To examine the relationship between duodenal bacterial numbers and a clinical response to antibiotics, SIBO and ARD were defined by nonoverlapping criteria. Quantitative duodenal juice bacteriology and indirect serum biochemical tests were used to assess small intestinal bacterial populations in 30 dogs with gastrointestinal disorders, including 9 with ARD. Serum total unconjugated bile acid (TUBA) concentrations were measured in all dogs, serum folate and cobalamin concentrations were measured in 29 of 30 dogs, and quantitative culture of duodenal juice was performed in 22 of 30 dogs. Serum TUBA concentrations also were measured in samples from 38 control dogs. Twenty of 22 affected (clinical) dogs in which quantitative bacteriology was performed were classified as having SIBO (>10(5) colony-forming units of total bacteria per milliliter of duodenal juice), but bacterial numbers did not differ significantly between dogs with ARD and dogs with other disorders. Increased folate (19/29), decreased cobalamin (16/ 29), or a combination (9/29) were common, but increased TUBA concentrations were documented in only 5 of 30 clinical dogs. Again, no significant differences were observed between dogs with ARD and those with other disorders, and a similar proportion (5/38) of controls had abnormally high TUBA concentrations. Finally, no significant differences were noted when duodenal bacteriology and TUBA concentrations were assessed before and during antibiotic therapy. These results question the utility of quantitative duodenal juice bacteriology and indirect biochemical marker tests for SIBO in the investigation of canine gastrointestinal disorders.     

Prevalence of Giardia duodenalis infection,co-morbidities and associated risk factors in dogs admitted to a veterinary teaching hospital in Israel

Giardia duodenalis causes gastro-intestinal (GI) disease and carries a zoonotic risk. The risk for infection depends on local prevalence and individual immunity, but clinical signs are inconsistent and the role of G. duodenalis in other GI diseases is unknown. The current study aims were to evaluate the prevalence of G. duodenalis in dogs presented to a university veterinary teaching hospital in Israel, investigate risk factors for infection and its associations with other diseases. Fecal samples from 163 dogs were tested for G. duodenalis by an antigen assay (FASTest® Giardia strip). Prevalence of G. duodenalis infection was 11.9 %. It was significantly associated with young age (≤1year) and canine parvoviral enteritis. Housing conditions, signs of GI disease or other systemic diseases were unassociated with infection. In conclusion, G. duodenalis infection prevalence is relatively low in the study population. Young age and diagnosis of co-infections are common, warranting screening for G. duodenalis.     

Treatment response and long term follow up in nineteen dogs diagnosed with chronic enteropathy in Australia

Chronic enteropathy (CE) in dogs is common worldwide, but little data is available from Australia. The aim of this study was to describe treatment response and long‐term outcome in a cohort of dogs with CE. Dogs were prospectively enrolled at Murdoch University and the University of Melbourne. After diagnostic investigation to rule out diseases other than CE, dogs underwent sequential therapeutic trials until achieving a clinical response (diet then antibiotics, and finally immunosuppressants). Success was defined as 75% reduction of clinical severity for a minimum of five weeks. A total of 21 dogs were enrolled, and 19 completed the study. One dog was euthanised for lack of response to treatment and one excluded for lack of owner compliance. Most dogs responded to diet (n = 10), followed by antibiotics (n = 7) and immunosuppressants (n = 2). Long‐term remission (median 21.1 months, [3.0‐44.7]) was achieved in eight out of ten dietary responders without additional treatment. In contrast, only two dogs with antibiotic response remained in long‐term remission, of which one needed on‐going antibiotic treatment. Longer term remission was achieved in the two dogs treated with immunosuppressants with on‐going low dose therapy. This study concludes that most dogs referred for CE in Australia respond to dietary treatment (even after previous dietary interventions), and remission is long‐term compared to dogs treated with an antibiotic. Furthermore, the need for long‐term antibiotics in some dogs to maintain response may lead to antibiotic resistance. This study supports adequate dietary trials for CE in dogs, and a need for alternative second‐line treatments.     

Food hypersensitivity reactions in Soft Coated Wheaten Terriers with protein-losing enteropathy or protein-losing nephropathy or both: gastroscopic food sensitivity testing, dietary provocation, and fecal immunoglobulin E

The purpose of this study was to evaluate Soft Coated Wheaten Terriers (SCWTs) affected with protein-losing enteropathy (PLE) or protein-losing nephropathy (PLN) or both for allergy to food. We performed gastroscopic food-sensitivity testing, a provocative dietary trial, and measurement of fecal immunoglobulin E (IgE) in 6 SCWTs affected with PLE or PLN or both. Positive gastroscopic food-sensitivity test reactions were noted in 5 of 6 dogs. Positive reactions were found to milk in 4 dogs, to lamb in 2 dogs, and to wheat and chicken each in 1 dog. Adverse reactions to food (diarrhea, vomiting, or pruritus) were detected in all 6 dogs during the provocative dietary trial. Adverse reactions were found to corn in 5 dogs, to tofu in 3 dogs, to cottage cheese in 2 dogs, to milk in 2 dogs, to farina cream of wheat in 2 dogs, and to lamb in 2 dogs. Serum albumin concentrations significantly decreased and fecal alpha1-protease inhibitor concentration significantly increased 4 days after the provocative trial when compared with baseline values. Antigen-specific fecal IgE varied throughout the provocative trial, with peak levels following ingestion of test meals. We conclude that food hypersensitivities are present in SCWTs affected with the syndrome of PLE/PLN. Mild inflammatory bowel disease was already established in the 6 SCWTs of this report at the time of study, making it impossible to determine if food allergies were the cause or result of the enteric disease.     

Blood neutrophil-to-lymphocyte ratio (NLR) as a diagnostic marker in dogs with chronic enteropathy

Few routinely available biomarkers are clinically useful in assessing dogs with chronic enteropathy (CE) and aid in CE subclassification. The diagnostic potential of the blood neutrophil-to-lymphocyte ratio (NLR) has not been evaluated in canine CE. We evaluated the NLR in 93 dogs with CE (no steroid treatment for ≥2 wk prior) and tested for an association with clinical, clinicopathologic, and histologic characteristics and also with CE subclassification. NLR was significantly higher in CE dogs with severe clinical disease than dogs with mild clinical disease (p = 0.047). Hypoalbuminemia (p < 0.001), but not hypocobalaminemia, was associated with higher NLRs. NLR was correlated with fecal alpha₁-proteinase inhibitor concentrations (ρ = 0.47) and the serum-to-fecal alpha₁-proteinase inhibitor ratio (ρ = –0.48; both p < 0.001) but not with serum or fecal inflammatory markers nor with the overall histologic score (all p > 0.05). Dogs with steroid- or other immunosuppressant-responsive (IRE) or nonresponsive enteropathy (NRE) had significantly higher NLRs (median: 7.3) than dogs with food-responsive enteropathy (FRE; median: 3.0; p = 0.003), and a NLR ≥5.5 best distinguished both groups of dogs. No difference in NLR was detected between dogs with IRE and dogs diagnosed with NRE. These findings suggest that leukogram changes (i.e., NLR) could be clinically useful in canine CE, and that neutrophils might play a role in the systemic inflammatory response associated with canine CE. The NLR can be easily assessed on routine hematology and can potentially aid in the subclassification of dogs with CE based on the response to treatment.     

Upregulation of toll-like receptors in chronic enteropathies in dogs

BACKGROUND: Inflammatory bowel disease (IBD) is thought to result from a dysregulated interaction between the host immune system and commensal microflora. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs), but their role in enteropathies in dogs is unknown. HYPOTHESIS: That there is a dysregulation of TLRs recognizing bacterial MAMPs in dogs with IBD. ANIMALS: Sixteen healthy beagles and 12 dogs with steroid-treated (ST) and 23 dogs with food-responsive (FR) diarrhea. METHODS: Prospective, observational study. mRNA expression of canine TLR2, 4, and 9 was evaluated by quantitative real-time RT-PCR in duodenal and colonic biopsies obtained before and after standard therapy. Samples from control dogs were taken at necropsy, with additional biopsies of stomach, jejunum, ileum, and mesenteric lymph node in 6 dogs. RESULTS: There were significant differences (P< or = .017) in expression of TLR2, 4, and 9 between the 6 sampled locations in healthy control dogs (lymph node > small intestine > or = colon). Before therapy, ST expressed more mRNA than control dogs for all 3 receptors (P < .05). There were no significant differences between pretreatment and posttreatment values, even though 32/35 dogs improved clinically. No associations were found when comparing receptor mRNA expression with either histology or clinical activity scores. CONCLUSIONS AND CLINICAL IMPORTANCE: Bacteria-responsive TLR2, 4, and 9 are upregulated in duodenal and colonic mucosa in IBD. This might lead to increased inflammation through interaction with the commensal flora. The absence of significant changes after therapy despite clinical improvement might point toward the existence of a genetic predisposition to IBD as described in human IBD.     

Perinuclear antineutrophilic cytoplasmic antibody and response to treatment in diarrheic dogs with food responsive disease or inflammatory bowel disease

The goal of this study was to investigate the correlation between perinuclear antineutrophilic cytoplasmic antibody (pANCA) and clinical scores before and after treatment in diarrheic dogs with food-responsive disease (FRD) or inflammatory bowel disease (IBD). pANCA serology was evaluated prospectively by indirect immunofluorescence in 65 dogs with signs of gastrointestinal disease, and if positive, pANCA antibody titers were determined. Thirty-nine dogs with FRD responded to a novel diet, and 26 dogs with IBD were treated with corticosteroids. The severity of clinical signs was scored by means of a canine IBD activity index (CIBDAI). At initial examination, a significantly (P = .002) higher percentage of dogs were pANCA-positive in the FRD group (62%) compared with the IBD group (23%). pANCA titers were significantly higher (P = .003) before treatment in the FRD group (median titer 100) compared with the IBD group (median titer 1). However, there was no difference in pANCA titers between the groups after respective treatments because dogs in the IBD group had a significant increase in pANCA titer after treatment. The CIBDAI score decreased significantly (P < .001) after treatment in both groups (74% moderate to severe in FRD dogs before versus 8% after treatment; 85% moderate to severe in IBD dogs before versus 32% after treatment). There was no correlation between pANCA status in FRD or IBD dogs before treatment and scores for CIBDAI, endoscopy, or histopathology before or after treatment, except for the endoscopic duodenal score in dogs with FRD after treatment (P = .03). A positive pANCA test before therapy may aid in the diagnosis of FRD.     

The Clinical Efficacy of Dietary Fat Restriction in Treatment of Dogs with Intestinal Lymphangiectasia

Background

Intestinal lymphangiectasia (IL), a type of protein‐losing enteropathy (PLE), is a dilatation of lymphatic vessels within the gastrointestinal tract. Dietary fat restriction previously has been proposed as an effective treatment for dogs with PLE, but limited objective clinical data are available on the efficacy of this treatment.

Hypothesis/Objectives

To investigate the clinical efficacy of dietary fat restriction in dogs with IL that were unresponsive to prednisolone treatment or showed relapse of clinical signs and hypoalbuminemia when the prednisolone dosage was decreased.

Animals

Twenty‐four dogs with IL.

Methods

Retrospective study. Body weight, clinical activity score, and hematologic and biochemical variables were compared before and 1 and 2 months after treatment. Furthermore, the data were compared between the group fed only an ultra low‐fat (ULF) diet and the group fed ULF and a low‐fat (LF) diet.

Results

Nineteen of 24 (79%) dogs responded satisfactorily to dietary fat restriction, and the prednisolone dosage could be decreased. Clinical activity score was significantly decreased after dietary treatment compared with before treatment. In addition, albumin (ALB), total protein (TP), and blood urea nitrogen (BUN) concentration were significantly increased after dietary fat restriction. At 2 months posttreatment, the ALB concentrations in the ULF group were significantly higher than that of the ULF + LF group.

Conclusions and Clinical Importance

Dietary fat restriction appears to be an effective treatment in dogs with IL that are unresponsive to prednisolone treatment or that have recurrent clinical signs and hypoalbuminemia when the dosage of prednisolone is decreased.