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The concentration of immunoglobulins in faecal extracts was investigated as a method of assessing the production of immunoglobulins by the gut mucosa of 137 dogs. There were significant correlations between the concentrations in faecal extracts and the concentrations produced in duodenal organ cultures. Seventy-six German shepherd dogs had significantly lower median immunoglobulin A (IgA) concentrations in their faecal extracts than 63 controls of various breeds. Sixteen of the German shepherd dogs had IgA concentrations below the 95 per cent confidence limit of the control population and six had no demonstrable faecal IgA. The faecal concentrations of immunoglobulin G and albumin were significantly higher in the German shepherd dogs than in the controls, but their immunoglobulin M concentrations were similar.

Many genes influencing mammalian coat colours are well conserved. While genes responsible for pelage phenotypes in one species provide strong evidence for a candidate gene in a different species, the X-linked orange phenotype of the domestic cat is unique within mammals. The orange locus (O) undergoes X-inactivation, producing females that express both wildtype black (wt) and orange (variant) phenotypes when heterozygous (tortoiseshell). The orange locus has not yet been localized on the X chromosome. Tortoiseshell male cats have been identified but have been shown to be sex chromosome trisomies (XXY). To localize the cat orange locus, 10 feline-derived X-linked microsatellites were analysed in two extended cat pedigrees consisting of 79 and 55 individuals, respectively, segregating for the orange phenotype. Linkage analyses excluded close association of orange in the vicinity of the nine informative X-linked microsatellites. One marker was not polymorphic within either family. Several markers suggested exclusion (Z < -2.0) at distances of 7.5-33 cM. Exclusion analyses suggested a possible location for orange a 14 cM region near Xcen. Recombination distances of markers in the segregating feline pedigrees were reduced as compared with the feline interspecies backcross family. Thus, the presented pedigrees may be useful as reference families for the domestic cat because more accurate recombination rates for domestic cats can be determined.

Samples of faeces from 57 dogs with acute diarrhoea, 82 dogs with chronic diarrhoea, 34 clinically healthy household dogs and 88 kennelled control dogs were analysed by hybridisation, using DNA probes to detect enteropathogenic Escherichia coli (EPEC) and enterotoxigenic E coli (ETEC), verocytotoxin-producing E coli (VTEC), enterohaemorrhagic E coli (EHEC), enteroinvasive E coli (EIEC) and enteroaggregative E coli (EAggEC). Samples of duodenal juice from 60 of the 82 dogs with chronic diarrhoea were also examined. Significantly more of the dogs with diarrhoea were excreting EPEC (acute 35.1 per cent, chronic 31.7 per cent) and VTEC (acute 24.6 per cent, chronic 28 per cent) than the kennelled dogs (EPEC 17.1 per cent, VTEC 0 per cent) or the household control dogs (EPEC 6 per cent, VTEC 5.9 per cent). Enteropathic E coli was also detected in the duodenal juice of 23 of 60 (38.3 per cent) of the dogs with chronic diarrhoea. The EPEC attaching and effacing A (eaeA) gene and the verocytotoxin 1 (VR1) gene coding for VTEC were often found together. There was good agreement between in vitro studies and hybridisation for the detection of eaeA and VT1. Isolates from the dogs with diarrhoea adhered significantly more to Hep-2 cells, and VT1-positive strains from the dogs with diarrhoea consistently killed more than 50 per cent of Vero cells.

IgA is the predominant immunoglobulin class in mucosal secretions and secretory deficiencies may predispose to chronic enteropathies. The polymeric immunoglobulin receptor (pIgR) facilitates the transport of IgA across the epithelial border. Critical to the transport of IgA by pIgR is the presence of a polypeptide joining chain (J-chain) linking the IgA monomers of the dimeric IgA molecule. In this study we examine the difference in expression of mRNA transcripts for pIgR, alpha-chain and J-chain by real-time reverse-transcription polymerase chain reaction (RT-PCR) in endoscopic biopsies from the duodenum of dogs with and without chronic diarrhoea. One-step, real-time RT-PCR was used to quantify the level of expression of transcripts for the housekeeper gene G3PDH, pIgR, alpha-chain and J-chain. There was no significant difference in expression of any transcript between dogs with (n=11) and without (n=8) chronic diarrhoea. Expression of alpha-chain mRNA in both groups had a similar bimodal distribution, as individuals either expressed relatively 'high' or 'low' levels of this transcript. The secretion of IgA by plasma cells is under the control of Th-2 cytokines, therefore the finding of 'high' and 'low' levels of alpha-chain expression may reflect different levels of these cytokines in duodenal mucosa.

The relationship between stool character and whole gut transit time (WGTT), which is the average time for the passage of material through the lumen of the alimentary tract from ingestion to defecation, was studied in eight control dogs and 12 dogs with non-specific dietary sensitivity. Dogs were fed four diets in a cross-over design, and faecal quality was assessed daily and WGTT determined using plastic pellets. Faecal quality was unaffected by diet in the control dogs. Dogs with dietary sensitivity produced looser faeces compared with the control dogs, and this was significant for two of the diets. There was no significant effect of diet on mean WGTT within or between groups. Minimum WGTT, which was the interval to the first appearance of markers in faeces, was shorter in sensitive dogs compared with controls, and this was significant for two of the four diets. There were significant, inverse relationships between minimum WGTT and both mean faeces score and percentage unacceptable defecations. These data suggest that rapid transit of certain dietary components may impact negatively on stool quality and contribute to loose faeces in dogs with non-specific dietary sensitivity.

Objective: To determine relationships between fecal consistency and colonic microstructure and absorptive function in dogs with and without nonspecific dietary sensitivity., Animals: 12 dogs with nonspecific dietary sensitivity (affected) and 9 healthy dogs (controls)., Procedure: Affected dogs were fed 4 test diets and control dogs, 3 diets for 4 weeks each in a crossover design. Fecal consistency was assessed daily. At the end of each feeding period, electrolyte and water transport were assessed, and colonic biopsy specimens were obtained for histologic examination and measurement of crypt water uptake by use of confocal microscopy., Results: Feces were consistently looser in affected dogs. In control dogs, we detected net colonic absorption of sodium and chloride and secretion of potassium and bicarbonate. Absorption of sodium and chloride was less in affected dogs, compared with controls, indicating that electrolyte transport was disrupted in affected dogs. This disruption was accentuated during feeding of diets associated with significantly poorer fecal consistency (ie, loose feces). Fecal consistency was inversely correlated with crypt water absorption, which was reduced in affected dogs. Colonic crypts were shorter and less dense in affected dogs fed diets associated with poor fecal consistency, compared with affected dogs fed other diets or with control dogs., Conclusions and Clinical Relevance: Colonic transport function is a major determinant of fecal consistency in dogs. Dogs with nonspecific dietary sensitivity are particularly susceptible to diet-induced changes in absorptive function. Such changes are associated with damage to colonic microstructure, disrupted electrolyte transport, and failure to dehydrate luminal contents.

Cats have a requirement for dietary protein two to three times that of omnivores and herbivores. This was reported to be due to the hepatic catabolic enzymes of this species being set to a permanently high level and, therefore, showing little adaptation to low dietary protein. A major mechanism for adapting to dietary protein in other species is amino acid oxidation (hereafter referred to as protein oxidation), and the objective of this study was to determine whether protein oxidation in cats was correlated with protein intake. Net protein and net fat oxidation in six adult cats were studied directly from gas exchanges using indirect calorimetry, after feeding moderate protein (MP; 35% energy) and high protein (HP; 52% energy) diets. Protein oxidation was significantly higher (P < 0.05) when cats were fed the HP diet (28.4 plus minus 0.7 mg/min) rather than the MP diet (20.4 plus minus 0.8 mg/min). Fat oxidation was significantly higher (P < 0.05) when cats consumed the MP diet (9.0 plus minus 0.7 mg/min) rather than the HP diet (4.7 plus minus 0.5 mg/min). Protein oxidation was significantly correlated (linear regression, R(2) = 46.0, P < 0.05) with protein intake such that the mean ratio of 18-h oxidation: 18-h intake was 1.2 on both diets. Fat oxidation was significantly correlated (linear regression, R(2) = 18.9, P < 0.05) with fat intake such that the mean ratio of 18-h fat oxidation: 18-h fat intake was 1.1 (MP) and 0.9 (HP). This study demonstrated that cats adapt net protein oxidation at these levels of protein intake, and the reason for the high dietary protein requirement of this species is, therefore, unclear.

Objective: To develop a noninvasive method for the in vivo assessment of flatulence in dogs., Animals: 8 adult dogs., Procedure: Rectal gases were collected via a perforated tube held close to each dog's anus and attached to a monitoring pump fitted with a sensor that recorded hydrogen sulfide concentrations every 20 seconds. Patterns of flatulence were monitored for 14 hours after feeding on 4 days, and within- and between-dog variation was assessed over 4 hours on 4 consecutive days. Rate of hydrogen sulfide production (flatulence index) and frequency and number of emissions were evaluated as potential indicators of flatus characteristics. An odor judge assigned an odor rating to each flatulence episode, and the relationship between that rating and hydrogen sulfide concentration was determined., Results: Flatulence patterns varied within and between dogs. Variation was most pronounced for flatulence index; mean coefficients of variance within dogs over time and between dogs on each day were 75 and 103%, respectively. Flatus with hydrogen sulfide concentrations > 1 parts per million could be detected by the odor judge, and severity of malodor was highly correlated with hydrogen sulfide concentration. Odor ratings were accurately predicted by use of the equation 1.51 X hydrogen sulfide concentration(0.28)., Conclusions and Clinical Relevance: The technique described in this report appears to provide sensitive, reliable, and relevant data and will enable further studies of the factors that influence flatulence in dogs. Use of this technique also has the potential to aid in investigations of colonic physiology and pathology.

Objective: To determine whether feeding activated charcoal, Yucca schidigera, and zinc acetate would ameliorate the frequency and odor characteristics of flatulence in dogs., Design: In vitro screening of active agents followed by a randomized controlled trial., Animals: 8 adult dogs., Procedure: A fecal fermentation system was used to assess the effects of activated charcoal, Yucca schidigera, and zinc acetate alone and in combination on total gas production and production of hydrogen sulfide, the primary determinant of flatus malodor in dogs. All 3 agents were subsequently incorporated into edible treats that were fed 30 minutes after the dogs ate their daily rations, and the number, frequency, and odor characteristics of flatulence were measured for 5 hours, using a device that sampled rectal gases and monitored hydrogen sulfide concentrations., Result: Total gas production and number and frequency of flatulence episodes were unaffected by any of the agents. Production of hydrogen sulfide in vitro was significantly reduced by charcoal, Yucca schidigera, and zinc acetate by 71, 38, and 58%, respectively, and was reduced by 86% by the combination of the 3 agents. Consumption of the 3 agents was associated with a significant decrease (86%) in the percentage of flatulence episodes with bad or unbearable odor and a proportional increase in the percentage of episodes of no or only slightly noticeable odor., Conclusions and Clinical Relevance: Results suggest that activated charcoal, Yucca schidigera, and zinc acetate reduce malodor of flatus in dogs by altering the production or availability of hydrogen sulfide in the large intestine.

Objective: To evaluate intestinal permeability and absorption in healthy cats in association with diet and normal intestinal microflora., Animals: 6 healthy domestic shorthair cats., Procedure: A sugar solution containing D-xylose, 30-methyl-D-glucose, L-rhamnose, lactulose, and 51Cr-EDTA was administered intragastrically to healthy cats, and urinary excretion of ingested sugars was determined 5 hours after administration. After the same cats had received metronidazole for 1 month, the study was repeated. A final study was performed while cats were maintained on a new diet differing in composition and processing., Results: Lactulose-to-rhamnose ratios, reflecting intestinal permeability, were higher in cats, compared with values for humans or dogs, and values obtained before and after metronidazole administration (mean +/- SEM; before, 0.40 +/- 0.08; after, 0.45 +/- 0.09) were not significantly different. Intestinal absorption also was unaltered after antibiotic administration, and the xylose-to-glucose ratio was 0.70 +/- 0.03 before and 0.71 +/- 0.06 after metronidazole administration. Sugar recovery did not differ significantly while cats were maintained on canned or dry food., Conclusions and Clinical Relevance: Reference ranges were established for the percentage urinary recovery of orally administered D-xylose, 3-0-methyl-D-glucose, L-rhamnose, lactulose, and 51Cr-EDTA obtained after 5 hours in healthy cats. The intestines of cats appear to be more permeable than those of other species, although the normal bacterial microflora does not appear to influence the integrity or function of the feline intestine, because values obtained for the measured variables before or after antibiotic administration were not significantly different. In addition, differences were not detected when the diet was completely altered.

Objective: To determine whether a colony environment predisposes healthy cats to high bacterial counts, including counts of obligate anaerobes, in the duodenum and whether increased numbers of bacteria could be found in the duodenum of cats with signs of chronic gastrointestinal tract disease., Design: Prospective study., Animals: 20 healthy control cats (10 from a colony environment and 10 pet cats) and 19 cats with a history of chronic gastrointestinal tract disease., Procedure: Undiluted duodenal fluid was quantitatively and qualitatively assessed by bacteriologic culture under aerobic and anaerobic conditions. Serum concentrations of cobalamin and folate were also measured., Results: Significant differences were not detected in the numbers of bacteria found in the duodenum of cats housed in a colony environment, compared with pet cats fed an identical diet prior to sampling. All healthy cats were, therefore, combined into 1 control group. Compared with healthy cats, cats with clinical signs of gastrointestinal tract disease had significantly lower counts of microaerophilic bacteria, whereas total, anaerobic, and aerobic bacterial counts were not significantly different. None of the cats with disease had total bacterial counts higher than expected from the range established in the control cats. Differences were not detected in regard to serum folate or cobalamin concentrations between diseased and healthy cats., Conclusions and Clinical Relevance: These findings indicated that healthy colony cats and pet cats have high numbers of bacteria in the duodenum, including high numbers of obligate anaerobes. Our findings also suggest that bacterial overgrowth in the small intestine is not a common clinical syndrome in cats with chronic nonobstructive gastrointestinal tract disease.

Objective: To determine effects of oral administration of metronidazole on the number and species of duodenal bacteria and selective nutrients of cats., Animals: 6 healthy domestic shorthair cats., Procedure: Undiluted duodenal fluid was obtained for quantitative and qualitative bacterial culture to determine species and number of bacteria in healthy cats. Blood samples were assayed for taurine, total protein, albumin, cobalamin, and folate concentrations. Cats then were given metronidazole (20 mg/kg of body weight, PO, q 12 h) for 1 month, after which bacterial cultures and serum assays of nutrients were repeated. Nine months after cessation of antibiotic treatment, duodenal bacteria were re-evaluated and serum was assayed for total protein, albumin, cobalamin, and folate concentrations., Results: Oral administration of metronidazole caused a significant decrease in aerobic and anaerobic bacterial counts in the duodenum of healthy cats, accompanied by emergence of Streptococcus spp and Corynebacterium spp. Serum concentrations of cobalamin and albumin increased when duodenal bacterial counts were decreased, although changes in folate or taurine concentrations were not detected. Measured variables did not differ, when comparing results obtained before and 9 months after cessation of metronidazole., Conclusions and Clinical Relevance: Oral administration of metronidazole decreased the number of aerobic bacteria and altered indigenous flora in the small bowel of cats. Normal duodenal flora appeared to be stable, because species of bacteria were re-established by 9 months after cessation of metronidazole. Bacterial flora appeared to have an impact on nutrients, because albumin and cobalamin increased during antibiotic administration and returned to preadministration concentrations after cessation of the antimicrobial.

PCRs were developed to detect 11 Escherichia coli virulence genes. Primers amplified the respective genes without cross-reaction with other genes. Specificity was maintained in multiplex reactions; excellent amplification of target genes was possible with a minimum of four multiplex reactions. These reactions successfully identified genes in E. coli from the feces of four dogs.

Objective: To establish a model for inheritance of gluten-sensitive enteropathy (GSE) in Irish Setters., Animals: 44 dogs of a 6-generation family of Irish Setters with GSE and 7 healthy Irish Setters., Procedure: Phenotype of each dog was determined after oral administration of gluten in the weaning diet, using morphometric evaluation of jejunal biopsies (all generations) and measurement of small intestinal permeability by use of a lactulose-rhamnose permeation test (generations 1, 2, and 3). Overall probability for each of 4 genetic models of inheritance (autosomal recessive, autosomal dominant, sex-linked recessive, and sex-linked dominant) accounting for segregation of partial villus atrophy within the entire family was calculated., Results: The autosomal recessive model was most tenable and was 56,250 times more likely to account for segregation of partial villus atrophy than the autosomal dominant model, assuming disease prevalence of 0.8%. Both sex-linked models were untenable. These conclusions were robust to the error attached to estimation of disease prevalence. High intestinal permeability without morphometric jejunal abnormalities in 4 of 20 dogs in the 3 youngest generations suggested heterogeneity of lesions associated with GSE., Conclusions: Genetic transmission of GSE is under the control of a single major autosomal recessive locus.

Nutritional modulation of insulin-like growth factors (IGF) and their binding proteins (IGFBP) is well established. The effect of nutritional restriction on the serum IGF/IGFBP system of adult cats was investigated to evaluate serum IGF-I as a biochemical marker of nutritional status. Assays for measuring feline serum IGF and IGFBP were validated and normal ranges established in a study population of 46 healthy nonobese adult cats. Serum concentrations of IGF-I and IGF-II correlated significantly with body weight (r = 0.75, P < 0. 0001 and r = 0.34, P < 0.03, respectively). Serum IGFBP profiles were similar to other species, including humans, dogs and guinea pigs. IGFBP-3 was the predominant binding protein reflecting IGF-I concentrations and body size. Serum IGFBP-2 concentrations were high relative to the normal human serum pool (NHS) control. Food withdrawal for 18 h followed by refeeding did not alter circulating IGF or IGFBP concentrations, including IGFBP-1, in nine cats. Short-term dietary restriction of nine adult cats to supply initially 56% (56%M) and then 42.5% (42.5%M) of calculated maintenance energy requirements for 14 d resulted in a significant weight loss (P < 0.01). However, serum IGF-I concentrations fell significantly (-51%, P < 0.01) only with 42.5%M restriction. Serum IGF-II, IGFBP, insulin and albumin concentrations were not altered during the study. We conclude that nutrition does modulate the adult feline IGF/IGFBP system, but to a lesser extent than in other species. Further evaluation is required before serum IGF-I can be used for the assessment of nutritional status in adult cats.

In situ hybridization (ISH) has found numerous applications in biology and medicine. However, its use to demonstrate expression of cytokines within the canine small intestine has not been described. Digoxigenin-labelled riboprobes complementary to mRNA encoding canine IFNgamma and IL10 were used to demonstrate expression of these cytokines within formalin-fixed, paraffin-embedded sections of jejunum obtained from healthy control Irish setter (IS) dogs (n = 4), gluten-sensitive IS in remission (n = 7), and beagles with high enteric bacterial populations (n = 5). Proportional areas of cells within the lamina propria showing one of three mutually exclusive staining intensities were measured, as well as the total stained area. Intensity categories were chosen arbitrarily to represent cells showing weak, moderate or dense staining (grades 1-3 respectively), reflecting increasing expression of mRNA. Control and gluten-sensitive IS showed similar total and grade-by-grade areas of expression of IFNgamma and IL10 in the lamina propria (p>0.05), in contrast to beagles, which showed greater total and grade 1 areas of expression of IFNgamma, and greater total, grade 1 and grade 2 areas of expression of IL10, than both groups of IS (p<0.05). Epithelial expression of both cytokines was demonstrated in beagles and IS, but differences between groups for each cytokine were not apparent (p>0.05). This study has validated the use of in situ hybridization for the detection of IFNgamma and IL10 mRNA within canine intestinal biopsies, andhas shown heightened jejunal expression of both cytokines in beagles with high enteric bacterial populations.

In order to assess an endoscopic collection procedure, populations of bacteria in duodenal fluid from seven adult cats were compared in paired samples obtained by endoscopy and direct needle aspiration during laparotomy. Each sample of duodenal juice was subjected to quantitative and qualitative culture of bacteria under both aerobic and anaerobic conditions. There were no significant differences in total numbers or individual species of bacteria comparing the two collection procedures. These findings indicate that collection of duodenal juice by endoscopy using the procedure described provides a representative sample of small bowel fluid for the assessment of the bacterial flora. Therefore, there appears to be no need for more invasive or complicated sampling techniques when quantitative and qualitative culture of duodenal juice is indicated as part of an investigation of small bowel disease in cats.

Gluten sensitive enteropathy (GSE) in Irish setter dogs has been proposed as an animal model for human celiac disease (CD), in which the major histocompatibility complex (MHC) class II alleles HLA DQA1*0501 and DQB1*0201 play an important role. To investigate whether an orthologous MHC class II region is involved in canine GSE, we undertook a linkage study in two large families of gluten sensitive Irish setter dogs. A total of 44 dogs in these pedigrees were genotyped for DQA1, DQB1 and C.2202 alleles, along with 30 unrelated healthy Irish setters. No genetic linkage between the DQ or C.2002 loci and GSE was detected. In contrast to CD, susceptibility to canine GSE does not appear to be determined by variation within the MHC class II gene cluster. Therefore, canine GSE may not be an appropriate model for CD, but nevertheless remains an important disease for advancing knowledge of pathological processes in the intestine.

Objective: To characterize histologic, biochemical, and ultrastructural changes in the intestine of Irish Setters susceptible to gluten-sensitive enteropathy (GSE) during controlled oral challenge exposure with gluten after weaning., Animals: Six gluten-sensitive and 12 healthy Irish Setters and 3 healthy Greyhounds., Procedure: Jejunal biopsy specimens were taken at 4 and 12 months of age from the 6 gluten-sensitive Irish Setters, which had been reared on a gluten-free diet to which a controlled dose of gluten (0.5 g/kg of body weight/d) was added. Control specimens were obtained at 4 (n = 5) and 12 (7) months of age from the healthy Irish Setters, which had been fed a conventional gluten-containing diet, and at 4 months of age from the healthy Greyhounds fed the controlled dose of gluten. The specimens were subjected to histologic and ultrastructural examinations and assay of brush border enzymes., Results: Gluten-sensitive Irish Setters developed abnormalities characteristic of GSE at 4 months. Abnormalities were comparable to changes not seen previously until 12 months in dogs with GSE fed a conventional gluten-containing diet. In addition, microvilli were stunted and irregular, and a few were vesiculated and reduced in number; the glycocalyx was reduced or absent. By 12 months of age, there was improvement in morphologic and biochemical parameters, indicating partial recovery despite continued exposure to gluten., Conclusions: Relative early onset of intestinal damage, compared with that previously reported, and subsequent partial recovery suggestive of oral tolerance to gluten may be attributable to oral administration of gluten as a purified extract rather than in dietary cereal, but alternative explanations include differences in environment or genetic susceptibility to gluten.

Objective: To determine whether biosynthesis of aminopeptidase N (ApN), alkaline phosphatase (AP), and total microvillus membrane protein is altered in Irish Setters with gluten-sensitive enteropathy (GSE)., Animals: A litter of 6 Irish Setters with GSE and 3 healthy Greyhounds., Procedures: Explants obtained from affected dogs at 4 and 12 months were maintained in vitro and were compared with material from healthy control dogs. Biosynthesis of ApN and AP was monitored by incorporation of [35S]methionine and immunoprecipitation of these enzymes., Results: Jejunal explants from affected Irish Setters had significantly higher rates of biosynthesis of total protein, microvillus membrane protein, AP, and ApN, compared with control tissue. Two forms of ApN with apparent molecular mass of 155 and 135 kd and 4 forms of AP with apparent molecular mass of 210 to 260, 150, 130, and 105 kd were identified in total membrane fractions from control and affected dogs., Conclusions: Reduced activities of ApN and AP in dogs with GSE are not attributable to decreased synthesis of these proteins and document enhanced synthesis of microvillar membrane proteins, which may be a compensatory response to enterocyte damage. The 150-kd form of AP was most prominent in tissue from the most affected dogs, probably representing an early form of this enzyme. In contrast, the 105-kd form was most intense in tissue from controls and less intense in tissue of affected dogs.

Eight gluten-sensitive Irish setters underwent a gluten challenge to investigate changes in the insulin-like growth factor (IGF) axis. In the first study, they were challenged with an acute intraduodenal administration of tryptic-peptic gluten digest and then maintained on dietary gluten for three months. In the second study, the challenge came solely from dietary gluten fed for three months. After the acute intraduodenal administration of gluten, serum IGF-I levels decreased significantly by 21.8 per cent, (P = 0.01) on day 3 after challenge and then returned to normal. There was also a decrease (52.5 per cent, P < 0.03) in the levels of serum IGF-binding protein-3 (IGFBP-3) until day 14 after challenge but they had returned to normal by day 28. In two dogs IGFBP-3 levels decreased through specific serum protease activity. There were no changes in serum IGF-I or IGFBP levels during the second study after the dietary gluten challenge alone, or in four non-gluten-sensitive beagles studied as controls during the acute intraduodenal/dietary gluten challenge.

A combined test of intestinal permeability using lactulose (L) and rhamnose (R), and absorptive function using xylose (X) and 3-O-methylglucose (G), was carried out at four, six, eight and 16 weeks of age in 22 healthy control and six gluten-sensitive Irish setter (IS) dogs fed a diet containing a controlled dose of gluten from weaning. Comparisons were made with two groups of 12 healthy control dogs of breeds other than IS, one fed the same diet as the setters and the other fed a gluten-free diet. Gluten-sensitive IS showed a rise in permeability (mean [SEM] urinary L/R) from 0.23 (0.07) at four weeks to 0.39 (0.05) at eight weeks, remaining at 0.36 (0.04) at 16 weeks. These results were significantly higher in gluten-sensitive than control IS at six, eight and 16 weeks, compatible with jejunal biopsy lesions characteristic of gluten-sensitive enteropathy demonstrated in affected dogs at 16 weeks. Urinary L/R ratios of control dogs of breeds other than IS peaked at six weeks 0.27 (0.02), and were significantly higher than those of control IS at six and eight weeks, demonstrating differences in permeability between Irish setter dogs and other breeds at this age. There were no significant differences in urinary X/G ratios at six, eight and 16 weeks of age between any of the groups of dogs challenged with gluten. Urinary L/R and X/G ratios were similar in the control dogs of breeds other than IS fed gluten-containing and gluten-free diets. These findings indicate that intestinal permeability testing of puppies during controlled oral gluten challenge provides a practical screening test for gluten sensitivity in Irish setter dogs at an early age.

A reference range was determined for a combined test of differential sugar permeability, using lactulose (L) and rhamnose (R), and intestinal function, using D-xylulose (X) and 3-O-methylglucose (G), in clinically healthy adult Irish setter dogs. Urinary L/R ratios in 48 Irish setters from one to 12 years old varied from 0.03 to 0.18, with a mean (SEM) of 0.10 (0.01); X/G ratios varied from 0.46 to 0.81, with a mean (SEM) of 0.59 (0.01). There were no significant differences between L/R or X/G ratios of dogs of different sex (P > 0.2) or age (P > 0.5), using analysis of covariance. Lactulose/rhamnose ratios of > or = 0.18 and X/G ratios > or = 0.43 were considered normal, defined by the respective mean +/- 2SD. Repeatability was established by performing three permeability and function tests at monthly intervals in twelve of the dogs. Analysis of repeated L/R and X/G ratios by means of linear models procedure revealed no significant differences between measurements made on successive occasions (P > 0.15), confirming the repeatability of this test.

In man and rodents, cells of the gastrointestinal immune system include B and T lymphocytes, granulocytes, macrophages and dendritic cells. Abnormalities in leucocyte numbers and function have been described in diseases of humans, such as coeliac disease and inflammatory bowel disease. The purpose of this study is to describe the normal distribution of T cells and MHC Class II expression in the small intestine of clinically normal dogs, to allow subsequent comparison with disease states. Full-thickness sections of duodenum, jejunum and ileum from seven young adult beagle dogs were immediately snap-frozen following euthanasia. Avidin-biotin-enhanced immunocytochemistry was used to detect expression of canine CD3, CD4, CD8 and MHC Class II antigens. Positively stained lamina propria cells were quantified using an eyepiece graticule, and positively stained intraepithelial cells by counts per 100 epithelial cells. In the lamina propria, the density of all leucocyte subsets was significantly increased towards the villus tip for all regions (p < 0.05). There was no apparent difference in the distribution of CD3+, CD4+ and CD8+ leucocytes between the three portions of the small intestine. The ratios of CD4+ cells to CD8+ cells in the lamina propria and epithelium were 59:41 and 15:85, respectively. Subtractive analysis suggested that 50-55% of CD3+ intraepithelial cells were CD4-CD8-. MHC Class II expression was apparent upon lamina propria cells with a dendritic morphology, as well as round cells. Epithelial MHC Class II expression was apparent in 7/7 ileal sections, compared with only 1/7 duodenal and 1/7 jejunal sections. This study shows that the small intestinal mucosa of the dog contains similar leucocyte populations to those found in other species, and suggests that these cells may play similar roles in gastrointestinal immunity.

This study has identified a naturally occurring, specific deficiency of a brush border aminopeptidase N (ApN) in the small intestines of five clinically healthy dogs. ApN activity in mucosal homogenates of dog small intestine was reduced significantly in deficient animals (13.4 (1.1) nmol min-1 mg-1 protein, n = 5, P < 0.002) compared to healthy control dogs (95.1 (6.7), n = 22). Alkaline phosphatase, gamma-glutamyl transferase, zinc-resistant alpha-glucosidase, maltase, sucrase and lactase in the ApN deficient dogs exhibited comparable activities to those in the control dogs. Microvillar membranes were analysed by one- and two-dimensional electrophoresis. ApN was represented by a single 145kDa band in all control dogs, identified by immunoblotting and immunoprecipitation. Protein maps from deficient dogs were normal apart from the virtual absence of an ApN spot and there were no apparent abnormalities in the glycosylation of microvillar proteins. The findings suggest that intestinal ApN deficiency in these dogs is a primary lesion involving diminished expression of an otherwise normal enzyme protein.

We demonstrate that rhamnose, 3-O-methyl-D-glucose, D-xylose and lactulose may be quantified accurately in blood by HPLC and pulsed amperometric detection, thus enabling studies of intestinal permeability and function to be carried out using plasma samples. Prior to HPLC, the endogenous glucose was enzymatically modified to gluconic acid and the protein precipitated. The precision of the quantification of the sugars in plasma (CV: 2.2-5.7%; 8.7-10.6% at very low concentrations) compared well with the quantification in urine. The results for groups of 8 dogs with small intestinal bacterial overgrowth and 12 dogs with inflammatory bowel disease were shown to be significantly different from a group of 20 normal control dogs (P < 0.001), demonstrating the test's value as a diagnostic tool. The normal ranges in blood 2 h post oral administration were determined to be 0.05-0.17 for the lactulose/rhamnose ratio and 0.45-0.65 for the xylose/3-O-methylglucose ratio. This method may be employed advantageously when the collection of urine in intestinal permeability and function tests is difficult.

Information about genetic variation within the canine major histocompatibility complex (MHC) class II genes is limited. In common with most other vertebrate species the canine MHC, or DLA, includes genes which are homologous to human DR, DQ, and DP. Recently, at least one functional DLA DQ gene-pair has been characterized, but so far systematic screening efforts have been lacking. In the present study, we sequenced both cDNA and genomic clones derived from DLA DQ genes of Irish setter dogs. This breed was of interest, since it shows a high prevalence of gluten sensitive enteropathy (GSE), which may be a useful animal model for celiac disease (CD) of man. Interestingly, few of the alleles found in Irish setters were identical to those previously detected in other breeds. Three novel DLA DQA and four novel DLA DQB alleles were discovered in 19 unrelated dogs. Strong association between certain HLA DQ alleles and CD of man prompted us to screen the DQ alleles of members of a family of gluten-sensitive Irish setter dogs. No haplotypes or alleles were shared by all affected dogs, but one frequent haplotype in this family was also detected in an unrelated gluten-sensitive Irish setter; this haplotype was absent in the healthy dogs. This observation warrants further investigation by screening the DQ alleles of a large population of unrelated gluten-sensitive Irish setters.

This study investigated whether gluten-sensitive enteropathy (GSE) in Irish setter dogs was associated with underlying structural abnormalities of microvillar membrane proteins. Jejunal biopsies taken from eight-month-old GSE-affected dogs reared on a normal, gluten-containing diet exhibited partial villous atrophy and contained more intra-epithelial lymphocytes than controls. The morphological abnormalities were reversed by feeding a gluten-free diet for five months and the changes were accompanied by an increase in the mucosal activity of the microvillar hydrolases, particularly aminopeptidase N and dipeptidyl aminopeptidase IV, which reverted to pre-treatment levels after a gluten challenge. Two-dimensional electrophoresis of microvillar membrane proteins isolated from GSE-affected dogs revealed an essentially normal protein map that was comparable to controls. The exception was an intense 85 kDa protein spot that diminished when the affected dogs were fed a gluten-free diet and re-intensified after a gluten challenge.

The surface properties of various Escherichia coli isolates associated with diarrhoeal illness were compared by aqueous partitioning between polyethylene glycol (PEG) and Dextran phases. Two well characterised strains of enteropathogenic E. coli (EPEC) were found to be very hydrophobic, based on the critical polymer concentration. EPEC strain E2348 cured of the EPEC adherence factor (EAF) plasmid had much reduced surface hydrophobicity. Partitioning of a series of diarrhoeagenic E. coli strains demonstrated that the majority of EAF+ EPEC strains were significantly more hydrophobic than EAF- EPEC strains. E. coli strains defined as enteroaggregative on the basis of hybridisation with a specific DNA probe showed much greater heterogeneity in their partitioning behaviour, possibly indicating that the AAF/I pili were not expressed in all strains. The E. coli K-12 strain used as a transformation host for adhesion studies had very low surface hydrophobicity but had a detectable negative charge. No alteration in these properties was observed when transformed with EPEC and recombinant plasmids known to specify adherence to tissue culture cells.

Small intestinal bacterial overgrowth (SIBO) has been reported to occur commonly in dogs with signs of chronic intestinal disease. There are usually few intestinal histological changes, and it is uncertain to what extent bacteria cause mucosal damage. The aim of this study was to apply a differential sugar absorption test for intestinal permeability and function to the objective assessment of intestinal damage in dogs with SIBO. Studies were performed on 63 dogs with signs of chronic small and, or, large bowel disease, in which SIBO (greater than 10(5) total or greater than 10(4) anaerobic colony forming units/ml) was diagnosed by quantitative culture of duodenal juice obtained endoscopically. None of the dogs had evidence of intestinal pathogens, parasites, systemic disease or pancreatic insufficiency. differential sugar absorption was performed by determining the ratios of urinary recoveries of lactulose/rhamnose (L/R ratio, which reflects permeability) and D-xylose/3-O-methylglucose (X/G ratio, which reflects intestinal absorptive function) following oral administration. Dogs with SIBO comprised 28 different breeds, including 13 German shepherd dogs. SIBO was aerobic in 18/63 dogs (29 per cent), and anaerobic in 45/63 (71 per cent). Histological examination of duodenal biopsies showed no abnormalities in 75 per cent, and mild to moderate lymphocytic infiltrates in 25 per cent of the dogs. The L/R ratio was increased (greater than 0.12) in 52 per cent, and the X/G ratio reduced (less than 0.60) in 33 per cent of the dogs. Differential sugar absorption was repeated in 11 dogs after their four weeks of oral antibiotic therapy. The L/R ratio declined in all 11 dogs (mean +/- SD pre: 0.24 +/- 0.14; post: 0.16 +/- 0.11; P < 0.05), but changes in the X/G ratio were more variable. These findings show that SIBO is commonly associated with mucosal damage not detected on histological examination of intestinal biopsies, and that changes in intestinal permeability following oral antibiotics may be used to monitor response to treatment.

The normal gastrointestinal tract contains an enormous number of aerobic and anaerobic bacteria which normally enjoy a symbiotic relationship with the host but can have adverse effects with local and systemic consequences. The small intestine constitutes a zone of transition between the sparsely populated stomach and the luxuriant bacterial flora of the colon. Regulation of the intestinal flora depends on complex interactions between many factors including secretion of gastric acid, intestinal motility, biliary and pancreatic secretions, local immunity, the surface glycocalyx and mucus layer, and diet. Microbial interactions are also important, and can involve alterations in redox potential, substrate depletion and production of substances such as bacteriocins that inhibit bacterial growth. The beneficial effect of the normal enteric flora include the competitive exclusion of potentially pathogenic organisms, and the production of nutrients such as short-chain fatty acids (which represent an important energy source for the colonic mucosa) and vitamins. Detrimental effects of the enteric flora include competition for calories and essential nutrients, particularly by bacteria located in the small intestine, and a capacity to damage the mucosa, in some circumstances causing or contributing to inflammatory bowel disease. These problems can be accentuated by interference with the physiological regulation of intraluminal bacteria allowing overgrowth by a normal resident, or colonisation by transient pathogens. The pathophysiological consequences may involve direct damage to the intestinal mucosa, and bacterial metabolism of intraluminal constituents, for example forming deconjugated bile acids and hydroxylated fatty acids which stimulate fluid secretion. Additional problems arise if there is interference with the mucosal barrier since this can result in increased passage of bacteria and bacterial products stimulating mucosal inflammation, while bacterial translocation can result in bacteraemia and septicaemia. Problems associated with bacterial pathogens are illustrated by the properties of the spectrum of pathogenic Escherichia coli, some of which facilitate long-term colonisation by adherence to the surface or invasion of enterocytes.

Seven simple sugars, commonly used in intestinal function tests, were simultaneously administered intravenously to dogs and their urinary excretion was measured to assess their potential for metabolic degradation. Lactose, lactulose and 3-O-methyl-D-glucose were excreted unchanged, but small proportions of palatinose and sucrose were not, and were assumed to have been metabolised. More than half of the administered D-xylose and a quarter of L-rhamnose was not excreted and was assumed to have been metabolised. These findings may be significant for the interpretation of differential sugar absorption tests and the D-xylose tolerance test.

A combination of mild proteolytic digestion and selective growth stimulation has been used to isolate and propagate adult rat intestinal epithelial cells with a finite life span. Growth of these cells on a variety of matrices and on mesenchymal cells has resulted in the expression of brush border enzymes including sucrase-isomaltase, aminopeptidase N, and alkaline phosphatase. Examination of the cells at the electron microscopic level has revealed that although these cells express key brush border enzymes, they do not have a fully formed brush border. These findings suggest that the expression of brush border enzymes and structural proteins represent distinct stages of enterocyte differentiation that are under separate transcriptional and temporal control.

To examine the postnatal development of equine small intestine, biopsy specimens of jejunal mucosa from 8 ponies, between 6 and 28 weeks old, were subjected to analytical subcellular fractionation and assay of organelle marker enzymes. Fractionation revealed a reduction in the particulate brush border component of beta-galactosidase (lactase) activity between 6 and 28 weeks, and a corresponding increase in soluble activity, although the reduction in mean specific activity was not significant. There also was a decrease in the proportion of brush border to soluble aminopeptidase N activity, a relative loss of brush border gamma-glutamyltransferase activity, and a considerable decrease in the specific activity of alkaline phosphatase throughout the gradient fractions. In contrast, there were marked increases in activities of alpha-glucosidase (maltase) and sucrase in the older ponies, accompanied by considerable changes in the intracellular distribution of particulate alpha-glucosidase activity, which was predominantly associated with endoplasmic reticulum at 6 weeks, whereas the large increase in activity observed by 28 weeks was clearly associated with the brush border. The modal density of brush borders also increased with age, suggestive of an increase in the glycoprotein-to-lipid ratio of the microvillar membrane. In contrast to these brush border changes, there was relatively little alteration in the activities or density distributions of marker enzymes for endoplasmic reticulum, basolateral membranes, mitochondria, or lysosomes. These findings indicate that maturation of equine intestinal epithelium during the first few months of life results in major changes in the properties and enzyme composition of enterocyte brush borders.

The short-term kinetics of Zn turnover were studied in Irish setters with gluten-sensitive enteropathy and control dogs following intravenous injection of 0.25 mg 96.5% enriched 70ZnCl2. The 70Zn enrichment of serum was found closely to obey two-compartment kinetics and the derived two-compartment decay equation was used to calculate the size and turnover of the two initial rapidly exchanging pools of body Zn. In normal Irish setters isotopic Zn initially equilibrates with a pool (a) of size 1.27 (SD 0.46) mumol/kg and then with a second pool (b) of size 6.83 (SD 1.72) mumol/kg. The fractional turnover of pool (b) was approximately one eighth that of pool (a). Enteropathic dogs showed no reduction in the size of either rapidly exchangeable Zn pool, reduction in serum Zn concentration or abnormality in Zn balance and hence these results do not support the possibility of an underlying Zn deficiency in this disorder.

Fifteen dogs with signs of small and, or, large bowel disease that responded clinically to an exclusion diet were studied, using differential sugar absorption as an objective parameter of the mucosal response to the diet. Intestinal permeability and function were assessed by determining the urinary excretion ratios of lactulose/rhamnose and xylose/3-O-methylglucose, respectively, following oral administration of a mixture of these four sugars. Five dogs, all retrievers, were tentatively diagnosed as having dietary hypersensitivity, based upon resolution of clinical signs and normalisation of high intestinal permeability following an exclusion diet and recurrence of signs (in four of five dogs) upon challenge with the original diet. The fifth dog did not become symptomatic when challenged, but intestinal permeability increased. The remaining 10 dogs were diagnosed as having food intolerance, based upon clinical improvement on an exclusion diet, relapse on challenge with their original diet, but lack of improvement in intestinal permeability. These findings suggest that a differential sugar absorption test may be useful to determine the reasons for clinical response to exclusion diets. Demonstration of increased intestinal permeability with subsequent normalisation following an exclusion diet may be useful in the diagnosis of dietary hypersensitivity, while persistent abnormalities in intestinal permeability are suggestive of underlying intestinal disease and food intolerance.

Dogs are particularly susceptible to development of glucocorticoid-induced hepatopathy, but the mechanisms are not well understood. We investigated the pathogenesis of glucocorticoid hepatopathy by examining sequential morphologic and biochemical changes in the liver of dogs during steroid administration. Six adult Beagles were given prednisolone acetate (4mg/kg of body weight, once daily for 24 days IM). Serum samples and percutaneous liver biopsy specimens were obtained before the start of the study (treatment day [TD] 0) and at TD 5, 10, 15, and 25. There were significant (P < 0.05) and progressive increases in serum activities of alkaline phosphatase, gamma-glutamyltransferase, and alanine transaminase. Light microscopic changes in liver biopsy specimens included progressive hepatocellular swelling and vacuolation. Electron microscopy revealed glycogen accumulation, peripheral displacement of organelles, and prominent dilatation of bile canaliculi, compared with findings at TD 0. Liver biopsy specimens taken at TD 25 had significantly (P < 0.05) increased activities of the plasma membrane enzymes, alkaline phosphatase and gamma-glutamyltransferase, and 5'-nucleotidase was significantly (P < 0.001) decreased. Subcellular fractionation on reorientating sucrose density gradients revealed high-density peaks of alkaline phosphatase and gamma-glutamyltransferase, compatible with a specific increase in the biliary canalicular component of the enzyme activities. Neutral alpha-glucosidase activity was shifted to the denser fractions, indicative of an increase in the proportion of rough to smooth endoplasmic reticulum and consistent with enhanced synthesis of plasma membrane proteins. There also was evidence for progressive increase in fragility of intracellular organelles, particularly lysosomes. These findings indicate that glucocorticoid hepatopathy in dogs is associated with progressive alterations not only to the plasma membrane, but also to other subcellular organelles.

The effects of pokeweed lectin (PWL) and concanavalin A (Con A) on microvillar membrane (MVM) proteins during the organ culture of rabbit ileal explants for 24 hours were compared with the known effects of enteropathogenic Escherichia coli (EPEC). PWL resulted in the accelerated release of brush border enzymes into the culture medium, accompanied by decreased tissue activities and an increase in the total activity present in the tissue and culture medium. Con A had less effect on the release and tissue activities of brush border enzymes and the total activity was not increased. Sucrose density gradient centrifugation of the culture medium showed that MVM enzymes were predominantly particulate, consistent with their release as vesicles. Centrifugation of ileal explants showed that PWL, but not Con A, resulted in a decrease in the modal density of the brush border which was consistent with a lower glycoprotein-to-lipid ratio. These findings suggest that PWL, in common with EPEC, may cause the disruption and vesiculation of microvilli and the compensatory stimulation of MVM protein synthesis.

We used organ culture of jejunal mucosal explants obtained from ponies aged between 2 and 12 months to study enterocyte damage by group A strains of equine rotavirus. Electron microscopy of jejunal explants maintained for < or = 48 h in the presence of organ culture medium alone showed that enterocytes were structurally intact and had a densely packed brush border and overlying mucus. Similarly, examination of explants maintained in the presence of rotavirus for 48 h revealed no apparent ultrastructural abnormalities. However, obvious replication and assembly of virus in enterocytes had occurred following exposure to equine rotavirus strains H2 (three of eight occasions), trypsin-activated H2 (two of four), L338 (five of five), and EQ124 (two of two), although brush borders and intracellular organelles appeared unaltered. Despite the absence of obvious ultrastructural damage, analytical subcellular fractionation showed that rotavirus infection can result in decreased activities of microvillar membrane enzymes, with a particular loss of the main brush border peak of modal density 1.21 g/ml. These findings demonstrate that organ culture of small intestine may be applied to the investigation of rotavirus pathogenicity; they further suggest that certain strains impair enterocyte function by affecting the turnover of microvillar membrane proteins.

A method for analysing microgram amounts of microvillar membranes by two-dimensional electrophoresis (protein mapping) is described, and has been used to characterize the microvillar proteins of the small intestine of German shepherd, corgi, and beagle dogs. Detergent-solubilized microvillar membranes were radiolabelled with 14C and separated by isoelectric focussing followed by SDS-PAGE. Proteins were detected fluorographically and glycoproteins by lectin-affinity staining. The microvillar hydrolases alkaline phosphatase and dipeptidyl aminopeptidase IV were identified by active-site labelling and aminopeptidase N by immunoprecipitation. Changes following pancreatic duct diversion were consistent with accumulation of pro-sucrase-isomaltase and diminished expression of the sucrase and isomaltase subunits. Cytoskeletal proteins were concentrated in the core fraction remaining after extraction of microvillar membranes with Triton X-100. There were no consistent differences between dogs of different breed, and the canine protein maps were similar to the human.

Small intestinal bacterial overgrowth (SIBO) was diagnosed by quantitative bacterial culture of duodenal juice samples obtained endoscopically in 41 of 80 dogs that were admitted with chronic diarrhea, vomiting, or weight loss. Thirteen dogs had aerobic bacterial overgrowth, most frequently comprising Escherichia coli, staphylococci, and enterococci, and 28 dogs had mixed anaerobic overgrowth, most frequently including Clostridium and Bacteroides spp. Affected dogs comprised 23 breeds, including 10 German Shepherd Dogs and median age at diagnosis was 2 years (range, 6 months to 11 years). High serum folate and low serum cobalamin concentrations had fair specificity (79 and 87%, respectively), but low sensitivity (51 and 24%, respectively) in detecting SIBO. Histologic examination of duodenal biopsy specimens did not reveal abnormalities (26/41 dogs), or revealed mild to moderate lymphocytic (12/41) or eosinophilic (2/41) infiltrates, or lymphosarcoma (1/41). Oral antibiotic treatment was effective in 77% (23/30 dogs), but prolonged treatment (> 4 weeks) was required to control signs and prevent recurrence in 50% (15/30). Corticosteroids were used alone in a dog with eosinophilic enteritis and in combination with antibiotics in 4 dogs with marked gastrointestinal lymphocytic/plasmacytic infiltrates. This study suggested that SIBO may be observed in dogs of many breeds, without an obvious primary cause, and that, although results of indirect tests may be suggestive of SIBO, bacterial culture of duodenal juice samples remains necessary for definitive diagnosis.

Repeated episodes of diarrhoea were seen in 4 laboratory beagles after experimental renal surgery and feeding a modified diet. Small intestinal bacterial overgrowth (SIBO) was suspected by exclusion of other causes and measurement of plasma folate. SIBO was confirmed by quantitative duodenal bacteriology. Beagles with SIBO can show no clinical signs, experimental stress and dietary change may have been reasons why these 4 beagles exhibited clinical signs with SIBO. Despite normal gut histology an increase in gut permeability was found using sugar absorption tests. This increased permeability had the potential to cause variations in drug absorption during experimental studies.

The quantification of monosaccharides and disaccharides used as probes in intestinal function and permeability tests can be technically demanding, detracting from the value of this approach to the indirect assessment of intestinal damage. In this study, a procedure is described for the simultaneous quantification of rhamnose, lactulose, 3-O-methyl-D-glucose and xylose in urine by HPLC using an anion exchange column with pulsed amperometric detection. This method is relatively fast and simple to perform, requiring no pre-treatment of urine samples or post-column derivatization. Accuracy and precision of determinations are illustrated by analytical recoveries (mean percentage +/- S.D., CV., n = 30) for multiple batch analyses of a diluted urine sample containing 20 mg/l of rhamnose (100 +/- 6.8, 6.2%), lactulose (100 +/- 6.1, 5.5%), 3-O-methyl-D-glucose (98 +/- 5.9, 5.5%) and xylose (104 +/- 7.1, 6.5%). Linearity of standard curves indicated that the lower limit for accurate quantification was 0.1 mg/l for all four sugars. Urinary recoveries following oral administration of these sugars to control dogs were determined as a baseline for the investigation of intestinal damage in this species and comparison of chromatograms illustrated enhanced permeability in dogs with gluten-sensitive enteropathy.