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Small intestinal bacterial overgrowth and enhanced intestinal permeability in healthy Beagles
Abstract
The small intestine of healthy adult Beagles was examined to determine whether subclinical abnormalities might exist that would be relevant to the use of Beagles in pharmacologic studies. Duodenal juice was obtained for qualitative and quantitative bacteriologic examinations; jejunal mucosa was taken for morphologic and biochemical investigation, and intestinal permeability was assessed by quantification of 24-hour urinary excretion of 51Cr-labeled EDTA after its oral administration. Comparisons were made with findings in healthy adult dogs of other breeds that served as controls. Small intestinal bacterial overgrowth was found in 14 of the 21 Beagles examined, and represented a mixed flora that included obligate anaerobic bacteria in 8 dogs and exclusively aerobic bacteria in 6 dogs. Intestinal permeability (percentage urinary recovery of 51Cr-labeled EDTA; mean +/- SEM) was considerably higher (P < 0.01) in Beagles with anaerobic overgrowth (37.6 +/- 3.2%) or aerobic overgrowth (30.5 +/- 4.8%), compared with Beagles with no overgrowth (17.3 +/- 1.6%) and with controls (11.1 +/- 1.0%). In Beagles, significant (r = 0.54, P = 0.03) correlation was observed between 24-hour urinary recovery of 51Cr-labeled EDTA and bacterial numbers in duodenal juice. Morphologic changes in jejunal mucosa were minimal, and specific activities of brush border enzymes were not significantly decreased, apart from aminopeptidase N, but activities of lysosomal and endoplasmic reticular marker enzymes were higher in the 3 groups of Beagles with anaerobic, aerobic, or no overgrowth, compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)
... Small intestinal bacterial overgrowth (SIBO) is becoming a significant problem in dogs and can result in enhanced intestinal permeability, diarrhea, and weight loss (Batt et al., 1992;Willard et al., 1994). Dogs that appear perfectly healthy can have small intestinal bacterial overgrowth (Batt et al., 1992). ...
... Small intestinal bacterial overgrowth (SIBO) is becoming a significant problem in dogs and can result in enhanced intestinal permeability, diarrhea, and weight loss (Batt et al., 1992;Willard et al., 1994). Dogs that appear perfectly healthy can have small intestinal bacterial overgrowth (Batt et al., 1992). Given these findings, it may be more important to examine the type of microbes inhabiting the small intestine rather than sheer numbers. ...
... Causes for the development of SIBO may include abnormalities in gastric acid secretion, intestinal peristalsis, ileocecal valve function, intestinal Ig secretion, and mucus barrier (Rutgers et al., 1996). Because it has been shown that high concentrations of microbial populations can be present in the small intestine of dogs that appear to be healthy (Batt et al., 1992), the balance of small intestinal microbial species seems to be more important than sheer numbers. Although Willard et al. (1994) did not measure specific microbial species, these researchers reported an improvement in small intestinal ecology of FOS-supplemented dogs having SIBO, with decreases (P < 0.05) in aerobic and anaerobic bacteria in tissue samples and aerobic bacteria in the duodenal/jejunal fluid samples. ...
... Gastrointestinal permeability is affected by factors within the lumen, such as pH and inflammation, as well as extraluminal factors including blood endotoxin levels and a genetic predisposition towards increased gut permeability (Sehesteed et al., 1999;Batt et al., 1992;Deitch et al., 1989;Stenson, 1999). ...
... Animals are also susceptible to SIBO after a course of antibiotics (Li, 1999). In the small intestine, Batt et al. (1992) showed that gastrointestinal permeability is increased during bacterial overgrowth in dogs, although pH was not measured. The pH of the gastrointestinal tract is known to decrease during SIBO in other species, however (Li, 1999). ...
... Gut inflammation. Chronic genetic conditions, such as inflammatory bowel disease in humans, can cause inflammation in the intestinal tract (Stenson, 1999) as can bacterial overgrowth due to stasis of the intestines (Batt et al., 1992;Li, 1999) or malabsorption of nutrients (Bown et al., 1974;Rutgers et al., 1996), and some toxins (Buchanan et al., 1991). This inflammation is thought to cause increased permeability in the small intestine (Batt et al., 1992;Fink et al., 1991;Krueger et al., 1986;Nusrat et al., 2000;Rutgers et al., 1996;Salzman et al., 1994;Shao et al., 2001). ...
The objective of this study was to evaluate the impact of post-ruminal starch digestion on inflammatory response in dairy cattle. Six cull, nonpregnant, nonlactating, multiparous cannulated Holstein dairy cows (BW 804±101 kg) were fed a high forage diet ad libitum starting 15 d before the infusion period. Cows were infused abomasally 12 h per day for 3 days with approximately 8 L d-1 of 0.9% saline solution (CTRL; 2 cows) or a 0.9% saline suspension of 4 kg of corn starch with 4 g xanthan gum (ST; 4 cows) using peristaltic pumps. Fecal samples, blood samples, and other measures were taken every 4 h (offset daily by 1 h). Data were analyzed as repeated measures with cow within treatment as a random variable. Fecal pH data showed 2 distinct responses to ST (ST1 and ST2). Fecal pH of all cows averaged 6.90 before infusion. During infusion days, fecal pH remained at 7.0±0.25 for CTRL, but declined in ST cows to 5.1 (ST1) and 4.9 (ST2) by the end of day 2, and diverged to 5.3 (ST1) and 4.6 (ST2) by the end of day 3. The increase in fecal pH for ST1 cows during day 3 suggests an increase in small intestinal digestion of starch, whereas a continuing decline in fecal pH for ST2 cows suggests that they did not adapt similarly. Fecal pH differed among CTRL, ST1, and ST2 (P<0.01). Blood values for haptoglobin decreased for CTRL and ST1 with day of infusion, but increased for ST2 with day 3 values of 12.3, 7.8, and 24.5 mg dl-1, respectively (P=0.04). Fibrinogen mg dl-1 tended to differ by treatment x infusion day, rising from 109 to 198, 143 to 188, and 221 to 250 between day 1 and 3 of infusion for CTRL, ST1, and ST2, respectively (P=0.11). Neither ceruloplasmin (P=0.38) nor alpha-acid glycoprotein (P=0.46) differed by treatment. Hematocrit tended to be greater for CTRL than ST (32.9 v 29.1%; P=0.08). Rectal temperature showed treatment x day effects with increases from day 1 to 3 of 38.5 to 38.7, 38.4 to 39.0, and 38.9 to 39.1°C for CTRL, ST1, and ST2, respectively (P<0.01); CTRL tended to be less than ST (P=0.09). Respiration rates did not differ (P=0.46). The basis for differing cow responses to post-ruminal starch load requires further evaluation.
https://www.ars.usda.gov/research/publications/publication/?seqNo115=251120
... Gastrointestinal permeability is affected by factors within the lumen, such as pH and inflammation, as well as extraluminal factors including blood endotoxin levels and a genetic predisposition towards increased gut permeability (Sehesteed et al., 1999;Batt et al., 1992;Deitch et al., 1989;Stenson, 1999). ...
... Animals are also susceptible to SIBO after a course of antibiotics (Li, 1999). In the small intestine, Batt et al. (1992) showed that gastrointestinal permeability is increased during bacterial overgrowth in dogs, although pH was not measured. The pH of the gastrointestinal tract is known to decrease during SIBO in other species, however (Li, 1999). ...
... Gut inflammation. Chronic genetic conditions, such as inflammatory bowel disease in humans, can cause inflammation in the intestinal tract (Stenson, 1999) as can bacterial overgrowth due to stasis of the intestines (Batt et al., 1992;Li, 1999) or malabsorption of nutrients (Bown et al., 1974;Rutgers et al., 1996), and some toxins (Buchanan et al., 1991). This inflammation is thought to cause increased permeability in the small intestine (Batt et al., 1992;Fink et al., 1991;Krueger et al., 1986;Nusrat et al., 2000;Rutgers et al., 1996;Salzman et al., 1994;Shao et al., 2001). ...
... Both enteric flora and genetic background of these dogs merited particular attention. The beagles had greater numbers of total and anaerobic bacteria than the IS, compatible with previous studies in beagles which have suggested that they tolerate a subclinical small intestinal bacterial overgrowth (SIBO) (Batt et al., 1992;Morris et al., 1994). Increased expression in beagles of both IFNg and IL10 may have been the consequence of direct interaction of bacteria with gut-associated lymphoid tissue (GALT), or over-exposure of the lamina propria to luminal antigens allowed across the intestinal epithelium by the increase in permeability afforded by bacterial interactions with enterocytes (Batt et al., 1992;Morris et al., 1994;Garcia-Lafuente et al., 1997). ...
... The beagles had greater numbers of total and anaerobic bacteria than the IS, compatible with previous studies in beagles which have suggested that they tolerate a subclinical small intestinal bacterial overgrowth (SIBO) (Batt et al., 1992;Morris et al., 1994). Increased expression in beagles of both IFNg and IL10 may have been the consequence of direct interaction of bacteria with gut-associated lymphoid tissue (GALT), or over-exposure of the lamina propria to luminal antigens allowed across the intestinal epithelium by the increase in permeability afforded by bacterial interactions with enterocytes (Batt et al., 1992;Morris et al., 1994;Garcia-Lafuente et al., 1997). The IFNg may have been a marker of an immunogenic, Th1 response to these antigens, while the IL10 may have been a marker of a reactive, contra-inflammatory response. ...
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 IFN gamma 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 IFN gamma, and IL10 in the lamina propria (p>0.05), in contrast to beagles, which showed greater total and grade 1 areas of expression of IFN gamma, 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 IFN gamma and IL10 mRNA within canine intestinal biopsies, and has shown heightened jejunal expression of both cytokines in beagles with high enteric bacterial populations.
... Both enteric flora and genetic background of these dogs merited particular attention. The beagles had greater numbers of total and anaerobic bacteria than the IS, compatible with previous studies in beagles which have suggested that they tolerate a subclinical small intestinal bacterial overgrowth (SIBO) (Batt et al., 1992;Morris et al., 1994). Increased expression in beagles of both IFNg and IL10 may have been the consequence of direct interaction of bacteria with gut-associated lymphoid tissue (GALT), or over-exposure of the lamina propria to luminal antigens allowed across the intestinal epithelium by the increase in permeability afforded by bacterial interactions with enterocytes (Batt et al., 1992;Morris et al., 1994;Garcia-Lafuente et al., 1997). ...
... The beagles had greater numbers of total and anaerobic bacteria than the IS, compatible with previous studies in beagles which have suggested that they tolerate a subclinical small intestinal bacterial overgrowth (SIBO) (Batt et al., 1992;Morris et al., 1994). Increased expression in beagles of both IFNg and IL10 may have been the consequence of direct interaction of bacteria with gut-associated lymphoid tissue (GALT), or over-exposure of the lamina propria to luminal antigens allowed across the intestinal epithelium by the increase in permeability afforded by bacterial interactions with enterocytes (Batt et al., 1992;Morris et al., 1994;Garcia-Lafuente et al., 1997). The IFNg may have been a marker of an immunogenic, Th1 response to these antigens, while the IL10 may have been a marker of a reactive, contra-inflammatory response. ...
... Small intestinal bacterial overgrowth (SIBO), defined as >10 5 bacteria per ml of duodenal juice, has been associated with IgA deficiency (72,73,78,79). Enteric bacteria can synthesize folate, and many can bind cobalamin (Vitamin B12) within the lumen. ...
... Lymphocytic-plasmacytic enteritis may be a direct consequence of small intestinal bacterial overgrowth (73,78). Enhanced permeability and histologic damage to jejunal mucosa was associated with confirmed SIBO in clinically normal Beagles (79). Antibiotic treatment to correct SIBO led to marked improvement in histologic lesions in a German Shepherd (78). ...
Primary immunodeficiencies are congenital defects that affect formation or function of the immune system. Congenital immunodeficiency should be considered as a differential diagnosis for repeated infections in a young animal. Defects in the immune system may lead to complete or partial loss of immunity. Some animals with mild immunodeficiency can be managed with long-term antibiotic therapy.
... Both enteric flora and genetic background of these dogs merited particular attention. The beagles had greater numbers of total and anaerobic bacteria than the IS, compatible with previous studies in beagles which have suggested that they tolerate a subclinical small intestinal bacterial overgrowth (SIBO) (Batt et al., 1992;Morris et al., 1994). Increased expression in beagles of both IFNg and IL10 may have been the consequence of direct interaction of bacteria with gut-associated lymphoid tissue (GALT), or over-exposure of the lamina propria to luminal antigens allowed across the intestinal epithelium by the increase in permeability afforded by bacterial interactions with enterocytes (Batt et al., 1992;Morris et al., 1994;Garcia-Lafuente et al., 1997). ...
... The beagles had greater numbers of total and anaerobic bacteria than the IS, compatible with previous studies in beagles which have suggested that they tolerate a subclinical small intestinal bacterial overgrowth (SIBO) (Batt et al., 1992;Morris et al., 1994). Increased expression in beagles of both IFNg and IL10 may have been the consequence of direct interaction of bacteria with gut-associated lymphoid tissue (GALT), or over-exposure of the lamina propria to luminal antigens allowed across the intestinal epithelium by the increase in permeability afforded by bacterial interactions with enterocytes (Batt et al., 1992;Morris et al., 1994;Garcia-Lafuente et al., 1997). The IFNg may have been a marker of an immunogenic, Th1 response to these antigens, while the IL10 may have been a marker of a reactive, contra-inflammatory response. ...
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.
... One possibility is that, given the very high sugar intake, there may have also been extensive microbial metabolism of [U-13 C]glucose. Glucose is normally efficiently absorbed in the small intestine, but small intestinal bacterial overgrowth [29,30], possibly induced by high sugar diets [31], can result in a portion of the glucose being fermented instead. Finally, the PPP flux is based on the sugar phosphates that are recycled back to fructose-6-P and glucose-6-P and does not take into account those pentose-P equivalents that were recruited for nucleotide biosynthesis. ...
Dietary glucose and fructose are both efficiently assimilated by the liver but a comprehensive
measurement of this process starting from their conversion to sugar phosphates, involvement
of the pentose phosphate pathway (PPP), and conversion to glycogen and lipid storage products,
remains incomplete. Mice were fed a chow diet supplemented with 35 g/100 mL drinking water of a
55/45 fructose/glucose mixture for 18 weeks. On the final night, the sugar mixture was enriched
with either [U-13C]glucose or [U-13C]fructose, and deuterated water (2H2O) was also administered.
13C-isotopomers representing newly synthesized hepatic glucose-6-phosphate (glucose-6-P), glycerol-
3-phosphate, and lipogenic acetyl-CoA were quantified by 2H and 13C NMR analysis of post-mortem
liver glycogen and triglyceride. These data were applied to a metabolic model covering glucose-6-P,
PPP, triose-P, and de novo lipogenesis (DNL) fluxes. The glucose supplement was converted to
glucose-6-P via the direct pathway, while the fructose supplement was metabolized by the liver
to gluconeogenic triose-P via fructokinase–aldolase–triokinase. Glucose-6-P from all carbohydrate
sources accounted for 40–60% of lipogenic acetyl-CoA and 10–12% was oxidized by the pentose
phosphate pathway (PPP). The yield of NADPH from PPP flux accounted for a minority (~30%) of
the total DNL requirement. In conclusion, this approach integrates measurements of glucose-6-P, PPP,
and DNL fluxes to provide a holistic and informative assessment of hepatic glucose and fructose
metabolism.
... Our strict study inclusion criteria (disease free animals with normal diagnostic tests) resulted in a small population of healthy control dogs, that were younger and had discrepancy in genetic background compared to case population. The majority of control dogs were of mixed-breed origin and were considered a better choice versus agematched, research reared Beagles which might have evidence of intestinal disease [84]. It is important to acknowledge that environmental factors (age, genetic background and dietary history) may have possibly biased our results. ...
Canine inflammatory bowel disease (IBD) is a group of chronic gastrointestinal disorders, the pathogenesis of which remains elusive, but it possibly involves the interaction of the intestinal immune system with luminal microbiota and food-derived antigens. Mucosal cytokines profiles in canine IBD have been investigated mainly in small intestinal disease, while data on cytokine profiles in large intestinal IBD are limited. The objective of this study was to measure colonic mucosal and cytobrush sample messenger (m)RNA expression of interleukin (IL)-1β, IL-2, IL-12p40, IL-23p19, tumor necrosis factor-alpha (TNF-α) and chemokine C‐C motif ligand (CCL28) in dogs with IBD and healthy controls using quantitative real-time polymerase chain reaction (PCR), and assess their correlation with clinical disease activity, endoscopic and histopathologic score. Dogs with IBD had a significantly increased mRNA expression of IL-1β, IL-23p19 and CCL28 in the colonic mucosa, compared to healthy controls. None of the selected cytokines had significantly different mRNA expression in the colonic cytobrush samples between the two groups or between the colonic mucosa and cytobrush samples of dogs with IBD. Finally, there was a statistically significant correlation of clinical disease activity with endoscopic activity score and fibrosis and atrophy of the colonic mucosa in dogs with large intestinal IBD. IL-1β, IL-23p19 and CCL28 could play a role in the pathogenesis of canine large intestinal IBD. Colonic cytokine expression does not correlate with clinical disease activity and/or endoscopic score. However, clinical signs reflect the severity of endoscopic lesions.
... However, inclusion of healthy control dogs of mixed breeds was considered preferable to inclusion of age-matched Beagles bred for use in research; such Beagles sometimes have evidence of intestinal disease. 53 Therefore, it is possible that some of the differences in gene expression were attributable to differences in age between the 2 groups of dogs. Nevertheless, the tissue sampling and microchip array analysis methods used in the present study yielded results that indicated patterns of gene expression in dogs with CE were broadly similar to those in humans with IBD, including expression of genes regulating immune and inflammatory responses, metabolism, cell proliferation, and epithelial structure and function. ...
... Batt and coworkers found bacterial overgrowth in 14 of 21 healthy beagles with only minimal morphologic changes in jejunal mucosa. 43 Furthermore, activities of lysosomal and endoplasmic reticulum marker enzymes were higher in the beagles compared with other healthy controls. Owing to limited access to other HCD, healthy Beagle dogs serving as placebo controls in pharmacologic studies were used. ...
Background
Cyclooxygenase-2 (COX-2) is a key enzyme in the synthesis of pro-inflammatory prostaglandins and 5-lipoxygenase (5-LO) is the major source of leukotrienes. Their role in IBD has been demonstrated in humans and animal models, but not in dogs with chronic enteropathies (CCE).HypothesisCOX-2 and 5-LO are upregulated in dogs with CCE.AnimalsFifteen healthy control dogs (HCD), 10 dogs with inflammatory bowel disease (IBD), and 15 dogs with food-responsive diarrhea (FRD).Methods
Prospective study. mRNA expression of COX-2, 5-LO, IL-1b, IL-4, IL-6, TNF, IL-10 and TFG-β was evaluated by quantitative real-time RT-PCR in duodenal and colonic biopsies before and after treatment.ResultsCOX-2 expression in the colon was significantly higher in IBD and FRD before and after treatment (all P < .01). IL-1b was higher in FRD in the duodenum after treatment (P = .021). TGF-β expression was significantly higher in the duodenum of HCD compared to FRD/IBD before treatment (both P < .001) and IBD after treatment (P = .012). There were no significant differences among groups and within groups before and after treatment for IL-4, IL-6, TNF, and IL-10. There was a significant correlation between COX-2 and IL-1b in duodenum and colon before treatment in FRD and IBD, whereas 5-LO correlated better with IL-6 and TNF. IL-10 and TGF-β usually were correlated.Conclusions and Clinical ImportanceCOX-2 is upregulated in IBD and FRD, whereas IL-1b and TGF-β seem to be important pro- and anti-inflammatory cytokines, respectively. The use of dual COX/5-LO inhibitors could be an interesting alternative in the treatment of CCE.
... Our strict study inclusion criteria (disease free animals with normal diagnostic tests) resulted in a relatively small population of healthy control dogs. Some of these dogs were of mixed-breed origin and were considered a better choice versus age-matched, research reared Beagles which may have evidence of intestinal disease (Batt et al., 1992). Other control dogs were young laboratoryreared Beagles which also were clinically normal and free of intestinal inflammation on histopathologic examination. ...
... However, inclusion of healthy control dogs of mixed breeds was considered preferable to inclusion of age-matched Beagles bred for use in research; such Beagles sometimes have evidence of intestinal disease. 53 Therefore, it is possible that some of the differences in gene expression were attributable to differences in age between the 2 groups of dogs. Nevertheless, the tissue sampling and microchip array analysis methods used in the present study yielded results that indicated patterns of gene expression in dogs with CE were broadly similar to those in humans with IBD, including expression of genes regulating immune and inflammatory responses, metabolism, cell proliferation, and epithelial structure and function. ...
To characterize mucosal gene expression in dogs with chronic enteropathy (CE).
18 dogs with CE and 6 healthy control dogs.
Small intestinal mucosal biopsy specimens were endoscopically obtained from dogs. Disease severity in dogs with CE was determined via inflammatory bowel index scores and histologic grading of biopsy specimens. Total RNA was extracted from biopsy specimens and microchip array analysis (approx 43,000 probe sets) and quantitative reverse transcriptase PCR assays were performed.
1,875 genes were differentially expressed between dogs with CE and healthy control dogs; 1,582 (85%) genes were downregulated in dogs with CE, including neurotensin, fatty acid-binding protein 6, fatty acid synthase, aldehyde dehydrogenase 1 family member B1, metallothionein, and claudin 8, whereas few genes were upregulated in dogs with CE, including genes encoding products involved in extracellular matrix degradation (matrix metallopeptidases 1, 3, and 13), inflammation (tumor necrosis factor, interleukin-8, peroxisome proliferator-activated receptor γ, and S100 calcium-binding protein G), iron transport (solute carrier family 40 member 1), and immunity (CD96 and carcinoembryonic antigen-related cell adhesion molecule [CEACAM] 18). Dogs with CE and protein-losing enteropathy had the greatest number of differentially expressed genes. Results of quantitative reverse transcriptase PCR assay for select genes were similar to those for microchip array analysis.
Expression of genes encoding products regulating mucosal inflammation was altered in dogs with CE and varied with disease severity. Impact for Human Medicine-Molecular pathogenesis of CE in dogs may be similar to that in humans with inflammatory bowel disease.
... Also, we could not verify conclusively whether they had been used in other trials prior to arrival at ISU. Furthermore, it has been suggested that research-derived Beagles may harbor an altered intestinal microbiota and may have evidence of intestinal disease [30]. Therefore, we chose to enroll a smaller number of dogs of mixed-breed and of random source origin to serve as the control group. ...
Canine idiopathic inflammatory bowel disease (IBD) is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing.
Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6) and dogs with moderate IBD (n = 7) or severe IBD (n = 7) as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001). Proportions of Fusobacteria (p = 0.010), Bacteroidaceae (p = 0.015), Prevotellaceae (p = 0.022), and Clostridiales (p = 0.019) were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044) and Acinetobacter (p = 0.040), were either more abundant or more frequently identified in IBD dogs.
In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation.
... Intestinal permeability, as measured by 51 Cr-EDTA or differential sugar absorption, can be abnormal in ARD and can improve after antibiotic treatment. 48,49 However, these findings are not pathognomonic for either secondary SIBO or idiopathic ARD. Moreover, measurement of intestinal permeability is not readily available in practice. ...
Antibiotic-responsive diarrhea (ARD) is an idiopathic syndrome causing chronic diarrhea in young, large-breed dogs. Why antibiotics are effective in controlling diarrhea is not understood, and whether small intestinal bacterial numbers are truly increased is now doubted, but previous focus on the condition being small intestinal bacterial overgrowth has hampered the understanding of this condition. The name ARD simply defines the condition, and studies are now looking at the interaction of small intestinal bacteria and the mucosa to try to understand why it occurs.
... LAB have been cited to be part of human (Fuller, 1991;Goldin, 1990;Holzapfel et al., 2001;Reid, 2001;Schrezenmeir and de Vrese, 2001;Sghir et al., 2000) and animal (Batt et al., 1991;Benno et al., 1992;Fujisawa and Mitsuoka, 1996;Perdigón et al., 2001;Rodríguez et al., 2003;Schrezenmeir and de Vrese, 2001) microbiota. The neonates receive their microbiota primarily in labor and later from the environment (Edwards and Parrett, 2002;Fuller, 1989;Fuller and Gibson, 1998;Metchnikoff, 1908). ...
... dog breeds examined and that they do not reflect changes specific to CE in GSD. Previous studies have used healthy Beagles as controls, which may represent an even poorer choice of control group as many Beagles have evidence of intestinal disease (Batt et al., 1992). However, the control group used in this study showed comparable relative mRNA expression of TLR2, TLR4 and TLR9 to previously published data from healthy Beagles (Burgener et al., 2008;McMahon et al., 2010). ...
The pathogenesis of chronic enteropathies in dogs likely involves an interaction between the intestinal immune system and luminal intestinal bacteria. German shepherd dogs (GSD) are particularly predisposed to chronic enteropathies. The present study sought to evaluate expression patterns of certain pattern recognition receptors of the innate immunity (Toll-like receptors, TLR), clinical disease activity and histopathological severity in GSD with chronic enteropathies. Mucosal biopsies were collected from the duodenum, colon and ileum of 13 affected GSD and 10 healthy greyhounds as controls. Dogs were objectively assessed using published scoring systems for clinical and histological severity of disease. Diversity of the duodenal microbiota was assessed by construction of 16S rRNA gene libraries. Expression of TLR2, TLR4, TLR5 and TLR9 in biopsies of the duodenum, ileum and colon was assessed by quantitative real-time PCR. TLR4 expression was increased in all intestinal segments in GSD, however, TLR5 expression was very low compared to the healthy dogs. The microbiota in the duodenum of GSDs was significantly different to that of the greyhounds, with an over-representation of 16S rRNA gene sequences belonging to the classes of Bacilli, and Erysipelotrichi, and to the orders of Lactobacillales, Actinomycetales and Erysipelotrichales. These findings could point to a distinct pathogenesis of chronic enteropathies in GSD, with differentially high and low expression of TLR4 and TLR5, respectively, and increased proportions of specific members of the Lactobacillales potentially playing a role.
... Microbiological culture of duodenal juice obtained endoscopically or at laparotomy is needed to confirm the diagnosis of SIBO, and should demonstrate greater than l o 5 total or greater than lo4 obligate anaerobic cfu/ml in dogs (Burrows andothers 1995, Rutgers andothers 1995a). A definite association between these numbers of bacteria, clinical disease and mucosal damage has been established in dogs (Batt and McLean 1987, Batt and others 1988, 1992, Rutgers and others 1995a) although higher numbers may be found in apparently clinically healthy dogs for reasons discussed above (Willard and others 1994). The most frequent isolates typically include enterococci and E coli in dogs with aerobic overgrowth, and clostridia in dogs with anaerobic overgrowth. ...
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.
... The bacterial overgrowth observed in association with NSAID administration 7±11, 25 also participates in the changes of permeability. 26,27 On the basis of the above mentioned biochemical, physiological and microbiological changes occurring in the gastrointestinal mucosa after NSAIDs ingestion, various properties of probiotic strains could exert protective effects against NSAID-induced injury: (i) ...
Chronic nonsteroidal anti-inflammatory drug (NSAID) ingestion strongly affects the gastrointestinal mucosa as a first stage before ulceration. Some Lactobacillus strains may stabilize the mucosal barrier by increasing mucin expression, reducing bacterial overgrowth, stimulating mucosal immunity and synthetizing antioxidant substances; these events are altered in NSAID-associated gastroenteropathy.
To determine whether ingestion of the probiotic Lactobacillus GG (LGG) protects the gastrointestinal mucosa against indometacin-induced alterations of permeability.
Four gastrointestinal permeability tests were carried out in random order in 16 healthy volunteers: (i) basal; (ii) after indometacin; (iii) after 5 days of living LGG ingestion before indometacin administration; (iv) after 5 days of heat-killed LGG ingestion before indometacin administration.
Indometacin significantly increased basal sucrose urinary excretion (29.6 mg [17.1-42.1] vs. 108.5 mg [68.2-148.7], P=0.0030) (means [95% CI]) and lactulose/mannitol urinary excretion (1.03% [0.73-1. 32] vs. 2.93% [1.96-3.90], P=0.00012). Heat-killed LGG did not modify the indometacin-induced increase of gastrointestinal permeability, while live bacteria significantly reduced the alteration of gastric (47.8 mg [31.1-64.6], P=0.012) but not intestinal permeability induced by NSAID.
Regular ingestion of LGG protects the integrity of the gastric mucosal barrier against indometacin, but has no effect at the intestinal level.
... In vitro microbial fermentation of starches by ileal bacteria can be substantial and is affected by differences in starch source, fraction, and processed form. healthy, adult beagles (Batt et al., 1992) and healthy, domestic short-hair cats (Johnston et al., 1993). Most components of cereal grains and potato are digested primarily via small intestinal enzymatic degradation . ...
Two in vitro experiments were conducted to evaluate the ability of small intestinal bacteria of dogs to ferment native and extruded cereal grains and potato starch and cereal grain and potato flours. Substrates included barley, corn, potato, rice, sorghum, and wheat. In addition to testing native grains and flours, extruded substrates also were tested. Substrates were extruded at low temperatures (LT; 79 to 93 degrees C) and high temperatures (HT; 124 to 140 degrees C) using a Wenger extruder (model TX-52). Substrates varied widely in concentrations of rapidly digestible starch (RDS), slowly digestible starch (SDS), resistant starch (RS), and total starch (TS). Extrusion of most substrates at HT vs LT resulted in increased RDS and decreased RS concentrations. Organic matter disappearance (OMD) values attributed to microbial fermentation for a 5-h period were as high as 27% for native extruded substrates (LT potato starch) and 39% for potato flour. Average OMD was higher for cereal and potato flours than for native extruded substrates (29.9 vs 25.4%). Average molar percentages of short-chain fatty acids produced from all substrates fermented for 5 h were 73, 14, and 13% (acetate, propionate, and butyrate, respectively). Average lactate production for substrates ranked as follows: flours > native and extruded cereal grains and potato starch (0.33 and 0.18 mmol/g OM, respectively). In vitro microbial fermentation of starches by ileal bacteria can be substantial and is affected by differences in starch source, fraction, and processed form.
This study aimed to assess the morphometry of enterocytes as well as the goblet cell-to-enterocyte ratio in different intestinal segments of dogs with chronic enteropathies (CE). Histopathological intestinal samples from 97 dogs were included in the study (19 healthy juveniles, 21 healthy adults, 24 dogs with protein-losing enteropathy (PLE), and 33 CE dogs without PLE). Healthy adult small intestinal enterocytes showed progressively reduced epithelial cell height in the aboral direction, while juvenile dogs showed progressively increased epithelial cell height in the aboral direction. CE dogs had increased epithelial cell height in the duodenum, while PLE dogs had decreased epithelial cell heights compared to healthy adult dogs. Both the CE and PLE dogs showed decreased enterocyte width in the duodenal segment, and the ileal and colonic enterocytes of CE dogs were narrower than those of healthy adult dogs. CE dogs had a lower goblet cell-to-enterocyte ratio in the colon segment compared to healthy dogs. This study provides valuable morphometric information on enterocytes during canine chronic enteropathies, highlighting significant morphological enterocyte alterations, particularly in the small intestine, as well as a reduced goblet cell-to-enterocyte ratio in the colon of CE cases compared to healthy adult dogs.
In its broadest terms, intestinal bacterial translocation can be defined as the passage of bacteria (both live and dead) and bacterial products (such as exotoxins, endotoxins, and cell wall fragments) from the intestinal lumen to otherwise sterile extraintestinal sites. Investigators studying immune mechanisms at mucosal surfaces have long recognized that epithelial uptake and processing of intestinal antigens is a complex process, needed not only to establish the immune status of the host, but to continually regulate the immune response (both inductive and suppressive) to intestinal antigens [1]. The transmigration of intestinal bacteria was initially viewed with skepticism by many physicians; however, in recent years, the existence of bacterial translocation (BT) has become generally accepted among clinicians, although the clinical significance of this process remains a subject of debate.
This chapter describes the normal biochemical processes of intestinal secretion, digestion, and absorption. The digestive system is composed of the gastrointestinal (GI) tract, or the alimentary canal, salivary glands, the liver, and the exocrine pancreas. The principal functions of the gastrointestinal tract are to digest and absorb ingested nutrients, and to excrete waste products of digestion. Most nutrients are ingested in a form that is either too complex for absorption or insoluble, and therefore, indigestible or incapable of being digested. Within the GI tract, much of these substances are solubilized and further degraded enzymatically to simple molecules, sufficiently small in size, and in a form that permits absorption across the mucosal epithelium. This chapter explains in detail the mechanisms of salivary secretions, compositions of saliva, and the functions of saliva. The chapter also elaborates properties of bile as well as the synthesis of bile acids. The chapter explores the pathogenesis of the important gastrointestinal diseases of domestic animals, and the biochemical basis for their diagnosis and treatment. The chapter concludes with a discussion on disturbances of gastrointestinal function such as vomition, acute diarrheas, malabsorption, bacterial overgrowth, and ulcerative colitis.
This overview summarizes research performed during the last decade that has had an impact on the diagnosis and management of exocrine pancreatic insufficiency in small animals. Procedures for the investigation of dogs with clinical signs suggestive of malabsorption are presented as a step-by-step approach; particular attention is paid to the diagnosis of pancreatic insufficiency prior to the investigation of small intestinal disease. Procedures applicable to cats are indicated. Secondary pathophysiologic changes in the small intestines of dogs with pancreatic insufficiency are summarized and related to the management of the disease. Some insight into the underlying mechanism of pancreatic acinar atrophy in dogs and the direction for future research are discussed.
Roux-en-Y gastric bypass (RYGB) interferes considerably with the anatomy and physiology of the gastrointestinal tract. The study of intestinal permeability can provide important information regarding changes in the structure and function of the mucosal barrier after the procedure.
The urinary excretion rates of lactulose and mannitol after oral intake of both substances were evaluated. We also evaluated the lactulose/mannitol excretion ratio. Tests were performed during the preoperative period (T0), at the first postoperative month (T1), and at the sixth postoperative month (T6).
The study included 16 morbidly obese patients. The excretion rate of mannitol was significantly lower at T1 compared with T0 and T6 (p = 0.003). There was no significant difference in the excretion rates of lactulose or in the lactulose/mannitol ratio during the three periods. Six patients (37.5 %) exhibited a considerable increase in the excretion rate of lactulose at T6 (4-73 times higher than the preoperative value), accompanied by proportional variations in the lactulose/mannitol ratio.
The significant increase in mannitol excretion rate from T1 to T6 most likely reflects the occurrence of intestinal adaptation (mucosal hyperplasia), which would tend to minimize the malabsorption of macronutrients. A subgroup of patients who undergo RYGB exhibit pronounced increase in their intestinal permeability (assessed by the lactulose/mannitol ratio and the lactulose excretion rate) at T6.
Vergleich von Zellulose, Pektin und Guar als diätetische Faserquellen im Mischfutter für Hunde
Zellulose, Guar und Pektin wurden zu jeweils 10% (ursprüngliche Substanz) als Faserquelle in Hundefutter eingesetzt, das als weitere Komponenten getrocknete Grieben, mikronisierten Reis, Sojaöl und ein vitaminiertes Mineralfutter enthielt. Vier adulte Hunde standen zur Verfügung, die von den jeweiligen Mischungen 18–21 g TS/kg KM/Tag aufnahmen. Neben möglichen Effekten auf die Kotkonsistenz wurden die scheinbare Verdaulichkeit der Rohnährstoffe sowie von Natrium und Kalium, die renale Ausscheidung von Stickstoff und Parameter zur Charakterisierung nutritiver Effekte auf die Intestinalflora (Zusammensetzung der fäkalen Mikroflora, flüchtige Fettsäuren und Laktat in den Fäzes, H2-Exhalationstest, renale Indikanexkretion) erfaßt.
Die Kotkonsistenz zeigte sich bei Verwendung von Zellulose fest-geformt (Wassergehalt der Fäzes 61,1 ± 1,8%), während Pektin und insbesondere Guar zu einer weich-schmierigen Kotbeschaffenheit führten (fäkale Wassergehalte 70,5 ± 3,6 bzw. 74,1 ± 2,9%). Die scheinbare Verdaulichkeit der Trokkensubstanz war bei Zellulosezusatz signifikant vermindert (80,3 ± 2,0%), bei Verwendung von Pektin bzw. Guar wurden 89,6 ± 1,7 bzw. 88,1 ± 1,3% ermittelt. Eine geringere Na-Nettoabsorption (86% gegenüber > 94%) wurde bei Verabreichung der zellulosehaltigen Diät festgestellt, während Guarzusatz die scheinbare Verdaulichkeit von Kalium verminderte (84% gegenüber > 91 % in den Vergleichsperioden). Guar und Pektin induzierten im Vergleich zu Zellulose höhere fäkale Keimgehalte, teilweise auch zunehmende Stoffwechselaktivitäten der Intestinalflora.
Aus vorliegender Untersuchung ist abzuleiten, daß Hunde ‘lösliche’ Faserquellen nur begrenzt tolerieren, ansonsten sind unerwünschte Effekte auf die Kotqualität nicht auszuschliessen.
Background:
The detailed pathological phenotype of diet-responsive chronic enteropathy (CE) and its modulation with dietary therapy remain poorly characterized.
Hypothesis/objectives:
Key mucosal lesions of diet-responsive CE resolve with dietary therapy.
Methods:
This was a prospective observational study of 20 dogs with diet-responsive CE. Endoscopic duodenal biopsies collected before and 6 weeks after the start of a dietary trial were assessed by means of qualitative and quantitative histopathological, immunohistochemical, and ultrastructural criteria. Control duodenal biopsies were obtained from 10 healthy Beagle dogs on 1 occasion.
Results:
Compared with control dogs, the CE dogs had higher villus stunting scores and higher overall WSAVA scores, a lower villus height-to-width ratio, and higher lamina propria density of eosinophils. The CE dogs also had ultrastructural lesions of the mitochondria and brush border. In common with other studies in which the disease and control populations are not matched for breed, age, sex, and environment, these comparisons should be interpreted with caution. Comparing biopsies collected at presentation and 6 weeks after starting the dietary trial, mean lamina propria mononuclear cell score and lamina propria densities of eosinophils and mononuclear cells decreased. Dietary therapy also improved ultrastructural lesions of the mitochondria and brush border, eliciting a decrease in intermicrovillar space and an increase in microvillus height.
Conclusions and clinical importance:
In dogs with diet-responsive CE, the remission of clinical signs with dietary therapy is associated with subtle decreases in lamina propria density of eosinophils and mononuclear cells, and resolution of ultrastructural lesions of the enterocyte.
Maximum breath hydrogen excretion after the oral administration of xylose to 11 healthy cats ranged from 0.13 ml/hour to 0.47 ml/hour, with a mean of 0.18 ml/hour. After oral administration of xylose, breath hydrogen excretion in five cats with chronic diarrhoea and, or, vomiting was significantly different (P<0.001) compared with healthy cats. Increased breath hydrogen excretion occurred before xylose was given and at all measurement times after its administration to the sick cats (P<0.05), indicating carbohydrate malassimilation. In four sick cats, large increases in breath hydrogen excretion occurred, with maximum values ranging from 1.21 to 1.56 ml/hour, but in one cat the maximum value was only 0.28 ml/hour. Plasma xylose concentrations in cats with chronic diarrhoea and, or, vomiting were not significantly different from healthy cats (P>0.05) and thus did not demonstrate carbohydrate malassimilation. A hiatus hernia was seen on radiographic views of the thorax and abdomen of one cat with chronic vomiting. Inflammatory bowel disease was found in three of the five sick cats after upper gastrointestinal endoscopic examination and mucosal biopsy. Clostridium species were isolated in increased numbers from the cats with chronic diarrhoea and, or, vomiting (P<0.005), after quantitative bacterial culture of small intestinal fluid specimens obtained endoscopically. Clostridium species were isolated from all five cats with chronic diarrhoea and, or, vomiting but from only one of eight healthy cats. However, whether a specific bacterial pathogen caused the increased breath hydrogen excretion found in these cats could not be determined from this study.
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 105 total or greater than 104 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 18 German shepherd dogs. SIBO was aerobic in 18/63 dogs (29 per cent), and anaerobic in 45/63 (71 per cent). Histological examination of duode-nal 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.
Objective —To provide values for gastrointestinal permeability and absorptive function tests (GIPFTs) with chromium 51 ( ⁵¹ Cr)-labeled EDTA, lactulose, rhamnose, d -xylose, 3-O-methyl- d -glucose, and sucrose in Beagles and to evaluate potential correlations between markers.
Animals —19 healthy adult male Beagles.
Procedures —A test solution containing 3.7 MBq of ⁵¹ Cr-labeled EDTA, 2 g of lactulose, 2 g of rhamnose, 2 g of d -xylose, 1 g of 3-O-methyl- d -glucose, and 8 g of sucrose was administered intragastrically to each dog. Urinary recovery of each probe was determined 6 hours after administration.
Results —Mean ± SD (range) percentage urinary recovery was 6.3 ± 1.6% (4.3% to 9.7%) for ⁵¹ Cr-labeled EDTA, 3.3 ± 1.1% (1.7% to 5.3%) for lactulose, 25.5 ± 5.0% (16.7% to 36.9%) for rhamnose, and 58.8% ± 11.0% (40.1% to 87.8%) for 3-O-methyl- d -glucose. Mean (range) recovery ratio was 0.25 ± 0.06 (0.17 to 0.37) for ⁵¹ Cr-labeled EDTA to rhamnose, 0.13 ± 0.04 (0.08 to 0.23) for lactulose to rhamnose, and 0.73 ± 0.09 (0.60 to 0.90) for d -xylose to 3-O-methyl- d -glucose. Median (range) percentage urinary recovery was 40.3% (31.6% to 62.7%) for d -xylose and 0% (0% to 0.8%) for sucrose.
Conclusions and Clinical Relevance —Reference values in healthy adult male Beagles for 6 of the most commonly used GIPFT markers were determined. The correlation between results for ⁵¹ Cr-labeled EDTA and lactulose was not as prominent as that reported for humans and cats; thus, investigators should be cautious in the use and interpretation of GIPFTs performed with sugar probes in dogs with suspected intestinal dysbiosis.
Permanent jejunal fistulas enable easy, noninjurious, repeated and direct administration to and collection from the small intestines of conscious laboratory dogs. This study aimed at identifying potential alterations in the small intestinal morphology and function of this canine model after the surgery required to establish the fistulas. Assays of serum folate and cobalamin and (51)Cr-EDTA permeability tests were performed before and 4 wk after experimental jejunoplasties in 14 laboratory beagle dogs. Serum folate concentrations (mean ± SD) before (12.22 ± 1.80 μg/L) and after (14.14 ± 1.70 μg/L) jejunal surgery were within reference ranges for healthy dogs, although folate concentrations were higher after surgery. The cobalamin concentrations and the 6-h urinary excretion of (51)Cr-EDTA before (573.50 ± 150.04 ng/L and 6.75 ± 1.56%, respectively) and after (496.71 ± 164.22 ng/L and 6.41 ± 1.10%) were normal for healthy dogs, and no significant differences between pre- and postsurgical values were detected. The findings of the present study indicate that the small intestinal vitamin absorption and permeability of laboratory beagle dogs with jejunal fistulas remains unimpaired.
A 51-chromium-labeled ethylenediamine tetra-acetic acid ((51)Cr-EDTA) permeability blood test was validated as a method to assess damage to the small intestine in dogs. The test was performed by calculating various percentages from sera after an orally ingested dose solution. The aim of the current study was to determine whether the use of serum or plasma had any influence on the results of the test. A test solution with 3.7 megabecquerel (100 µCi) of (51)Cr-EDTA was delivered through an orogastric tube to 13 healthy laboratory Beagle dogs. From each dog, 2 concurrent blood samples were withdrawn from cephalic veins using clotting-factor activator tubes for serum and heparinized tubes for plasma. The samples (n = 26) were taken at 3 and 5 hr after administration of the test solution. Percentages of the orally ingested dose were then calculated in serum and plasma, and their relationship was assessed using correlation analysis. The mean ± standard deviation percentages in serum and plasma after 3 hr were 0.85 ± 0.43% and 0.88 ± 0.49%, respectively, whereas respective percentages in serum and plasma after 5 hr were 0.78 ± 0.52% and 0.81 ± 0.51%. The combined correlation coefficient between the percentages from the sera and plasma samples was excellent (R = 0.98). It was concluded that the (51)Cr-EDTA permeability test in blood may be performed using serum or plasma of dogs, and the choice between the 2 samples is one of convenience.
To develop and analytically validate a gas chromatography-mass spectrometry (GC-MS) method for the quantification of lactulose, rhamnose, xylose, 3-O-methylglucose, and sucrose in canine serum.
Pooled serum samples from 200 dogs. Procedures-Serum samples spiked with various sugars were analyzed by use of GC-MS. The method was analytically validated by determination of dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability.
Standard curves ranging from 0.5 to 500 mg/L for each sugar revealed a mean r(2) of 0.997. The lower detection limit was 0.03 mg/L for lactulose, rhamnose, xylose, and methylglucose and 0.12 mg/L for sucrose. The observed-to-expected ratios for dilutional parallelism had a mean +/- SD of 105.6 +/- 25.4% at dilutions of 1:2, 1:4, and 1:8. Analytic recoveries for the GC-MS assays of sugars ranged from 92.1% to 124.7% (mean +/- SD, 106.2 +/- 13.0%). Intra-assay coefficients of variation ranged from 6.8% to 12.9% for lactulose, 7.1% to 12.8% for rhamnose, 7.2% to 11.2% for xylose, 8.9% to 11.5% for methylglucose, and 8.9% to 12.0% for sucrose. Interassay coefficients of variation ranged from 7.0% to 11.5% for lactulose, 6.4% to 9.4% for rhamnose, 6.8% to 13.2% for xylose, 7.0% to 15.9% for methylglucose, and 5.5% to 9.4% for sucrose.
The GC-MS method described here was accurate, precise, and reproducible for the simultaneous measurement of sugar probes in canine serum.
Antibodies to heat shock proteins of the 65 kDa group were demonstrated in canine sera and synovial fluid. This paper reports these antibody measurements in three groups of dogs with joint disease and compares them with those of a control population. Dogs with rheumatoid arthritis (RA) showed higher anti-heat shock proteins (HSP) antibody levels, in both their sera and synovial fluids, compared to the control dogs and these antibodies were predominantly of the IgG and IgM class; there was a significant correlation between IgM anti-HSP65 and IgM-rheumatoid factors. There was also a significant correlation between anti-HSP65 and antibodies to canine distemper virus, but only of the IgM class and the relevance of these antibodies to the overall pathogenesis of canine RA and, in particular, to the presence of canine distemper virus within the joint, are discussed.
Orocaecal transit time (OCTT) was assessed in six healthy beagles by means of the breath hydrogen test (BH2T) and the sulphasalazine/sulphapyridine method (SLZ) after the administration of a test meal of canned food mixed with sulphasalazine. Orocaecal transit time was defined as the time taken from the oral administration of the test meal to the time when the first portion of the meal reached the colon. In five of the dogs the OCTTs assessed by the BH2T were shorter than those measured by the SLZ method by 30, 15, 45, 30 and 45 minutes. However, the median OCTT assessed by the BH2T (135 minutes, range 120 to 195 minutes) was not significantly different from that measured by the SLZ (180 minutes, range 150 to 210 minutes) and was highly correlated with it (r = 0.94, P = 0.016). The sixth dog maintained baseline hydrogen and plasma sulphapyridine readings throughout the monitoring period and the OCTT could not be measured.
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.
Sixteen IgA-deficient German Shepherd Dogs with small intestinal bacterial overgrowth were randomized into 2 groups. One group was fed a chicken-based kibble diet; the other was fed the same diet, but with 1% fructo-oligosaccharides supplemented at the expense of cornstarch. After being exposed to the diets for 46 to 51 days, the group that ate the supplemented diet had significantly (P = 0.04) fewer aerobic/facultative anaerobic bacterial colony-forming units in fluid from the duodenum/proximal part of the jejunum, as well as in the duodenal mucosa. We could not detect significant differences in the species of bacteria found in the intestine of these 2 groups of dogs. We conclude that at least some dietary carbohydrates can affect small intestinal bacterial populations in dogs with small intestinal bacterial overgrowth.
The use of the lactulose and rhamnose urinary excretion test was evaluated in dogs with gastrointestinal disease. Lactulose and rhamnose urinary excretion was measured in three groups of dogs: clinically healthy dogs and dogs with gastrointestinal disease with and without coexistent panhypoproteinaemia. A significant increase in both the percentage of lactulose:percentage of rhamnose urinary excretion ratio and the percentage of lactulose excretion was demonstrated in dogs with hypoproteinaemia when compared to the other two groups. The results suggest that the lactulose/rhamnose urinary excretion test may prove a useful adjunct to currently available tests for assessing small intestinal function, but lacks sensitivity in detecting small intestinal mucosal damage in the absence of villus atrophy.
Breath hydrogen excretion over a period of three hours was measured to evaluate carbohydrate malassimilation in healthy cats treated orally with antibiotics. Both an absorbable carbohydrate (xylose) and a non-absorbable carbohydrate (lactulose) were administered during the tests to evaluate the changes in the intestinal mucosa and the population of bacteria within the intestinal lumen. Overall, the effects of oxytetracycline and metronidazole on breath hydrogen excretion were not significantly different. However, the treatment effect with an antibiotic did significantly change breath hydrogen excretion after xylose administration (P < 0.05) within groups. Similarly, with each antibiotic, breath hydrogen excretion was affected significantly (P < 0.001) by the time after the administration of the carbohydrate. Treatment with each antibiotic also interacted significantly with this time effect (P < 0.05) within groups. After lactulose administration, there was a trend within groups for the type of antibiotic to interact with the treatment effect on breath hydrogen excretion (P = 0.09). After oxytetracycline treatment, more hydrogen was exhaled during the first 120 minutes after lactulose administration than in the pre-antibiotic test, whereas after metronidazole treatment, less hydrogen was exhaled between 60 and 180 minutes after lactulose, administration. After treatment with either oxytetracycline or metronidazole, more hydrogen was exhaled after xylose administration. Obligate anaerobes could be isolated from samples of small intestinal fluid obtained endoscopically after oxytetracycline treatment, but they could not be isolated after treatment with metronidazole.
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.
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.
To investigate the bacterial flora of the proximal part of the small intestine of healthy cats and determine the effect of sample collection method on results of bacteriologic culture.
25 healthy barrier-maintained specific-pathogen-free-derived cats.
Aspirated, undiluted samples of proximal small intestinal juice were obtained via oral endoscopy (UEA), and a second sample was collected after instillation of 1 ml of sterile saline solution (diluted, DEA). Undiluted juice also was obtained by direct needle aspiration (NA) from the intestinal lumen. Samples for quantitative and semiqualitative bacteriologic examination were grown aerobically and anaerobically.
Mean (range) log10 colony-forming units of total bacteria/ml were 6.2 (2.0 to 8.3) for NA, 6.0 (2.0 to 7.9) for UEA, and 4.9 (2.0 to 7.5) for DEA samples. One cat had no growth (< or = 2.0 colony-forming units/ml) for samples obtained using all 3 methods, and another cat had no growth for the DEA sample only. Mean total aerobic, anaerobic, and bacterial counts were not significantly different between NA and UEA methods, but these techniques yielded significantly higher mean counts than did DEA samples (P < or = 0.002, ANOVA). As a percentage of the total bacteria isolated, anaerobes constituted a median 35, 32, and 50% of the NA, UEA, and DEA samples, respectively. Good correlation was found between the NA and UEA samples for total bacteria, aerobes, and anaerobes (r > or = 0.830).
Compared with human beings, healthy cats carry high numbers of bacteria in the proximal part of the small intestine. By comparison with NA samples, UEA samples accurately reflected bacterial populations in the small intestine, whereas DEA samples significantly underestimated these populations.
It is clear that the exact definition of small intestinal bacterial overgrowth (SIBO) needs to be reappraised in veterinary medicine. Antibiotic responsive enteropathies due to SIBO must be distinguished from those that are not associated with SIBO, such as those caused by a lack of immune tolerance. Once appropriate definitions and criteria for diagnosis are in place, the wide variety of diagnostic procedures that may facilitate the diagnosis can be evaluated with respect to their sensitivity and specificity, and statements about the prevalence and significance of this disorder can be made.
Although diarrhea as a consequence of dysfunction of the small intestine (SI) is a common presenting problem in practice, most cases are acute, nonfatal, and self-limiting, and they require only symptomatic support. Thus, the need for any investigation of SI function beyond physical examination is a clinical judgment made on the potential of the problem being persistent, contagious, or life-threatening. Acute, severe, or bloody diarrhea; concurrent signs of systemic illness; or chronic diarrhea (which is not self-limiting) indicate a need for more detailed investigations. In all cases, the fluid and electrolyte needs of the patient must be addressed even while the diagnostic effort is in progress.
German shepherd dogs (GSD) are predisposed to enteropathies such as inflammatory bowel disease (IBD) and small intestinal bacterial overgrowth (SIBO). The present study examined the role of cytokines in the immunopathogenesis of both conditions. Duodenal mucosal biopsies were taken from GSDs with small intestinal enteropathies (group 1; N = 16) or control dogs (group 2, N = 12). IL-2, IL-4, IL-5, IL-10, IL-12p40, IFN-gamma, TNF-alpha, and TGF-beta1 mRNA expression was determined by semiquantitative reverse transcriptase polymerase chain reaction. IL-2, IL-5, IL-12p40, TNF-alpha, and TGF-beta1 mRNA expression in group 1 dogs was significantly greater than in group 2 dogs (all P<0.01), but there were no significant differences between dogs with IBD or SIBO. Further, antibiotic treatment in five dogs with SIBO, resulted in reduced TNF-alpha and TGF-beta1 mRNA expression (P<0.05). Such alterations in cytokine mRNA expression suggest heightened immune responses within the duodenal mucosa in GSDs with either SIBO or IBD.
Small intestinal bacterial overgrowth (SIBO) has a high incidence in dogs and, as in humans, is difficult to diagnose. The aim of this study was to determine the diagnostic significance of serum unconjugated bile acid concentrations in dogs with bacterial overgrowth. Fasting sera were obtained from 23 dogs: 10 with culture-proven SIBO, 8 with indirectly diagnosed SIBO (normal pancreatic function but small intestinal disease associated with subnormal serum cobalamin and supranormal folate concentrations), and 5 healthy controls. Unconjugated bile acids were determined using gas chromatography-mass spectrometry after isolation by liquid-solid extraction and anion-exchange chromatography. Mean serum unconjugated bile acid concentrations were significantly elevated in dogs with SIBO (mean +/- SD: 0.91 +/- 1.03 micromol/liter), and in dogs with indirectly diagnosed SIBO (2.11 +/- 2.20 micromol/liter) compared to clinically healthy dogs (0.015 +/- 0.015 micromol/liter, P < 0.005). Cholic acid was the predominant unconjugated bile acid in the serum of dogs with SIBO. In conclusion serum unconjugated bile acid concentrations of healthy dogs are significantly lower than reported values for humans, and this fraction represents a relatively small proportion (0-2.3%; mean 0.8%) of the total bile acids in dogs. Unconjugated bile acids increased 10- to 20-fold in dogs with SIBO indicating the clinical utility of serum unconjugated bile acids for diagnosis of intestinal bacterial overgrowth in dogs.
To investigate the recovery of iodinated water-soluble contrast medium from small bowel with small morphological alterations, iohexol or iodixanol was instilled through an orogastric tube in rats 14 days after surgery that established a self-filling blind loop in the jejunum. This rat model induced small bowel bacterial overgrowth with only minor abnormalities observed on histology and scanning electron microscopy. Animals with end-to-end anastomosis of the jejunum or unoperated rats served as controls. Compared with unoperated animals, urinary recovery of iohexol and iodixanol was significantly higher in both groups that underwent surgery. Moreover, the contrast medium recovery was numerically higher in the self-filling blind loop group given iodixanol than in the end-to-end anastomosis group, although not statistically significant, P = 0.09. Our results indicate that iohexol and iodixanol may detect small barrier impairments in the intestines. Iodixanol, the largest of the two, may seem to differentiate better between normal and minimally impaired intestinal barrier.
The roles of extracellular and intracellular mechanisms in the degradation of brush border proteins have been investigated by studying the small intestinal mucosa of dogs with naturally occurring exocrine pancreatic insufficiency. Peroral jejunal biopsies were homogenised and the organelles separated by isopycnic centrifugation on continuous sucrose density gradients. The distributions of marker enzymes for the principal subcellular organelles were determined in the gradients and related to the specific activities in the homogenates. There were increased activities of the brush border carbohydrases zinc-resistant alpha-glucosidase, maltase and sucrase in the pancreatic insufficient animals, but no change in lactase activity. The activity of gamma-glutamyl transferase was also higher in the affected group; the activities of two other brush border enzymes, alkaline phosphatase and leucyl-beta-naphthylamidase, however, were unaltered. These findings with an increase in the modal density of the brush border from 1.20 to 1.22 are consistent with an enhanced glycoprotein content of the microvillus membrane. There were also rises in the activities of lysosomal enzymes. N-Acetyl-beta-glucosaminidase activity was increased in the soluble fractions and the percentage latent enzyme activity was reduced, findings indicative of an increased fragility of the lysosomal membrane. There were no marked alterations in the activities or density gradient distributions of marker enzymes for the other organelles, stressing the specificity of the changes in the brush borders and lysosomes. These findings are compatible with the degradation of certain exposed brush border proteins by pancreatic proteases and suggest that when this is defective, intracellular degradative mechanisms may be stimulated.
Dogs with naturally occurring aerobic or anaerobic bacterial overgrowth have been examined before and after antibiotic therapy in order to assess reversibility of damage to the jejunal mucosa. Histological changes in peroral jejunal biopsies were relatively minor before and after treatment, but sucrose density gradient centrifugation revealed specific biochemical abnormalities that responded to antibiotic therapy. Aerobic overgrowth was initially associated with a marked loss of the main brush border component of alkaline phosphatase activity; this recovered following treatment, suggesting that aerobic bacteria may cause reversible damage to the hydrophobic region of the brush border membrane. In contrast, anaerobic overgrowth was initially associated with a marked reduction in brush border density, indicative of a considerable fall in the glycoprotein-to-lipid ratio of the membrane. Density increased from 1.17 to 1.21 g/ml after antibiotic therapy, consistent with recovery from this relatively severe damage to the brush border caused by anaerobic bacteria. Reductions in soluble and peroxisomal catalase activities which could compromise mucosal protection against free radicals in dogs with aerobic overgrowth, and a loss of particulate malate dehydrogenase activity indicative of mitochondrial disruption in dogs with anaerobic overgrowth, were also reversed after treatment. These findings indicate that aerobic and anaerobic bacterial overgrowth can result in contrasting but potentially reversible damage to the jejunal mucosa which would not be detected by conventional investigative procedures.
Determinations of serum folate and cobalamin concentrations in dogs have proved of considerable value for the identification and characterisation of chronic small intestinal disorders, but the microbiological assays used are time-consuming and technically demanding. Dual isotope radio-assays are more convenient and have been developed for the determination of folate and cobalamin in human beings. This study has evaluated such an assay for the determination of serum folate and cobalamin concentrations in dogs by direct comparison with microbiological assays used previously. Assays were performed on samples from 77 dogs, including controls and animals with confirmed or suspected chronic small intestinal disease. Regression analysis demonstrated a significant relationship between the two assays for serum folate concentrations (R=0–85; P=0–0001) and a definite trend for radioassay to give lower results than bioassay. There was also a significant relationship between bioassay and radioassay data for serum cobalamin (R=0–91; P=0–0001) with comparable absolute values for these two assays. Radioassay of serum samples from 31 clinically healthy dogs gave control ranges of 3–7 to 8-8 4mUg/litre for folate and 205 to 490 ng/litre for cobalamin. These ranges were similar to those calculated by comparison with the established ranges for bioassay using regression analysis, which predicted ranges of 4-4 to 8-4 μg/litre and 217 to 398 ng/litre for radioassay of serum folate and cobalamin, respectively. These data indicate that a dual isotope radioassay of serum folate and cobalamin may be used for dogs, and emphasise the need for laboratories to validate and establish their own control ranges for different assays.
Portions of closed jejunal biopsies from the dog were homogenised and their organelles separated by isopycnic centrifugation on continuous sucrose density gradients. The distributions of marker enzymes for the principal organelles were determined using highly sensitive assay procedures. The following organelles, with assayed marker enzymes and modal densities between brackets were characterised: peroxisomes (catalase, 1.21); brush borders (zinc-resistant alpha-glucosidase, leucyl-beta-naphthyl-amidase, gamma-glutamyl transferase, alkaline phosphatase, 1.20); lysosomes (N-acetyl-beta-glucosaminidase, alpha-mannosidase, 1.19); mitochondria (malate dehydrogenase, 1.18); endoplasmic reticulum (Tris-resistant alpha-glucosidase, 1.16); basal-lateral membranes (5'-nucleotidase, 1.11) and cytosol (lactate dehydrogenase). Homogenisation in isotonic sucrose containing digitonin (0.12 mmol/litre) selectively disrupted lysosomes and increased the equilibrium density of brush border and basal-lateral membranes. This procedure will be used to study the subcellular pathology of naturally occurring intestinal disease in the dog.
Subcellular biochemical changes in the jejunal mucosa have been compared in dogs with either aerobic or anaerobic bacterial overgrowth to explore relationships between composition of the flora and mucosal damage. Affected animals comprised 17 German shepherd dogs with chronic diarrhea or weight loss, or both. Analysis of duodenal juice demonstrated aerobic overgrowth in 10 cases, most frequently comprising enterococci and Escherichia coli, and obligate anaerobic overgrowth in 7 cases, most frequently including Clostridia spp. Histologic changes were minimal; however, examination of peroral jejunal biopsy specimens by sucrose density gradient centrifugation revealed specific biochemical abnormalities. In the dogs with aerobic overgrowth, there was a selective loss of brush border alkaline phosphatase activity, and gamma-glutamyl transferase activity was increased, whereas activities of disaccharidases and aminopeptidase N were unaltered. In contrast, anaerobic overgrowth was associated with a reduction in brush border density, indicative of a considerable fall in the glycoprotein-to-lipid ratio of the brush border membrane, whereas brush border enzyme activities were unaltered. There was a loss of peroxisomal catalase activity in dogs with aerobic overgrowth, and an indication of mitochondrial disruption in dogs with anaerobic overgrowth, but little evidence for damage to other subcellular organelles. These findings demonstrate that aerobic and anaerobic overgrowth may be associated with distinct but different mucosal abnormalities particularly affecting the brush border membrane.
Serum folate and vitamin B12 concentrations have been measured in 53 dogs presented for an investigation of malabsorption. Abnormal concentrations have permitted the differentiation of animals with small intestinal disease into three main groups, each with distinct biochemical abnormalities in the jejunal mucosa. The first group had reduced folate and vitamin B12 concentrations. Jejunal biopsies revealed marked villous atrophy and generalised biochemical abnormalities in the brush borders, lysosomes and endoplasmic reticulum. The second group had reduced folate but normal vitamin B12 concentrations and although histological changes were minimal there were specific biochemical changes confined to the brush borders. In the third group, increased folate and reduced vitamin B12 concentrations suggested a bacterial overgrowth in the proximal small intestine. Minor histological changes were accompanied by marked biochemical changes in brush borders and lysosomes. A group of animals with severe exocrine pancreatic insufficiency had increased mean folate but reduced mean vitamin B12 concentrations. These changes are consistent with bacterial overgrowth, but could be due to defective degradation of a B12-binding protein.
The microassay of intestinal disaccharidases
- T J Peters
- R M Batt
- J R Heath
Peters TJ, Batt RM, Heath JR, et al. The microassay of intestinal
disaccharidases. Biochem Med 1976 ; 15 : 145-148.
Effect of secreted Bacteroides
- S P Riepe
- J Goldstein
- D H Alpers
Riepe SP, Goldstein J, Alpers DH. Effect of secreted Bacteroides