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Department of Animal Sciences, University of Illinois, Urbana, IL 61801
2 To whom correspondence should be addressed. E-mail: gcfahey{at}uiuc.edu.
KEY WORDS: • immunity • immunoglobulins • intestine • dogs
Secretory immunoglobulin (Ig)3 A is present in mammals' mucosal membranes of the intestinal, respiratory, biliary, and genital tracts. It also is present in the circulatory system, although to a much lesser extent, as IgG is the primary form present in blood (1). The primary function of secretory IgA is part of a localized immune response that serves to prevent bacteria and viruses from attaching and invading enterocytes (2). Intestinal concentrations of IgA are of interest to clinicians, as they can be used to diagnose IgA deficiency or determine antigen-specific responses (3). Researchers also utilize IgA in the study of immune responses to functional ingredients (4,5). However, collecting small intestinal IgA samples can be a rather invasive process. To determine a suitable alternative to invasive collection methods, our objective was to elucidate the relationship between IgA concentrations in selected biological samples, and within a given specimen, to determine the variability of IgA concentrations due to time and method of sampling.
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MATERIALS AND METHODS |
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 TOP MATERIALS AND METHODS RESULTS AND DISCUSSIONLITERATURE CITED |
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Seven purpose-bred adult female dogs with hound bloodlines (average weight = 22.5 kg, range 20.5–28.2 kg) previously fitted with ileal cannulas according to the procedure described by Walker et al. (6) were used in this study. All dogs were healthy and had normal, formed stool consistency scores. Dogs were individually housed in 1.2 x 3.1-m floor pens in a temperature-controlled room (21°C) at the animal facility of the Edward R. Madigan Laboratory on the University of Illinois campus. A 16-h light/8-h dark cycle was used. Dogs were fed 400 g/d of a dry, extruded kibble diet formulated to meet or exceed nutrient requirements for adult dogs at maintenance (7). All dogs were allowed free access to water. Dogs were healthy and were not given an antigen challenge or other immunostimulant. All animal care procedures were approved by the University of Illinois Campus Laboratory Animal Care Advisory Committee before initiation of the experiment.
Sample collection and handling
All seven dogs were adapted to the diet for 10 d before a 3-d collection period. During this 3-d period, feces, ileal effluent, saliva, and blood were collected once daily on each of the three collection days. Ileal effluent was collected 2 h after the morning meal (fed at 0800 h each day), whereas blood and saliva were collected after a 12-h fast. In addition, on the third collection day, blood and saliva also were collected at 2 and 6 h postprandial, and ileal effluent was collected 12 h postprandial. At each sample collection, the order of dogs sampled was randomized and sampling was completed within 1 h to minimize the impact of variations in time on IgA concentrations.
On each of the three days, a single recently excreted fecal sample (<30 min after defecation) was collected from the floor of the pens. Feces were frozen at –20°C for subsequent freeze-drying in a Tri-PhilizerTM MP microprocessor-controlled lyophilizer (FTS Systems, Stone Ridge, NY) and grinding with mortar and pestle. Ileal effluent was collected (
15 g) by attaching a Whirl-Pak bag (Pioneer Container, Cedarburg, WI) to the cannula barrel using a rubber band, and allowing digesta to flow naturally through the simple T cannula. Before attachment of the bag, the interior of the cannula was scraped clean with a spatula and any digesta discarded. During collection of ileal effluent, dogs were encouraged to move around freely. Blood samples (5 mL) were collected via jugular puncture into nonheparinized evacuated tubes. Blood was allowed to clot at room temperature for 40 min before centrifuging at 2060 x g for 20 min at 4°C and serum was collected. To determine the effects of sampling methods on IgA concentration, saliva was collected by either swabbing the dog's mouth with a sterile cotton swab (CS), or by gently rubbing the oral cavity with a soft rubber spatula (RS). Samples were collected at the same time of day in a randomized order. For both methods, to avoid dilution of saliva, dogs were not stimulated to salivate and water was removed for 2 h. For the CS methods, saliva was removed from the cotton swabs by centrifugation at 13,000 x g for 10 min. For the RS method, saliva from the rubber spatulas was scraped into plastic collection tubes, diluted 1:2 with phosphate-buffered saline (PBS), and mixed on a magnetic stir plate for 10 min before centrifuging at 13,000 x g for 10 min to remove foreign particles. This dilution was necessary due to the high viscosity of the RS saliva. The supernatant from both methods was used for IgA determination.
Chemical analyses
Dry matter content of feces and ileal effluent was determined according to the Association of Official Analytical Chemists (AOAC) (8). Concentration of IgA in feces and ileal effluent (dry matter basis) was determined using a modification of the procedure described by Nara et al. (9). Briefly, 1 g of dried feces was mixed with 10 mL PBS for 30 min at room temperature before centrifugation at 20,000 x g for 30 min at 4°C. Freshly collected ileal effluent was diluted 1:2 with PBS, thoroughly mixed on a vortex, and centrifuged at 13,000 x g for 10 min. Fecal and ileal extracts, serum samples, and saliva supernatant were analyzed for IgA concentration using a radial immunodiffusion kit (ICN Biomedicals, Aurora, OH). The accuracy of this kit is reported to be ±10% according to the manufacturer's package insert.
Statistical analyses
Data were analyzed by the General Linear Models procedure of SAS (version 8.02, SAS Institute, Cary, NC). The statistical model included effects of animal and time of sample collection. Due to unequal sample sizes, least-squared means are reported. Treatment means were compared using the "PDiff" option of the "LSMeans" command of SAS. To determine any correlation among IgA concentrations in the selected biological specimens, Pearson's correlation coefficients were determined by executing Proc Corr of SAS. Although P < 0.05 was judged to be statistically significant, trends between P < 0.06 and P < 0.10 also are discussed.
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RESULTS AND DISCUSSION |
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TOPMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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5 times higher, whereas concentrations of IgA in ileal effluent are similar to those previously reported by Swanson et al. (5). This was not expected, as both the dog colony and the analytical methods are nearly identical between the two studies. Possible explanations for this difference may include faster fresh fecal collection and freezing in the current study or variability in intestinal IgA concentrations within the dog colony over time. The serum IgA concentrations reported in the current study are 7 times lower than those reported by Swanson et al. (5), but are similar to values reported for mongrel dogs by Reynolds and Johnson (10). Salivary IgA concentrations did not differ among the three collection days, ranging from 0.25 to 0.48 g/L for RS saliva and ranging from 0.03 to 0.47 for CS saliva. This is in contrast to Kikkawa et al. (11) that reported somewhat higher salivary IgA concentrations, and also determined no significant differences in salivary IgA concentration due to day-to-day variation. The results from this study suggest that, in nonimmunostimulated dogs, ileal IgA concentrations may be more subject to day-to-day variability as compared to fecal, salivary, and serum IgA concentrations.
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A positive correlation (r = 0.51) tended (P < 0.10) to exist between CS saliva and serum (0.36 ± 0.109 and 0.28 ± 0.012 g/L, respectively). This also was unexpected, as salivary IgA plays a role in the mucosal defense system, whereas serum IgA is part of the systemic immune response and is overshadowed by the presence of much larger quantities of serum IgG. German et al. (12) reported no correlation between canine serum and salivary IgA concentrations. Rinkinen et al. (15) reported no correlation between salivary and duodenal IgA concentrations, but indicated a negative correlation (r = –0.64, P < 0.01) between duodenum and serum IgA concentrations in dogs. Externest et al. (14) reported a positive correlation between salivary and serum IgA concentrations (r = 0.97) as well as salivary and ileal IgA concentrations (r = 0.97) in mice. No correlation existed between salivary and fecal IgA concentrations. This data suggest that salivary IgA may not be a reliable indicator of intestinal IgA concentrations.
In the gastrointestinal tract, IgA plays an important role in protection against bacterial invasion of the mucosa. Saliva and serum also contain measurable concentrations of IgA. This study provides information regarding the relationship of IgA concentrations among serum, salivary, ileal, and fecal samples, as well as the variability of IgA concentrations present in each specimen. Future research should continue to search for less invasive techniques to assess localized gastrointestinal immune function and further investigate the relationship of immunoglobulin concentrations in biological specimens in companion animals.
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FOOTNOTES |
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3 Abbreviations used: AOAC, Association of Official Analytical Chemists; CS, cotton swab; Ig, immunoglobulin; PBS, phosphate-buffered saline; RS, rubber spatula.
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LITERATURE CITED |
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1. Macpherson, A. J., Hunziker, L., McCoy, K. & Lamarre, A. (2001) IgA responses in the intestinal mucosa against pathogenic and nonpathogenic microorgansisms. Microbes Infect. 3: 1021–1035.[Medline]
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