• View in gallery

    Optical density readings (mean ± SD) obtained after co-incubation of Clostridium perfringens alpha toxin with DTOS (diamonds) or BSS (squares) at sequential adsorbent dilutions. The means of the ODs for all replicates for each adsorbent dilution were calculated. *For the DTOS dilutions, value differs significantly (P < 0.05) from that of a control sample containing toxin but no adsorbent substance (tox+/ads–). †For the BSS dilutions, value differs significantly (P < 0.05) from that of the tox+/ads– control sample. ‡Within a dilution, DTOS value differs significantly (P < 0.05) from BSS value.

  • View in gallery

    Optical density readings (mean ± SD) obtained after co-incubation of C perfringens beta toxin with DTOS (diamonds) or BSS (squares) at sequential adsorbent dilutions. The means of the ODs for all replicates for each adsorbent dilution were calculated. See Figure 1 for key.

  • View in gallery

    Optical density readings (mean ± SD) obtained after co-incubation of C perfringens beta-2 toxin with DTOS (diamonds) or BSS (squares) at sequential adsorbent dilutions. The means of the ODs for all replicates for each adsorbent dilution were calculated. See Figure 1 for key.

  • View in gallery

    Mean ± SD equine colostral IgG concentration remaining in samples (all replicates) after co-incubation with DTOS at dilutions of 1:4, 1:8, 1:16, and 1:32. *Value differs significantly (P < 0.05) from that of a control sample containing colostrum but no adsorbent substance.

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Adsorptive effects of di-tri-octahedral smectite on Clostridium perfringens alpha, beta, and beta-2 exotoxins and equine colostral antibodies

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  • 1 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.
  • | 2 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.
  • | 3 Animal Population Health Institute, Colorado State University, Fort Collins, CO 80523.
  • | 4 Animal Population Health Institute, Colorado State University, Fort Collins, CO 80523.
  • | 5 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.
  • | 6 Animal Population Health Institute, Colorado State University, Fort Collins, CO 80523.

Abstract

Objective—To determine the adsorptive capability of di-tri-octahedral smectite (DTOS) on Clostridium perfringens alpha, beta, and beta-2 exotoxins and equine colostral antibodies.

Sample Population—3 C perfringens exotoxins and 9 colostral samples.

Procedures—Alpha, beta, and beta-2 exotoxins were individually co-incubated with serial dilutions of DTOS or bismuth subsalicylate, and the amount of toxin remaining after incubation was determined via toxin-specific ELISAs. Colostral samples from healthy mares were individually co-incubated with serial dilutions of DTOS, and colostral IgG concentrations were determined via single radial immunodiffusion assay.

Results—Di-tri-octahedral smectite decreased the amount of each C perfringens exotoxin in co-incubated samples in a dose-dependent manner and was more effective than bismuth subsalicylate at reducing exotoxins in vitro. Decreases in the concentration of IgG were detected in samples of colostrum that were combined with DTOS at 1:4 through 1:16 dilutions, whereas no significant decrease was evident with DTOS at the 1:32 dilution.

Conclusions and Clinical Relevance—Di-tri-octahedral smectite effectively adsorbed C perfringens exotoxins in vitro and had a dose-dependent effect on the availability of equine colostral antibodies. Results suggested that DTOS may be an appropriate adjunctive treatment in the management of neonatal clostridiosis in horses. In vivo studies are necessary to fully assess the clinical efficacy of DTOS treatment.

Abstract

Objective—To determine the adsorptive capability of di-tri-octahedral smectite (DTOS) on Clostridium perfringens alpha, beta, and beta-2 exotoxins and equine colostral antibodies.

Sample Population—3 C perfringens exotoxins and 9 colostral samples.

Procedures—Alpha, beta, and beta-2 exotoxins were individually co-incubated with serial dilutions of DTOS or bismuth subsalicylate, and the amount of toxin remaining after incubation was determined via toxin-specific ELISAs. Colostral samples from healthy mares were individually co-incubated with serial dilutions of DTOS, and colostral IgG concentrations were determined via single radial immunodiffusion assay.

Results—Di-tri-octahedral smectite decreased the amount of each C perfringens exotoxin in co-incubated samples in a dose-dependent manner and was more effective than bismuth subsalicylate at reducing exotoxins in vitro. Decreases in the concentration of IgG were detected in samples of colostrum that were combined with DTOS at 1:4 through 1:16 dilutions, whereas no significant decrease was evident with DTOS at the 1:32 dilution.

Conclusions and Clinical Relevance—Di-tri-octahedral smectite effectively adsorbed C perfringens exotoxins in vitro and had a dose-dependent effect on the availability of equine colostral antibodies. Results suggested that DTOS may be an appropriate adjunctive treatment in the management of neonatal clostridiosis in horses. In vivo studies are necessary to fully assess the clinical efficacy of DTOS treatment.

Enteric clostridiosis in equine neonates can result in life-threatening disease and has been associated with a high mortality rate in the early neonatal period.1– 3 Clostridium perfringens is one of the most important enteric pathogens associated with the development of diarrhea in the first several weeks of a foal's life.1–6 In a 10-year retrospective university study,4 overall case fatality rate in foals with C perfringens–associated enterocolitis was 54%, whereas case fatality rate for foals with C perfringens type C infection was 83%. Clostridium perfringens, a gram-positive, spore-forming rod bacterium can be a clinically important enteric pathogen, yet it is ubiquitous in the environment and gastrointestinal tract of adult horses and foals.2,7,8 Additionally, it has been associated with enterocolitis in a variety of livestock, horses, and humans.5,8–12

The virulence associated with C perfringens appears to depend on the type of toxin produced. Clostridium perfringens isolates are classified into 5 genotypes (types A through E) on the basis of the ability to synthesize 1 or more major lethal toxins, including alpha, beta, epsilon, and iota toxins.2 Genotypes A and C are most commonly isolated from foals and have been associated with disease,1–4 whereas types B, D, and E have rarely been reported to cause disease in equine neonates.13,14 Alpha toxin is produced by all C perfringens genotypes, whereas beta toxin is only produced by types B and C.8 Recently, beta-2 (cpb2), which has been associated with all C perfringens genotypes (A through E), has been associated with disease of equine neonates and adult horses, as well as with necrotizing enterocolitis of calves and young pigs.15,16 Furthermore, Plampin et ala determined that concentrations of alpha, beta, and beta-2 exotoxins in fecal and intestinal samples obtained from foals with neonatal enteric clostridiosis were higher than concentrations in samples from healthy foals. Thus, in addition to the more widely studied enterotoxin of C perfringens, exotoxins of importance in equine neonatal enterocolitis appear to include alpha, beta, and beta-2 types.

The potent clostridial exotoxins induce inflammation and ultimately lead to destruction of the intestinal mucosal barrier. Supportive care is currently the mainstay of treatment. New therapeutic interventions are needed to decrease the morbidity and death associated with neonatal enteric clostridiosis. Products that are capable of binding to a variety of toxins may be effective in treatment of enteric clostridiosis. Di-tri-octahedral smectite,b a commercially available natural hydrated clay silicate consisting of sheets of aluminum and magnesium, has been shown to be effective in the management of infectious colitis in humans and other animal species.17–22 This effect has been attributed to adsorption of endotoxins and exotoxins from the gastrointestinal tract. Di-tri-octahedral smectite effectively adsorbs Clostridium difficile toxins A and B, C perfringens enterotoxin, Bacteroides fragilis toxin, and gram-negative endotoxin in vitro.23,24 However, to our knowledge, the effect of DTOS on C perfringens exotoxins, particularly alpha, beta, and beta-2 toxins, has not previously been evaluated.

In the study of this report, we evaluated the efficacy of DTOS binding to C perfringens exotoxins. Ultrapure water and BSSc were used as comparative control substances. Bismuth subsalicylate was chosen as an adsorbent control substance because it is another commercially available product that is widely used in the treatment of diarrhea in humans and other animals.25–28 Bismuth subsalicylate is thought to have antiendotoxic, weak antibacterial, and antiprostaglandin activities; however, the exact mechanism of these actions is unknown.26 Although BSS has been administered to equine neonates with enteric disease,29,30 its ability to bind toxins is unclear.

On farms where neonatal enteric clostridiosis is endemic, owners and farm managers may prophylactically administer adsorbents such as DTOS to foals during the first few hours of life. The goal of such prophylaxis is to successfully bind clostridial and other bacterial toxins prior to the induction of mucosal damage, thereby preventing or minimizing clinical disease. Because administration of adsorbents to foals coincides with the period of passive transfer of colostral antibodies,31–33 the effect of adsorbents on immunoglobulin (IgG) absorption must be evaluated to ensure that foal health is not compromised.

The purpose of the study reported here was to determine the adsorptive capability of DTOS for C perfringens alpha, beta, and beta-2 exotoxins and equine colostral antibodies. We hypothesized that DTOS would adsorb C perfringens exotoxins, but would not interfere with the availability of colostral antibodies.

Materials and Methods

Toxins—Alphad and beta-2e toxins were obtained as purified recombinants and were each used at a concentration of 2 μg/mL. Betaf toxin was obtained as a partially purified culture filtrate and prepared as a 1:5,000 dilution with ultrapure water (double-distilled water purified to 18 m7 of resistance). Toxin concentrations were chosen on the basis of results of pilot studies in which detection limits of the ELISA assays were evaluated.

Preparation of adsorbent substances—The commercially available adsorbents DTOSb and BSSc were diluted with ultrapure water to final dilutions of 1:4 through 1:131,072. Di-tri-octahedral smectite was used in a powder form and BSS was used in a liquid form as supplied by the manufacturer in preparation of the 1:4 through 1:32 final adsorbent dilutions. For preparation of the final adsorbent dilutions > 1:32, in which a sufficiently small quantity of adsorbent material was used, it was necessary to create pipettable slurries of DTOS and BSS with ultrapure water for improved accuracy of measurement. The range of dilutions (1:4 through 1:132,072) was selected on the basis of findings of pilot studies of DTOS toxin adsorption and extrapolation from current DTOS in vivo dosing recommendations.

Incubation of toxins with sequential dilutions of DTOS or BSS—A 180-μL volume of standardized toxin was added to each microcentrifuge tube. This was followed by the addition of a volume of adsorbent slurry that was calculated to result in the desired final adsorbent dilution of 1:4 through 1:131,072. An appropriate amount of ultrapure water was then added to each microcentrifuge tube to provide a final total volume of 250 μL. For each absorbance assessment, a control sample containing toxin but no adsorbent substance (tox+/ads–) was included. Tubes were vortexed to suspend the adsorbent material before being placed horizontally on a rocker for 30 minutes of incubation at room temperature (20 ± 2°C). Tubes were then immediately centrifuged at 8,000 × g for 5 minutes at room temperature. Clarified supernatants were transferred to sterile microcentrifuge tubes and placed on ice. Each experimental and control sample was tested for the presence of toxins via toxin-specific indirect ELISA.

ELISA technique—Toxin-specific indirect noncompetitive ELISA assays were used to detect C perfringens toxins by use of a technique described by Dennison et al.34 The 96-well ELISA platesg were prepared by addition of an equal volume of clarified sample supernatant and double-strength (0.4M) carbonate coating bufferh (pH, 9.6) to duplicate wells. The ELISA plates were then incubated at room temperature (20 ± 2°C) for 30 minutes on a shaker in a humidified chamber. A humidified chamber was created by placing the ELISA plates on moist paper towels in a covered plastic box. The ELISA plates were then washed 4 times with 200 μL of PBST. Wells were filled with 130 μL of 5% nonfat milk in PBST to block nonspecific reactions and incubated as described. Blocking solutions were tapped out of the plates, and 50 μL of the appropriate monoclonal antibodyi–k diluted to 1:200 in PBST was added to each well. After monoclonal antibody incubation and washing, plate wells were filled with 50 μL of conjugatel and incubated as before. Plates were then washed as described, and wells were filled with 100 μL of prepared chromagen solution and incubated in a stationary position in the dark at room temperature for 6 minutes. To prepare the chromagen solution, 12 mg of O-phenylenediaminem was mixed into 12 mL of substrate buffer (0.1M sodium citrate; pH, 5.0), and 4.8 μL of 30% hydrogen peroxide was added to each plate. Reactions were stopped by the addition of 50 μL of 2N sulfuric acid to each well. Optical densities were determined by use of an automatic plate spectrophotometern at a wavelength of 492 nm. The mean number of replicate assays performed for each adsorbent dilution was 5.

Two negative control samples were included in each assay. Wells that contained carbonate coating buffer without toxin were included to ensure monoclonal antibody was not binding to the ELISA plate, and wells that contained PBST in place of monoclonal antibody were included to ensure that the conjugate was not binding to the toxin. Positive control samples that were comprised of toxin diluted in carbonate coating buffer without the adsorbent substance were serially diluted on each plate to achieve standard curves. These standard curves were used to maintain interplate and inter-assay repeatability. As previously indicated, BSS was included as a comparative control adsorbent.

Sample collection for in vitro co-incubation of colostrum with DTOS—At the time of parturition, the udders of 9 healthy broodmares were hand stripped to provide 9 samples of colostrum (15 mL each). Colostral samples were labeled and stored in plastic conical tubes at −20°C until used. Approval by the institutional review committee on the care and use of client-owned animals (Animal Care and Use Committee Protocol No. 05-003A-01) at Colorado State University was obtained prior to collection of samples for the study. Client consent was obtained for sample collection.

Preparation of adsorbent material—Di-tri-octahedral smectite was used directly in the powder form as supplied by the manufacturer in preparation of the 1:4 through 1:32 final adsorbent dilutions. Clinically relevant dilutions were calculated on the basis of in vivo dosing recommendations for DTOS and the typical volume of colostral intake expected during the first 12 hours of a foal's life. These dilutions were determined by calculation of the amount of adsorbent material that would be mixed with an expected amount of neonatal gastrointestinal tract fluids in vivo. The manufacturer of DTOS recommends a dosing regimen of 3 tablespoons of DTOS powder in 30 mL of water administered orally every 8 hours. In our experiments, the weight of 3 tablespoons of DTOS powder was 38.1 g; when added to 30 mL of water, the volume of slurry was 49.7 mL. The amount of fluid (colostrum) ingested by a foal in the first 12 hours can range from 160 to 560 mL/h,35,36 and the mean ingested quantity for a healthy 45-kg foal is expected to be approximately 210 to 250 mL/h. The resulting dilutions were 1:4, 1:8, 1:16, and 1:32 assuming 1 to 12 hours of colostral ingestion.

Incubation of colostrum with sequential dilutions of DTOS—Colostral samples were thawed at room temperature, and baseline IgG concentrations in each sample were obtained via SRID assay.° Because of the high viscosity of colostrum, each sample was diluted with ultrapure water to a final dilution of 1:20 to optimize the SRID assay. A 720-μL volume of diluted colostrum was added to each microcentrifuge tube. This was followed by the addition of DTOS powder and ultrapure water calculated to result in the desired final dilutions of 1:4 through 1:32. An appropriate amount of ultrapure water was then added to each tube to provide a final total volume of 1 mL. For each absorbance assessment, a control sample containing colostrum but no adsorbent substance (col+/ads–) and 4 reference standards provided by the SRID assay manufacturer (200, 400, 800, and 1,600 mg of IgG/dL) were included. Tubes were vortexed to suspend the adsorbent material before being placed horizontally on a rocker for 1 hour of incubation at 37°C. Preliminary studies detected no differences in SRID assay results with variable incubation time periods from 30 minutes to as long as 20 hours. Tubes were then centrifuged at 8,000 × g for 5 minutes, and clarified supernatants were assessed for final IgG concentration via a standard SRID assay protocol. Single radial immunodiffusion plates were evaluated after a minimum of 18 hours of incubation at room temperature. Each experimental and control sample was tested in duplicate for the amount of IgG via SRID assay. The diameter of each ring of precipitation on the SRID plate was measured in millimeters and converted to a concentration of IgG (mg/dL) by use of a logarithmic scale. A standard curve was established by use of the reference values. Sample measurements were calculated by comparison with this standard curve.

Statistical analysis—For the toxin adsorption investigation, OD readings were obtained for each experimental sample and control sample. The mean OD from multiple replicates was calculated for each toxin at each adsorbent dilution and used for statistical analysis. For each dilution, ODs for samples with adsorbent material were comparedp with findings for their tox+/ads– control sample by use of paired Student t tests. For any given toxin, independent Student t tests were used to compare mean ODs for samples with DTOS versus samples with BSS at each adsorbent dilution. Values of P < 0.05 were considered significant.

For the investigation of in vitro co-incubation of colostrum with DTOS, mean values from duplicate analyses were used for the statistical analysis. The mean of the natural log (ln) for each adsorbent dilution was comparedq with findings for the col+/ads– control sample by use of a paired Student t test. Values of P < 0.05 were considered significant.

Results

Toxin adsorption investigation—Both DTOS and BSS resulted in a significant reduction in measurable amounts of all 3 toxins (alpha, beta, and beta-2), compared with the value in the appropriate tox+/ads– control sample. For alpha toxin, significant (P values ≤ 0.007) decreases in the amount of toxin were detected at dilutions of DTOS ranging from 1:4 through 1:4,096, compared with the control sample for each dilution (Figure 1). Significant (P values ≤ 0.025) decreases in amounts of alpha toxin were also detected at dilutions of BSS ranging from 1:4 through 1:512. Di-tri-octahedral smectite was significantly (P values ≤ 0.028) more effective than BSS in decreasing detectable amounts of alpha toxin at dilutions of adsorbent material ranging from 1:128 through 1:4,096. At other adsorbent dilutions, no significant differences in detectable amounts of alpha toxin in DTOS and BSS samples were identified.

Figure 1—
Figure 1—

Optical density readings (mean ± SD) obtained after co-incubation of Clostridium perfringens alpha toxin with DTOS (diamonds) or BSS (squares) at sequential adsorbent dilutions. The means of the ODs for all replicates for each adsorbent dilution were calculated. *For the DTOS dilutions, value differs significantly (P < 0.05) from that of a control sample containing toxin but no adsorbent substance (tox+/ads–). †For the BSS dilutions, value differs significantly (P < 0.05) from that of the tox+/ads– control sample. ‡Within a dilution, DTOS value differs significantly (P < 0.05) from BSS value.

Citation: American Journal of Veterinary Research 69, 2; 10.2460/ajvr.69.2.233

For beta toxin, there were significant decreases in the amount of toxin detected at dilutions of DTOS ranging from 1:4 through 1:4,096 (P values ≤ 0.008) and at dilutions of BSS ranging from 1:4 through 1:64 (P values ≤ 0.002), compared with the tox+/ads– control sample for each dilution (Figure 2). Di-tri-octahedral smectite significantly decreased detectable amounts of beta toxin at dilutions of 1:4 (P = 0.014) and from 1:16 through 1:4,096 (P values ≤ 0.024), compared with BSS. At other adsorbent dilutions, no significant difference in detectable amounts of beta toxin in DTOS and BSS samples was identified.

Figure 2—
Figure 2—

Optical density readings (mean ± SD) obtained after co-incubation of C perfringens beta toxin with DTOS (diamonds) or BSS (squares) at sequential adsorbent dilutions. The means of the ODs for all replicates for each adsorbent dilution were calculated. See Figure 1 for key.

Citation: American Journal of Veterinary Research 69, 2; 10.2460/ajvr.69.2.233

For beta-2 toxin, there were significant decreases in the amount of toxin detected at dilutions of DTOS ranging from 1:4 through 1:8,192 (P values ≤ 0.037) and at 1:32,768 (P = 0.012) and at dilutions of BSS ranging from 1:4 through 1:8,192 (P values ≤ 0.017), compared with the tox+/ads– control sample for each dilution (Figure 3). Comparison of DTOS and BSS revealed significant differences in absorption of beta-2 toxin at dilutions from 1:8 through 1:64 (P values ≤ 0.029), from 1:256 through 1:8,192 (P values ≤ 0.046), and at 1:32,768 (P = 0.019). At other adsorbent dilutions, no significant difference in detectable amounts of beta-2 toxin in DTOS and BSS samples was identified.

Figure 3—
Figure 3—

Optical density readings (mean ± SD) obtained after co-incubation of C perfringens beta-2 toxin with DTOS (diamonds) or BSS (squares) at sequential adsorbent dilutions. The means of the ODs for all replicates for each adsorbent dilution were calculated. See Figure 1 for key.

Citation: American Journal of Veterinary Research 69, 2; 10.2460/ajvr.69.2.233

In vitro co-incubation of colostrum with DTOS—Significant decreases in IgG concentration were detected in samples of colostrum combined with DTOS at dilutions of 1:4 (P = 0.002), 1:8 (P < 0.001), and 1:16 (P = 0.04), compared with col+/ads– control samples (Figure 4). However, there was no significant (P = 0.9) difference in IgG concentration in colostral samples combined with DTOS at a dilution of 1:32. The amount of detectable IgG in the colostral samples increased in a dose-dependent manner as the amount of DTOS decreased (ie, as the sample became more dilute).

Figure 4—
Figure 4—

Mean ± SD equine colostral IgG concentration remaining in samples (all replicates) after co-incubation with DTOS at dilutions of 1:4, 1:8, 1:16, and 1:32. *Value differs significantly (P < 0.05) from that of a control sample containing colostrum but no adsorbent substance.

Citation: American Journal of Veterinary Research 69, 2; 10.2460/ajvr.69.2.233

Discussion

Results of the present study revealed the extent of the adsorptive capabilities of DTOS when co-incubated with C perfringens alpha, beta, and beta-2 exotoxins in vitro. The adsorption of all toxins by DTOS was dose dependent, with maximal adsorption occurring at dilutions that far exceeded clinically relevant dilutions (ie, the ratio of adsorbent material to the total volume of gastrointestinal fluid predicted in vivo). Dose-dependent adsorption of all 3 clostridial exotoxins was also evident with the adsorbent control material BSS; however, at higher dilutions of adsorbent material, DTOS consistently reduced toxin amounts more than BSS, indicating increased potency for toxin adsorption of DTOS.

The findings of our study are in agreement with results of previous investigations20,23,24,37 that indicated that DTOS adsorbs endotoxins and exotoxins from the gastrointestinal tract of various species. In clinical trials in children with acute diarrhea, DTOS decreased the duration of diarrhea and reduced the frequency of stool excretion without major adverse effects.17,19,21,38 Smectite products have similar efficacy in the treatment of chronic, functional diarrhea of adult humans.22 Additionally, smectite products have shown promise in the treatment of rabbits, dogs, and calves with enterocolitis attributable to infection from a variety of etiologic agents including Escherichia coli, rotavirus, and cholera toxin.20,37,r Results of other research have indicated that DTOS prevents the development of diarrhea and improves survival in adult horses with colics or colitis.18 Hassel et als determined that horses with large colon disease that were treated after surgery with DTOS had a 33% less risk of developing postoperative diarrhea, compared with control horses treated with the same volume of water.

Previous work by Weese et al24 extrapolated the dilutional strength of DTOS to gastrointestinal contents on the assumption that the mean amount of diarrhea excreted by an adult horse with colitis per day was 56 L/d, which resulted in clinically relevant dilutions of 1:17 to 1:30. Those investigators detected partial binding of C perfringens enterotoxin and C difficile toxins A and B at DTOS dilutions of 1:2 to 1:16. Those dilutions are comparable to the 1:4 to 1:32 dilutions prepared in this study. In the present study, complete adsorption of all toxins by DTOS was evident at concentrations as dilute as 1:4,096. Adsorption of all toxins by DTOS occurred in samples that were far more dilute than that considered clinically relevant. On the basis of these findings, if C perfringens exotoxins are free and available for binding within the colonic lumen in clinical cases of enterocolitis, DTOS should hold promise for clinical application.

Although the mechanism of action of DTOS is not fully understood, it has been proposed that DTOS enhances the intestinal barrier function and effectively adsorbs toxins.39 Enteropathogenic bacteria and toxins cause disruption of the intestinal mucosal layer. Furthermore, in experiments involving rabbit ileum, smectite appeared to decrease mucolysis and thus decrease enterocyte destruction.40 Additionally, DTOS may effectively adsorb toxins from the gastrointestinal tract lumen, thereby preventing direct damage to the enterocytes and minimizing translocation of toxins into the bloodstream. Results of in vitro studies23,24 have indicated that DTOS effectively binds clostridial and Bacteroides fragilis toxins. Smectite products also adsorb viruses, bacteria, and toxins; protect the intestinal mucus layer from degradation; and provide some anti-inflammatory activity at the intestinal level.20,23,24,39,41 This was substantiated in our study by the impressive adsorption of all 3 clostridial exotoxins by highly dilute solutions of adsorbent material (1:4,096 for alpha and beta toxins and 1:32,768 for beta-2 toxin). These dilutions are far more dilute than conditions expected in the gastrointestinal tract of equine neonates and adult horses in vivo.

Because exposure to C perfringens is common in neonatal foals,2,3 prevention of neonatal enteric clostridiosis is difficult. Presently, there is no effective vaccine approved for use in horses. The efficacy of toxin adsorption by adsorbent materials determined in our study suggests that administration of DTOS or BSS may decrease the severity of clostridiosis in equine neonates and may be effective in conjunction with standard rehydration, antimicrobial, and antiinflammatory treatments in affected foals. Furthermore, smectite use is known to be safe and effective in the treatment of acute diarrhea in children.17,19,21,42 Although further in vivo studies are necessary to substantiate the body of clinical and anecdotal evidence that support the use of DTOS, it currently appears to be an effective adjunctive treatment of equine neonatal clostridiosis in combination with other supportive therapies. Because of the severity of enteric clostridiosis, particularly that associated with C perfringens type C infection, it seems practical and prudent to incorporate adsorbent materials into the treatment regimen of affected equine neonates.

However, it is critical in the early neonatal period that foals receive an adequate quantity of good quality colostrum. Thus, any agent that may interfere with antibody absorption from the intestinal tract can put foals at risk for failure of passive transfer and subsequent infection.43–45 The present study was undertaken to determine whether DTOS would interfere with the availability of immunoglobulins for absorption during this critical period. Our findings indicated that co-incubation of DTOS with colostral antibodies resulted in minimal binding at the 1:32 adsorbent dilution. However, decreased concentrations of colostral antibodies in the 1:4, 1:8, and 1:16 adsorbent dilutions were detected. Thus, considering the dilutional effects of ingested colostrum or milk along with fluid secretion within the gastrointestinal tract, the authors recommend that a period of at least 6 to 8 hours after ingestion of good quality colostrum is allowed to elapse before administration of DTOS to minimize the potential impact on availability of colostral antibodies for absorption.

In a limited field evaluation performed on a farm where neonatal clostridiosis was endemic, 9 healthy foals received DTOS at 6 hours after suckling. At 12 hours after suckling, serum IgG concentrations were assessed and revealed adequate absorption of immunoglobulins as determined via SRID assay° (n = 5; mean IgG concentration, 4,284 mg/dL; range, 3,329 to 6,152 mg/dL) or a semiquantitative immunoenzymatic testt (n = 4; IgG concentrations > 800 mg/dL).u On the basis of results of that study, it appears that if a mare's colostrum is of poorer quality (specific gravity < 1.060), as may occur in maiden mares or mares > 15 years old,43,44 it may be prudent to withhold DTOS treatment from a foal for a period > 6 to 8 hours after suckling to allow ample time for ingestion of colostrum or consider supplementation with good quality colostrum.

Limitations of the present study include the in vitro format used to investigate co-incubation of adsorbent material with either clostridial toxins or colostrum. In addition to the inherent limitations of in vitro studies, the experiment could not mimic the dynamic process of secretion, mixing, and absorption of fluids that would occur in live animals. Also lacking in a laboratory setting is individual animal variation in response to administration of oral adsorbent substances and other treatments (eg, fluids, antimicrobials, and antiinflammatory agents). Additionally, we used the SRID assay to evaluate colostral antibody concentrations. Because the SRID kit is not designed to be used with such a viscous material, dilutions of colostrum were necessary. This may have affected the accuracy of the test. Although the level of detectable toxins decreased when co-incubated with DTOS and BSS, results of the present study did not prove that the toxins were actually bound to the adsorbent material. We assumed that because the toxins were undetectable, they were bound to the adsorbent material and would be unavailable to cause clinical disease in a live animal. To further investigate this assumption, one could consider labeling the toxin molecules to identify whether they were truly bound to adsorbent material or inactivated; however, such an undertaking was beyond the scope of the present study. Similarly, if clostridial exotoxins are contained within the mucus glycocalyx layer on the surface of the small intestine, exposure of the toxin to adsorbent material may be limited. Despite these study limitations, our in vitro findings can be extrapolated to a clinical setting, but the need to validate these results through clinical trials must be recognized.

Administration of DTOS is a treatment option that holds promise for the amelioration of equine neonatal enteric clostridiosis. This disease of equine neonates is frustrating for owners and clinicians because severe illness and death may still occur despite early and aggressive medical treatment. Di-tri-octahedral smectite is a potentially useful adjunct to conventional management of clostridial diarrhea. Administration of DTOS is cost-effective, has no known risks or adverse effects, and is efficacious in the treatment of diarrhea associated with a variety of causes in both humans and other animal species. In vitro adsorption of clostridial toxins by DTOS is superior to another conventional antidiarrheal agent, BSS.

Concern for adequate transfer of colostral antibodies is of primary importance in establishing a competent immune system in foals. Our preliminary data suggest that as long as foals are allowed adequate ingestion of colostrum of sufficient quantity and quality, there is little risk for DTOS-associated failure of passive transfer. Therefore, the authors recommend that until further studies are completed, the administration of DTOS to neonates be withheld until at least 6 hours after the ingestion of good quality colostrum. Controlled clinical trials for definitive assessment of IgG concentrations and evaluation of the efficacy of DTOS adsorption of toxins are indicated to substantiate the in vitro effects identified in the present study.

ABBREVIATIONS

DTOS

Di-tri-octahedral smectite

BSS

Bismuth subsalicylate

PBST

PBS solution and 0.05% Tween-20

SRID

Single radial immunodiffusion

OD

Optical density

a.

Plampin EC, Ellis RP, Traub-Dargatz JL, et al. Detection of Clostridium perfringens alpha, beta and beta-2 toxins in foal feces (abstr), in Proceedings. 85th Annu Conf Res Workers Anim Dis 2004;126.

b.

Bio-Sponge powder, Platinum Performance Inc, Buellton, Calif.

c.

Bismuth subsalicylate, Biocode, VedCo Inc, St Joseph, Mo.

d.

Purified recombinant C perfringens his-alpha toxin, provided by Dr. J. Glenn Songer, Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Ariz.

e.

Purified recombinant C perfringens beta-2 toxin, provided by Dr. J. Glenn Songer, Department of Veterinary Science and Microbiology, The University of Arizona, Tucson, Ariz.

f.

Partially purified (culture filtrate) C perfringens Type C beta toxin, culture #4414 used to produce IRP 459, provided by Dr. Paul Hauer, Center for Veterinary Biologics, USDA, Ames, Iowa.

g.

Nunc-Immuno 96 microwell flat-bottom polystyrene plates, Nalge Nunc International, Rochester, NY.

h.

Carbonate coating buffer, Pierce Biotechnology, Rockford, Ill.

i.

Monoclonal antibodies to alpha (Mab 9A24B) toxin, provided by Dr. William Scheuchenzuber, Life Sciences Consortium, Pennsylvania State University, University Park, Pa.

j.

Monoclonal antibody to beta-2 (Mab 9E2B) toxin, provided by Dr. William Scheuchenzuber, Life Sciences Consortium, Pennsylvania State University, University Park, Pa.

k.

Monoclonal antibody to beta toxin (Mab 10A2), provided by Dr. Paul Hauer, Center for Veterinary Biologics, USDA, Ames, Iowa.

l.

Goat anti-mouse horseradish peroxidase conjugate (0.67 μg/mL), Rockland Immunochemicals Inc, Gilbertsville, Pa.

m.

O-phenylenediamine (OPD) chromagen, Sigma Chemical Co, St Louis, Mo.

n.

Multi Skan automatic plate spectrophotometer, Thermo Electron Corp, Waltham, Mass.

o.

Immunocheck, VMRD Inc, Pullman, Wash.

p.

SPSS, version 11.0, SPSS Inc, Chicago, Ill.

q.

Excel 2003, Microsoft Corp, Redmond, Wash.

r.

Droy-Lefaix MT, Nayetat H, Espinasse J, et al. Rotavirus infection in calves, protective effect of smectite (abstr). Dig Dis Sci 1986;(suppl 31):G68.

s.

Hassel D, Smith P, Nieto J, et al. Di-tri-octahedral smectite for the prevention of post-operative diarrhea in equine colic patients: results of a randomized clinical trial (abstr), in Proceedings. 12th Annu Am Coll Vet Surg Symp 2004;E11.

t.

SNAP Foal IgG, IDEXX Laboratories, Westbrook, Me.

u.

Farrand G, Northern Colorado Veterinary Service, Fort Collins, Colo: Personal communication, 2006.

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Contributor Notes

Dr. Lawler's present address is Colorado Equine Veterinary Services, 16850 Murphy Road, Peyton, CO 80831.

Supported in part by Platinum Performance Incorporated, Buellton, Calif, and the USDA's Cooperative State Research Education and Extension Services for the Colorado State University Program for Economically Important Animal Diseases (PEIIAD).

Presented in part as an abstract at the 24th Annual Meeting of the American College of Veterinary Internal Medicine, Louisville, June 2006.

The authors thank Cindy Hirota for technical assistance.

Address correspondence to Dr. Hassel.