Article

Pediatric Research (2002) 51, 370–376; doi:10.1203/00006450-200203000-00017

Oral Bovine Serum Concentrate Improves Cryptosporidial Enteritis in Calves

Elaine Hunt1, Qiang Fu2, Martha U Armstrong1, Derralyn K Rennix1, David W Webster1, Joseph A Galanko3,4, Wunian Chen2, Eric M Weaver5, Robert A Argenzio1,4 and J Marc Rhoads2,4

  1. 1Department of Anatomy, Physiology, and Radiological Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC, U.S.A.
  2. 2Department of Pediatrics, University of North Carolina, Chapel Hill, NC, U.S.A.
  3. 3School of Public Health, University of North Carolina, Chapel Hill, NC, U.S.A.
  4. 4Center for Gastrointestinal Biology and Disease, Chapel Hill, NC, U.S.A.
  5. 5Proliant, Inc., Ames, IA, U.S.A.

Correspondence: J. Marc Rhoads, Department of Pediatrics, University of North Carolina, Campus Box 7220, Chapel Hill, NC 27599-7220, U.S.A.; e-mail: rhoads@med.unc.edu

Received 17 November 2000; Accepted 4 May 2001

This study was supported by Proliant Inc., Ames, IA, U.S.A.

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Abstract

Cryptosporidium parvum produces a prolonged watery diarrhea unresponsive to conventional antimicrobials. Because of reported efficacy of antibody-based immunotherapy, we studied the effect of inexpensive, commercially available oral bovine serum concentrate (BSC) in experimental cryptosporidiosis. Twenty-four calves were treated with 57 g/d BSC (n = 12) or soy protein (n = 12) added to their standard whey protein-based milk replacer (227 g/2 L twice daily). Of the 24, 9 were also treated with l-glutamine (GLN), 8 g/L (50 mM) in the milk (5 calves in the BSC group and 4 in the soy group). Animals were inoculated with 108 cryptosporidium oocysts per os on d 8 of life and received oral rehydration on d 12–14. Eight uninfected controls were treated with BSC or soy protein. Fecal and urine volume and urinary Cr-EDTA excretion were measured. Animals were killed on d 18 of life. Cryptosporidiosis induced severe watery diarrhea lasting >9 d and produced a 25% increase in intestinal permeability, a 33% decrease in villous surface area, and a 40% reduction in mucosal lactase specific activity. Glutamine treatment had no effect on the diarrhea or any of the intestinal tests; and therefore pooled data were used to compare the 12 calves treated with BSC with the 12 treated with soy. In animals receiving BSC, peak diarrheal volume and intestinal permeability were reduced 33%, fewer oocysts were shed, intestinal crypts were significantly deeper, and villous surface area returned to normal by 9 d after infection (all p ≤ 0.05). BSC should be studied as a treatment for human cryptosporidiosis.

Abbreviations:

BSC, bovine serum concentrate; EGF, epidermal growth factor; Ig, immunoglobulin; TGF, transforming growth factor

Cryptosporidiosis is a major enteric pathogen found globally in infants with acute and chronic diarrhea, and is a cause of persistent diarrhea in 10%–20% of patients with AIDS. Cryptosporidium parvum is also the most widespread enteropathogen identified in neonatal calves. Ninety percent of American dairy farms harbor this coccidian, and 92% of asymptomatic adult cows have specific anti-C. parvum IgG, IgG1, IgG2, and IgM antibodies (1). Neonatal calves experience high morbidity, but low mortality with monoinfections, although mixed infections result in a much greater rate of mortality (2). The epidemic potential of this organism was realized in Milwaukee in 1993, when over 400,000 residents contracted the infection from contaminated municipal water sources (3). Presently, there is no available antimicrobial to kill cryptosporidium; in fact, there is no treatment to stimulate bowel repair from any infectious agent in infants with serious or chronic diarrhea.

We have shown that cryptosporidial diarrhea is both malabsorptive and secretory in nature, produced by partial mucosal destruction as well as elevated levels of prostaglandin E2 and prostacyclin, the latter of which activate enteric secretomotor neurons (4). We have also found beneficial effects of growth factors (present in serum and colostrum) in enhancing bowel repair in rotavirus diarrhea and after intestinal ischemic injury (5, 6). Recently, Lembcke (7) showed in Peruvian children recovering from acute diarrhea that BSC treatment improved fat and energy absorption compared with standard oral rehydration therapy. Because serum contains specific growth factors and stimulates cultured intestinal cell proliferation and migration (8), and because most cows have specific antibodies to C. parvum(1), we proposed that BSC will reduce the severity of cryptosporidiosis and enhance bowel repair in cryptosporidiosis.

The principal aim of these studies was to determine whether bovine serum concentrate enhances bowel repair in acute bovine cryptosporidial enteritis. We also determined whether BSC facilitated clearance of cryptosporidium, which would be of great benefit in immunocompromised patients.

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METHODS

Animals and challenge.

Experimental bull calves (n = 32, approximately 50 kg), isolated at birth from the herd and fed colostrum by the vendor farm, were obtained by 2 d of age. The basic calf diet was a commercially available 23% whey protein-based milk replacer (Purina, St. Louis, MO, U.S.A.). Calves were fed 454 g dry matter daily [227 g in two feedings, except during oral rehydration solution (ORS) feeding] delivered in two 2-L feedings offered in the morning and evening. On d 2–7, calves were fed 170.3 g milk replacer (23% protein) blended with 56.7 g BSC (Proliant Inc., Ames, IA, U.S.A.) or 56.7 g soy protein (Ardex AF by Archer Daniels Midland, Decatur, IL, U.S.A.). On d 8–10, calves were fed two feedings of ORS blended with 56.7 g BSC or soy protein. On d 11–13, calves were returned to the initial feeding regimen. Calves were assigned to one of six treatment groups, as follows:

  • uninfected control + isolated soy protein (n =4)
  • uninfected control + BSC (n = 4)
  • infected + soy protein (n =8)
  • infected + BSC (n =7)
  • infected + soy protein + l-glutamine (9 g/L) (n = 4)
  • infected + BSC + l-glutamine (9 g/L) (n = 5)

Cr-EDTA was given at a constant dose of 90 mg in all feedings and was measured in the daily urine as an indicator of intestinal damage and permeability (9). All diet components were premeasured and mixed just before feeding with 2 L warmed water. Calves were maintained on the assigned diets for 7 d before infection to establish steady state Cr-EDTA conditions (see below). All calves were fed via a nipple-bottle feeder their assigned treatment twice per day for 17 d.

Plasma proteins are commonly collected from abattoirs under hygienic conditions for use in specialty food products, biologicals, animal health, and pharmaceuticals. The U.S. Department of Agriculture (USDA) regulates the collection and processing of plasma proteins for edible uses. The bovine serum used in this study was inspected and approved for edible use. Briefly, calcium chloride is added to chilled plasma to activate the conversion of fibrinogen to fibrin. The fibrin is removed by centrifugation and the resulting serum (defibrinated plasma) is concentrated using ultrafiltration. The concentrated serum protein fraction is then spray-dried to maintain the functional characteristics of the proteins. Dried BSC is a fine, tan-colored powder containing approximately 80% protein, of which 60% is albumin and 25% is IgG. We have also measured significant levels of IGF-I (6000 ng/g protein) and TGF-β1 (90 ng/g) in BSC.

For the first 6 d, calves were housed in individual pens and received twice daily health exams and the assigned feedings. On d 7, calves were housed in an isolation room in individual metabolic cages that allowed the separation and collection of urine and feces. These output collections were weighed every 24 h, and the urine was sampled and saved for subsequent Cr analysis by flame atomic absorption spectrometry. Physical exams and fecal smears (for C. parvum grading) were continued daily. All data collected through the d 7 were considered baseline before infection with C. parvum.

On d 8 of age, calves received either 108 oocysts of C. parvum orally by bottle feeding or a sham inoculum of PBS substrate. C. parvum oocysts isolated from feces of infected calves were purchased from Pleasant Hill Farms (Troy, ID, U.S.A.) and were used within 12 wk of purchase. The Iowa isolate of C. parvum was originally obtained from H. Moon (Ames, IA, U.S.A.). Physical exams, fecal smears, and feedings were continued daily for the next 9 d postinfection. On d 4, 5, and 6 postinfection, both control and infected calves received oral rehydration supplement (Resorb, Pfizer, Exton, PA, U.S.A.) replacing the milk substrate but including the 57 g of assigned treatment and 90 mg of Cr-EDTA. On postinfection d 9, calves were killed with i.v. sodium pentobarbital (90 mg/kg after sedation with xylazine 0.1 mg/kg). Mucosal sections of ileum were taken for lactase activity determination and histologic analysis.

Fecal scores.

Stools were scored as follows: 1+: solid stools; 2+: mildly loose stools; 3+: thick liquid stools; 4+: watery diarrhea.

Oocyst shedding was quantified (1+–4+) as reported previously by the auramine 0 staining technique using a Zeiss fluorescent microscope (Zeiss, Welwyn Garden City, U.K.) (10).

Histology and morphometric analysis.

Segments of ileum, the site of peak intestinal damage in cryptosporidiosis (9), were fixed in situ by intraluminal injection of a 10% formalin solution. The fecal material was gently removed and the segment immersed in fixative. Formalin-fixed segments were embedded in paraffin and 5-μm-thick sections were cut and stained with hematoxylin and eosin for examination by light microscopy. Computer-assisted morphometric measurements were conducted with a video-imaging system (Nikon-FXA, Nikon, Melville, NY, U.S.A.). All samples were measured twice by each of two observers masked to the treatment group. Three to six well-oriented villi from sections of ileum (without underlying Peyer's patches) were measured at 100× magnification to determine mean villous height and width. Crypt depth was measured from three to four sites from the same sections. Villous surface areas for representative villi were calculated as reported previously (10).

Assays of mucosal lactase.

Intestinal lactase specific activity was measured by the technique of Dahlquist et al. (32) as previously described (11). Mucosa was scraped from the jejunal segments on a pane of glass over ice, using a glass slide; the scrapings were homogenized in iced buffer (0.25 mM lactose, 0.2 M Tris, 0.15 M, KC1, 2.5 mM, EDTA, pH 7.4 at 4°C). The homogenates were immediately frozen in aliquots at −70°C and were assayed within 2–3 wk. Five samples in the soy-treated group and three samples from the BSC-treated group were lost because of malfunction of the laboratory freezer.

Statistics.

We used a one-sided Wilcoxon test to determine whether there was any advantage of BSC compared with soy protein. The Wilcoxon test is a nonparametric test used to determine whether two groups are significantly different while not making assumptions about the distribution of the data. Data are expressed as means ± SE.

These studies were approved by the North Carolina State University College of Veterinary Medicine Committee to Review Applications for Vertebrate Animal User.

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RESULTS

Diarrheal output.

Although none of calves died, moderate diarrhea was observed in each calf investigated (score 3+ out of 4+ by visual analog score) at d 4–6. Diarrhea was mild (score 2+) for the first 3 d, became moderate (score 3+) for 3 d, with a mean daily volume of approximately 0.5 L), and then became mild again in both treatment groups between d 7 and 9.

Glutamine treatment had no significant effect on diarrheal volume. For example, calves treated with BSC with glutamine had 3.9 ± 1.5 total liters of stool, whereas calves treated with BSC without glutamine had 4.0 ± 0.7 L of stool. Additionally, analysis of intestinal mucosal histology revealed no significant differences in villous surface area or crypt depth in glutamine-treated calves, and glutamine had no beneficial effect on Cr-EDTA permeability. Therefore, all data presented reflect pooled data of BSC-treated compared with soy protein-treated groups.

BSC-treated calves had approximately a 33% reduction in volume of diarrhea (p ≤ 0.05) at the peak of illness (d 4–6 postinoculation)(Fig. 1), compared with soy protein-treated calves, although stools remained looser than normal and volume was about twofold greater than normal at the end of the study in both groups. There were no significant differences in urine output, comparing the two treatment groups.

Figure 1.
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Diarrheal output per animal on d 1–3, 4–6, and 7–9 postinoculation. Soy protein-supplemented group is shown in striped bars; BSC-treated group is shown in open bars. Means ± SEM. *p = 0.03 comparing soy protein and BSC-supplemented groups (n = 12 in each group).

Full figure and legend (228K)

Intestinal permeability.

Comparing uninfected animals supplemented with soy protein or BSC, there was no difference in permeability to chromium. BSC produced approximately a 30% decrease in total gut permeability at d 4–6 after inoculation, compared with permeability in soy-treated infected animals, as measured by urinary excretion of ingested chromium (p ≤ 0.001). Additionally, infected calves treated with BSC (at d 1–3 postinoculation) demonstrated reduced permeability (p = 0.02, Fig. 2) compared with the uninfected soy-treated and BSC-treated control groups.

Figure 2.
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Urinary chromium excretion as a measure of intestinal permeability in BSC- and soy-treated groups in 3-d intervals of the study. Soy protein-supplemented group is shown in striped bars; BSC-treated group is shown in open bars. Means ± SEM. *p ≤ 0.01 comparing the two groups on indicated day.

Full figure and legend (244K)

Ileal morphology.

Figure 3 shows representative histologic samples from ileum of normal and cryptosporidium-infected calves. We measured villus height, villus width, and crypt depth from all samples. Because villous surface area gives a more accurate picture of absorptive surface than villous height in cryptosporidiosis, where villus blunting is partial, we calculated villous surface area using an equation we previously published in a study of piglet cryptosporidiosis (10). Figures 3 and 4A depict an approximately 15% increase in villus surface area of ileum from infected calves fed BSC compared with soy-supplemented controls (p = 0.05 comparing soy- and BSC-treated groups). We also measured ileal crypt depth as an index of the proliferative response to infection and BSC treatment. Crypt depth of the ileum of infected calves at 9 d postinoculation was significantly increased, comparing BSC with soy protein treatment (Figs. 3 and 4B), with a 15% increase in crypt depth in the BSC-treated group compared with the soy group (p ≤ 0.05). The number of histologic samples used for villus/crypt measurement differed from the number of animals enrolled in the study because several histologic samples had no measurable villi that were Peyer's patch-free. (Note that villi above Peyer's patches are shorter than villi from patch-free mucosa.)

Figure 3.
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Ileal mucosa of normal and 9-d cryptosporidium-infected calves (hematoxylin and eosin; scale bar represents 10 μm). C-Soy: Ileum of control 18-d-old calf treated with soy protein supplement. C-BSC: Ileum of control calf treated with BSC. I-Soy: Infected ileum, soy-treated group. I-BSC: Infected ileum of calf treated with BSC. Ileal villi of BSC-treated infected calves were taller and thinner, consistent with less inflammation. Ileal crypts of BSC-treated infected calves were deeper than those of soy-treated controls.

Full figure and legend (307K)

Figure 4.
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(A) Calculated villus surface area of ileum from normal and infected calves (with soy or BSC treatment) at d 9 postinoculation. *p = 0.05 comparing soy group with BSC group of infected calves (n = 4 in each uninfected group, n = 8 in each infected group). Numbers of calves studied are the same for villous surface area and crypt depth. (B) Crypt depth of ileum from normal and infected calves (with soy or BSC treatment) at d 9 postinoculation. *p < 0.05 comparing soy group with BSC group of infected calves. Soy protein-supplemented group is shown in open bars; BSC-treated group is shown in striped bars.

Full figure and legend (434K)

Lactase specific activity.

BSC enhanced the specific activity of intestinal lactase in infected calves (p ≤ 0.05, comparing BSC-treated with soy protein-treated groups, Fig. 5). No significant difference in ileal lactase activity was found comparing normal calves treated with soy protein with those receiving BSC. Soy-treated infected calves had a 30% lower ileal lactase-specific activity than soy-treated controls (p = 0.03) at d 18 of life. This provided evidence that cryptosporidium-infected ileum had not fully recovered by d 9 postinoculation.

Figure 5.
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Ileal lactase activity from normal and cryptosporidiosis infected calves. *p ≤ 0.05 comparing BSC and soy-treated groups. +p ≤ 0.05 comparing soy-treated uninfected and infected groups. (n = 4 in each uninfected group, n = 7 from BSC-infected group, n = 9 from soy-infected group). Soy protein-supplemented group is shown in open bars; BSC-treated group is shown in striped bars.

Full figure and legend (241K)

Oocyst shedding.

In addition to growth factors, BSC contains Ig (as measured by radial immunodiffusion), which could reduce the duration or severity of infection. BSC reduced cryptosporidial oocyst shedding almost four fold at d 4–6 postinoculation (p = 0.03, Fig. 6). However, at other time points, including at the end of the study, the mean number of oocysts per high-power field and per stool volume was unchanged compared with controls. Animals from all treatment groups continued to shed oocytes at the end of the study, even though they were recovering from the diarrhea.

Figure 6.
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Number of cryptosporidial oocysts per high-power field (40×) on fecal microscopy, plotted as counts per 3-d interval after infection. *Indicates a significant reduction in oocyst counts on d 4–6 postinoculation (p ≤ 0.05, n = 12 in each group). Soy protein-supplemented group is shown in open bars; BSC-treated group is shown in striped bars.

Full figure and legend (228K)

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DISCUSSION

Cryptosporidium is a major threat to humans, producing prolonged episodes of acute diarrhea in infants, especially in daycare center outbreaks, and life-threatening watery diarrhea in immunocompromised teens and adults (12, 13). HIV-infected patients have been reported with infections lasting years and fecal volumes averaging 1.5 L/d (13). There is no antimicrobial agent proven to be effective in treating cryptosporidiosis. We chose to study a calf model of this infection. In this host at 1 wk of age, 85% of suckling calves and 52% of dairy calves excrete the parasite, and about half of these have diarrhea (14).

We proposed that BSC would be effective in treating this infection, because hyperimmune bovine colostrum has been previously shown to protect from infection and to produce a partial resolution of symptoms in infected individuals (15). Additionally, even though hyperimmunized cows were not used to prepare the BSC, most adult cattle have detectable antibody to C. parvum (1; Perryman LE, personal communication, August 2000). BSC is inexpensive, commercially available, and approved by the USDA for use as a food ingredient. BSC is currently available as a dietary supplement (Immunolin or NutraGammax) in health food stores. Spray-drying of BSC protects the structure of IgG, IgM, and IgA, but also of transferrin, albumin, TGF-β, and IGF-1. BSC has been used to assist the recovery of children with acute enteritis and is currently under investigation as a dietary additive to prevent infection in children at high risk for diarrheal disease (Brown KH, personal communication, September 1999).

BSC has also been shown to increase efficiency of dietary protein utilization in newborn piglets (16). Soy protein supplementation was chosen to control for possible nutritional effects of BSC because human studies have demonstrated equivalent growth in babies fed cow milk and soy milk-based formulas (17). As mentioned, slightly inferior rates of growth in exclusively soy-fed calves have been observed (18, 19), but in the current study we used bovine whey protein in the basal diet with soy or BSC added as a supplement, and the duration of the study was only 9 d. Thus, soy protein is a nutritionally similar supplement for comparison with BSC.

In our study, BSC successfully diminished fecal losses, reduced gut permeability, and enhanced ileal crypt depth and villous surface area. The diarrhea was moderate, with mean fecal volume at the peak of infection of 500 g/d in BSC-treated infected calves, 800 g/d in soy-fed calves, and 200 g/d in the preinfection baseline period. None of the calves died during the study. BSC not only improved the diarrhea but also reduced gut permeability and enhanced crypt depth and villous surface area after infection.

There are three potential mechanisms for the efficacy of BSC in reducing the severity of bovine cryptosporidiosis. First, Ig in the BSC may have facilitated a reduction in number of viable parasites. Previous studies in human cryptosporidiosis have shown efficacy of immune bovine colostrum in some studies (20) and lack of efficacy in others (21). In our study, reduced oocyst shedding in BSC-treated calves was seen, but oocysts remained in the stool at the end of the study. Previous animal studies showed that colostral antibodies detected in feces are not protective. In feces from infected animals treated with colostrum, anti-C. parvum, detectable IgA levels rose between d 7 and 14 postinfection (22). These coproantibodies coincided with falling oocyst output, but fecal antibodies were protective only at late time points after infection (16 wk later). BSC is defibrinated, dialyzed, and spray-dried but is not heated above 63°C, resulting in preservation of its Ig. One of our collaborators attempted to measure BSC-specific reactivity to sporozoite/oocyst proteins by ELISA, but ran into difficulties with dried immunoglobulins producing a high background activity. We have consistently measured titers to Escherichia coli and other bacteria and rotavirus, and have observed in vitro toxin neutralization by BSC, but BSC does not neutralize cryptosporidium and did not prevent infection in our study. In a recent study of BSC treatment of piglets inoculated with porcine rotavirus, BSC treatment did not affect virus shedding into the feces, but completely prevented the diarrhea; BSC treatment produced taller jejunal villi and greater intestinal lactase activity (23). These findings rule against an immunologic effect of BSC as the mechanism of efficacy in rotavirus enteritis.

Second, BSC could have facilitated intestinal repair via growth factors present in bovine serum. We demonstrated decreased intestinal permeability and increased crypt depth in the ileum of BSC-treated infected calves. In infectious enteropathies, stem cells in the intestinal crypts undergo mitosis to regenerate the damaged villi. In a rat intestinal cell line (IEC-6), we have found that BSC enhances the rate of thymidine incorporation and the rate of proliferation-independent cell migration across de-epithelialized surfaces, each about twofold compared with control responses without serum (8). This effect is partially inhibited by anti-TGFβ antibody (8). Such increases in epithelial cell migration could explain the decreased intestinal permeability to Cr-EDTA in vivo. Antibodies to IGF-1 and TGFβ, both of which stimulate intestinal cell migration, partially neutralize this in vitro effect of BSC (Chen W, Rhoads JM, unpublished observation). Interestingly, human volunteers who ingested C. parvum were found to have enhanced TGFβ mRNA expression in the small intestine at the onset of and during diarrhea (24). Of even more direct relevance to our findings with BSC, which contains TGFβ, Roche et al. (25) recently found that, when TGF-β was applied to intestinal epithelial cell monolayers, this growth factor prevented the increase in permeability seen when monolayers were subsequently experimentally infected with cryptosporidium.

A third potential mechanism for reduced severity of infection is that the BSC could have interfered with cryptosporidial attachment, reducing invasion and replication. This mechanism was not addressed in the present study, but the observation that Cr-EDTA permeability decreased on d 1–3 postinfection in the BSC-treated group suggest that parasites did attach early, resulting in villus damage. The observation that small numbers of cysts persisted in the stool in both treatment groups 9 d after inoculation also suggests that oocysts remained in the intestine during the experimental period.

The results of the present study compare favorably with previous animal studies of oral therapy with growth factors and/or nutrients for animal models of infant diarrhea. There have been multiple negative studies of enteral treatment for acute diarrhea, including studies from our group of glutamine, polyamines, fructo-oligosaccharides, and rice powder in piglets with rotavirus infection (26). In rabbit and piglet models of viral enteritis, EGF and TGFα stimulated villus height but did not reduce diarrheal volume (27, 28). In a more severe model, piglet ischemia-injured ileum, we found that glutamine plus TGFα stimulated mitogen-activated protein kinases and villous repair (6). We are not aware of reports of reduced gut permeability in vivo after treatment with EGF or TGFα during acute diarrhea, as seen with BSC in the current studies. Additionally, BSC had potent effects on recovery from diarrhea in a preliminary report of piglet rotavirus diarrhea (23).

In our studies, BSC treatment appears to have reduced intestinal permeability compared with infected soy-treated controls and compared with uninfected controls. There are at least two opposing factors contributing to this effect. One is a decrease in surface area caused by infection, which would reduce the number of paracellular pathways in the epithelium and reduce permeability across infected tissue. This does not explain why permeability is reduced in the BSC-treated group compared with the infected soy-treated group. The opposing factor is probably enhanced restitution in the BSC-treated group. Thus, the unaffected permeability in the soy group on d 1–3 reflects the decreased surface area superimposed on an increased permeability due to parasite damage, with the two effects canceling. In the BSC group, decreased surface area plus increased restitution would be additive and would decrease ileal permeability. In support of this explanation, in studies of intestinal cell monolayers, Guerrant's group has shown increased conductance after cryptosporidial infection (25), whereas in whole tissue we have consistently found that the conductance decreases after cryptosporidial infection produces a reduction in mucosal surface area (10).

Despite numerous human trials of oral therapy for infant diarrhea, very few treatments have been shown to reduce the volume or duration of diarrhea, and none has been shown to improve intestinal histology. Rice-based oral rehydration solutions have been shown to reduce diarrheal volume approximately 33% in patients with secretory diarrhea, such as cholera and enterotoxigenic E. coli(29). However, in infantile diarrhea, rice-based ORS has not been shown to have a significant effect (30). A promising new development is probiotic therapy, or the use of colonizing bacteria with beneficial effects. In three human trials, including a multicenter European trial in infants (31), administration of probiotics, such as Lactobacillus rhamnosis GG and Lactobacillus reuterii, have been shown to reduce the severity and duration of diarrhea. However, at the present time, these products are expensive, costing about $0.70 per capsule (1010 organisms), which is added to each 8-oz. bottle of oral rehydration. This would cost about $2.80/d when added to maintenance fluids in a 10-kg infant. BSC is available as a dietary supplement under the brand name ingredient Immunolin. The dietary supplement available in health food stores has had albumin removed, yielding a product that is 40% IgG, making it about 2.5-fold as rich in IgG as BSC. The efficacy of BSC is most likely determined by its growth factors; therefore, the optimal dose for treatment of cryptosporidial enteritis remains to be determined. The cost of BSC is relatively low, $0.02 to $0.07 per gram ($0.20–$0.70/d in a 10-kg child), compared with other alternatives such as probiotics. Depending on dose and efficacy, BSC is a promising new ingredient for bowel recovery.

The supply of BSC is substantial, particularly in countries with bovine-based protein consumption. The BSC used for these studies was processed and produced under USDA regulations for meat-based food ingredients. It is approved and is safe for use in foods. Despite the recent concerns in regard to bovine spongiform encephalopathy in Europe, no cases of bovine spongiform encephalopathy have been reported in the United States. Our data do not support the conclusion that BSC antibodies are protective, only that BSC facilitates bowel recovery.

In summary, oral BSC treatment, compared with soy protein supplementation, of newborn calves with cryptosporidial diarrhea reduced peak diarrheal volume, improved intestinal permeability, and facilitated crypt cell-mediated regeneration of villous surface. These significant effects and the low cost of BSC suggest that BSC should be studied in human trials of cryptosporidial diarrhea.

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Acknowledgements

The authors thank Dr. Robert Bagnell for use of the University of North Carolina Department of Pathology microscopy services and Marjorie Gray for technical assistance.