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Institute of Food, Nutrition, and Human Health, Massey University, Palmerston North, New Zealand
2To whom correspondence should be addressed. E-mail: F.M.Wolber{at}Massey.ac.nz.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • rotavirus • mouse model • whey protein concentrate
Rotavirus infection is the most common cause of diarrhea in young children (1,2) and nearly 500,000 children die each year as a result of severe rotaviral disease (3). Unlike many enteric pathogens, rotavirus causes symptomatic disease mainly in the young, although adults can be infected and will shed virus in the feces. Currently, no antirotavirus vaccines are available. There is therefore a great need to develop safe and effective preventatives for reducing childhood rotaviral infection as a means of improving pediatric health.
Both T- and B-lymphocytes play an important role in the control of rotavirus infection and in the subsequent clearance of the virus (4–7); probiotics, which enhance the immune response, were shown to be partially effective against rotavirus in animal models (8–12) and in humans (13). Lactadherin in human milk blocks rotavirus attachment or replication as do intestinal mucins and some cytokines in vitro (14–17). Oral administration of a sulfated sialyl lipid reduced rotavirus-induced diarrheal disease in suckling mice, and rotavirus-specific Ig was partially effective in oral daily doses of 3–10 g for 4 d in children with rotavirus infection (18–20). However, oral rehydration therapy remains the standard treatment (21,22); neither probiotics nor Ig are yet used in routine clinical practice (23,24).
Bovine milk and whey protein concentrates (WPC)3 contain antiviral and immunomodulatory components and were shown to enhance a variety of immune functions (23,25–31). The current study evaluated the effects of the proprietary whey protein concentrate IMUCARETM (WPC-IC) on rotaviral disease in neonatal mice (32–35).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Time-mated females were caged individually, provided with nesting materials, and monitored to identify time of littering. Within 12 h of birth, pups were culled or cross-fostered as necessary to provide each dam with 6 pups per litter. When pups were 8 d old, dams and litters were number-tagged and randomly assigned to the control group or the test group in a blinded fashion. Pups were allowed to nurse and to sample the diet that their dams consumed ad libitum; in addition, beginning from the age of 9 d, they received by gavage the dietary test substances once daily as described below.
The study and technical procedures were carried out with the full approval of the Massey University Animal Ethics committee, maintaining internationally recognized standards of laboratory animal heath and care.
Dietary supplements. From the age of 9 to 17 d, suckling mice received by oral gavage once daily the appropriate control [bovine serum albumin (BSA)] or test (whey protein concentrate) dietary supplement. The whey protein concentrate IMUCARETM (WPC-IC) was provided by New Zealand Milk (IMUCARETM is a trademark of New Zealand Milk Brands) and contained 80% protein, 8.6% carbohydrate, 4.9% fat, 4.6% moisture, and 3.3% ash. BSA (fraction V; Sigma-Aldrich) is a minor and nonimmunomodulatory component of WPC-IC. Tubes containing coded dietary supplements were reconstituted in the appropriate amount of sterile water and used within 4 h. Gavage operations were performed using a 24-gauge, 2.54-cm ball-ended animal feeding needle inserted into the esophagus and advanced to the cardia as identified by a premeasured demarcation on the canula.
To compensate for their increasing body weight, mice were fed an escalating dose of test or control supplement calculated to be 
3 g/kg metabolic body weight (BW0.75). From d 9 to 17, daily doses per mouse were 12.0, 12.9, 14.0, 15.3, 16.5, 17.2, 18.4, and 18.8 mg. Supplements were delivered in a volume of 50 µL (d 9–14) or 100 µL (d 15–17).
Twenty litters treated daily by gavage with BSA or with WPC-IC (n = 60 mice/group) were infected with rotavirus as described below. An additional 4 litters treated daily by gavage with BSA or with WPC-IC (n = 12 mice per group) were not infected with rotavirus and were assessed to ensure that supplemental gavage feeding from the age of 9 to 17 d had no deleterious effects on health; fecal samples collected from these mice during this time period were used to determine background levels in the rotavirus ELISA assay described below.
Rotavirus isolation and infection procedures. MA-104 cells (ATCC) were grown to confluence and infected with live rotavirus strain EDIM (36). Supernatants containing virus were harvested and centrifuged at 17,000 x g at 4°C for 1 h to remove cell debris. The virus titers of supernatants were assessed by ELISA (IDEIA kit #602011, DAKO) following the manufacturer’s instructions. Supernatants were centrifuged at 100,000 x g at 4°C for 20 h to concentrate the virus particles, and frozen at –80°C.
Twenty litters of mice treated daily with supplemental BSA or with WPC-IC (n = 60 mice per group) received by gavage at 11 d of age a single dose of 50 µL containing 105 rotavirus particles, delivered 0.5 h after the mice received their dietary supplement.
Rotaviral disease assessment. Mice were weighed daily and their body weights recorded. Daily weight gain as a percentage of body weight for individual mice was calculated as: [(Weight on day Z + 1 – weight on day Z) x 100]/weight on day Z.
Mice were individually held and pressed gently on the abdomen to encourage production of feces. Each fecal sample was assessed at the time of collection to determine the level of disease symptoms: feces with normal color (brown) and normal consistency were scored as "Normal"; feces with abnormal color (green or yellow-green) and normal consistency were scored as "Mild"; feces with normal color (brown) and abnormal consistency (watery) were scored as "Moderate"; feces with abnormal color (green or yellow-green) and abnormal consistency (watery) were scored as "Severe." Descriptive noncontinuous word scores were utilized because the sample size was not large enough to warrant parametric analysis of number scores. Such graded scoring systems are commonly used in animal studies to differentiate between normal brown and abnormal yellow/green feces or among solid, semiliquid, and liquid feces (37–40). Fecal samples collected on d 11, 13, 15, and 17, as available, were coded and stored frozen at –80°C for later analysis.
Viral shedding in feces (ELISA). Fecal samples collected during the study were graded by size as a percentage of a full-sized fecal pellet; such estimations were necessary because samples varied in size from partial, broken pellets to full-sized feces. To maintain consistency, a single researcher, who was unaware of treatment, graded all of the fecal samples (n = 381 total samples).
Fecal samples were reconstituted and processed through 2 freeze/thaw cycles, then assessed in duplicate by ELISA using standard procedures (9,26) in Nunc-Immuno plates (Nalge Nunc International). Rabbit anti-human rotavirus capture antibody, horseradish peroxidase-labeled rabbit-anti human rotavirus detector antibody, and substrate (TMB+ Substrate-Chromogen) were obtained from DAKO and used following manufacturer’s instructions. Optical density was assessed at 450 nm (ELX 808 Ultra Plate Reader, Biotek). Means of duplicate readings were normalized to calculate virus particles per full-sized fecal pellet, using the graded sample size and a 7-point standard curve of previously titrated purified virus. The mean background level in feces from uninfected mice was 10,000 virus particles per fecal pellet.
Dissection and tissue/fluid collection. Mice at the age of 18 d were anesthetized with isoflurane and blood was obtained by cardiac puncture into nonheparanized syringes for subsequent serum assays. Mice were killed by cervical dislocation. Gut fluid was collected by excising the small intestine from the pyloric junction to the ileocecal junction, applying pressure to remove the majority of the gut contents, and then rinsing the intestinal lumen with 1 mL of sterile PBS through and collecting the eluate. Gut fluids and serums were centrifuged at 1000 x g for 10 min, and the clear supernatants retained and stored at –80°C until use.
Specific antirotavirus Ig in serum and gut fluid (ELISA). ELISA assays were carried out as described above. After the wells were coated overnight with virus capture antibody, they were blocked, and purified virus at 108 particles/L was added to the wells. Diluted serum or gut fluid samples were added to duplicate wells. Data were normalized against a 7-point standard curve of isotype-matched Ig (purified murine IgG, IgG1, IgG2a, Serotec; murine IgA, Zymed Laboratories). Horseradish peroxidase-conjugated detector antibodies (sheep anti-mouse IgG, goat anti-mouse IgG1, goat anti-mouse IgG2a, and goat anti-mouse IgA) were purchased from Serotec and used following manufacturer’s instructions.
Statistical analysis.
Measurements from WPC-IC–fed mice vs. control mice were compared by Student’s t test or binomial proportion analysis to assess statistical significance, using the Primer of Biostatistics Program (PBP) version 3.02 (41). Serial assessments over time were analyzed by repeated-measures ANOVA using SYSTAT or by 
2 using PBP. In all cases, differences were considered significant at P < 0.05. Values in the text are means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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2). The number of days on which all mice had severe symptoms during the postinfection period was significantly lower in the WPC-IC–fed mice than in controls (Fig. 2B). Of the subsets of mice in each group that did experience severe diarrhea (control: 37/60; WPC-IC, 20/60), the time period between infection and symptom onset was significantly longer for WPC-IC mice (2.92 ± 0.22 d) than for control mice (2.6 ± 0.46 d; P = 0.006 by Student’s t test). Within these subsets, the duration of severe symptoms in individual mice (1.6 d) did not differ between the groups.
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Viral shedding. Overall postinfection viral load was not affected by diet. In mice for which 2 or more postinfection fecal samples were available, virus levels per full-sized fecal pellet were 266,000 ± 21,000 in the control group (n = 48 mice) and 269,000 ± 24,000 in the WPC-IC group (n = 40 mice).
Incomplete sets of fecal samples were available from mice, particularly during the preinfection period (n = 5–22/d) when production of feces was less common. As expected, viral shedding levels on d 9 to 11 were comparable to those measured in feces of uninfected mice, i.e., negligible. After infection on d 11, viral shedding was prominent in both groups during the period from d 13 to 17 (Fig. 3) and was significantly higher in the WPC-IC group on d 14. However, in the control group, viral shedding was >2 x 105 viral particles (>2 times the original infection dose) per fecal pellet 2 d after infection. In contrast, viral shedding in the WPC-IC group was <7 x 104 at this time point (P < 0.05). Similarly, on d 17, viral shedding was >2.9 x 105 in the control group and <1.7 x 105 in the WPC-IC group (P < 0.05). The kinetics of viral shedding differed between the 2 groups (P = 0.027 by repeated measures ANOVA).
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2 x 105 viral particles per fecal pellet), using twice the initial inoculum amount as the defining cutoff point. The majority of mice in both groups fell into the high-shedding category on d 14 and 15 (Fig. 4). However, the WPC-IC supplement reduced the period of high viral shedding from 5 to 2 d on a group basis. More than half of the control mice also shed high levels of virus on d 13, 16, and 17; significantly fewer WPC-IC–fed mice shed high levels of virus at these time points.
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5% of antigen-specific serum IgG in both groups (data not shown). Initial body weights did not differ between groups (P > 0.9; data not shown). Proportional weight gain over each 24-h period was greater in the WPC-IC group than in the control group but only on d 15 (data not shown). Over the 8-d test period, the weight gain in the control group was 2.74 ± 0.34 g/mouse, whereas the WPC-IC group gained 2.89 ± 0.31 g/mouse (P < 0.03).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Probiotic bacteria were shown to have a positive effect against rotavirus in a variety of models. Lactobacillus casei strain DN-144 001 decreased the incidence of diarrhea in rotavirus-infected neonatal rats, but this probiotic also caused diarrhea in noninfected rats: approximately one-third of normal 6-d-old rat pups produced liquid yellow-green diarrhea after consuming the probiotic product in fermented milk for 4 d (11). In contrast, WPC-IC did not produce diarrhea in noninfected neonatal mice in the current study. In a recent randomized, controlled, double-blind human study, the probiotic Bifidobacterium lactis HN019 significantly reduced bloody diarrhea of unknown etiology by 22% in children aged 1–3 y (44). Bifidobacterium pretreatment delayed the onset of diarrhea and hastened disease convalescence in rotavirus-infected mice (10) in a manner similar to that observed with WPC-IC in the current study. Compared with probiotics, WPC-IC has the advantages of stability and ease of storage because it does not have to be cultured daily or stored under stringent conditions.
The mechanism(s) by which WPC-IC reduced rotaviral disease is unclear. Both local and systemic antirotavirus antibody levels were unaffected by WPC-IC supplementation, although there was substantial variability among individual mice. Only 8 mice in each group were assessed for Ig levels due to the limited number of samples that could be collected in a single day; in retrospect, assaying a larger number of mice might have provided stronger data. The IgG1 and IgG2a subclasses made up only a small proportion of total serum antirotavirus IgG, suggesting that the majority was of other Ig subclasses. Predominantly IgG2b or IgG3 responses in mice were observed previously (45); alternatively, the IgG detection antibody used in the current study’s ELISA assay may have been less class specific than expected.
In studies elsewhere, supplemental feeding with WPC-IC enhanced antigen-specific antibody production (26). This was not observed in the current study. The WPC-IC may have enhanced the immune response to rotavirus challenge and reduced viral clearance time by increasing neutrophil or natural killer cell activity. It is also possible that a component of WPC-IC directly blocked rotaviral infection by preventing viral attachment to sialic acid or integrin receptors on gut enterocytes, as some cytokines were observed to do (15,18).
It was shown that 1-wk-old mice infected with rotavirus have decreased body weight, intestinal length, and amino acid uptake in the gut compared with normal mice (46). WPC-IC–fed mice gained slightly more body weight than did control mice during the study period. This finding was likely a reflection of the reduction in disease severity and duration, which would have ameliorated the nutrient loss associated with diarrhea.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BSA, bovine serum albumin; PBP, Primer of Biostatistics Program; WPC, whey protein concentrates; WPC-IC, WPC IMUCARETM.
Manuscript received 2 December 2004. Initial review completed 23 December 2004. Revision accepted 14 March 2005.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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