Diarrhea and severity of illness scoring
Study design
A total of 91 neonatal mice were cross-fostered and divided into 4 study groups as depicted in Table 1. A schematic design of the mouse model is shown in Figure 1. From day 4 until day 10 of age, pups of group D received daily 20 μl PBS containing rotavirus-specific antibodies, Gastrogard-R®, (kindly provided by Dr. Rosie Pereira) (1 mg/day) by oral gavage. At day 7 of age, pups of group A, C and D were inoculated by oral gavage with 10 μl RRV (CCID50 1 × 107.7) and mice of group B were inoculated with 10 μl PBS as a control. From day 8 until day 14, diarrhea and severity of illness was scored. From day 8 until day 27 feces were collected daily, the feces of all animals within one group were pooled each day. At day 16 of age, a blood sample was taken and pooled from all animals within one group. At day 17 of age, pups of group B, C and D were inoculated with 5 μl EDIM (400 μg rotavirus/ml) and mice of group A were inoculated with 10 μl PBS as a control. At day 27 of age a rotavirus-specific DTH was elicited by subcutaneous inoculation of UV-inactivated rotavirus in the ear pinnea, EDIM (1 μg/ml) in the left ear and RRV (CCID50 5 × 107) in the right ear. Non-inoculated mice of comparable age were also injected as a control. Twenty four hours later, the DTH responses were determined by measuring ear thickness using a digital micrometer (Mitutoyo, Veenendaal, The Netherlands) prior to collection of individual spleen and individual blood samples.
Diarrhea and severity of illness scoring
Daily evaluation, starting 1 day after RRV inoculation, for the presence of diarrhea (defined as having diarrheal stool after gentle palpation of the abdomen) was performed for each animal, and results were reported as the percent of animals having diarrhea in each group. Stool samples were collected daily, the feces of all animals within one group were pooled each day. The severity of illness was scored daily by assigning numeric values to the color of stool where a high score indicates severe illness (yellow = 3; yellow-brown = 2; brown = 1), degree of soiling (very soiled = 4; somewhat soiled = 1; no soiling = 0), and consistency (very liquid = 4; liquid = 3; solid = 1) of the stool. The severity score was calculated by dividing the total severity score by the total number of animals on each day after RRV immunization.
Detection of rotavirus in feces
A commercial ELISA kit (IDEIA; Dako diagnostics) for the detection of group A rotavirus in human fecal samples was used according to the manufacturer’s protocol. In short, precoated wells with rotavirus-specific polyclonal antibody were sampled with a reference EDIM stock with known concentration (300 ng/ml) which was used as a standard, stool samples (dilutions 5×, 10×, 50× and 100×), positive and negative control supplied in the kit. Then 100 μl rotavirus-specific polyclonal antibody-peroxidase labeled was added and incubated for 1 hour at RT. Wells were washed with 350 μl wash buffer and incubated with 100 μl of TMB substrate for 10 min. at room temperature (RT). The reaction was stopped by adding 100 μl of 0.46 mol/L sulfuric acid and the absorbance was measured at 450 nm on a microplate reader (BioRad, Hemel Hempstead, UK). The ELISA cutoff was the mean absorbance of the negative control at 450 nm plus a factor of 0.1. The absorbance of the positive control should be in the range indicated at the validation inlay in the kit. Concentration of rotavirus in the samples was expressed in ng/ml.