Bovine milk derived skimmed milk powder and whey protein concentrate modulates Citrobacter rodentium shedding in the mouse intestinal tract

Collection of milk for preparation of SMP and WPC

Milk for in-house SMP and WPC products was obtained from Jersey and Friesian cows grazed on pasture and located on one farm in the central North Island region of New Zealand, as part of a commercial milking herd. The cows were at mid lactation and milked on a twice daily regime. Individual cows were screened for IgA levels, as a representative marker of selected bioactive protein content. For preparation of the SMP, milk was collected on two occasions from three cows with high levels of IgA in their milk (SMP high) and three cows with low levels of IgA (SMP Low). Milk was pooled from each group and stored at 2–4 °C until processed. For processing of milk into WPC a larger volume was required. Milk was collected on two occasions, on two consecutive days, from 18 cows producing high levels of IgA (WPC high) in their milk. This group included the cows milked for the SMP product. Milk was pooled and stored at 2–4 °C until processed.

Milk processing and reconstitution

Preparation and administration of C. rodentium

A nalidixic acid-resistant strain of C. rodentium (DBS100) was obtained from P. Fineran, University of Otago, and was grown overnight in Luria-Bertani (LB; Fort Richard Laboratories, Auckland, New Zealand) broth at 37 °C, then transferred into LB broth containing 50 μg/mL nalidixic acid (Sigma-Aldrich, Auckland, New Zealand), and streaked onto an LB-agar plate (Fort Richard Laboratories, Auckland, New Zealand) containing 50 μg/mL nalidixic acid. Single nalidixic acid-resistant colonies were serially selected a further two times (Wiles et al., 2004). A selected single circular colony of C. rodentium was then grown overnight in LB broth, pelleted by centrifugation at 500 g for 15 min, and resuspended in phosphate-buffered saline (PBS; pH 7.2, Oxoid; ThermoFisher Scientific, Auckland, New Zealand). Mice were orally inoculated using a gavage needle containing 150 μL of C. rodentium in PBS (2 × 10⁹ bacteria/inoculum) (Bouladoux, Harrison & Belkaid, 2017) which was freshly prepared for each oral gavage. The stock suspension was diluted and plated onto nalidixic acid-containing LB agar, incubated overnight at 37 °C and the viable count enumerated to confirm the number of viable bacteria inoculated.

Mouse feeding trials

All animal manipulations and procedures were approved by the Ruakura Animal Ethics Committee (RAEC #13644, #13701). Female BALB/c mice were bred within the Ruakura Small Animal facility at AgResearch in Hamilton, New Zealand. All mice used in these studies were housed under quarantine and in micro-isolator cages throughout the experimental period. Mouse chow was available ad libitum.

Two trials were conducted: (1) the SMP trial and (2) the WPC trial. For each trial mice (aged 8–10 weeks) were randomised into seven treatment groups of 16 mice and a control (no infection) group of eight mice. Animals were housed in cages of up to four animals per cage.

The daily liquid intake volumes were recorded and an average per/mouse estimated (intake per group divided by number of mice per cage Table 2). Fluid intake was measured by weighing the bottles each day to determine the volume of liquid remaining. After weighing, the bottles were replaced with fresh liquid. The control groups were given water to drink. The treatment animals had WPC or SMP as their only source of liquid. The bottles containing WPC and SMP were replaced daily with freshly mixed powder/water. To ensure consistency, sufficient powder was pre-weighed and aliquoted prior to the experiment, for the duration of the trial. The correct amount of water was added for mixing each day. Treatment groups were fed their designated liquid for 10 days before inoculation of C. rodentium by gavage, and then until trial end. Animals were denied food overnight prior to gavage but their milk treatments remained. On inoculation day all treatment groups were inoculated with C. rodentium in PBS (pH 7.2), and the control (no infection) group was inoculated with PBS only.

The mice were weighed at the trial start, then seven days before bacterial inoculation, immediately before inoculation, daily for the first four days after inoculation, and then every second day thereafter, until end of trial. The behaviour and stance of each mouse was monitored daily throughout the trial for signs of discomfort using a health score (activity; posture; coat; breathing).

Analysis of faecal shedding

Citrobacter rodentium colonization was assessed by enumerating viable bacteria from faecal samples, based on the assumption that shedding of C. rodentium in the faeces parallels colonization (Mundy et al., 2005). Faecal pellets were collected at the start of the trial, immediately before inoculation, and then every second day after inoculation until trial end (10 days following inoculation for SMP trial and 14 days for WPC trial). Pellets were collected by placing each mouse in a separate aerated container until they had passed two to three faecal pellets. Faecal pellets were weighed and then homogenised in PBS to achieve a suspension of 100 mg pellet per mL. A series of dilutions of the suspension were plated onto LB-agar containing nalidixic acid. The colonies (viable count) present after incubation at 37 °C for 24 h were enumerated and reported as cfu/g faeces.

Chemicals and reagents

Hydrochloric acid (CAS 7647-01-0), NaOH (CAS 1310-73-2) and Nalidixic acid (CAS 389-08-2) were obtained from Sigma-Aldrich (Auckland, New Zealand).

ELISA kits were obtained from Bethyl Laboratories (Montgomery, TX, USA), and PBS tablets from Oxoid; ThermoFisher Scientific (Auckland, New Zealand).

Statistical design

Comparisons between treatment groups were performed using analysis of variance (ANOVA) using GenStat (Genstat for Windows 17th Edition; VSN International, Hemel Hempstead, UK). Post hoc analysis used Fisher’s unprotected least significant difference (ULSD). Error bars illustrate standard errors of differences of means (sed).

Characterisation of SMP and WPC products

Following pilot scale processing, all reconstituted blended SMPs contained 3.2% protein, 4.4% lactose, but had no detectable fat. Levels of IgA, IgG and Lf in blended liquids and the commercially sourced colostrum and SMP products are listed in Table 1. Blended SMPs contained IgA ranging from 0.07 to 0.35 mg/mL, IgG ranging from 0.28 to 0.58 mg/mL and Lf ranging from 0.03 to 0.1 mg/mL. Commercial SMP had undetectable levels of IgA, IgG and Lf, while commercial colostrum only had detectable IgG (0.07 mg/mL).

Reconstituted WPC contained less than 0.2% fat, and lactose was not detectable. Levels of IgA, IgG and Lf in blended liquids and the commercial WPC are listed in Table 1. Blended WPCs contained IgA ranging from 0.35 to 1.66 mg/mL, IgG ranging from 0.58 to 2.36 mg/mL and Lf ranging from 0.02 to 0.45 mg/mL. When reconstituted to 3.2% protein, the commercial WPC contained 0.43 mg/mL IgA, 1.10 mg/mL IgG and 0.08 mg/mL Lf.

For the animal trials, the maximum estimated daily dose of IgA, IgG and Lf was more than four times higher in the WPC treatments compared with the SMP treatments (Table 2). Animals fed commercial WPC received IgA, IgG and Lf concentrations that were intermediate between the mid-low and mid-high WPC treatment groups.

Mouse weights and liquid intake during the trial

Skimmed milk powder feeding commenced 10 days prior to inoculation with C. rodentium (oral gavage) and continued for 10 days after, which was the expected time of peak shedding as determined by a pilot trial (Fig. S1). Towards the end of the trial it was apparent that peak shedding differed slightly from the pilot trial. The WPC trial was performed following the SMP trial and the time was extended to provide more extensive data on the period following peak C. rodentium shedding.

Weight measurements (Figs. S3 and S4) and liquid intake (Table 2) indicated the infection did not adversely affect the health or growth of the mice over the course of the experiment. Mice were weighed every day for 4 days and then every second day to monitor infection-induced weight loss due to poor appetite and/or dehydration. There was no significant differences between start weight of groups in the SMP trial or groups in the WPC trial. The average weights of water-fed infected mice was significantly lower than water fed no-infection controls on days 2 (p = 0.02) and day 3 (p = 0.02) for SMP trial and on day 2 of the WPC trial (p = 0.03) (Figs. S3 and S4). Any weight loss was transient and animals’ weight quickly returned to normal. The non-infected mice that were given water steadily gained weight (Figs. S3 and S4). Overall, animals drinking SMP and WPC consumed a higher volume of liquid compared with those mice drinking water, which may suggest increased palatability of milk diets (Table 2). The intake of water for the no infection control groups were similar between the WPC and SMP trials (p = 0.3) but water intake in groups infected with C. rodentium was different (average water + infection for SMP trial and WPC trial was 2.9 mL/mouse and 3.2 mL/mouse, respectively; p = 0.03, Student’s t-test, equal variance).

Effect of SMP on C. rodentium shedding over time

On Day 0, all groups received a bacterial inoculation or sham PBS inoculation via oral gavage. No C. rodentium was detected in sham PBS inoculated mice. The average viable count of C. rodentium present in the faecal pellets of the mice for each of the treatment groups over the course of the infection period are presented in Fig. 1A. For SMP fed mice, differences were observed between water controls and the high SMP, mid-high SMP, low SMP and commercial SMP treatment groups on day 8 and water controls and low SMP treatment group on day 10 (Fig. 1A). The mean C. rodentium viable count for the each of the SMP groups (except for the mid-low treatment) was significantly lower than water-fed infected controls (ANOVA followed by Fisher’s ULSD). We could not detect significant differences in bacterial viable counts with regard to levels of IgA, IgG or Lf in the SMP, or colostrum treatment.

Effect of WPC on C. rodentium shedding over time

In the WPC trial, animals in the WPC treatment groups excreted equal or increased levels of faecal C. rodentium compared to water-fed infected controls. This trial was extended to observe progression of infection. Significant differences were observed between high SMP and commercial WPC (day 4–day 12, p < 0.02 ANOVA followed by Fisher’s ULSD), Fig. 1B and Table S3). No C. rodentium was detected in sham PBS inoculated mice.

Evaluating the effect of milk matrix on C. rodentium shedding over time

The second trial also enabled us to compare treatments that delivered similar amounts of selected bioactive but in a different product matrix (SMP versus WPC). For example, Table 1 shows IgA content of high SMP to be 0.35 mg/mL which sits between mid-low and low WPC which contain 0.53–0.22 mg/mL IgA respectively. Mid-high SMP contains 0.23 mg/mL IgA, and low WPC 0.22 mg/mL IgA. Table 2 shows estimated selected bioactive IgA intake for SMP mid-high fed mice to be 47 μg/g/mouse/day which is comparable to WPC low, at 48 μg/g/mouse/day.

The WPC trial included a High SMP treatment group where the consumption of IgA, IgG and Lf (65, 107 and 18 μg/gram mouse/day respectively) was intermediate between the mid-low WPC (109, 219 and 25 μg/gram mouse/day respectively) and Low WPC (48, 163 and 4 μg/gram mouse/day) treatment groups. Lower C. rodentium counts (cfu) (as determined by viable counts) were observed from animals receiving High SMP, but not for those receiving the mid-Low and Low WPC treatments. In addition, the High SMP group had significantly less C. rodentium counts (cfu) compared to the commercial WPC fed animals (Fig. 1B), which were the treatment groups containing the most similar selected bioactive content (IgA, IgG and Lf at 88 μg, 224 μg and 16 μg/gram mouse/day respectively).

Effect of SMP or WPC on magnitude (maximum viable count) of C. rodentium faecal shedding

All animals in each trial received the C. rodentium within 1 h of each other, however, maximum excretion did not occur on the same day, either within groups, or between groups. Therefore, we compared the maximum C. rodentium viable count from faecal pellets for each of the groups up to day 10 (enabling direct comparison of the two trials). These data illustrate a reduction in bacterial shedding in the groups given the SMP preparation compared to water (Fig. 2A). Conversely in the WPC trial, C. rodentium enumerated from the WPC treatments were not significantly different from the infection water-fed control (Fig. 2B; Table 3). However, the High SMP group in the WPC trial had reduced bacterial shedding, approaching statistical significance (ANOVA p = 0.01, Fishers ULSD, Water (infection), ab; High, mid-high, mid-low, bc; low, c; SMP, a; commercial WPC, c, where mean values with unlike letters are significantly different (p < 0.05)). The SMP treatment was significantly different (less C. rodentium shedding) compared to other treatment groups.