Enriched bovine IgG fraction prevents infections with Enterohaemorrhagic Escherichia coli O157:H7, Salmonella enterica serovar Enteritidis, and Mycobacterium avium

Abstract A bovine IgG‐enriched whey fraction contains antibodies against various bacterial antigens. We investigated the protective effects of a bovine whey fraction preparation against infections with Enterohaemorrhagic Escherichia coli O157:H7, Salmonella enterica serovar Enteritidis, and Mycobacterium avium in mouse models. After infection with these pathogens, the IgG‐enriched fraction or skim milk was given ad libitum at a 5% solution instead of water. The mice given the IgG‐enriched fraction were significantly resistant to orally challenged EHEC O157:H7 (LD50: 4.0 × 105 CFU/mouse) infections compared with the mice given skim milk (LD50: <1.5 × 102 CFU/mouse). The mice given the IgG‐enriched fraction were also significantly resistant to orally challenged S. Enteritidis (LD50: 5.0 × 106 CFU/mouse) infections compared with the mice given skim milk (LD50: <2.5 × 101 CFU/mouse). When the mice were nasally infected with M. avium, the numbers of the bacteria in lungs of mice given the IgG‐enriched fraction were significantly lower than those given skim milk 2 and 3 weeks after infection. These results strongly indicate that oral administration of the bovine IgG‐enriched whey fraction protects mice against food‐borne infection and also that it partially protects mice against respiratory tract infection.

countries. Typhoid (enteric) fever and its potential complications have a significant impact on children in developing countries.
Several milk preparation products have been reported effective as a prophylactic and treatment for food-borne infections (Ulfman, Leusen, Savelkoul, Warner, & Neerven, 2018). Bovine colostrum preparations obtained from cows immunized with antigens of various human gastrointestinal infections were called "hyperimmunized milk" (Golay, Ferrara, Felber, & Schneider, 1990). This milk is characterized by high antibody activities against the specific organisms. Several clinical trials indicate that immune cow colostrum can help in shortening the duration of gastrointestinal infections (Ulfman et al., 2018). The second generation is colostrum derived from healthy nonimmunized pasture-fed cows. Early pioneering studies showed colostrum immunity against Salmonella infection in calves (Griffiths, 1969;Royal, Robinson, & Duganzich, 1968), and more recent investigations showed that an immunoglobulin preparation from nonimmunized cows contains high levels of antibodies and neutralizing activity against Vero/ Shiga toxin of EHEC O157:H7 (Lissner, Schmidit, & Karch, 1996).

| Mice
Male Balb/c mice at 8 weeks of age were purchased from Oriental Yeast Co., and were used in the experiments.

| IgG-enriched whey fractions and reagents
The IgG-enriched whey fraction (IgG25 + ; Aotearoa Co.) were prepared from milk of pasture fed, nonimmunized healthy New Zealand cows by New Zealand Dairy Group by centrifugation to remove fat and condensation of immunoglobulin. The cows were never exposed to insecticidal drugs, nor did they receive antibiotics or growth hormones. The composition of the powder of colostrum was as follows (w/w): 84.7% protein, <8.4% fat, 7.0 mg/g lactoferrin, and 327.7 mg/g immunoglobulin. Antibodies, detected by an indirect enzyme-linked immunosorbent assay (ELISA) using plates coating carious microorganisms, were defined as positive when absorbance in ELISA for a 0.1% (v/w) powder solution was >1.0 and 4 times higher than that of skim milk.  (Funatogawa et al., 2002). Skim-milk powder was obtained from Meiji Seika Co. The powder was dissolved into sterile water plates (100 μg/ ml). at a 5% solution. Streptomycin sulfate (Meiji Seika) was dissolved in drinking water (5 g/L) or nutrient agar plates (100 μg/mL).

| Bacterial strains
Streptomycin-resistant E. coli O157:H7 strain (O157-SM R ) (Fukuda et al., 1998) was provided by Dr. Yoshichika Arakawa, Nagoya University. These organisms were grown in brain heart infusion broth at 37 ℃ for 24 hr. The viable numbers of these organisms were determined using quantitative cultivation on nutrient agar plates supplemented with streptomycin (50 μg/ml) for O157-SM R . Salmonella enterica serovar Enteritidis No. 11 F1 was provided by Dr. Masayasu Nakano, Jichi Medical School. These organisms were grown in brain heart infusion broth at 37℃ for 24 hr. Mycobacterium avium MINO was provided by Dr. Toshitaka Goto, Miyazaki University.

| Infection protocols
Groups of mice (n = 5 or 6 per group) were given drinking water that did or did not contain streptomycin for 7 days before administrations of E. coli O157-SM R and S. enterica serovar Enteritidis No. 11 F1. The grown E. coli and S. Enteritidis organisms were washed by saline and suspended in saline. The bacterial suspensions (E. coli: 2 × 10 1 to 2x10 6 , S. Enteritidis: 2.5 × 10 1 to 2.5 × 10 6 CFU/mouse) were administrated orally into mice. The IgG-enriched whey fraction or skim milk was given ad libitum at a 5% solution instead of water from 1 hr after bacterial challenges. Mice infection with E. coli O157-SM R was performed as described previously (Funatogawa et al., 2002). The humane endpoint was set up as 20% body weight loss or inability to access food or water after bacterial infection.
Bacterial suspensions of M. avium MINO were administrated into mice at 40 μl of 1.6 × 10 4 CFU/mouse intranasally after anesthetizing with 400 μl of 2.5% tribromoethanol intraperitoneally. After administration, drinking water was replaced by a solution containing the IgG-enriched whey fraction or skim milk. Lungs were removed from mice at 1, 2, and 3 weeks after infection, and homogenized, and then viable numbers of M. avium in the homogenate were determined using Middlebrook 7H11 agar (Difco Laboratories, MS).

| Statistical analysis
Statistical significance was determined by Student's t test for bacterial counts and by the chi-square test for mortality.

| Ethics approval
Ethical approval was obtained from Ethical Committee of Tochigi Prefectural Institute of Public Health and Environmental Science (approval numbers: H30-000731).

| Chemical and bacterial analysis of IgGenriched milk
The

| Effects of pretreatment of streptomycin on Salmonella Enteritidis infection
Mice were given drinking water with streptomycin (5 μg/ml) or without streptomycin for 7 days. After given nothing per os for 7 hr, S. Enteritidis was orally inoculated in mice at indicated doses. After inoculation, these mice were given drinking water without streptomycin and fed normally, and were observed for survival for 4 weeks ( Table 1). The medium lethal doses (LD50 values) were 1 × 10 3 CFU/ mouse in streptomycin-pretreated mice and >1 × 10 6 CFU/mouse in nontreated mice (Table 1).

| IgG-enriched fraction protect mice to S. Enteritidis and EHEC infection
When inoculated 2.5 × 10 1 or 2.5 × 10 2 CFU of S. Enteritidis in mice, all mice survived in the group of the IgG-enriched whey fraction administration ( Figure 1). The survival rates of mice decreased in the group in the dose-dependent manner of the numbers of inoculated S. Enteritidis from 2.5 × 10 3 to 2.5 × 10 6 CFU/mouse, with LD 50 of 5 × 10 5 CFU/mouse (Figure 1), whereas no mouse survived in the group of skim-milk administration when inoculated from 2.5 × 10 1 to 2.5 × 10 6 CUF/mouse, with LD 50 of< 2.5 × 10 1 CUF/mouse (Figure 1).
When inoculated 2 × 10 1 or 2 × 10 2 CFU of EHEC in mice, all mice survived in the group of the IgG-enriched whey fraction administration ( Figure 1). The survival rates of mice decreased in the group in the dose-dependent manner of the numbers of inoculated S. Enteritidis from 2 × 10 3 to 2 × 10 6 CFU/mouse, with LD 50 of 4 × 10 5 CFU/mouse (Figure 2). When inoculated 2 × 10 1 and 2 × 10 2 CFU of EHEC in mice, 2 of 6 and 3 of 6 mice survived in the group of skimmilk administration, respectively (Figure 1). No mouse survived in the group of skim-milk administration when inoculated from 2 × 10 3 to 2 × 10 7 CUF/mouse, with LD 50 of<1.5 × 10 2 CUF/mouse (Figure 2).

| IgG-enriched fraction inhibited the growth of M. avium in lungs of mice
When inoculated with 1.6x10 4 CFU/mouse of M. avium, the numbers of CUF in lungs did not increase from 1 to 3 weeks after inoculation in the group of the IgG-enriched whey fraction administration, whereas the numbers of CFU in lungs increased from 1 to 3 weeks after inoculation in a time-dependent manner in the group of skimmilk administration (Figure 3). As the results, the numbers of CFU 1.0 x 10 6 0/5 3/5 a Mice were given drinking water with streptomycin (SM; 5 μg/ml) or without SM for 7 days. S. Enteritidis was orally inoculated in mice at indicated doses. After inoculation, these mice were given drinking water without streptomycin and fed normally, and were observed for survival for 4 weeks.
F I G U R E 1 Protective effects of the IgG-enriched whey fraction (IgG25 + ) against S. Enteritidis infection in mice. Mice were given drinking water that contained streptomycin for 7 days before administrations of S. Enteritidis No. 11 F1. S. Enteritidis was administrated orally into mice (2.5 × 10 1 to 2.5 × 10 6 CFU/mouse). The IgG-enriched whey fraction (in dark gray) or skim milk (in light gray) was given ad libitum at a 5% solution instead of water from 1 hr after bacterial challenges. The humane endpoint was set up as 20% body weight loss or inability to access food or water after bacterial infection F I G U R E 2 Protective effects of the IgG-enriched whey fraction (IgG25 + ) against Escherichia coli O157-SM R infection in mice. Mice were given drinking water that contained streptomycin for 7 days before administrations of E. coli O157-SM R . E. coli was administrated orally into mice (2 × 10 1 to 2 × 10 6 CFU/mouse). The IgG-enriched whey fraction (in dark gray) or skim milk (in light gray) was given ad libitum at a 5% solution instead of water from 1 hr after bacterial challenges. The humane endpoint was set up as 20% body weight loss or inability to access food or water after bacterial infection in lungs in the group of the IgG-enriched whey fraction administration were significantly smaller than those in the group of skim-milk administration.
The IgG-enriched preparations did not show direct bactericidal effects in vitro, when S. Enteritidis and EHEC were incubated with more than 7% of the IgG-enriched whey fraction solutions (data not shown).

| D ISCUSS I ON
Bovine IgG-enriched preparations protect bacterial infections on several levels via enhancing clearance of pathogens, preventing colonization, and modulating immune functions (Funatogawa et al., 2002;Ulfman et al., 2018). Our previous in vitro study using sections made from the cecum wall demonstrated that 5% solution of immunoglobulin-enriched bovine colostrum significantly blocked the attachment of EHEC O157 to mucous membrane and inhibits the colonization of the bacteria (Funatogawa et al., 2002).
This is the first report that oral administration of bovine IgG-

| CON CLUS ION
Oral administration of a bovine IgG-enriched whey fraction protects mice against food-borne infections with Enterohaemorrhagic Escherichia coli O157:H7, Salmonella enterica serovar Enteritidis, and Mycobacterium avium, and it partially protects mice against respiratory tract infection.

ACK N OWLED G M ENTS
This study was supported by a grant of AOTEAROA Co., Ltd.

CO N FLI C T O F I NTE R E S T
M.T. works for Aotearoa Co., Ltd.

E TH I C A L S TATEM ENT
The protocols and procedures of animal experiments in this study were ethically reviewed and approved by Ethical Committee of

Tochigi Prefectural Institute of Public Health and Environmental
Science (approval numbers: H30-000731). This work does not involve any human studies.