In 2017, the agricultural output of Hokkaido was 1,276,2 billion yen corresponding 13% of the whole value in Japan. Of which, the livestock production was 727.9 billion yen corresponding 57% of the total agricultural output of Hokkaido. In Hokkaido, production rates of livestock and milk account for 21% and 51% of the total productions in Japan, respectively, which is reason why it is called the livestock kingdom.
In dairy industry, calf diarrhea has been causing considerable economic losses because of calf mortality, weight loss in surviving calves all over the world, including Hokkaido. Several probiotics have been used as preventive or supportive therapy for dairy cattle and neonatal calf for a long time, and previous reports indicated the benefit calf performance and health [14–25]. Fermented milk, an old probiotics for dairy cattle, provides several clinical benefits for calf breeding, and has been widely used as economical and safe probiotics [26–28]. However, the efficacy of fermented milk is often inconsistent, and vary in the fed-calf performance because lack of the unifying protocol for the preparation. In addition, there is no direct evidence of the efficacy of fermented milk against infectious diseases. In this study, in order to examine the efficacy of probiotics, we conducted the experimental infection of BRV for clinical evaluation of the probiotics-fed calves, and showed milk replacer-based probiotics reduces severity and tissue damage of the intestinal tract in calf diarrhea by rotavirus. This is first direct evidence of efficacy of milk replacer-based probiotics against infectious disease in calf.
Fermented milk includes the rich amino acids required for calf growth, and has been used for many years in the dairy industry [26–28]. A recent report indicates that the fermented milk enhanced nutritional parameters such as height, weight, and body performance . In addition, the feeding of fermented milk reduced in diarrhea mobility and mortality . However, fermentation of whole fresh milk is easily influenced temperature condition, and the preparation is needed to take long time for the fermentation. Furthermore, the worst drawback of fermented milk is to be easy to contaminate coliform during the fermentation. Indeed, the poor quality of fermented milk often causes diarrhea by contaminated coliform in neonatal calves. Thus, in this study, we firstly tried to establish the protocol of milk replacer-based fermented milk without a risk contamination of coliform. The coliform was not detected in the FMR produced by our protocol. The number of LAB in the FMR efficiently increased compare to fermented whole fresh milk, and the maximum bacteria load reached at 2 days in the cultivation. By the comparative component analysis, quality of the FMR derived 3.5-fold concentrated FMR was better than those of 7-fold concentrated FMR. Thus, we used 3.5-fold concentrated FMR for the evaluation of efficacy against diarrhea caused by BRV infection.
In the clinical experiment, meanwhile control calves consistently showed severe watery diarrhea during the trial, the FMR-fed calves showed diarrhea, but the water content of feces was low and stable, and the diarrhea score kept low or recovered. In addition, the amount of milk intake decreased temporarily, but recovered immediately in the FMR-fed calves. The pathological score for enteritis tended to be lower in the FMR-fed calves than that of the control calves. Interestingly, although the protocols and used LAB for fermentation were different, similar efficacy of fermented milk in rotaviral diarrhea have been reported in children [29–31] and sucking rats [32, 33]. In case of fermented milk made by using human Lactobacillus sp strain GG, the fermented milk promoted recovery from rotavirus diarrhea via augmentation of the local immune defense by specific IgA response to virus . Although the mechanism of the efficacy of FMR is still unknown, FMR might induce similar local immune defense. Further studies on the mechanism of the efficacy of FMR might help to improve the disease control in calf by probiotics.
In the present study, three lactic acid bacteria mixture including Streptococcus faecalis T-110 strain, Clostridium butyricum TO-A strain, Bacillus mesentericus TO-A strain, a probiotics used widely for dairy cattle in Japan, was used to prepare the FMR. The beneficial effects of probiotics have been reported in inflammatory bowel diseases, including the suppression of pathogenic enteric bacteria. In human, the probiotics have been clinically used for the treatment of patients with symptoms owing to imbalanced intestinal flora, such as refractory diarrhea, constipation and abdominal distension. Furthermore, several previous studies showed the effectiveness of probiotic therapy using the three LAB mixture, such as in both clinical and bacteriological responses in the patients with vaginosis and vaginitis , the reduction of postoperative infectious complications after pancreaticoduodenectomy , the prevention of relapse in patients with inactive ulcerative colitis , the improvement of the clinical symptoms and endoscopic findings in patients with ulcerative colitis refractory to conventional therapy . Interestingly, Huang et al. reported seven-days administration of the LAB mixture demonstrated high efficacy in infants and children with severe gastroenteritis by salmonella and rotavirus . Indeed, the incidence of severe gastroenteritis was significantly reduced in the lactic acid bacteria mixture-treated groups. As result, the total diarrhea duration was significantly shorter for children treated with the lactic acid bacteria mixture. After lactic acid bacteria mixture administration, there were significantly less intervention group patients with severe diarrhea at intervention day 3. In addition to this report, Chen et al. also reported that the probiotics mixture reduced the severity of diarrhea and length of hospital stay in children with acute diarrhea . Interestingly, Hayakawa et al. reported that administration of the probiotics containing Streptococcus faecalis T-110 strain, Clostridium butyricum TO-A strain, Bacillus mesentericus TO-A strain to sows and/or their neonates improves the reproductive performance, incidence of post-weaning diarrhea and histopathological parameters in the intestine of weaned piglets . In addition, Inatomi et al. reported that administration of the probiotics to porcine epidemic diarrhea (PED)-vaccinated sows were healthier, transferred PED-specific antibodies via colostrum to piglets, had greater litter weight at birth, and reduced mortality during suckling . Furthermore, more recently, Tsukahara et al. reported that the probiotics also improved reproductive performance of unvaccinated farmed sows infected with porcine epidemic diarrhea virus . However, there is no information concerning efficacy of the FMD richly containing Streptococcus faecalis T-110 strain, Clostridium butyricum TO-A strain, Bacillus mesentericus TO-A strain in dairy cattle or calf. Further research is necessary to evaluate the potential of FMR to improve health in calf production.
Although the immunological mechanism of the clinical efficacy of probiotics is still unclear, Isono et al. examined whether the probiotics affect Toll-like receptor 4 (TLR4), which play a role in continuous inflammation of the intestinal mucosa in inflammatory bowel diseases, expression in human colonic epithelial cells in vitro, and showed that butyrate produced by C. butyricum TO-A specifically downregulated TLR4 mRNA level in human colonic epithelial cells . TLR4-mediated recognition of microbial components triggers aberrant IL-12p40 production by myeloid cells, leading to the development of enterocolitis. The report suggested that the development and persistence of colitis could be prevented by suppression of TLR4 expression in intestinal epithelial cells. On the other hand, Hua et al. reported the several immunological functions of three lactic acid bacteria mixture such as induction of Th1 immune response, downregulation of pro-inflammatory cytokines (TNF-α), upregulation of anti-inflammatory cytokine (IL-10), enhancement of CD11b, HLA-DR, CD4, CD45RA, CD25, CD44 and CD69 expression . Although the detailed mechanism of the reduction severity of diarrhea in fed calves is still unknown, FMR may restore the beneficial intestinal flora and enhance host protective immunity such as down-regulation of pro-inflammatory cytokines and up-regulation of anti-inflammatory cytokines. Further investigation is needed.
In this study, as an alternative way, we evaluated the possibility of LAB-MR for calf breeding. However, LAB-MR was not fermented under the tested several conditions (data not shown). L. plantarum HOKKAIDO strain was isolated from well-pickled vegetables in Hokkaido, and used for the fermentation of soymilk . L. plantarum HOKKAIDO strain, a plant lactobacillus, might be difficult to metabolize the animal carbohydrate in the milk, resulted in not growth in the cultivated milk. However, interestingly, direct use of the high-concentrated LAB-MR-fed calves reduced the severity of diarrhea as well as FMR-fed calves, meanwhile a low concentrated LAB-MR-fed calf was not effective to rotaviral diarrhea. As immunological function of L. plantarum HOKKAIDO strain, it is seemed that the lactobacillus induced IL-8, IL-12, IP-10, important cytokines for activation of cell mediated immunity, form human dendritic cell lines [46, 47]. Although it was other L. plantarum strain, orally administered L. plantarum No.14 reduce inflammatory cytokine production by circulating exosomes in the mice model . In addition, the L. plantarum strain strongly induced the gene expression of Th1-type cytokines by suing pig model . The mechanism of the preventive effects of L. plantarum HOKKAIDO strain was also still unknown, these finding might suggest that the HOKKAIDO strain improves immune function including the activation of cell mediated immunity. Further research is necessary to identify underlying mechanisms and to evaluate the potential of direct use of the unfermented milk replacer.
In conclusion, the feeding of two milk replacer-based probiotics, FMR and high-concentrated LAB-MR, reduce BRV-induced diarrhea and tissue damage to the intestinal tract, and suppress the clinical symptoms of acute enteritis by maintaining milk intake. This direct evidence could be helpful to the development of potential novel methods for the control of diarrhea in neonatorum calf. Indeed, our retrospective investigation indicated the FMR feeding reduced the incidence of diarrhea in a dairy farm with a high incidence of enteritis due to mixed infection with rotavirus and C. parvum. Although there is no direct evidence of efficacy of FMR against C. parvum infection, the feeding with fermented milk was reported as a protective factor against the shedding of C. parvum under field conditions . In addition, the feedings with FMR reduced the percentage of calves that required therapy and the amount of treatments needed against digestive diseases.