#14-075

Complete

Date Full Report Received

04/28/2016

Date Abstract Report Received

04/28/2016

Investigation

Institution:
Primary Investigator:
Bacterial resistance to antibiotics is considered to be one of the most important problems currently encountered in animal and human health. According to the World Health Organization, “Our grandparents lived during an age without antibiotics; so could many of our grandchildren”, if the problem is not addressed immediately. The major generator of bacterial genes encoding antibiotic resistance is the overuse of antibiotics in both human medicine and animal agriculture. In food-producing animals, the major concern is the use of antibiotics as routine feed additives to prevent diseases and promote faster animal growth. The focus of our research program is to investigate natural non-antibiotic feed additives against porcine postweaning enteric colibacillosis to limit the development of bacterial resistance to antibiotics.

Enteric colibacillosis, caused by Escherichia coli, is the most common bacterial disease of neonatal and postweaning piglets. Neonatal colibacillosis is controlled to a certain degree by maternal colostral immunity. However, the control of postweaning colibacillosis is problematic in the modern swine industry. To ensure profitability, piglets are weaned at ~3 weeks of age (when their immune system is still weak). Consequently, postweaning diarrhea (PWD) caused by E. coli is a major health problem associated with high morbidity & mortality that is currently controlled in part by antibiotic feed additives.

Pathogenesis of postweaning diarrhea is only partially understood and multiple predisposing factors seem to be involved. However, the sudden termination of milk intake by relatively young piglets having naïve (weak) immune system has been considered to be one of the most important factors resulting in reduced exposure of piglets to immediate and long-term protection mediated by various defense components present in the sow’s milk. This sudden termination of lactogenic protection results in decreased resistance to infectious diseases. Intestinal colonization by E. coli is the first necessary step in the pathogenesis of enteric colibacillosis. This colonization is mediated by fimbriae (bacterial hands) attaching to intestinal receptors (handles).
Our research group isolated various glycoproteins from porcine milk, which are recognized by the E. coli fimbria (bacterial hands) as its receptor analogue (false handle). Once E. coli binds to these receptor analogues (false handle), it is prevented from attaching to intestinal receptors (real handles), which is a necessary step for colonization and development of diarrhea. We demonstrated that these receptor analogues (false handle) are carbohydrate portions of milk proteins; identification and subsequent production of these carbohydrates (sugars) could potentially be used as non-antibiotic feed additives against colibacillosis. During our investigation of the identity of these complex carbohydrates, we discovered that certain strains of Lactococcus lactis consistently and reproducibly inhibited adherence of E. coli to cultured porcine enterocytes in vitro. Based on this discovery, we hypothesized for this project that L. lactis added to post-weaning diet as a probiotic has potential to prevent intestinal colonization by E. coli.
To test this hypothesis, we first had to determine whether it is safe to feed piglets high doses of live L. lactis bacteria. Accordingly, three groups of weaned piglets were fed non-medicated starter diet (control group), and starter died sprinkled with low dose (1010cfu/kg) and high dose (1011cfu/kg) of live probiotic L. lactis bacteria. No adverse effects (e.g. diarrhea, depression, weight loss) were noticed in any of the experimental groups and both low dose and high dose were colonized by L. lactis bacteria 1-2 days after initiation of probiotic diet.
The second major objective was to determine whether exposure of piglets to L. lactis may reduce colonization by enterotoxigenic E. coli. In order to perform this experiment, we had to make sure before each experimental trial that all the experimental piglets possess receptors for F4 positive E. coli (this was done by PCR).
First we performed a pilot study using 6 piglets (3/group) to determine if our infection/colonization model works. Piglets were weaned at day 21 and fed non-medicated starter diet (control) and non-medicated starter diet with probiotic bacteria (1010 cfu/kg) (probiotic group). After 3 days, both groups were challenge with F4 positive E. coli (2×109 cfu) (administered directly into the stomach). All piglets were colonized by E. coli bacteria and probiotic group seemed to clear the infection faster than control group (within 4 days). However, none of the animals developed diarrhea. Accordingly, for the main experiment we doubled the infectious dose of E. coli.
Main experiment was performed on 16 piglets (8/group) weaned at 21 days. Control group was fed a non-medicated starter diet and probiotic group the same diet sprinkled with live L. lactis bacteria (1010/kg). At day three after initiation of probiotic diet, both groups were challenged with the double dose of F4 positive E. coli. Piglets were observed three times per day and fecal samples were collected daily until euthanasia at day 7 post-infection. Both groups were successfully infected/colonized by E. coli but none of the piglets developed diarrhea or had any gastrointestinal pathology. To determine if probiotic L. lactis interfered with the extent of colonization of intestines by E. coli, both bacteria were quantified in the feces of individual piglets and compared statistically between groups. Unfortunately, we did not see any differences between two experimental groups, which is contrary to our previous results obtained in vitro and to the results of the pilot study.
Conclusion and potential future study: Under experimental conditions implemented during the main experiment, we could not demonstrate any protective effects of probiotic L. lactis bacteria against colonization by E. coli. Based on this findings, it is not recommended to use of L. lactis as probiotic for prevention of porcine post-weaning diarrhea at this stage. At the same time, feeding live L. lactis bacteria did not seem to have any negative effects on weaned piglets and based on limited data generated by our pilot study, L. lactis seemed to interfered with colonization of E. coli in vivo. Accordingly, we are considering to test the effects of various doses of L. lactis and E. coli on colonization/infection in the near future.