Isolating Lactobacillus from Soil Samples to Develop in vitro Methodology to Produce a Probiotic for Control of Necrotic Enteritis in Chickens

Necrotic enteritis is a serious disease in the global poultry industry, estimated to cost US$6 billion per annum due to control strategies and loss of production. It is a multifactorial disease which is most commonly associated with the increased growth of Clostridium perfringens in the chicken’s gastrointestinal tract. Due to the increasing risk of antibiotic resistance globally, numerous countries have prohibited the prophylactic use of antibiotic growth promoters (AGPs) in the poultry industry, resulting in an increase in infections. Probiotics offer a potential solution; live bacterial feed supplements which aid the maintenance of a healthy microbiota and reduce pathogen morbidity and mortality. This study aimed to investigate whether soil is an appropriate sample source for the commonly used probiotic genera, Lactobacilli. A range of in vitro antagonistic assays were conducted to detect inhibitory activity towards the poultry pathogen C. perfringens. A total of 600 soil samples from around Lincoln, UK and 381 samples sourced from chicken gastrointestinal tracts were screened for 11 Lactobacillus spp. using a multiplex polymerase chain reaction (PCR). From these, 21 Lactobacilli isolates were identified as five different species (L.fermentum, L. paracasei, L. gasseri, L. delbrueckii and L. salivarius). These samples were then tested for antagonistic effect against C. perfringens ATCC 13124, using a range of assays. Two of the assays (antagonistic plug and co-culture assays) displayed quantitative data which indicated samples NHFS045 and HP199T had the greatest antagonistic effect. These isolates were then tested against 11 different strains of C. perfringens sampled from chicken intestines from a previous study so as to test antagonistic range. Lactobacillus samples (NHFS045 and HP199T) were able to inhibit the growth of all 11 C. perfringens strains. The tolerance of the Lactobacillus isolates to low pH conditions was also tested. Both were able to grow at pH 4, indicating that they would tolerate intestinal pH conditions. The results of this study show that potential probiotic bacteria with an inhibitory effect towards the poultry pathogen C. perfringens can be found in soil. This is a far quicker and easier sample source compared to a chicken’s intestine; the current most common source for probiotic bacteria. Additionally, this study has identified the two antagonistic assays which should be conducted before in vivo trials (antagonistic plug and co�culture assays) so as to quantify and compare antagonistic effectiveness. Subsequently, the antagonistic range assay demonstrated that the Lactobacillus isolates inhibited C. perfringens isolates which were resistant against five widely used antibiotics (tetracycline, bacitracin, tylosin, penicillin and erythromycin). This indicates that these Lactobacillus isolates would be a more suitable treatment. All experimentation has shown that HP199T and NHFS045 isolates are suitable candidates for a poultry probiotic and should therefore move onto in vivo trials