Main topic : Animal Health
Expression of CRISPR-associated endonuclease in the cecal microbiota of fattening pigs raised without antibiotics
BUTHASANE P. 1, ROYTRAKUL S. 2, PHAONAKROP N. 2, TUNGSAGOOL P. 3, BUTHASANE W. 1, AM-IN N. 1, SURIYAPHOL G. 1
1 Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand; 2 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok, Thailand; 3 Faculty of Agro-Industry, Kasetsart University, Bangkok, Thailand
Owing to the increased problem of antimicrobial resistance (AMR) in farm animals, the Department of Livestock Development, Thailand, has launched a campaign for antibiotic-free (ABF) animals in livestock production1. Fattening ABF pigs did not receive any antibiotics during their lifetime. However, they received oral probiotics containing Lactobacillus spp. at the age of 1-3 days to improve growth performance, intestinal morphology, the immune system, and gut health2. The controls (CTRL) were fattening pigs raised in the ordinary industrial system. They received prophylactic antibiotics via intramuscular injection or being added to a basic feed for a short period with a pre-slaughter withdrawal time. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) system, which functions in defense against viruses, was found to be widespread in lactic acid bacteria, particularly Lactobacillus3-4. Hence, the aim of the present study was to investigate the expression of CRISPR-associated endonucleases in the cecal microbiota of ABF pigs, compared to that of CTRL pigs, using a metaproteomic approach.
Cecal samples of ABF (n = 18) and CTRL groups (n = 20) were obtained from a private slaughterhouse located in Chonburi, Thailand. The content and the mucus were randomly collected from 5 positions of each cecum sample using a biopsy punch, mixed with a 1.5 mL RNAlater solution and stored at −80 °C until analysis. Protein samples were extracted and quantified, prior to trypsin digestion. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was analyzed in a linear positive mode with a m/z of 150-2200 using an Ultimate 3000 Nano/Capillary LC System (Thermo Fisher Scientific) coupled to a Hybrid quadrupole Q-Tof impact II (Bruker Daltonics). Protein profiles of CRISPR-associated endonuclease were analyzed using MaxQuant v 184.108.40.206 together with the UniProt database. t-test or Mann-Whitney U Test in R program was used for statistical analysis.
In the ABF group, CRISPR-associated endonucleases Cas1 and Cas9 were prominently observed in Ruminococcus and Lactobacillus, respectively (Fig 1.). In addition, Lactobacillus proteins had beneficial effects on gut health with no marked expression of antimicrobial, biofilm and metal resistance proteins.
CRISPR-associated endonuclease Cas9, the protective mechanism of bacteria for virus defense, was highly expressed in Lactobacillus in the ABF group. ABF pigs may have more beneficial microorganisms in the gut than CTRL pigs.