Isolation,Identification Of Canine-derived Lactobacillus Plantarum And Bacillus Subtilis And Preliminary Exploration Of The Prevention Effects On Canine Diarrhea

Probiotics are living microorganisms that can exert a beneficial effect on the health of the host by ingesting a sufficient amount.It has the functions of maintaining the balance of the host’s intestinal flora,regulating the body’s immune response,and promoting the digestion and absorption of nutrients.It has been extensively researched and applied to varying degrees in human medical clinics,livestock and poultry,aquaculture and pets.At present,Lactobacillus and Bacillus are two types of probiotics that are widely studied and applied in animal health and disease prevention and control.Diarrhea in dogs is a symptom of intestinal disease often seen in dogs.The disease most commonly occurs in puppies,with an incidence of about 30%.Diarrhea in dogs usually leads to malnutrition,depression,abdominal pain,and in severe cases gastroenteritis,sepsis,and even death.It has been proved that intestinal microflora disorder is very easy to cause diarrhea.The disease is usually treated with antibiotics in clinic.The use of antibiotics accelerates the imbalance of intestinal microbiota and exacerbates the emergence of bacterial resistance.Thus,probiotics with the ability to prevent and control targeted diseases have become a research hotspot of scholars in the world.In this study,we used selective medium to isolate and screen canine-derived Lactobacillus and Bacillus strains with antibacterial activity and good resistance to stress from feces of healthy puppies by spread plate technique.Then,based on the screening results,the isolates RW1 and YW1 were selected to be used in vivo test to preliminarily explored whether the isolate regulate diarrhea induced by senna in poodles.Firstly,Anaerobic cultivation and isolation of Lactobacillus using MRS medium by utilizing the characteristics of facultative anaerobic,and the isolated strains were identified through morphology,physiological and biochemical characteristics and 16S rRNA gene sequence analysis.The isolated strain RW1 was determined to be Lactobacillus plantarum.Considering the heat-resistant characteristics of Bacillus,nutrient agar medium was used to isolate and screen Gram-positive strains similar to Bacillus strains.Finally,the isolated strains YW1 was determined to be Bacillus subtilis.Then the isolated strains were analyzed for some biological characteristics such as acid resistance,bile salt resistance,growth characteristics,in vitro antibacterial effect and drug sensitivity.The results show that Lactobacillus RW1 and Bacillus YW1 strain possess good acid resistance and tolerance to bile salt.The results of the Oxford Cup method to measure the antibacterial activity in vitro showed that these two strains have different degrees of antagonism against common pathogenic bacteria.The results of drug susceptibility tests showed that these two isolates were sensitive to most antibiotics.However,there were different degrees of resistance to aminotrimethamine and furazolidone.Then,in this study,RW1 and YW1 strains were mixed in a ratio of 1:1 by vacuum freeze-drying to prepare a diploidy powder preparation with bacterial content of 5×10~9 CFU/g.The poodle were given oral dichromatic powder at a dose of 1g/kg for 14 d,followed by oral dichromatic powder at a dose of 2mL/kg for 1 g/mL in the sensa leaves of the poodle for 3 d,during which the dichromatic powder was continuously administered.Clinical manifestations showed that the degree of diarrhea was significantly reduced in the oral administration of diphenylcholine powder group.The clinical manifestations showed that the diarrhea degree of the oral combination bacteria powder group was significantly reduced.The qPCR results of the three intestinal flora showed that the number of Lactobacillus in poodle taking taking oral combination bacteria powder increased compared with the positive control group,the number of pathogenic bacteria such as E.coli decreased significantly decreased significantly,and the number of Enterococci have also been reduced by a certain amount.The above results indicate that the RW1+YW1 diploidy powder can regulate intestinal flora to a certain extent,and improve the degree of diarrhea in dogs.