| Urolithiasis is a common urinary tract disease in cats,and its incidence accounts for about 15%-23% of cat urinary tract diseases.The types of stones in urolithiasis mainly include: calcium oxalate stones,magnesium ammonium phosphate stones,cystine,xanthine,blood clots,etc.The two most common types of stones in cat urolithiasis are struvite and calcium oxalate.Different from other stones,calcium oxalate stone is hard in texture and it could be treated by surgery.It has a high recurrence rate which not only brings physical pain to animals but also takes spiritual and economical pressure to pet owners.Therefore,we urgently need to find a way to prevent calcium oxalate stones in cats.The production of oxalic acid mainly includes exogenous food intake and endogenous liver metabolism.Due to the lack of enzymes that could degrade oxalic acid in cats,the metabolic pathways of oxalic acid are mainly through the digestive tract and urinary tract.Therefore,it increases the concentration of oxalic acid excreted by digestive tract,and then reduces the concentration of oxalic acid excreted by urinary tract.And the formation of calcium oxalate crystals or stones are reduced,thereby effectively preventing the occurrence of calcium oxalate stones.Studies have found that there is a relationship between intestinal flora and various metabolic diseases such as obesity,coronary heart disease,and hypertension.But there are few studies on the prevention of calcium oxalate stones.Therefore,this paper screened out bacteria that degraded oxalic acid by isolating cat fecal bacteria and screening existing probiotics in the laboratory.And we established the mouse model of calcium oxalate stones and the cat food-borne high oxalate model to verify screening bacteria for preventing calcium oxalate stones in cats.Test 1: Screening of oxalic acid-degrading bacteriaBy referring to a large number of literature materials,we screened a total of 15 bacteria from three species including Lactobacillus,Bacillus subtilis and Enterococcus faecalis to screen for oxalate degrading bacteria.MRS medium,Enterococcus faecalis medium and TSB medium were used in the experiment.And 10 mmol/L sodium oxalate was added to the medium to prepare a medium for screening oxalic acid-degrading bacteria.The bacteria were inserted into the medium,and the growth curve of the bacteria in the screening medium for 12 h was recorded.After two days of constant temperature incubation at 37 °C,oxalate ion detection kit was used to detect the remaining oxalic acid concentration in the screening medium,and the oxalic acid degradation efficiency of each bacterium was calculated.Two bacteria that could effectively degrade oxalic acid were screened.A strain of Lactobacillus plantarum and a strain of Bacillus subtilis will be used in subsequent experiments.Test 2: Exploration on the preventive effect and concentration safety of oxalic acid degrading bacteria on calcium oxalate calculus mouse model.We took 10-week-old male Kunming mice as test animals and the mouse model of calcium oxalate calculi was established by intraperitoneal injection of 80 mg/kg glyoxylic acid.The effect of two strains of bacteria screened in test 1 on the formation of calcium oxalate stones in mice was investigated by feeding the bacteria solution.During the test,the body weight changes of each group of mice were recorded every day,and we drown the body weight change trend chart.All mice were processed after28 days,and relevant samples were collected for subsequent experimental analysis.The test results showed that the test concentrations of Lactobacillus plantarum and Bacillus subtilis did not affect the normal vital signs of mice,indicating that the concentration of the test bacteria solution was a safe concentration.Through hematological analysis of all experimental groups,it was found that both Lactobacillus plantarum and Bacillus subtilis could effectively reduce the concentration of serum creatinine,blood urea nitrogen and blood oxalic acid in mice,and ensure the normal renal function of mice.The results of the pathological tissue sections of mice kidneys showed that compared with the kidneys of the mice in the stone model group,the kidney damage and the number of crystals in the mice in the bacterial fluid intervention group were reduced.It showed that both Lactobacillus plantarum and Bacillus subtilis could effectively affect the formation of calcium oxalate stones in mice and reduce the damage to the kidneys caused by calcium oxalate stones.Since calcium oxalate stones could cause oxidative stress in kidney tissue,we detected oxidative stress damage indicators in mice kidney tissue: total superoxide dismutase（SOD）activity and malondialdehyde（MDA）content.The results showed that compared with the stone model group,both Lactobacillus plantarum and Bacillus subtilis effectively alleviated the oxidative stress in kidney tissue caused by calcium oxalate stones.Therefore,by feeding the mouse calcium oxalate calculus model with the bacterial solution in test 1,it could be determined that both Lactobacillus plantarum and Bacillus subtilis could effectively prevent the formation of calcium oxalate calculi in mice.The preventive effect was basically the same.The use concentration was safe and had no obvious side effects.Test 3: Establishment of a model of cat food-derived excessive intake of oxalic acid and exploration of the effect of bacteria on preventing calcium oxalate stonesBy adding 1% sodium oxalate by weight to the cat’s feed,a model of excessive oxalic acid intake was established in the cat’s food.The body weights of all cats in the test groups were recorded daily,and the weight change trend chart was drawn.After the end of the test,clinical examinations were performed on all the test cats: the detection of serum creatinine,blood urea nitrogen and blood oxalic acid,the microscopic examination of urine sediment,and the Doppler ultrasonography of the cat’s kidney and bladder.According to the conclusions drawn in test 2,we used the bacterial concentration in Experiment 2 to feed the bacterial intervention group in test 3.The hematological results showed that,similar to the results of test 2,both Lactobacillus plantarum and Bacillus subtilis could effectively reduce the concentration of serum creatinine,blood urea nitrogen and blood oxalic acid in cats.And the concentrations did not reduce the weight of cats,and had no obvious toxic and side effects on cats.Ultrasound examination showed that the renal echo in the model group was significantly increased.The outline of the kidney was not smooth,and there were a lot of sand-like hyperechoic substances in the bladder.In the bacterial intervention group,the renal echo was slightly elevated or normal.And the outline and size of the kidney were normal.There was no or a small amount of sediment-like hyperechoic material in the bladder.By collecting the urine of cats and examining the urine sediment,it was found that a large number of calcium oxalate crystals were seen in the model group,and there were no crystals or a small amount in the bacterial intervention group.Therefore,both Lactobacillus plantarum and Bacillus subtilis could effectively prevent calcium oxalate stones caused by excessive oxalic acid intake in cats.In summary,both Lactobacillus plantarum and Bacillus subtilis could prevent calcium oxalate stones in mice and cats and the effect was basically the same.Therefore,the test results suggest that both Bacillus subtilis and Lactobacillus plantarum were expected to be potential drugs for preventing calcium oxalate stones in cats. |
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