| According to the report of the center of Minnesota Urolith, in 1981, calcium oxalate calculi represented only 5% of canine uroliths whereas struvite was detected in 78%. However, in 2007,41% of the urolithiasis in dogs was calcium oxalate calculi, while struvite represented 40%. In order to reduce struvite crystal formation, oxalic acid and its salts are widely distributed in dry commercially prepared dog food to make acidification of urine. Calcium oxalate calculi represent the most common type of stones in humans. Increased intestinal absorption of oxalate may lead to hyperoxaluria with significantly enhanced risk of urinary stone formation. Like human, increased dietary oxalate results in increased urinary oxalate and calcium oxalate relative supersaturation in healthy adult dogs.Unlike other types of stones, calcium oxalate stones, once formed, can only be removed by surgical methods. It has brought great pain to the dogs. The calcium oxalate stones could be prevented by reducing the amount of oxalic acid of the intestinal tract.Previous studies reported that lactic acid bacteria could reduce oxalate both in vitro and in vivo. However, only a few studies have reported the isolation of oxalate-degrading lactic acid bacteria. In addition, unlike O. formigenes, lactic acid bacteria are "generalists." The effect of the glucose concentration on the oxalate-degrading capacities of "generalists" is unclear, and further experiments are required to resolve this issue. Moreover, in these studies, the oxalate-degrading capacities of isolated strains were only evaluated individually. To our knowledge, few studies have been conducted on the oxalate-degrading capacities of mixtures of strains of different species.Investigation of canine urolithiasis of field cases was conducted. The compositions of urolith samples obtained from some clinics were analyzed. These results provided theoretical basis for controlling and preventing urolithiasis for clinical veterinary. Also, we want to build the animal model of hyperoxaluria and calcium oxalate calculi which were caused by diet intake too much oxalate, to isolate and identified a range of oxalate-degrading lactic acid bacteria which owe the stabile oxalate-degrading capacity and could be used as probiotics, and to use them in vivo.Experiment 1 The investigation of urolithiasis and composition analysis of canine urinary calculi in Nanjing Area The investigation of field cases of canine urolithiasis was obtained form 3 representative animal hospitals in Nanjing area in recent three years. In the same time, the composition of 146 canine urinary stones was studied by chemical qualitative analysis. The results indicated that:Four types of calculi were found on the base of their main constituents:52.55%(76/146) were magnesium ammonium phosphate (struvite),17.81%(26/146) were urat,2.74%(4/146) were calcium phosphate, 23.29%(34/146) were calcium oxalate,4.11%(6/146) were composite stone. It was analysed that the characters on the location of the calculi, breed, average easy-infected age, genders and food about the canine with urolithiasis, which provided clinical basis for controlling and preventing urolithiasis in Nanjing areas.Experiment 2 Isolation and identification of oxalate-degrading bacteria Lactobacillius were isolated on MRS-vancomycin agar, while Enterococcus were isolated on bile aesculin agar. Fecal samples were collected from 36 dogs of three different breeds. After assessing the colony morphology, gram reaction, and catalase activity, the gram-positive and catalase-negative strains were selected as lactic acid bacteria. The isolated colonies were re-streaked on MRS agar to ensure their purity. The pure isolates were transferred to MRS-ox agar, and the strains that survived were selected for the next experiment. The oxalate-degrading capacities of isolates were detected in the MRS-ox broth by ion chromatography. And all of the isolated strains were identified by a commercial biochemical assay (V1TEK compact 2, Biomerieux, French). Forty-seven strains of lactic acid bacteria included 22 isolates of Lactobacillius,6 isolates of Lactococcus,4 isolates of Leuconostoc, and 15 isolates of Enterococcus. One representative isolate each from Leuconostoc mesenteroides (RL75), Lactococcus garvieae (CD2), and Lactococcus subsp. lactic (CS21) as well as two representative isolates from Enterococcus faecium (CL71 and CL72) and three representative isolates from Enterococcus faecalis (CD 14, CS62, and CD 12) degraded more than 5% of the oxalate present. These eight strains showed significant oxalate degradation (P<0.05) in comparison with the sodium oxalate media control. These 8 isolates were selected for the next experiment.Experiment 3 Oxalate-degrading capacities of the isolates under different glucose concentrations The glucose consumptions of 8 isolates were observed in the MRS broth with different glucose concentrations, and a glucose concentration of 2.5 g-L-1 was regarded as insufficient, and a concentration of 20 g-L-1 was considered to be sufficient for the next experiment. There were 12 samples in each group of the same glucose concentration:each individual isolate, a mixture of the three Lactococcus, a mixture of the five Enterococcus, a mixture of eight isolates, and a medium blank. Isolates were grown in MRS broth for 24 h. The culture broths were inoculated at 5% into corresponding media containing 20 mmol·L-1 sodium oxalate and different concentrations of glucose. In comparison with the control medium, all of the individual isolates and mixtures of isolates could degrade oxalate in all three groups (P< 0.05). The oxalate degradation rates of CL72 were significantly increased (P< 0.05) among the three glucose concentrations. The oxalate degradation rate of RL75 in the medium containing 2.5 g·L-1 glucose was significantly higher than that in the medium containing 20 g·L-1 glucose and the medium containing 0 g·L-1 glucose (P<0.05). No significant differences were observed in the oxalate degradation rates of CS62, CD 14, and CS21 among the three glucose concentrations (P> 0.05). The other isolates showed higher oxalate degradation rates in media containing 2.5 g·L-1 or 20 g·L-1 glucose. The oxalate-degrading capacities of the isolates were isolate dependent. There may be different relationships between oxalate metabolism and glucose for different types of bacteria. For partial of lactic acid bacteria, the oxalate degrading rate was mainly influenced by bacterial count. The mixture of all isolates showed higher oxalate-degrading capacity than the individual isolates and other mixtures. Except RL75, the other 7 isolates were chosen for next experiments.Experiment 4 The potential probiotic effection, safety evaluation, and in vivo gastric transit of seven isolated oxalate-degrading LAB The potential probiotic effect of seven oxalate-degrading LAB were futher evaluated. Resistance to low pH environment, resistance to bile salts, antibiotic susceptibility, acute toxicity, and assessment of gastric transit of 7 isolates in vivo were performed. Isolates were resuspended in the MRS broth at 107 CFU·mL-1, and the pH of MRS broth was adjusted to 2.0-3.0. All of the isolates survived in the pH 2.0 MRS broth for 30 min, and in the pH3.0 MRS broth for 4 h. Among them, CL72 and CS21 had better resistance to low pH environment than other isolates. Isolates were inoculated at 107CFU·mL-1 in the MRS broth with 0.3% and 1% w/v ox-bile purified added, and their survival measured at intervals of 0 h,12 h, and 24 h. All of the isolates survived in the MRS broth with 0.3% ox-bile for 24 h, and in the MRS broth with 1% ox-bile for 12 h. Antibiotic susceptibility for isolates were assayed with the agar diffusion disk method recommended. Different isolates had different antibiotic susceptibility. All of the isolates were sensitive to Penicillins, Fluoroquinolones, Rifampicin, Glycopeptides, and Nitrofurans. One hundred and forty Kunming mouse of clean grade of each sex were equally divided into 7 groups, fed the different eight strains at dose based on the maximum achievable concentration (>1010 CFU·mL-1) and the maximum dose volume (15 g·Kg-1). We observed no adverse effects and no deaths after 20 days. Necropsy showed no anomalous findings. Isolates were cultured onto MRS agar plates containing rifampicin to gain the rifampicin-resistant isolates. The rifampicin-resistant isolates were resuspended to a dose of 109 CFU·mL-1. Five mL resuspended isolates were administered to dogs for 10 days. Canine faecal pellets were collected prior to feeding (Day 0) and on Days 1,5,10,15, and 20. The CFU·g-1 was determined by plating onto MRS agar containing rifampicin, in order to facilitate uncomplicated identification of the rifampicin-resistant isolates from all other LAB. Prior to feeding rifampicin-resistant isolates, no rifampicin-resistant isolates were detected on culture plates. No significant difference was observed between transit levels on d 1,5, or 10 (P>0.05). Ten days after the experiments, the rifampicin-resistant isolates also could be isolated from the faecal pellets. Significant differences were observed between the faecal pellets on d 10,15, or 20 (P<0.05). CL72 and CS21 were selected for the next experiment. These 2 isolates were identified based on 16S rRNA sequence analysis. Contrasting the 16S rRNA sequence in the data base (GenBank+EMBL+DDBJ+PDB), the result showed that the CL72 belong to Enterococcus faecium, and CS21 belong to Lactococcus subsp. lactic. Sequence analysis of CL72 and CS21 were submitted to GenBank. The access numbers in data base (GenBank+EMBL+DDBJ+PDB) were JF895185 (CL72) and JF895186 (CS21), individually. These results were consistent with the results of commercial biochemical assay. These two isolates were chosen for next experiment.Experiment 5 Establishment of hyperoxaluria and calcium oxalate calculus models in canie Two concentrations of oxalate were used to build the animal models. The urine calcium, urine oxalate, urine BUN, urine CA, serum BUN, and serum CA were detected on d 0,15, and 30. And when the experiment finished, the histopathological changes of kidney were monitored. The result showed that the urine specific gravity of 1% oxalate group significantly increased on d 15 (P< 0.05), while the urine specific gravity of 0.5% oxalate group significantly increased on d 30 (P<0.05). The urine pH of both oxalate concentrations group significantly decreased on d 15 (P<0.05). On d 15 and d 30, the serum calcium significantly decreased when compared to the control group (P<0.05); and the serum calcium of 1% oxalate group significantly decreased than 0.5% group (P< 0.05). The serium BUN and serium CA of 0.5% group showed no difference among d 0,15, and 30 (P>0.05); while the serum BUN and CA of 1% significantly increased on d 15 (P< 0.05). On d 15, the urine calcium of 1% group significantly increased (P<0.05) when compared to the control group; while on d 30, the urine calcium of two group both increased significantly (P<0.05). On d 15 and d 30, the urine oxalate concentration of both oxalate concentration group significantly increased (P<0.05), and the urine oxalate concentration of 1% group were significiantly higher than that of 0.5% group. On d 15, the urine BUN and the urine CA showed no difference with the control group (P>0.05); while on d 30, the urine BUN and CA of 1% oxalate group showed significiantly increase (P< 0.05). The crystal in 1% oxalate group were piling and connecting. The epithelial cells were swelling, degeneration, necrosis, lumen significiantly expansed.Experiment 6 In vivo oxalate degradation of selected oxalate-degrading bacteria in a canine model Thirty local dogs were randomly allotted to groups A, B, C, D, E. Group A was designed as control group and only fed with commercial canine food,0.5% oxalate was provided in group B,0.5% oxalate+Enterococcus faecium (CL72) was provided in group C,0.5% oxalate+Lactococcus subsp. lactic (CS21) was provided in group D, and 0.5% oxalate+mixture was provided in group E. Blood and urine samples were collected to analyze the concentration of Ca, BUN, CA, oxalate. The results showed that during the experiment, the urine specific gravity of the three oxalate-degrading LAB groups showed no difference(P>0.05). The urine oxalate concentration in CL72 and mixture isolate group showed significiant decreased than the 0.5% oxalate group. The urine BUN, urine CA, serum BUN, serum CA in all of the three oxalate-degrading LAB showed no difference with the 0.5% oxalate group (P>0.05). On d 30, the urine calcium of the three oxalate-degrading LAB group showed significiant decrease (P<0.05), while the serum calcium showed no difference (P>0.05). |
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