| Diarrhea of young calves is one of the most prevalence diseases and happened in yaks(Bos grunniens) which mainly caused by pathogenic Escherichia coli(E. coli), it has been associated with significant economic losses in yak operations. In pastoral area, there is a lack of basic veterinary and relative medical equipments. Meanwhile, yak is a wild animal and the normal drug delivery technology such as intravenous injection(i.v.), intravenous fluid therapy is difficult to apply and promote. In general, it’s hard for herdsman to master clinical operation including i.v., intramuscular injection(i.m.), intragastric(i.g.), and it is inconvenience to use powers and injection with extra medical appliances in yaks. What’s more, it may cause yak stress response and security risk for herdsman. Thus, developing oral gel medicine to use in the production of yaks, it can be easy used and reduce the animal stress. In short, it satisfied with the animal welfare of needle-free, and provides convenience for the treatment of diarrhea in yaks.The present study was conducted to investigate antimirobial susceptibility and mutidurg resistant of 41 E. coli isolated form free-range diarrhea young yaks using disk diffusion method. Based on the results, we screen out the Gentamicin(GN) which belongs to aminoglycoside with low resistant and high sensitivity. Meanwhile, we determine the aminoglycoside-modifying enzyme(AME) genes and sequence. According to the orthogonal arrays related L9(34), four main adjuvant materials including amidulin, carboxymethylcellulose sodium(CMC-Na), glycerol and Tween-80 were chosen to prepare a new preparation of gel. To screen the pharmaceutical formulation, the sample was evaluated by the appearance, properties, viscosity and stability, and preliminary quality research was conducted. At the same time, the gel security test was carried out in SD rats with different GN gel dosage depending on other references, and clinical trials were conducted in yaks. The results are as follows: 1. Antibiotic susceptibility and multidrug resistant in E.coli isolated form diarrhea yong yaks fecesAntibiotic susceptibility was investigated in 41 E.coli isolated form diarrhea yaks by standard disc diffusion method against 21 different antibiotics. The result showed that the aminoglycoside including Neomycin, Gentamicin, Amikacin and Kanamycin with a high sensitivity of 80.49%, 82.93%, 92.68 and 70.73%, respectively; meanwhile, amikacin is the most sensitive and no strain was resistant to Neomycin. However, the sensitivity of fluoroquinolones including norfloxacin, ciprofloxacin and ofloxacin were lower than 60%, and the resistant of ciprofloxacin and ofloxacin is up to 43.90%. The sensitivity of β-lactam antibiotics varied from 2.44% in Cefradine to 85.37%. All the Tetracycline antibiotics resistant were higher than 50%. There is diversity of sensitivity and resistant to E.coli about other antibiotics. At the same time, 60.97%(25/41) strains were determined as multidrug resistant, and the highest strain was resistant to 17 different drugs. However, 21.95%(9/41) strains were sensitive to all kinds of antibiotics. According the resistant spectrum, E.coli trains with more than ten-resistant resisted to fluoroquinolones, tetracycline and β-lactam antibiotics. It showed that the yak resource E.coli had multiple drug resistance and cross resistance, which can provide a reference to use drug scientific and rational in the local pastoral areas. 2. Detection and sequence analysis of Aminoglycoside-modifying enzymes gene in E.coliPCR amplifications and sequence of Aminoglycoside-modifying enzymes(AMEs) genes, including ant(4’)-Ia, aad B, aph(3’)-III, aph(3’)-IIa C, aac(3)-II, aac(6)-Im, and aaac(6’)-Ie-aph(2’)-I were performed with specific primers. During the test, Aminoglycoside nucleotidyl transferases(ant), aminoglycoside phosphotransferases(aph) and bifunctional aminoglycoside modifying enzyme were discovered in E.coli. In this study, however, we did not find the aminoglycoside acetyltransferases(aac). All strains carried the aac(3)-II gene in our study, and the positive rate of aph(3’)-III and aac(6’)-Ie-aph(2’)-Ia gene were 17.07% and 80.43%. When blast in NCBI about aac(3)-II and aph(3’)-III, no other Colibacter in any other animal source is similar to this strain isolated from yaks. However, E.coli have the same sequence(99% similarity) of aph(3’)-III in other fifteen bacterium including Streptococcus, Campylobacter coli, Enterococcus faecium, Gallibacterium I, Staphylococcus. The present study indicated that E.coli isolated from yaks with phenotypic resistance towards aminoglycoside without relativity presence of AMEs, also AMEs exist in the strains which without resistant to aminoglycoside. This is the first study that detected and sequence analysis of Aminoglycoside-modifying enzymes gene in E.coli isolated from yaks. 3. Manufacture Gentamicin oral antidiarrheic gelAccording to the orthogonal arrays related L9(34), four main adjuvant material including amidulin, CMC-Na, glycerol and Tween-80 were chosen to prepare nine samples. To screen the pharmaceutical formulation, the samples were evaluated by the appearance, properties, viscosity and stability. The optimal prescription contains amidulin 15, and 0.9% CMC-Na, 20% of glycerol, 0.2% Tween-80, and then added potassium sorbate, alt and edible milk flavor to the preparation, sterilized water up to 100 g. Microbiological assay through agar diffusion was developed to measure the concentration of GN in oral gels, and the RSD of intra-day and inter-day were less than 3%, the recovery rate of GN is more than 97% by adding quantities, it’s demonstrate that the method is precise and accurate. Thus, Microbiological assay is satisfactory for quantitation of in vitro antibacterial activity of Gentamicin. It is easy to prepare the GN oral gels, and the optimal production had exquisite appearance, fragrance and moderate viscosity. Though the characters of gel had tiny changed when they were stored in different temperature and humidity, yet the content of the GN remained stable at more than 95% concentration. And when stored in 15℃ for 180 days, the characters and concentration of oral gel is stable. In conclusion, the preparation of oral gel is convenient and feasible. 4. Safety testing of GN oral gel in SD rats and clinical trials in yaksAdult, female SD rats weighing 180 to 210 g were selected to evaluate the safety of GN oral gel with four groups including 12 mg/kg, 24 mg/kg, 48 mg/kg and control group. Besides control group, all rats were administrated GN oral gels two times by intragastric at 8 a.m. and 9p.m everyday sustaining 15 days. The weight gain in group 12 mg/kg, 24 mg/kg were 22.67 g and 23.33 g, which were lower than control group(P ﹥ 0.05). However, the weight gain(6g) in 48 mg/kg group was significant lower than that in control group(38.0g)(P﹤0.05), and the concentration of serum BUN and CRE were significant higher than control group(P﹤0.05) at 10 days later. All the viscera indexes were similar between control and positive group, and other serum contents were normal(P﹥0.05).Using GN oral gels treated diarrhea yaks with the dosage of 8mg/kg. During the treatment, oral gels were administrated two times a day for 3 days, and monitored for a week. Results show that the oral gels were effective to treatment of diarrhea in yaks with a efficiency of 75.00%, what’s more it is easy for use and had the similar efficiency(76.9%) treated with GN injection(P﹥0.05). The concentration of serum contents changed between sick and normal yaks, the concentration of Ca2+, P, Cl-, Na+, TP, ALB, ALP, ALT and AST in diarrhea yaks were significant lower than normal ones(P﹤0.05). After treatment, however, the Ca2+ and Alb returned to be normal level, and other biochemistry parameters were recovered gradually. Moreover, after administrated the same GN oral gels, there were not any adverse symptoms happened in healthy yaks, and the biochemistry parameters haven’t changed during the trail. In conclusion, gentamycin oral gel is safty and efficient for yaks with a recommended dosage of 8mg/Kg. |
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