南通地区家兔球虫病流行病学调查、防治研究

发布时间：2018-06-10 18:54

本文选题：家兔球虫病 + 流行病学调查　；参考：《扬州大学》2014年博士论文

【摘要】：球虫病是家兔常见和危害严重的寄生虫病,断奶后至3月龄的幼兔感染率可达100%,死亡率可达70%。本研究在南通地区球虫病流行病学调查和虫种鉴定基础上,以SPF级新西兰兔为对照,观察球虫感染普通级家兔的肝、肠病理结构及肝功能指标变化,评价磺胺喹VA林、氯苯胍、妥曲珠利三种抗球虫药物的治疗效果及其与抗生素氟苯尼考的相互作用,在基因、蛋白水平阐明抗球虫药物对家兔体内药物代谢酶CYP450的影响,以期为抗生素和抗球虫药物的合理配伍提供理论支持,为进一步提高家兔球虫病防治效果打下基础。一、南通地区家兔球虫病流行病学调查为了对南通地区家兔球虫病进行系统的流行病学调查,从4个商品兔场(A、B、C、E)和1个SPF级兔场(D)采集粪样共75份,用常规方法进行球虫卵囊检查。检查结果发现,4个商品兔场均有球虫感染,且为多种球虫混合感染,SPF级兔场无球虫感染,符合现行的实验动物国家标准；在60份球虫阳性粪样中,感染强度最大的OPG值为38.75×104,最小的为14.75×104；在调查发现的10种兔艾美耳属球虫中,最常见的球虫是穿孔艾美耳球虫(占27.8%),其次为小型艾美耳球虫(占15.1%)和肠艾美耳球虫(占10.2%)；A兔场优势虫种为肠艾美耳球虫(占23.6%),B、C和E兔场优势虫种均为穿孔艾美耳球虫(分别占35.2%、28%和37.2%)。二、球虫感染兔的肝肠病理结构及肝功能变化在普通级家兔普遍感染球虫的现实条件下,为了真实反映球虫感染引起的组织病理和肝功能指标变化,取4月龄球虫感染的普通级新西兰兔和未感染球虫的SPF级新西兰兔各6只,耳缘静脉采集血液,经全自动生化分析仪测定谷丙转氨酶(ALT)、谷草转氨酶(AST)、白蛋白(ALB)、谷氨酰氨转移酶(GGT)、碱性磷酸酶(ALP)等肝功能指标。麻醉后颈静脉放血,采集肝脏、十二指肠标本制备病理切片。与SPF级新西兰兔相比,球虫感染普通级新西兰兔外周血的谷丙转氨酶、碱性磷酸酶活性显著升高(p0.05),谷草转氨酶和谷丙酰胺转移酶活性极显著升高(p0.01),白蛋白含量显著降低(p0.05)。组织病理学观察结果显示：SPF级新西兰兔的肝、肠细胞核排列有序、均一；球虫感染普通级新西兰兔的肝脏有明显病变,部分肝细胞坏死或者增生,肝细胞排列紊乱,肠壁及内膜变薄,肠粘膜层内有感染的卵囊,肠道呈现局部坏死并逐渐形成空泡。超微结构观察结果显示：SPF级新西兰兔的肝细胞结构正常,十二指肠微绒毛排列整齐,长短基本一致,无明显炎症细胞浸润；球虫感染普通级新西兰兔的部分肝细胞结构破坏,有很多坏死结构形成的空泡状小体,十二指肠微绒毛脱落,断裂,表面长短不一。这些研究结果提示,普通级商品兔不仅球虫感染普遍,而且引起不同程度的组织病理变化,影响其作为实验动物的价值。三、三种抗球虫药的治疗效果观察为了比较不同抗球虫药的防治效果,将40只、2月龄左右自然感染球虫的新西兰兔随机分为空白对照组、磺胺喹嗯啉治疗组、氯苯胍治疗组和妥曲珠利治疗组,每组10只。空白对照组家兔饲喂不添加药物的正常颗粒饲料,磺胺喹VA啉治疗组按250mg/kg浓度将磺胺喹VA啉混入颗粒饲料中,连用5天,间隔5天后,再次用药5天；氯苯胍治疗组按100mg/kg浓度将氯苯胍混入颗粒饲料中,连用14天；妥曲珠利治疗组按10mg/kg浓度将妥曲珠利混入颗粒饲料中,连用2天,间隔8天后,再次用药2天；自由采食、饮水。以实验后第10、20、30d的球虫感染强度(OPG)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、白蛋白(ALB)、总胆红素(TBIL)、谷氨酰氨转移酶(GGT)、碱性磷酸酶(ALP)、日增重等为检测指标,将药物治疗组与空白对照组所得数据进行t检验,判定试验药物治疗兔球虫病效果。结果表明：氯苯胍在特定时间段有一定的抗球虫效果,但效果甚小；妥曲珠利、磺胺喹VA啉能显著抑制球虫卵囊的产生和排出,改善试验兔的肝功能指标,降低幼兔死亡率,有效提高兔只增重和饲料报酬的作用。四、三种抗球虫药对家兔氟苯尼考药物动力学的影响将32只新西兰兔随机分为对照组、磺胺喹嗯啉(SUL)处理组、氯苯胍(ROB)处理组和妥曲珠利(TOL)处理组,每组8只。对照组家兔饲喂正常颗粒饲料,磺胺喹VA啉处理组饲喂含250mg/kg磺胺喹VA啉的颗粒饲料,氯苯胍处理组饲喂含66mg/kg氯苯胍的颗粒饲料,妥曲珠利处理组饲喂含2mg/kg妥曲珠利的颗粒饲料,连续饲喂20天。第21天时,每组动物按25mg/kg体重左侧耳静脉注射氟苯尼考(FFC),注射后24h内收集心脏血液,用高效液相色谱测定静脉注射氟苯尼考的血药浓度,3P97药代动力学软件处理数据。研究结果显示：家兔静脉注射25mg/kg氟苯尼考后,其体内药代动力学特征符合一室开放模型；与FFC对照组相比,FFC+ROB组tl/2Ke和AUC值显著下降,Ke和CL值明显上升,而FFC+SUL和FFC+TOL组未出现这样的变化,提示氟苯尼考与氯苯胍联合用药时会发生药物代谢间相互作用。此外我们还发现,与FFC对照组相比,FFC+TOL组的Vd值差异显著(p0.05),而其他两组的Vd值差异不显著。五、三种抗球虫药对家兔肝CYP3A6表达及酶活性的影响 24只新西兰白兔随机分为空白对照组、磺胺喹VA啉处理组、氯苯胍处理组和妥曲珠利处理组,每组6只。空白对照组家兔饲喂正常颗粒饲料,磺胺喹嗯啉处理组饲喂含250mg/kg磺胺喹VA啉的颗粒饲料,氯苯胍处理组饲喂含66mg/kg氯苯胍的颗粒饲料,妥曲珠利处理组饲喂含2mg/kg妥曲珠利的颗粒饲料,连续饲喂20天。在第21天,无菌取出动物部分肝脏提取RNA和蛋白样品,分别进行Real-Time RT-PCR和Western blot检测。结果显示：磺胺喹VA啉和妥曲珠利处理组家兔的CYP3A6mRNA水平显著上调(p0.05),氯苯胍处理组家兔的CYP3A6mRNA水平无显著变化(p0.05)；磺胺喹嗯啉和妥曲珠利处理组家兔的CYP3A6蛋白表达显著上调(p0.05),氯苯胍处理组家兔虽有一定程度上调,但差异不显著(p0.05)。用LC-MS/MS测定家兔肝脏微粒体中CYP3A探针底物代谢物1-羟基-咪达唑仑的生成情况,结果显示：磺胺喹嗯啉、氯苯胍和妥曲珠利处理组家兔的肝脏CYP3A酶活性与对照组相比均有极显著提高(p0.01)。这些结果表明：家兔连续服用磺胺喹VA啉、氯苯胍和妥曲珠利三种抗球虫药物,均能显著诱导家兔肝CYP3A酶活性。
[Abstract]:Coccidiosis is a common and serious parasitic disease in rabbits. The infection rate of young rabbits after weaning to 3 month old is up to 100%. The mortality rate can reach 70%.. Based on the epidemiological investigation and insect species identification of coccidiosis in Nantong area, the liver, intestinal pathological structure and liver function index of rabbits infected with coccidiosis were observed with SPF New Zealand rabbits as the control. To evaluate the effect of three kinds of anti coccidiosis drugs on the sulfamethazine VA forest, chlorobenzidine, and doberoli, and the interaction with the antibiotic florfenicol, and the gene and protein levels to clarify the effect of anti coccidiosis drugs on the drug metabolizing enzyme CYP450 in rabbits, in order to provide theoretical support for the rational compatibility of antibiotics and anti coccidiosis drugs. It will lay a foundation for further improving the control effect of coccidiosis in rabbits.
An epidemiological survey of rabbit coccidiosis in Nantong area
In order to carry out a systematic epidemiological survey on rabbit coccidiosis in Nantong area, 75 feces samples were collected from 4 commodity rabbit farms (A, B, C, E) and 1 SPF class rabbits (D). The oocyst examination of coccidia was carried out by conventional methods. The results showed that the 4 commodity rabbits were infected with coccidiosis, and were mixed with coccidiosis, and there were no coccidian infection in the SPF class. In the current national standard of experimental animals, among the 60 coccidiosis positive feces, the highest OPG value was 38.75 x 104 and the smallest was 14.75 x 104. Among the 10 species of Eimeria, the most common coccidia were perforated Eimeria (27.8%), followed by small Eimeria (15.1%) and Eimeria Enterococcus (Eimeria Enterococcus). 10.2%); the dominant species in A rabbit farm were Eimeria intestines (23.6%), and B, C and E rabbits were all Eimeria perfora (35.2%, 28% and 37.2% respectively).
Two, pathological changes of liver and intestine and liver function in rabbits infected with coccidia
In order to reflect the histopathology and liver function changes caused by the infection of coccidiosis, 4 month old New Zealand rabbits infected with coccidiosis and 6 SPF New Zealand rabbits without coccidiosis were collected, and the blood was collected from the auricular vein, and the alanine transaminase (A) was measured by the automatic biochemical analyzer. LT) the liver function indexes such as AST, ALB, GGT and ALP. After anaesthesia, the blood of the cerneck, the liver and the duodenum were collected for pathological section. Compared with the SPF New Zealand rabbits, the coccidiosis was infected with the ALP activity in the peripheral blood of the normal New Zealand rabbits and the alkaline phosphatase activity was significant. Increase (P0.05), the activity of transaminase and alprolactam transferase increased significantly (P0.01), and the content of albumin decreased significantly (P0.05). Histopathological observation showed that the liver of New Zealand rabbits of grade SPF was arranged orderly and homogeneous, and the liver of the Coccidioides infected ordinary New Zealand rabbits had obvious pathological changes, some liver cells were necrotic or the liver cells were necrotic or necrotic. The liver cell arrangement disorder, the intestinal wall and intima thinning, the infected oocysts in the intestinal mucosa, the intestinal necrosis and the gradual formation of vacuoles in the intestinal mucosa. The ultrastructure observation showed that the liver cell structure of the SPF New Zealand rabbits was normal, the duodenal microvilli was arranged neatly, the length of the duodenum was basically the same, there was no obvious inflammatory cell infiltration; coccidia feeling was not obvious. Some of the liver cell structures of New Zealand rabbits are damaged, there are many vacuolated bodies formed by necrotic structure, and the duodenal microvilli fall off, break and the surface is different. These results suggest that the common grade rabbit is not only common in the infection of coccidiosis, but also causes different degrees of histopathological changes, affecting it as an experimental animal. Value.
Observation on the therapeutic effect of three, three kinds of anti coccidiosis drugs
To compare the control effect of different anti coccidiosis drugs, 40 New Zealand rabbits were randomly divided into the blank control group, the Sulfamethoxine treatment group, the chlorobenzguanidine treatment group and the doberoli treatment group, with 10 rats in each group. The blank control group was fed with normal pellet feed without adding drugs, and the sulfametholine VA group was treated in the treatment group. 250mg/kg concentration mixed Sulfamethoxine VA into pellet feed for 5 days, 5 days after interval, and again for 5 days. Chlorobenzene guanidine group mixed chlorobenzene guanidine into granular feed according to 100mg/kg concentration, and used for 14 days. The TTL group was mixed with dobi pearl in granular feed according to the concentration of 10mg/kg, for 2 days and 2 days after 8 days. Free feeding and drinking water. In the experiment, the infection intensity (OPG) of coccidiosis (ALT), cereal transaminase (ALT), glutamine (AST), albumin (ALB), total bilirubin (TBIL), glutamyl aminotransferase (GGT), alkaline phosphatase (ALP) and daily weight gain were tested, and the data of the drug treatment group and the blank control group were tested by t test, and the test was determined. The effect of drug treatment on rabbit coccidiosis showed that chlorobenzidine had a certain anti coccidiosis effect in a certain period of time, but the effect was very small, and the effect of DHL and sulfaquine VA can significantly inhibit the production and discharge of coccidiococcium oocyst, improve the liver function index of experimental rabbits, reduce the mortality of young rabbits and improve the effect of rabbit weight gain and feed reward.
Effects of four, three coccidiosis drugs on the pharmacokinetics of florfenicol in rabbits
32 New Zealand rabbits were randomly divided into the control group, the sulfanilamide quinoline (SUL) treatment group, the chlorobenzidine (ROB) treatment group and the TOL treatment group, with 8 rabbits in each group. The control group was fed with normal pellet feed, the sulfaquine VA treatment group was fed with 250mg/kg sulfametholine VA in the pellet feed, and the chlorobenzidine treatment group was fed with 66mg/kg chlorobenzene guanidine. Feed, the DDR treatment group was fed with 2mg/kg tugal pearl pellet feed for 20 days. At twenty-first days, each group was injected with florfenicol (FFC) in the left ear vein of 25mg/kg weight. The blood was collected in 24h after injection. The blood concentration of florfenicol was measured by high performance liquid chromatography, and the 3P97 pharmacokinetic Software Department The results showed that after the intravenous injection of 25mg/kg florfenicol in rabbits, the pharmacokinetic characteristics of the rabbits were in accordance with the one chamber open model. Compared with the FFC control group, the values of tl/2Ke and AUC in the FFC+ROB group decreased significantly, and the Ke and CL values increased significantly, while the changes in the FFC+SUL and FFC+TOL groups did not appear, suggesting the combination of florfenicol and chlorobenzidine. In addition, we found that compared with the FFC control group, the Vd value of the FFC+TOL group was significantly different (P0.05), but the difference in the Vd value of the other two groups was not significant.
Effects of five, three coccidiosis drugs on CYP3A6 expression and enzyme activity in rabbit liver
24 New Zealand white rabbits were randomly divided into blank control group, Sulfamethoxine VA treatment group, chlorobenzene guanidine treatment group and dordo pearl treatment group, 6 rabbits in each group. The blank control group was fed normal pellet feed, the sulfamethoxaline treatment group was fed with 250mg/kg sulfametholine VA particle feed, and the chlorobenzidine treatment group was fed with 66mg/kg chlorobenzene guanidine fed granule feeding For 20 days, the RNA and protein samples were extracted from the liver of the animals for 20 days for 20 days. The results showed that the level of CYP3A6mRNA in the rabbits with Sulfamethoxine VA and the treatment group was significantly up (P0.05) (P0.05). There was no significant change in the CYP3A6mRNA level of the rabbits treated with chlorobenzidine (P0.05); the expression of CYP3A6 protein in the rabbits with sulfamethoxazoline and the treatment group was significantly up (P0.05). Although the rabbits in the chlorobenzidine treatment group were up to a certain extent, the difference was not significant (P0.05). The CYP3A probe substrate metabolite 1 in the rabbit liver microsomes was determined by LC-MS/MS. The results of the formation of - hydroxy midazolam showed that the activity of CYP3A enzyme in the liver of the rabbits was significantly higher than the control group (P0.01). These results showed that the continuous use of three kinds of anti coccidiosis drugs in the rabbits were the continuous use of sulfametholine, chlorobenzidine, and the three kinds of anti coccidiosis drugs. The activity of CYP3A enzyme in rabbit liver.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：S858.291

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