本文選題：家兔球蟲(chóng)病 + 流行病學(xué)調(diào)查　； 參考：《揚(yáng)州大學(xué)》2014年博士論文

【摘要】：球蟲(chóng)病是家兔常見(jiàn)和危害嚴(yán)重的寄生蟲(chóng)病,斷奶后至3月齡的幼兔感染率可達(dá)100%,死亡率可達(dá)70%。本研究在南通地區(qū)球蟲(chóng)病流行病學(xué)調(diào)查和蟲(chóng)種鑒定基礎(chǔ)上,以SPF級(jí)新西蘭兔為對(duì)照,觀(guān)察球蟲(chóng)感染普通級(jí)家兔的肝、腸病理結(jié)構(gòu)及肝功能指標(biāo)變化,評(píng)價(jià)磺胺喹VA林、氯苯胍、妥曲珠利三種抗球蟲(chóng)藥物的治療效果及其與抗生素氟苯尼考的相互作用,在基因、蛋白水平闡明抗球蟲(chóng)藥物對(duì)家兔體內(nèi)藥物代謝酶CYP450的影響,以期為抗生素和抗球蟲(chóng)藥物的合理配伍提供理論支持,為進(jìn)一步提高家兔球蟲(chóng)病防治效果打下基礎(chǔ)。 一、南通地區(qū)家兔球蟲(chóng)病流行病學(xué)調(diào)查 為了對(duì)南通地區(qū)家兔球蟲(chóng)病進(jìn)行系統(tǒng)的流行病學(xué)調(diào)查,從4個(gè)商品兔場(chǎng)(A、B、C、E)和1個(gè)SPF級(jí)兔場(chǎng)(D)采集糞樣共75份,用常規(guī)方法進(jìn)行球蟲(chóng)卵囊檢查。檢查結(jié)果發(fā)現(xiàn),4個(gè)商品兔場(chǎng)均有球蟲(chóng)感染,且為多種球蟲(chóng)混合感染,SPF級(jí)兔場(chǎng)無(wú)球蟲(chóng)感染,符合現(xiàn)行的實(shí)驗(yàn)動(dòng)物國(guó)家標(biāo)準(zhǔn)；在60份球蟲(chóng)陽(yáng)性糞樣中,感染強(qiáng)度最大的OPG值為38.75×104,最小的為14.75×104；在調(diào)查發(fā)現(xiàn)的10種兔艾美耳屬球蟲(chóng)中,最常見(jiàn)的球蟲(chóng)是穿孔艾美耳球蟲(chóng)(占27.8%),其次為小型艾美耳球蟲(chóng)(占15.1%)和腸艾美耳球蟲(chóng)(占10.2%)；A兔場(chǎng)優(yōu)勢(shì)蟲(chóng)種為腸艾美耳球蟲(chóng)(占23.6%),B、C和E兔場(chǎng)優(yōu)勢(shì)蟲(chóng)種均為穿孔艾美耳球蟲(chóng)(分別占35.2%、28%和37.2%)。 二、球蟲(chóng)感染兔的肝腸病理結(jié)構(gòu)及肝功能變化 在普通級(jí)家兔普遍感染球蟲(chóng)的現(xiàn)實(shí)條件下,為了真實(shí)反映球蟲(chóng)感染引起的組織病理和肝功能指標(biāo)變化,取4月齡球蟲(chóng)感染的普通級(jí)新西蘭兔和未感染球蟲(chóng)的SPF級(jí)新西蘭兔各6只,耳緣靜脈采集血液,經(jīng)全自動(dòng)生化分析儀測(cè)定谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)、白蛋白(ALB)、谷氨酰氨轉(zhuǎn)移酶(GGT)、堿性磷酸酶(ALP)等肝功能指標(biāo)。麻醉后頸靜脈放血,采集肝臟、十二指腸標(biāo)本制備病理切片。與SPF級(jí)新西蘭兔相比,球蟲(chóng)感染普通級(jí)新西蘭兔外周血的谷丙轉(zhuǎn)氨酶、堿性磷酸酶活性顯著升高(p0.05),谷草轉(zhuǎn)氨酶和谷丙酰胺轉(zhuǎn)移酶活性極顯著升高(p0.01),白蛋白含量顯著降低(p0.05)。組織病理學(xué)觀(guān)察結(jié)果顯示：SPF級(jí)新西蘭兔的肝、腸細(xì)胞核排列有序、均一；球蟲(chóng)感染普通級(jí)新西蘭兔的肝臟有明顯病變,部分肝細(xì)胞壞死或者增生,肝細(xì)胞排列紊亂,腸壁及內(nèi)膜變薄,腸粘膜層內(nèi)有感染的卵囊,腸道呈現(xiàn)局部壞死并逐漸形成空泡。超微結(jié)構(gòu)觀(guān)察結(jié)果顯示：SPF級(jí)新西蘭兔的肝細(xì)胞結(jié)構(gòu)正常,十二指腸微絨毛排列整齊,長(zhǎng)短基本一致,無(wú)明顯炎癥細(xì)胞浸潤(rùn)；球蟲(chóng)感染普通級(jí)新西蘭兔的部分肝細(xì)胞結(jié)構(gòu)破壞,有很多壞死結(jié)構(gòu)形成的空泡狀小體,十二指腸微絨毛脫落,斷裂,表面長(zhǎng)短不一。這些研究結(jié)果提示,普通級(jí)商品兔不僅球蟲(chóng)感染普遍,而且引起不同程度的組織病理變化,影響其作為實(shí)驗(yàn)動(dòng)物的價(jià)值。 三、三種抗球蟲(chóng)藥的治療效果觀(guān)察 為了比較不同抗球蟲(chóng)藥的防治效果,將40只、2月齡左右自然感染球蟲(chóng)的新西蘭兔隨機(jī)分為空白對(duì)照組、磺胺喹嗯啉治療組、氯苯胍治療組和妥曲珠利治療組,每組10只�？瞻讓�(duì)照組家兔飼喂不添加藥物的正常顆粒飼料,磺胺喹VA啉治療組按250mg/kg濃度將磺胺喹VA啉混入顆粒飼料中,連用5天,間隔5天后,再次用藥5天；氯苯胍治療組按100mg/kg濃度將氯苯胍混入顆粒飼料中,連用14天；妥曲珠利治療組按10mg/kg濃度將妥曲珠利混入顆粒飼料中,連用2天,間隔8天后,再次用藥2天；自由采食、飲水。以實(shí)驗(yàn)后第10、20、30d的球蟲(chóng)感染強(qiáng)度(OPG)、谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)、白蛋白(ALB)、總膽紅素(TBIL)、谷氨酰氨轉(zhuǎn)移酶(GGT)、堿性磷酸酶(ALP)、日增重等為檢測(cè)指標(biāo),將藥物治療組與空白對(duì)照組所得數(shù)據(jù)進(jìn)行t檢驗(yàn),判定試驗(yàn)藥物治療兔球蟲(chóng)病效果。結(jié)果表明：氯苯胍在特定時(shí)間段有一定的抗球蟲(chóng)效果,但效果甚�。煌浊槔�、磺胺喹VA啉能顯著抑制球蟲(chóng)卵囊的產(chǎn)生和排出,改善試驗(yàn)兔的肝功能指標(biāo),降低幼兔死亡率,有效提高兔只增重和飼料報(bào)酬的作用。 四、三種抗球蟲(chóng)藥對(duì)家兔氟苯尼考藥物動(dòng)力學(xué)的影響 將32只新西蘭兔隨機(jī)分為對(duì)照組、磺胺喹嗯啉(SUL)處理組、氯苯胍(ROB)處理組和妥曲珠利(TOL)處理組,每組8只。對(duì)照組家兔飼喂正常顆粒飼料,磺胺喹VA啉處理組飼喂含250mg/kg磺胺喹VA啉的顆粒飼料,氯苯胍處理組飼喂含66mg/kg氯苯胍的顆粒飼料,妥曲珠利處理組飼喂含2mg/kg妥曲珠利的顆粒飼料,連續(xù)飼喂20天。第21天時(shí),每組動(dòng)物按25mg/kg體重左側(cè)耳靜脈注射氟苯尼考(FFC),注射后24h內(nèi)收集心臟血液,用高效液相色譜測(cè)定靜脈注射氟苯尼考的血藥濃度,3P97藥代動(dòng)力學(xué)軟件處理數(shù)據(jù)。研究結(jié)果顯示：家兔靜脈注射25mg/kg氟苯尼考后,其體內(nèi)藥代動(dòng)力學(xué)特征符合一室開(kāi)放模型；與FFC對(duì)照組相比,FFC+ROB組tl/2Ke和AUC值顯著下降,Ke和CL值明顯上升,而FFC+SUL和FFC+TOL組未出現(xiàn)這樣的變化,提示氟苯尼考與氯苯胍聯(lián)合用藥時(shí)會(huì)發(fā)生藥物代謝間相互作用。此外我們還發(fā)現(xiàn),與FFC對(duì)照組相比,FFC+TOL組的Vd值差異顯著(p0.05),而其他兩組的Vd值差異不顯著。 五、三種抗球蟲(chóng)藥對(duì)家兔肝CYP3A6表達(dá)及酶活性的影響 24只新西蘭白兔隨機(jī)分為空白對(duì)照組、磺胺喹VA啉處理組、氯苯胍處理組和妥曲珠利處理組,每組6只。空白對(duì)照組家兔飼喂正常顆粒飼料,磺胺喹嗯啉處理組飼喂含250mg/kg磺胺喹VA啉的顆粒飼料,氯苯胍處理組飼喂含66mg/kg氯苯胍的顆粒飼料,妥曲珠利處理組飼喂含2mg/kg妥曲珠利的顆粒飼料,連續(xù)飼喂20天。在第21天,無(wú)菌取出動(dòng)物部分肝臟提取RNA和蛋白樣品,分別進(jìn)行Real-Time RT-PCR和Western blot檢測(cè)。結(jié)果顯示：磺胺喹VA啉和妥曲珠利處理組家兔的CYP3A6mRNA水平顯著上調(diào)(p0.05),氯苯胍處理組家兔的CYP3A6mRNA水平無(wú)顯著變化(p0.05)；磺胺喹嗯啉和妥曲珠利處理組家兔的CYP3A6蛋白表達(dá)顯著上調(diào)(p0.05),氯苯胍處理組家兔雖有一定程度上調(diào),但差異不顯著(p0.05)。用LC-MS/MS測(cè)定家兔肝臟微粒體中CYP3A探針底物代謝物1-羥基-咪達(dá)唑侖的生成情況,結(jié)果顯示：磺胺喹嗯啉、氯苯胍和妥曲珠利處理組家兔的肝臟CYP3A酶活性與對(duì)照組相比均有極顯著提高(p0.01)。這些結(jié)果表明：家兔連續(xù)服用磺胺喹VA啉、氯苯胍和妥曲珠利三種抗球蟲(chóng)藥物,均能顯著誘導(dǎo)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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：S858.291

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