培养基和换液时间对E.tenella子孢子在宿主细胞体外培养中发育的影响

探索培养基和换液时间对E.tenella子孢子在宿主细胞体外培养的影响,为E.tenella损伤机制及抗球虫药的研究提供体外模型。本实验研究了不同换液时间和不同培养基对E.tenella子孢子在鸡胚盲肠上皮细胞体外培养中发育情况的影响。结果表明:(1)在相同培养基下(MEM199培养基或RPMR1640),与3h换液相比,4h换液更适于E.tenella子孢子的入侵;4h换液后培养至24h(或48h),E.tenella子孢子发育更好。(2)相同培养时间下,与RPMR1640培养基相比MEM199培养基更适于E.tenella子孢子的发育。     

E.tenella鸡胚盲肠上皮细胞体外培养的影响因素

研究E.tenella子孢子的纯化方法、接种剂量以及细胞生长状态对E.tenella在鸡胚盲肠上皮细胞体外培养中的影响,为E.tenella鸡胚盲肠上皮细胞体外培养模型的建立提供依据。以子孢子回收率和细胞感染率为指标,对DE52纤维素层析法、蔗糖密度梯度离心法和双层目筛过滤法纯化子孢子的效果进行比较;测定了子孢子接种剂量和细胞生长状态对虫体感染率的影响,并对各发育阶段的虫体进行观察。结果表明,采用双层目筛过滤法可得到纯净的子孢子,并且回收率和接种细胞后24、48h的虫体感染率均显著(P0.05)和极显著(P0.01)高于DE52纤维素层析法和蔗糖密度梯度离心法;在48孔细胞培养板中,子孢子的最佳感染剂量为每孔10万个;鸡胚盲肠上皮细胞覆盖率为85%~90%且贴壁细胞融合率在30%以上时接种子孢子,各时间段虫体的入侵率较高,且可以观察到E.tenella主要发育阶段的虫体。本研究获得了能使E.tenella在鸡胚盲肠上皮细胞完成整个内生发育的子孢子纯化方法、接种剂量和细胞生长状态。     

柔嫩艾美耳球虫细胞培养液对鸡胚盲肠上皮细胞活性的影响

为了探讨鸡柔嫩艾美耳球虫(E.tenella)分泌物及代谢产物对鸡胚盲肠上皮细胞活性的影响,用E.tenella裂殖生殖期细胞培养液培养鸡胚盲肠上皮细胞24 h,通过MTT法检测细胞活性,HE染色观察细胞形态变化。结果:用含25%的48 h及72 h攻虫培养液的混合培养基可使鸡盲肠上皮细胞的活性显著下降(P0.01)。表明E.tenella裂殖生殖期的分泌物及代谢产物可降低未感染鸡胚盲肠细胞的活性。     

鸡球虫体外HCT-8细胞培养模型的建立

由柔嫩艾美耳球虫(Eimeria tenella)引起的球虫病严重损害鸡的肠道健康,给养鸡业造成巨大的经济损失。E.tenella寄生在鸡盲肠上皮细胞,体外培养可在鸡原代肾细胞内发育成卵囊完成内生发育过程,而在其他传代细胞系内只能完成部分生殖阶段,为了延长其在细胞内的发育进程,建立了鸡球虫HCT-8细胞培养模型。将新鲜制备的E.tenella子孢子接种HCT-8细胞,制备细胞爬片,分别进行HE染色和DAPI染色,观察子孢子在细胞内的发育状况。结果表明,子孢子在接种后6 h即可入侵HCT-8细胞,随后子孢子发育为滋养体至48 h形成第一代裂殖体,至54h第一代裂殖子释放,于72 h形成第二代裂殖体,在144 h第二代裂殖子释放出来,继续培养至168h未见到卵囊形成,而试验虫株接种鸡体后在144～168 h即可观察到大量未孢子化卵囊排出体外,据此确定子孢子在该细胞内的发育过程结束。本研究所建立的鸡球虫细胞培养模型将子孢子发育至第二代裂殖生殖阶段,将发育阶段向前推进了一个进程,为鸡球虫相关研究工作的开展提供技术平台。     

鸡胚盲肠上皮细胞原代培养与鉴定

为研究鸡E.tenella的宿主细胞--盲肠上皮细胞体外培养技术,为E.tenella损伤机制及抗球虫药的研究提供体外模型,分别用胰蛋白酶法、胶原酶Ⅰ法、嗜热菌蛋白酶法、嗜热菌蛋白酶+胶原酶Ⅰ法和中性蛋白酶Ⅰ+胶原酶Ⅺ法分离纯化原代鸡胚盲肠上皮细胞,通过测定细胞活力、细胞团块比例、总细胞产量和细胞团块产量,筛选出鸡胚盲肠上皮细胞最佳分离方法,并进行了纯化和培养,对培养细胞进行了鉴定.结果表明:嗜热菌蛋白酶消化、低速离心去除单细胞、差速贴壁除去成纤维细胞,为最佳分离和纯化盲肠上皮细胞的方法;分离纯化的细胞接种后分别于第3-5天、第10-11天进入对数生长期,可存活14 d以上;经形态学观察、碱性磷酸酶染色、扫描电镜鉴定为盲肠上皮细胞,所分离的细胞培养至第4、7、11天时上皮细胞比例分别为81.67%、84.33%和72.00%.用该法可获得纯度较高的原代鸡胚盲肠上皮细胞.     

鸡胚盲肠上皮细胞的传代培养

本研究旨在探讨鸡胚盲肠上皮细胞传代培养条件及其特性,为E.tenella损伤机制及抗球虫药的研究提供体外模型。分别用胰酶-EDTA联合消化和分步消化2种方法分离纯化原代鸡胚盲肠上皮细胞,通过测定贴壁细胞覆盖率选择细胞传代的适宜消化方式;筛选了传代细胞培养液中L-GLN和胰岛素的最佳浓度,通过贴壁差进一步纯化盲肠上皮细胞,探索其合适的培养条件。通过形态学观察、透射电镜、免疫组织化学技术、组织化学技术、糖原染色、流式细胞术的方法对传代细胞特性进行了鉴定。结果表明:胰酶-EDTA联合消化、15min贴壁差纯化除去成纤维细胞,72.5mg.L-1 L-GLN、0.05mg.L-1胰岛素和5%FBS的传代细胞培养液更有利于盲肠上皮细胞的生长;经形态学和透射电镜观察,AKP、Vimentin及PAS染色,所培养的传代细胞具有典型的上皮细胞特征,在传代后24～72h盲肠上皮细胞纯度达95%以上,贴壁细胞覆盖率达85%以上;细胞凋亡测定表明,细胞连传5代,活性较好,第6代细胞凋亡率显著增加,贴壁细胞覆盖率降低。本研究提示用该法传代可获得稳定、数量大、活性高、纯度高的鸡胚盲肠上皮细胞。     

柔嫩艾美耳球虫病鸡盲肠上皮细胞凋亡的分析

从细胞凋亡的角度探讨柔嫩艾美耳球虫(E.tenella)对鸡盲肠黏膜的损伤机理,为鸡球虫病的防治提供理论依据.用E.tenella孢子化卵囊感染雏鸡,于感染后不同时间段取盲肠组织,运用常规病理组织学、超微病理学和原位末端标记技术对感染E.tenella病鸡肓肠黏膜上皮细胞和肠腺上皮细胞进行观察和分析.结果表明:鸡感染E.tenella的第2天,盲肠黏膜上皮细胞凋亡数即开始增加,感染后第4～6天是病变最严重且具有特征性的阶段,主要表现为中、后段盲肠黏膜深层严重出血,大最黏膜上皮脱落,严重者可见整个黏膜几乎完全脱落,肠腺破坏,其表面被覆盖大量脱落的变性、坏死的上皮细胞和红细胞,残存的黏膜上皮细胞和肠腺上皮细胞凋亡速度加快,大多数上皮细胞处于凋亡状态,凋亡指数极显著(P≤0.01)高于空白对照组,凋亡细胞体积缩小,细胞线粒体肿胀,严重时破裂成大空泡,染色质致密边集或断裂成核碎片,形成膜包裹性凋亡小体.结果提示E.tenella感染鸡后盲肠上皮细胞凋亡加重,以裂殖生殖阶段最为明项,且与损伤程度和病程密切相关.     

不同浓度表皮生长因子(EGF)对鸡胚盲肠上皮细胞生长的影响

为了研究不同浓度表皮生长因子(EGF)对鸡胚盲肠上皮细胞生长的影响,通过体外培养鸡胚盲肠上皮细胞,添加不同浓度表皮生长因子(EGF,0、10、20、30、40 ng/mL)对鸡胚盲肠上皮细胞生长的影响,并对体外培养的鸡胚盲肠上皮细胞进行了碱性磷酸酶鉴定,细胞形态学观察贴壁率,测定生长曲线。结果显示:加入20 ng/mL的EGF,对鸡胚盲肠上皮细胞增殖的促进作用是极显著的(P0.01)。表明EGF作为重要的细胞因子之一,参与调节并促进鸡胚盲肠上皮细胞体外生长。     

鸡胚接种柔嫩艾美耳球虫和堆形艾美耳球虫活卵囊的免疫保护试验

给18日龄鸡胚接种一定剂量的柔嫩艾美耳球虫(Eim eria tenella)和/或堆形艾美耳球虫(E.acervulina)孢子化卵囊,出雏后在无球虫环境中笼养,1~10日龄每天收集各组粪便样本,计数克粪便卵囊数(OPG),并于14日龄时以大剂量同源孢子化卵囊攻虫,以相对增重率(RWG)、饲料转化率(FCR)、相对卵囊产量(ROP)评价免疫保护效果。结果显示,以E.tenella或E.acervulina卵囊免疫18日龄鸡胚,其卵囊排出的潜隐期及达到峰值的时间与1日龄雏鸡接种组相一致,有相似的排卵囊曲线,提示其诱导免疫的建立是在出雏后开始建立的。攻虫后各免疫组的RWG由攻虫对照组的31.9%~51.7%提高到了76.5%~83.6%,RCR由攻虫对照组的4.11~4.89改善为2.72~2.96,ROP降至4.7%~23.5%。结果表明以一定剂量E.tenella和E.acervulina卵囊单独或混合经羊膜腔免疫18日龄鸡胚都可以建立起针对出雏后14日龄同源攻虫的良好免疫保护力。比较混合免疫E.tenella和E.acervulina卵囊组与单一接种E.tenella或E.acervulina卵囊组的免疫效果发现,混合免疫组的各项指标均稍优于后者。     

堆型艾美耳球虫与柔嫩艾美耳球虫卵囊最短孢子发育时间的测定

将收集到的新排出的堆型艾美耳球虫(E.acervulina)与柔嫩艾美耳球虫(E．tenella)卵囊于29℃静态系统中孢化。以30min为间隔，对卵囊孢子发育的4个阶段在显微镜下进行观察。以1h为间隔，用发育中的卵囊连续接种无球虫鸡，以确定最短孢子发育时间(MST)。对影响孢子发育的外部因素进行了初步探讨。用堆型艾美耳球虫比较不同卵囊浓度，不同悬液深度在卵囊孢化过程中第20h时子孢子形成的卵囊百分率。E.acervulina与E.tenella的MST分别为11b与18b。卵囊浓度和悬液深度与孢子发育时间呈明显反比关系。     

Heartworm in dogs in Canada in 1984

In late December 1984, 1853 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1984 and 35% of them responded. Veterinarians reported that 97,794 dogs were blood-tested to check for microfilariae and 1417 dogs (1.45% of those tested) were found with heartworm. Another 34 dogs were amicrofilaremic, but were diagnosed as having heartworm disease, to give the total number diagnosed in 1984 as 1451 (1.48%). Heartworm was reported from all provinces except Prince Edward Island and Newfoundland but most (1310) of the cases were in Ontario. In Quebec, 126 cases were reported mostly from west of Montreal.

Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 27% of the cases were observed with clinical signs of heartworm disease and 72% had a history of not having left Canada. Southwestern Ontario continued to be the primary focus of the infection.

     

Heartworm in dogs in Canada in 1985

In late December 1985, 1485 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1985 and 44% of them responded. Veterinarians reported that 137,300 dogs were blood-tested to check for microfilariae and 1210 dogs were found with heartworm. Another 36 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number diagnosed in 1985 as 1247 (0.91%).

Heartworm was reported from all provinces except Prince Edward Island, Newfoundland and Saskatchewan but most (1126) of the cases were in Ontario. Southwestern Ontario continued to be the primary focus of the infection in Canada. From Quebec, 91 cases were reported mostly from and around Montreal. From Manitoba, 19 cases were reported from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 28% of the cases were observed with clinical signs of heartworm disease and 78% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1988

In late November 1988, 1581 small and mixed animal clinics and institutional veterinarians across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1988, and 46% of them responded. Veterinarians reported that 181,577 dogs were blood-tested for heartworm disease and 367 dogs were found with D. immitis microfilariae. Another 60 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1988 as 441 (0.24%).     

Heartworm in dogs in Canada in 1989

In late November 1989, 1732 clinics and institutional veterinarians were sent a questionnaire to assess the status of Dirofilaria immitis, and 51.7% responded. Of 247,716 dogs tested, 394 had D. immitis microfilariae and 51 were amicrofilaremic for a total of 445 cases and heartworm prevalence of 0.17%. Most (408) of these dogs had no preventive medication and the prevalence among dogs tested and unprotected was 1.01%. That prevalence was considerably higher in endemic areas. Thirty-seven dogs with heartworm had preventive medication. Heartworm was most frequent in companion dogs over three years of age maintained outdoors in rural areas. About 75% of the cases had never left Canada, 26% had clinical signs and 125 were not treated.

Heartworm was reported from British Columbia, Manitoba, Ontario, Quebec, Nova Scotia and Newfoundland, but 383 cases were in Ontario. South-western Ontario was the primary focus of infection. There were 33 cases in Quebec and 24 in Manitoba, mainly found in and around Metropolitan Montreal and Winnipeg respectively.

     

Heartworm in dogs in Canada in 1987

In late November 1987, 1246 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1987, and 50% of them responded. Veterinarians reported that 165,428 dogs were blood tested for heartworm disease and 511 dogs were found with D. immitis microfilariae. Another 78 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1987 as 589 (0.35%).     

Heartworm in dogs in Canada in 1986

In late December 1986, 1224 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1986; 46% of them responded. Veterinarians reported that 150,989 dogs were blood-tested for microfilariae and 869 dogs were found with heartworm. Another 65 dogs were amicrofilaremic but diagnosed with heartworm disease and one was found with heartworm at necropsy to give the total number diagnosed in 1986 as 935 (0.62%).

Heartworm was reported from Manitoba, New Brunswick, Ontario and Quebec, but most (810) of the cases were from Ontario. South-western Ontario continued to be the primary focus of the infection in Canada. There were 103 cases reported from Quebec, mostly from and around Montreal, and 21 cases from Manitoba, from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 33% of the cases were observed with clinical signs of heartworm disease and 81% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1991

In late November 1991, 1883 clinics in Canada were sent a questionnaire to assess the status of Dirofilaria immitis in dogs in 1991 and there was a 60.0% response. There were 344,031 dogs tested for heart-worm (HW), 627 were found infected and the prevalence of HW infection was 0.18%. There were 417 dogs with HW in Ontario, 116 in Manitoba, 38 in Quebec, 53 in British Columbia, three in Alberta, and one in Nova Scotia. In British Columbia, all of the infected dogs but one were from the Okanagan valley which, as from 1991, is a new focus of infection in Canada. Most dogs with HW had not been on preventive medication in 1990, and the prevalence among dogs tested and unprotected was 0.59%. That prevalence was considerably higher in endemic areas. Companion dogs, over three years of age and maintained primarily outdoors in rural areas, were most frequently infected. One cat was diagnosed with D. immitis and 33 dogs had Dipetalonema reconditium.     

Heartworm in dogs in Canada in 1983

In late December 1983, 2 800 veterinarians across Canada were sent a questionnaire in order to assess the status of heartworm disease in Canada in 1983 and 26% of them responded. Veterinarians reported that 59 504 dogs were blood-tested to check for microfilariae and 771 dogs (1.30% of those tested) were found with Dirofilaria immitis. Heartworm disease was diagnosed in all provinces except New Brunswick and Newfoundland but most (733) of the cases were in Ontario.

Heartworm disease was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 31% of the cases were observed with clinical signs of heartworm disease and 64% had a history of not having left Canada. Southwestern Ontario continues to be the focus of the infection and most of the dogs there had not left the province previously.