Effects of mosquito hemocytes on normal and degenerated oocysts of Plasmodium yoelii and their role in oocyst melanization

Anopheles stephenst, infected with Plasinodium yoelii, was fed with 10% sucrose solution contatning 1% difluoromethylornithine (DFMO) to induce oocyst degeneration of the malaria parasites. On the 11th day after infection, the effects of mosquito hemocytes On the normal and degenerated oocysts were observed under transmission electron microscope. In the control group, no hemocyte could be found around the normally-developed oocysts. In the DFMO-treated group, all the oocysts underwent degeneration in various degrees and some of them were melanized. All the oocysts were attached by one or more hemocytes of the kind of granulocytes.There were many granules with microtubular structure in the cytoplasm of the hemocytes and in the space between the hemocyte and the oocyst. The findings suggest that the degeneration of oocysts can attract the hemocytes to attach around them and the latter can release granules and possibly other substances to cause encapsulation and melanization of the oocvsts.

Prevalence of Cryptosporidial Oocysts in Soil of Recreational Parks in Abuja, Nigeria

Contamination of soil with cryptosporidial oocysts poses a great health risk to people especially in individuals who are immune-compromised. Exposure to contaminated soil can cause diarrhoea in humans especially in children who play around the parks and may not wash their hands before handling food. The prevalence of cryptosporidial oocyst in soils of 4 recreational parks in Abuja, Nigeria was investigated. Four hundred soil samples were collected from various locations within the recreational parks and examined for the presence of cryptosporidial oocyst using modified acid fast staining technique. In nine (2.3%) of the samples, 8 were from park A and 1 was from park B contained cryptosporidial oocyst. There was significant association (χ2 = 20.1;df = 2;p = 0.00004) between prevalence of Cryptosporidium oocysts and level of hygiene of the parks. It was concluded that soil in recreational parks may be a source of infection with Cryptosporidium oocysts.

A Protocol for a Highly Consistent, High Level Production <i>in Vivo</i>of <i>Plasmodium falciparum</i>Oocysts and Sporozoites

Investigation of the intimate relationship between the human malaria parasite Plasmodium falciparum and its Anopheles vector requires the reliable production and isolation of successive sexual stages of the parasite from infected mosquitoes. Such an advance in propagation would benefit a range of molecular, cellular, immunochemical and transmission-blocking research studies. Parasite cultivation, mosquito rearing, infection and subsequent dissection of mosquitoes are all highly technical procedures that require both skill and experience to perform with competence. Furthermore, to produce mosquitoes of an appropriate age to infect during the short period in which parasites are viable for infection demands precise planning in order to coordinate the interacting life cycles of the parasite and vector. Here, a protocol is described for the complete development of P. falciparum within Anopheles stephensi. A very consistent, high level production in vivo of P. falciparum oocysts and sporozoites is demonstrable by dissection of the mosquito midgut and salivary glands, respectively.

The Effects of Washing Conditions on the Coccidian Oocyst Collection in Chicken Manure

To optimize the production process of occidium vaccine and provide the basis and methods for the production of live vaccine,this experiment uses the collected liquid with different centrifugal forces and centrifugation time,to measure and compare the recovery,purity,collection time and costs of coccidian oocyst,for the purpose of exploring the impact of washing conditions on coccidian oocyst collection in chicken manure.The results show that for the centrifugal machine with the radius of 17.2 cm,the best washing centrifugal rotational speed is 2500 rpm /min,that is,the best washing centrifugal force RCF = 1201.85 g,and the centrifugation time is 12 min.

对氯间甲酚及复方对氯间甲酚对鸡柔嫩艾美耳球虫卵囊的抑杀效果

为验证苯酚类消毒剂对鸡球虫卵囊的抑杀作用,采用不同浓度的单体和复方对氯间甲酚消毒剂与未孢子化的柔嫩艾美耳球虫卵囊共孵育,检测其对卵囊孢子化率的影响;与孢子囊共同孵育,通过子孢子脱囊率评价其对子孢子释放的干扰。孢子化卵囊经过不同浓度消毒剂直接浸泡、粪便干扰下浸泡或喷雾处理后经口接种雏鸡,通过OPG和盲肠病变记分探究消毒剂处理对卵囊的繁殖力和鸡的致病性的影响。结果显示,2种消毒剂均可使孢子化率和子孢子脱囊率显著下降,且3种浓度的复方对氯间甲酚与3%氨水对照组(PC)的消毒效果无显著差异。直接浸泡处理试验结果显示,2%和3%复方对氯间甲酚(HTS2和HTS3)具有更为显著的抑杀效果,与PC无显著差异;粪便干扰试验结果显示,粪便作用下这两种消毒剂对球虫卵囊仍有理想的抑杀效果;喷雾处理试验结果显示,3%对氯间甲酚(HT3)对球虫卵囊抑杀效果最好,显著优于PC组(P<0.05),而2%对氯间甲酚(HT2)、HTS2和HTS3组的抑杀效果与PC组无显著差异。研究结果表明,不同浓度下这2种消毒剂对孢子化和未孢子化的柔嫩艾美耳球虫卵囊都有一定的抑杀作用,综合比较,复方对氯间甲酚消毒剂具有更强的抑杀球虫卵囊效果,具有代替氨水成为鸡球虫消毒剂的潜能。

柔嫩艾美耳球虫贵阳株对6种抗球虫药物的耐药性试验

为探究柔嫩艾美耳球虫(Eimeria tenella,E.tenella)贵阳株对6种抗球虫药物的耐药性,试验将1日龄黄羽蛋鸡144只随机分为地克珠利组(D组)、妥曲珠利组(T组)、莫能菌素组(M组)、盐酸氯苯胍组(Y组)、癸氧喹酯组(G组)和磺胺氯吡嗪钠组(H组)6个给药组及感染模型组(C组)和空白对照组(K组),每组18只鸡,在14日龄时,对6个给药组分别连续给药8 d;15日龄时,除K组外,其余各组一次口服接种E.tenella贵阳株孢子化卵囊2×10^(4)个,观察各组雏鸡临床症状,计算雏鸡增重、相对增重率、料肉比、盲肠病变记分、每克盲肠内容物卵囊数和存活率、最适抗球虫活性百分数、盲肠病变记分减少率、相对卵囊产量和抗球虫指数(ACI),综合判断E.tenella贵阳株对6种抗球虫药物的耐药性。结果表明:口服接种E.tenella贵阳株孢子化卵囊3 d后,除K组外,各组雏鸡均表现出食欲下降、排血便等临床症状;D组、G组和Y组雏鸡的食欲、饮水及精神状态等较C组稍好,症状较轻。D组增重和相对增重率最大,增重显著高于C组(P<0.05);且料肉比最低,显著低于C组(P<0.05)。D组、Y组和G组盲肠病变记分显著低于C组和H组(P<0.05);D组、T组、M组、Y组和G组每克盲肠内容物中的卵囊数显著低于C组和H组(P<0.05)。M组存活率为90%,其他各组均为100%;D组、G组、Y组ACI>180,D组最佳(ACI=196.60)。E.tenella贵阳株对地克珠利无耐药性,对盐酸氯苯胍和癸氧喹酯轻度耐药,对妥曲珠利中度耐药,对磺胺氯吡嗪钠和莫能菌素完全耐药。说明贵阳地区在防治E.tenella病时,应将地克珠利作为首选药物,盐酸氯苯胍和癸氧喹酯作为备选药物(抑制球虫在体内增殖的效果较好),停止使用妥曲珠利、磺胺氯吡嗪钠和莫能菌素,并注意轮换、交叉、联合用药,避免产生耐药性。

不同杀虫剂对鸡柔嫩艾美耳球虫卵囊孢子化的影响

为了解不同杀虫剂对鸡柔嫩艾美耳球虫卵囊孢子化的影响,为鸡球虫病的防治提供参考。试验采用不同浓度的大灭25CS(1∶50、1∶100)、保安定500EC(1∶2500、1∶5000)、大灭G 15.1%ZC(1∶100、1∶200)、剑心200SC(1∶800、1∶1600)分别对鸡柔嫩艾美耳球虫卵囊作用24、48 h,观察其对球虫卵囊孢子化的影响。结果表明,不同杀虫剂对鸡柔嫩艾美耳球虫卵囊的孢子化均有一定的抑制作用,但其抑制作用随着时间的推移有所减弱;不同杀虫剂使鸡柔嫩艾美耳球虫孢子化卵囊的外观结构出现皱褶、凹陷等变化。这些试验结果为鸡柔嫩艾美耳球虫病的预防提供了新的思路。

Occurrence of Cryptosporidium oocysts in edible frogs (Rana species) sold for human consumption in Hanwa frog market, Zaria, Kaduna State, Nigeria

Objective: To determine the occurrence of Cryptosporidium oocysts in edible frogs (Rana spp.) sold at the Hanwa frog market, Zaria, Kaduna State, Nigeria. Methods: A total of 117 frogs (Rana spp.) were randomly collected based on consent and availability at the market. The intestinal contents from the frogs were examined by staining flotation and sedimentation smears with modified Ziehl-Neelsen staining technique and microscopy of the oocysts. Results: Overall, 35.9% of frogs sampled from the Hanwa frog market were positive for Cryptosporidium oocysts. There were more Cryptosporidium oocysts detected by sedimentation test (28.2%) than flotation test (23.9%). There was no significant statistical association between sex of frogs and oocyst detection (χ2 = 0.5349, P > 0.05). Also, there was no correlation between the weights of frogs and the sizes of the Cryptosporidium oocysts detected (r = 0.0109, P > 0.05). Nevertheless, female frogs (40%) and frogs within the weight range 170–219 g were more infected with Cryptosporidium (66.7%). Oocysts size range 6.10–7.00 μm had the highest frequency of 10 (23.8%). By size 28.2% of the oocysts detected suggested infection with Cryptosporidium parvum and Cryptosporidium maleagridis. Conclusions: This study has established that edible frogs (Rana spp.) sold at the Hanwa frog market for human consumption were infected with Cryptosporidium spp. which constitutes a valid public health risk especially for immunocompromised individuals.

集约化羊场绵羊球虫感染情况调查与分析

应用饱和盐水浮集法和麦克马斯特虫卵计数法对河南省洛宁县某集约化羊场384只绵羊粪便样品进行球虫感染情况调查。结果发现,球虫总感染率为69.5%(267/384),哺乳羔羊、断奶羔羊、哺乳母羊和怀孕母羊感染率分别是52.6%(61/116)、94.5%(104/110)、65.8%(52/79)和63.3%(50/79)。检出的11种艾美耳球虫及其感染率分别是小型艾美耳球虫27.1%、类绵羊艾美耳球虫40.6%、苍白艾美耳球虫25.5%、浮氏艾美耳球虫16.7%、槌型艾美耳球虫33.1%、巴库艾美耳球虫27.9%、阿撤他艾美耳球虫18.5%、温布里吉艾美耳球虫16.4%、错乱艾美耳球虫1.8%、马尔西卡艾美耳球虫3.6%、颗粒艾美耳球虫6.8%。多为2~5种混合感染,混合感染率77.9%(208/267)。类绵羊艾美耳球虫和槌型艾美耳球虫为优势种。哺乳羔羊和断奶羔羊阳性样品每克粪便中的卵囊数极显著高于哺乳母羊和怀孕母羊(P<0.01)。结果表明,球虫感染在集约化绵羊养殖场十分普遍,应高度重视哺乳羔羊和断奶羔羊球虫病的预防控制。

不同处理措施对牛粪样中隐孢子虫卵囊杀灭效果观察

取一例牛隐孢子虫阳性粪样分别采用1%甲醛溶液(A组)、1%二氯异氰脲酸钠溶液(B组)、2%烧碱溶液(C组)以及密封厌氧发酵(D组)处理,经5 d、10 d、15 d、20 d后分别取样采用蔗糖漂浮、涂片以及改良抗酸染色和镜检检查相应样本中隐孢子虫卵囊数量。结果A组4个时间节点卵囊数量分别为3.5枚、0.5枚、0枚、0枚;B组4个时间节点卵囊数量分别为1.75枚、0.25枚、0枚、0枚;C组4个时间节点卵囊数量分别为7.5枚、5.25枚、2.75枚、0.75枚;D组4个时间节点卵囊数量分别为9.75枚、6.50枚、4.75枚、3.00枚;而对照组4个时间节点卵囊数量分别为10.50枚、8.50枚、8.00枚、7.25枚。结果表明:4个试验组对牛隐孢子虫卵囊均有不同程度抑杀效果,其中B组效果相对较好。

Growth performance,nutrient digestibility,intestinal morphology,cecal mucosal cytokines and serum antioxidant responses of broiler chickens to dietary enzymatically treated yeast and coccidia challenge

Background There is a growing search for natural feed additives to alleviate the deleterious effects of coccidia infection in poultry production.This study aimed to investigate the effect of enzymatically treated yeast(ETY) on the growth performance,nutrient digestibility,intestinal morphology,antioxidative status,and cecal mucosa cytokines of coccidia-challenged broiler chickens.Methods From d 1 to 14 post hatching,480 broiler chickens were allocated to 3 corn-soybean meal-based experimental diets with increasing concentrations of ETY(0,1,or 2 g/kg).The experiment was designed as a randomized complete block design with body weight(BW) used as a blocking factor.On d 14 post hatching,the birds were re-randomized within each of the 3 experimental diets.Each of the 3 diet groups was split into a challenge or no-challenge group.This resulted in a 3 × 2 factorial arrangement of treatments.The coccidia challenge was administered on d 15 by an oral gavage.Results Dietary ETY improved(P < 0.05) the G:F of birds on d 21 regardless of the challenge state and linearly increased(P < 0.01) the apparent ileal digestibility of dry matter(DM),nitrogen,and gross energy(GE).The coccidia challenge decreased(P < 0.05) BW gain and feed intake of broiler chickens and reduced(P < 0.01) the total tract retention of DM,GE,and nitrogen.The coccidia challenge increased(P < 0.01) the mRNA gene expression of TNFα,IL-1β,IL-10,and IL-6 in the cecal mucosa.There was a tendency(P ne = 0.08) for ETY to linearly reduce IL-1β expression.Additionally,ETY supplementation increased(P < 0.05) the geexpression of OCLN.Serum catalase increased(P < 0.05) with dietary ETY in broiler chickens on d 21.Dietary ETY linearly increased(P < 0.05) the ileal villus height to crypt depth ratio,and ileal goblet cell density in broiler chickens.The ileal and excreta oocyst counts decreased(P < 0.01) with increasing supplementation of dietary ETY in coccidia-challenged broiler chickens on d 21.Conclusions Dietary ETY enhanced nutrient utilization and augmented intestinal development in broiler chickens.However,dietary ETY did not completely attenuate the adverse effects of a coccidia challenge in broiler chickens.

7种抗球虫药对柔嫩艾美耳球虫荣昌株的疗效试验

旨在探究7种抗球虫药对柔嫩艾美耳球虫(Eimeriatenella)荣昌株的治疗效果。采用笼饲法,将180只罗曼粉雏公鸡饲喂至13日龄随机分为9个试验组,即沙咪珠利组(EZL)、地克珠利组(DIC)、磺胺氯吡嗪钠组(SUS)、尼卡巴嗪组(NIC)、氯羟吡啶组(CLP)、氨丙啉组(AML)、癸氧喹酯溶液组(DQ)、感染对照组(PC)和空白对照组(NC),每组20只鸡,除NC组外,其余各组鸡在14日龄经口接种5×104个柔嫩艾美耳球虫荣昌株孢子化卵囊,各用药组于接种前一天混饲或饮水给药直至试验结束。试验期间观察各组鸡只的临床表现,通过其增重情况、存活率、病变值、卵囊值等计算药物组的抗球虫指数(ACI)。结果显示,EZL和DQ组鸡几乎未表现出临床症状,且在盲肠病变计分和卵囊产量明显低于其他各试验组,其ACI分别为184.00和189.50;SUS和NIC组鸡相对增重率较高,其抗球虫指数分别为136.00和124.06;DIC、CLP和AML组鸡的各项指标均较差,其ACI分别为123.50、106.50和88.38。EZL和DQ对柔嫩艾美耳球虫荣昌株敏感,DIC、SUS和NIC效果较差,CLP和AML无效。

鸡柔嫩艾美耳球虫单卵囊分离技术的构建及致病性研究

构建了一种新的鸡柔嫩艾美耳球虫单卵囊分离技术 ,并对杨陵柔嫩艾美耳球虫地理株进行了分离 ,对继代增殖的纯种卵囊进行了致病性试验。结果表明 ,该单卵囊分离技术简单易行 ,单卵囊感染成功率可达4 0 % ;该虫株对雏鸡有很强的致病性 ,经口接种 1× 10 5个孢子化卵囊 ,致死率高达 80 %。

不同剂量和免疫次数对鸡毒害艾美耳球虫免疫保护力的影响

以相对增重率(RBWG)、饲料转化率(FCR)和盲肠相对卵囊产量(ROP)为指标,评价了1次或2次分别以1×103个或3×103个毒害艾美耳球虫(E.necatrix)孢子化卵囊(SO)/鸡剂量免疫接种后特异性免疫力的产生和持续时间。笼养岭南黄肉雏鸡于3日龄1次或3日龄和10日龄2次分别接种不同剂量的E.necatrix SO后,于17日龄(2次免疫组为24日龄)、34日龄和50日龄时以14×104SO/鸡剂量的同源卵囊攻虫,结果表明,免疫后2周卵囊接种鸡已产生坚强免疫力,1次免疫接种者(17日龄)高、低2个不同剂量组的RBWG、FCR和ROP分别为95.5%、2.32、24.54%和101.4%、2.32、21.1%;而2次免疫组(24日龄)的上述指标则分别为106.5%、2.44、9.3%和98.2%、2.38、7.48%。至34日龄和50日龄攻虫时,这些指标均有不同程度的下降,且随着时间延长,下降更为明显,但2次免疫处理组的这些指标均明显优于1次免疫者;同一次处理的不同剂量组间无明显差异。结果揭示,雏鸡口服免疫接种一定剂量的E.necatrix孢子化卵囊后可迅速诱导鸡体特异性免疫力的产生,但免疫保护力在笼养条件下持续时间较短。

用PCR技术检测水中隐孢子虫

介绍了一种检测水中隐孢子虫的巢式PCR方法:根据Cryptosporidiumparvum18srRNA基因序列选用了两对引物,特异性扩增了该基因可变区中1056bp和435bp目标片段。加标试验表明,该方法的检测限为10个卵囊。

家鹅球虫种类调查及人工感染试验

对江苏省扬州、盐城地区的鹅球虫种类进行了调查和人工感染试验.结果表明,鹅球虫感染率为74.4%.共鉴定出6种球虫,即:Eimeriaanseris、E.fulva、E.hermani、E.nocens、E.stigmosa、Tyzzeriaparvula.12日龄无球虫感染仔鹅感染5×104个混合卵囊即发生死亡.通过单种卵囊接种试验获得了5种球虫的纯种卵囊并对它们的一些生物学特征进行了观察,为这些球虫的生活史和致病性研究奠定了基础.

微小隐孢子虫卵囊DNA提取及用于PCR检测

目的采用3种方法提取微小隐孢子虫卵囊DNA,并用于PCR检测以进行比较。方法微小隐孢子虫卵囊经多次冻融加热破壁后,采用螯合树脂(Chelex-100)、酚/氯仿和基因组DNA纯化系统试剂盒3种方法提取微小隐孢子虫卵囊DNA,并根据微小隐孢子虫基因序列(L16996)设计一对寡核苷酸引物,分别对3种方法制备模板进行PCR扩增分析。Chelex-100提取的DNA也用于观察PCR检测的敏感性。结果3种方法制备的微小隐孢子虫卵囊模板用于PCR检测均获得1条446 bp条带,Chelex-100提取的DNA用于PCR检测的敏感性至少达0.5个卵囊。结论3种方法提取的微小隐孢子虫卵囊DNA均可用于PCR检测,Chelex-100法是一种高效而快速的微量提取DNA方法,适用于对隐孢子虫DNA的检测。

隐孢子虫牛源分离株的分离和鉴定

目的分离和鉴定采自徐州自然感染奶牛粪便中的隐孢子虫卵囊。方法在徐州某奶牛场采集经改良抗酸染色镜检确认为隐孢子虫感染的奶牛粪便,用不连续Sheather’s蔗糖密度梯度离心法纯化卵囊。采用Chelex-100方法提取卵囊基因组DNA,设计引物扩增小亚基核糖体RNA(SSU rRNA)基因和卵囊囊壁蛋白(COWP)基因,分别克隆到pGEM-T和pGEM-T Easy载体,测定核苷酸序列,运用BLAST和MEGA软件进行序列同源性和种系发生分析。结果分离的隐孢子虫卵囊个体大小为(7．4±0．32)μm×(5．4±0．21)μm,长宽比为1．3±0．07(n=20)。徐州牛源隐孢子虫与Gen- Bank公布的安氏隐孢子虫比较,SSU rRNA和COWP基因序列同源性分别为100％和99％。种系发生分析显示该株隐孢子虫与安氏隐孢子虫处于同一分支。结论由徐州自然感染奶牛粪便中分离获得的隐孢子虫是安氏隐孢子虫。

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。

隐孢子虫卵囊纯化方法的改进研究

用蒸馏水代替PBS稀释Sheather's蔗糖溶液,并测定不同稀释度的蔗糖溶液密度,使用不同密度的蔗糖溶液分离纯化安氏隐孢子虫、贝氏隐孢子虫和猪隐孢子虫卵囊,计算纯化后的卵囊回收率和卵囊活性。安氏隐孢子虫、贝氏隐孢子虫和猪隐孢子虫卵囊纯化后的总回收率分别为47.5%、49.6%和41.7%;孔雀绿染色活性检测的活性率分别为97.9%、96.7%和95.1%;37℃水浴1h后的脱囊率分别为87.9%、80.0%和81.7%。