广东省乳牛隐孢子虫病的流行病学调查

对广东省10个奶牛场进行了隐孢子虫病的流行病学调查，并按大致10％的采样率采集了1087头乳牛的新鲜粪便，以饱和蔗糖漂浮法和改良抗酸染色法检查隐孢子虫卵囊，其中检出卵囊的阳性牛92头，阳性率为8．46％；有7个场检出隐孢子虫卵囊，场阳性率为70％。这7个奶牛场的卵囊阳性检出率分别是10．00％、7．19％、6．67％、9．80％、6．72％、12．76％和6．72％。调查发现，乳牛隐孢子虫的阳性率和感染强度与乳牛年龄呈负相关关系，而且可能与气候有关；所检出的隐孢子虫卵囊经形态学鉴定为鼠隐孢子虫(Crptosporidium muris)。     

隐孢子虫卵囊部分特性研究

本试验对小球隐孢子虫卵囊、鼠隐孢子虫卵囊以及新发现的隐孢子虫亚型卵囊的抗酸染色特性进行了观察，发现只有小球隐孢子虫卵囊具有酸染色特性。     

水牛隐孢子虫病的调查及实验感染的研究

以硫酸锌漂浮——耐酸滤过漏斗法,对181头1～7月龄水牛犊作卵囊纯化涂片镜检。结果发现自然感染的隐孢子虫2种:微小隐孢子虫(Cryptosporidium　parvum)和鼠隐孢子虫(Cryptosporidi-um muris),皆为本省首次报道。水牛犊感染率结果:以3月龄牛犊感染率为最高(18.3％),2月龄次之(15.1％),7月龄最低(2.3％),以后随月龄增长而呈下降趋势。小白鼠人工感染成功,第10d粪检均查找到2种隐孢子虫卵囊,但受试动物均未出现腹泻、厌食和消瘦等症状。     

安徽省黄牛隐孢子虫病流行病学调查

为查明安徽省黄牛隐孢子虫感染情况 ,取该省 6个县 (市 )进行了黄牛隐孢子虫感染情况的调研 ,在 42头黄牛的粪样中查到了隐孢子虫卵囊 ,其感染率为 1 5 2 7% (42 /2 75)。经鉴定 ,所获虫体为鼠隐孢子虫 (Cryptosporidiummuris)和小隐孢子虫 (Gryptosporidiumparvum)。黄牛隐孢子虫感染存在年龄和地区性差异 ,但与性别无关。     

应用PCR诊断隐孢子虫病

应用聚合酶链反应（ Ｐ Ｃ Ｒ）建立了一种诊断人及牛等哺乳动物隐孢子虫病的方法。试验采用甘油漂浮 Ｇ３ 耐酸漏斗过滤法纯化隐孢子虫卵囊，以液氮冻融法制备模板 Ｄ Ｎ Ａ，根据隐孢子虫 １８ Ｓｒ Ｒ Ｎ Ａ 序列设计 Ｐ Ｃ Ｒ 引物建立其诊断方法。该方法特异性强，可检出鼠隐孢子虫（ Ｃｒｙｐｔｏｓｐ ｏｒｉｄｉｕｍ ｍ ｕｒｉｓ）和小球隐孢子虫（ Ｃ．ｐａｒｖｕｍ ）卵囊；敏感性高，每克粪便可检出 ４００ 个卵囊。初步应用结果表明，所建立的 Ｐ Ｃ Ｒ 方法适合于人、牛等哺乳动物隐孢子虫病的临床诊断和流行病学调查。     

人源隐孢子虫小白鼠感染模型的建立

为评价2种常用免疫抑制方法对小白鼠排人源隐孢子虫卵囊规律的影响,将19日龄小白鼠随机分成2组,第1组通过饮水给予地塞米松,第2组采用导胃管灌服地塞米松。第7 d,2组小白鼠灌胃接种人源隐孢子虫,每只小白鼠感染量为1.5×106个卵囊。两组均在感染当天就有卵囊排出,感染后第6 d粪便卵囊计数明显增加,随之出现3个高峰期,此后逐渐下降。免疫抑制组小白鼠在卵囊持续期不断有死亡但无腹泻症状。试验结果表明,用这2种免疫抑制方法,均能成功感染人源隐孢子虫,但通过灌服地塞米松可使小白鼠获得更高的OPG(每克粪便中的卵囊数)值和更长的排卵囊持续期。     

水貂隐孢子虫病流行病学调查及虫种的初步鉴定

为初步了解水貂隐孢子虫病的流行情况,作者于2005年11月份用饱和蔗糖溶液漂浮法检查了河北省肃宁县某水貂养殖场的469份粪便样品。结果,8份粪便样品为隐孢子虫阳性,总感染率为1.71%(8/469)。其中,白貂感染率为2.15%(5/233)、灰貂感染率为2.08%(1/48)、黑貂感染率为1.06%(2/188)。所查的8份隐孢子虫阳性样品均来自5~6月龄的水貂,表明幼龄水貂容易感染隐孢子虫病而老龄水貂不易感染。另外,8份阳性样品多数来自雄性水貂,显示水貂的隐孢子虫感染可能存在性别的差异性。根据卵囊形态和大小将水貂隐孢子虫初步鉴定为小球隐孢子虫(Cryptosporidium parvum)。同时,利用所收集的隐孢子虫卵囊进行了小白鼠感染试验,结果表明水貂源隐孢子虫不感染免疫抑制状态下的昆明系小白鼠。     

固始鸡源隐孢子虫人工感染试验

为了解来源于地方鸡品种的隐孢子虫分离株致病特点,对收集到的河南固始鸡源隐孢子虫经鹌鹑传代纯化后,进行动物感染。结果:固始鸡源隐孢子虫分离株无论在正常还是免疫抑制情况下均不能感染小鼠,但能成功感染海兰雏鸡,出现明显的呼吸道症状及法氏囊病变。剖检发现虫体主要寄生在法氏囊、气管和泄殖腔等部位。根据卵囊形态学及寄生部位等特点,本试验分离的隐孢子虫种类鉴定为贝氏隐孢子虫(Crypto-sporidium baileyi)。增大感染剂量,可使雏鸡排卵囊高峰期提前,排卵囊量增大,持续期延长;免疫抑制剂的使用也可使高峰期提前,持续期延长,但会造成试验动物死亡率增高。雏鸡临床症状、剖检病变和增重减少均与感染剂量呈正相关,免疫抑制剂的使用会加重此影响。     

双抗夹心─ELISA诊断兔隐孢子虫病初步研究

本文报道了应用双抗夹心─ＥＬＩＳＡ检测兔粪便中隐孢子虫卵囊抗原方法的建立。试验所用抗体为抗小球隐孢子虫（Ｃ．ｐａｒｖｕｍ）卵囊壁的单克隆抗体，经对２０头份兔粪便样本分别进行抗酸染色和双抗夹心─ＥＬＩＳＡ试验．结果抗酸染色法检出８份有隐孢子虫卵囊，而ＥＬＩＳＡ法除对抗酸染色阳性的８份粪样判为阳性外，还对抗酸染色阴性的１份粪样判为阳性；并不与兔球虫粪便发生类属反应。此外，本试验还在稀释液中加入ＥＤＴＡ，并增加了反应温度，使得试验在抗体包被反应板并封闭完成后３０分钟结束整个检测过程。     

合肥市奶牛隐孢子虫病流行病学调查

为查明合肥市某奶牛场奶牛隐孢子虫感染情况,采用饱和白糖溶液漂浮法对该场381头奶牛的粪样进行检查,在52头奶牛的粪样中查到了隐孢子虫卵囊,其感染率为13.62%(52/381)。经鉴定,所获虫体为鼠隐孢子虫Crtptosporidium.muris。     

Activity of decoquinate against Cryptosporidium parvum in cell cultures and neonatal mice

Cryptosporidium parvum is an apicomplexan parasite that is an important cause of diarrhea in neonatal calves and humans. No treatment is currently available for neonatal calves. We have recently learned from colleagues in the pharmaceutical industry that dairy practitioners are sometimes using decoquinate for the treatment of neonatal bovine cryptosporidiosis. Therefore, the present study was undertaken to determine whether the clinical observations in calves can be substantiated by laboratory investigation. Oocysts of the KSU-1 isolate of C. parvum were used to infect human ileocecal epithelial cells in vitro to measure the efficacy of treatment using an ELISA based assay. No activity was observed at 10 or 50microM decoquinate, but at 100microM an 8% inhibition of development was seen. Oocysts of the AUCp-1 isolate of C. parvum were then used to infect suckling mice. The numbers of oocysts observed in suckling mice treated with 2.5 or 5.0mg/kg decoquinate were not significantly different from untreated control suckling mice (p0.05). The results of our study suggest that decoquinate should have little efficacy for treatment of neonatal bovine cryptosporidiosis if administered once per day and that any clinical improvement observed in treated calves may be due to factors unrelated to decoquinate's effect on C. parvum.     

Infectivity of Cryptosporidium parvum isolated from asymptomatic adult goats to mice and goat kids.

An experimental study was carried out in neonatal goat kids to examine the infectivity of Cryptosporidium oocysts, pattern of oocyst shedding and morphological changes in the intestine during the infection. Cryptosporidium oocysts isolated from adult asymptomatic goats, and identified as C. parvum by polymerase chain reaction (PCR) were used in this study. Of three 4-day-old goat kids, two were orally infected with C. parvum oocysts (10(5) oocysts in 10 ml PBS/kid). One goat kid given 10 ml PBS only by the oral route served as a control. Cryptosporidium oocysts were detected in the faeces of one infected kid on day 3 post-inoculation (pi) whereas in the other 6 days pi. The faecal oocyst counts gradually increased and the peak counts in the two kids were 2 x 10(6)g(-1) (on day 12 pi) and 3.2 x 10(6)g(-1) (on day 14 pi). The increase in faecal oocyst output coincided with diarrhoea in an infected kid from days 10-17 pi. Although the oocyst excretion declined gradually after the peak, both infected kids excreted oocysts until euthanized on days 20 and 22 pi. Light and scanning electron microscopic investigations of the ileum revealed the endogenous stages on the brush border of the enterocytes, infiltration of neutrophils and mononuclear cells into the lamina propria, atrophy, stunting and fusion of villi. For purposes of comparison, goat Cryptosporidium oocysts were inoculated orally (10(3) oocysts/mouse) to eight, 1-week-old mice. All experimental mice excreted oocysts from day 3 pi, and four infected mice continued to excrete oocysts up to day 42 pi. The experimental infection described in goat kids resembled the natural disease in terms of oocyst excretion, clinical signs and intestinal pathology. The ability of oocysts excreted by asymptomatic goats, to infect goat kids and mice is likely to have a major impact on the epidemiology of cryptosporidiosis in livestock and man.     

Viability and infectivity of Cryptosporidium parvum oocysts detected in river water in Hokkaido,Japan

The viability and infectivity of Cryptosporidium parvum (C. parvum) oocysts, detected in water samples collected from river water in Hokkaido, were investigated using Severe Combined Immunodeficient (SCID) mice. The water samples collected from September 27 through October 10, 2001 by filtration using Cuno cartridge filters were purified and concentrated by the discontinuous centrifugal flotation method. From 1.2 x 10 (5) liters of the raw river water, approximately 2 x 10(4) oocysts were obtained and designated as Hokkaido river water 1 isolate (HRW-1). Oocyst identification was carried out using microscopic and immunological methods. Six 8-week-old female SCID mice were each inoculated orally with 1 x 10 (3) oocysts. Infection was successfully induced, resulting in fecal oocyst shedding. Oocysts were then maintained by sub-inoculation into SCID mice every 3 months. Infectivity was evaluated by making comparisons with two known C. parvum stocks, HNJ-1 and TK-1, which were bovine genotypes detected in fecal samples from a cryptosporidiosis patient and young cattle raised in Tokachi, Hokkaido respectively. The oocyst genotypes were determined from a small subunit ribosomal RNA (SSU-rRNA) gene by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis. No significant differences were observed in the average number of oocysts per gram of feces (OPG) in any of the isolates. Our data indicates that the C. parvum oocysts detected in the sampled river water were of C. parvum genotype 2. Moreover, our data on the continued isolation, detection and identification of the C. parvum isolates is consistent with the available epidemiological data for the Tokachi area.     

Cryptosporidium parvum: investigation of sporozoite excystation in vivo and the association of merozoites with intestinal mucus

Enteric cryptosporidiosis was studied in the small intestine of five-day-old sucking mice after infection with 10(6) Cryptosporidium parvum oocysts. It was shown that excystation and the majority of subsequent endogenous stages occurred predominantly in the ileum. During the first three days of infection the number of merozoites collected in ileal washings increased over 100-fold to approximately 10(6) merozoites per mouse on the third day. In contrast to control mice, wash fluid from infected mice contained numerous strands of dislodged mucus. Estimates of mucus in the ileal washings of infected mice were similar to those made in controls until day 4 after infection when they increased and remained high throughout the remainder of the experiment. This study describes a method whereby ileal mucus washings from C parvum infected infant mice could be used as a rich source of merozoites.     

Cryptosporidium: a water-borne zoonotic parasite

Of 155 species of mammals reported to be infected with Cryptosporidium parvum or C. parvum-like organisms most animals are found in the Orders Artiodactyla, Primates, and Rodentia. Because Cryptosporidium from most of these animals have been identified by oocyst morphology alone with little or no host specificity and/or molecular data to support identification it is not known how many of the reported isolates are actually C. parvum or other species. Cryptosporidiosis is a cause of morbidity and mortality in animals and humans, resulting primarily in diarrhea, and resulting in the most severe infections in immune-compromised individuals. Of 15 named species of Cryptosporidium infectious for nonhuman vertebrate hosts C. baileyi, C. canis, C. felis, C. hominis, C. meleagridis, C. muris, and C. parvum have been reported to also infect humans. Humans are the primary hosts for C. hominis, and except for C. parvum, which is widespread amongst nonhuman hosts and is the most frequently reported zoonotic species, the remaining species have been reported primarily in immunocompromised humans. The oocyst stage can remain infective under cool, moist conditions for many months, especially where water temperatures in rivers, lakes, and ponds remain low but above freezing. Surveys of surface water, groundwater, estuaries, and seawater have dispelled the assumption that Cryptosporidium oocysts are present infrequently and in geographically isolated locations. Numerous reports of outbreaks of cryptosporidiosis related to drinking water in North America, the UK, and Japan, where detection methods are in place, indicate that water is a major vehicle for transmission of cryptosporidiosis.     

The homologous and interspecies transmission of Cryptosporidium parvum and Cryptosporidium meleagridis

Experimental infections have been performed in several mammals and birds to determine the host's specificity for Cryptosporidium parvum (of human and bovine origin) and for Cryptosporidium meleagridis (isolated from broiler chicken). The C. parvum infection (of human origin) was established also in calves (Bos taurus), lambs (Ovis aries), mice (Mus musculus), and rats (Ratus norvegicus), but not in broiler chickens (Gallus domestica). The C. parvum infection (of bovine origin), was achieved in calves, lambs, dogs (Canis familiaris), cats (Felis domesticus), rabbits (Orytolagus cuniculus), mice, rats, and guinea-pigs (Cavia porcelus) but not in broiler chickens. We have demonstrated that C. meleagridis can produce infection in broiler chickens and in 5 mammalian species (calves, pigs, rabbits, rats, mice) but not in guinea-pigs.     

Infectivity to experimental rodents of Cryptosporidium parvum oocysts from Siberian chipmunks (Tamias sibiricus) originated in the People's Republic of China

We isolated Cryptosporidium parvum-type oocysts from naturally infected siberian chipmunks which originated in the People's Republic of China and examined the infectivity to rodents as experimental animals. The naturally infected chipmunks did not show any clinical symptoms. The oocysts were 4.8 x 4.2 microm on average in size. They were ovoid and morphologically similar to the C. parvum oocysts isolated from human and cattle. Experimental rodents were inoculated with 1.6 x 10(6) original oocysts each. SCID mice began to shed oocysts on day 7 and the OPG value was 10(5) from 50 days. The oocysts were found from ICR mice on days 13 and 16 by only sugar flotation method, however, any oocysts were not detected from the rats, guinea pigs and rabbits until 30 days. Two infected SCID mice were necropsied on days 100 and 102 and examined for coccidian organisms. Merozoites and oocysts were found in the low part of jejunum and ileum, however, no parasites were detected in the stomach. Consequently, it was considered that the present species was C. parvum and was probably genotype 2 from result of infectivity to rodents.     

Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea

OBJECTIVE: To determine duration of infection and association of infection with diarrhea for dairy calves with naturally acquired cryptosporidiosis and giardiosis. DESIGN: Cohort study. ANIMALS: 20 Holstein calves on a single dairy farm. PROCEDURE: Fecal samples were collected 3 times/wk for the first 45 days after birth, then weekly until calves were 120 days old and examined for Giardia duodenalis cysts and Cryptosporidium parvum oocysts. Calves were monitored for diarrhea during the first 45 days after birth; during each episode of diarrhea, fecal samples were examined for parasitic, bacterial, and viral pathogens. RESULTS: All 20 calves shed Giardia cysts and Cryptosporidium oocysts at some time during the study. Mean ages at which Giardia cysts and Cryptosporidium oocysts were first detected were 31.5 and 16.3 days, respectively. Mean number of Giardia cysts in feces remained high throughout the study, whereas Cryptosporidium occysts decreased to low or undetectable numbers 2 weeks after infection. Eighteen calves had a total of 38 episodes of diarrhea during the first 45 days after birth. Giardia duodenalis was the only pathogen identified during 6 (16%) episodes, C parvum was the only pathogen identified during 9 (24%) episodes, and G duodenalis and C parvum were identified together during 10 (26%) episodes. CONCLUSIONS: Prevalences of giardiosis and cryptosporidiosis were high in these calves, and both parasites were associated with development of diarrhea. Cryptosporidium parvum was an important pathogen when calves were < 1 month old, but G duodenalis was more important when calves were older. Calves cleared C parvum infections within 2 weeks; however, G duodenalis infections became chronic in these calves.     

Prevalence of Cryptosporidium parvum infection and pattern of oocyst shedding in calves in Japan

Cryptosporidium parvum infection and the pattern of oocyst shedding were observed in calves. A total of 480 fecal samples were collected from 30 calves (age, < or =30 days) over a period of 10 months from June 1998 to March 1999. A sucrose centrifugal flotation technique revealed 28/30 (93%) calves were passing Cryptosporidium oocysts. Oocyst shedding was first detected on the sixth day after birth, with 8% of the calves testing positive. This rate increased day by day and reached approximately 80% by day 15. Oocyst shedding varied from 1 to 13 days, with a mean of 7 days. Calves infected with C. parvum had a significantly higher rate of diarrhea (33%) than non-infected calves (8%) (P<0.05), suggesting C. parvum infection as the likely cause. The mean number of oocysts excreted by calves < or =30 days old was approximately 6x10(7) per gram of feces. These results indicated that one calf would excrete some 6x10(11) oocysts in the first month after birth, taking both the quantity of feces in a day and the period of excretion into consideration. Accordingly, it is clear that calves are important in the spread of cryptosporidiosis to calves and humans.     

Immunization protocols against Cryptosporidium parvum in ovines: protection in suckling lambs

Ovine colostrum and milk from immunized ewes were tested for their ability to prevent cryptosporidiosis in the lambs experimentally infected with 10(6) oocysts of Cryptosporidium parvum at 36-48 h of age (day 0 post-infection). All lambs became infected and developed clinical cryptosporidiosis. However, lambs fed by immunized dams have shown shedding involved, significantly, fewer oocysts and lasted for a shorter period than in control lambs. In addition, diarrhoea was less severe. The best results emerged in lambs of ewes immunized by intramuscular injection of an emulsion of 2 ml of Freund's complete adjuvant and 2 ml of C. parvum antigen in sterile phosphate buffered saline solution, administrated four weeks before parturition, together with an intramammary infusion of 25 microg of antigen in 2 ml of sterile PBS emulsified in 2 ml of Freund's incomplete adjuvant, which showed the highest anti-C. parvum titres in lacteal secretions. In their case, the onset of output of oocysts was delayed by two days, the patent period was shortened by three days, their diarrhoea continued for only three days, and the quantity of oocysts shed decreased by 77%. The outcome was that at the end of the study they had a live weight gain of 2 kg more than the lambs in the control group. These results indicate that lactogenic immunoprophylaxis should help mitigate the financial losses caused by cryptosporidiosis in small ruminants, as well as reducing the risk of infection of humans through the decreased contamination of the environment with oocysts.