Prevalence of Giardia sp. Cryptosporidium parvum and Cryptosporidium andersoni (syn. C. muris) [correction of Cryptosporidium parvum and Cryptosporidium muris (C. andersoni)] in 109 dairy herds in five counties of southeastern New York

A cross-sectional study was undertaken to determine the prevalence of Giardia sp. (G. duodenalis group), Cryptosporidium parvum and Cryptosporidium andersoni (C. muris) [corrected] in dairy cattle in three different age groups, and to evaluate the association of age and season with prevalence. One hundred and nine dairy farms, from a total of 212 farms, in five counties of southeastern New York volunteered to participate. On these farms, 2943 fecal samples were collected from three defined age groups. The farms were randomly assigned for sampling within the four seasons of the year. Each farm was visited once during the study period from March 1993 to June 1994 to collect fecal samples. Demographic data on the study population was collected at the time of sampling by interviewing the farm owner or manager. At collection, fecal samples were scored as diarrheic or non-diarrheic, and each condition was later related to positive or negative infection with these parasites. Fecal samples were processed using a quantitative centrifugation concentration flotation technique and enumerated using bright field and phase contrast microscopy. In this study, the overall population prevalence for Giardia sp. was 8.9%; C. parvum, 0.9%; and C. muris, 1.1%. When considering animals most at the risk of infection (those younger than 6 months of age) Giardia sp. and C. parvum was found in 20.1 and 2.4% of the animals, respectively. Giardia sp. and C. muris were found in all age groups. There was no significant seasonal pattern of infection for any of these parasites.     

安氏隐孢子虫PCR诊断试剂盒的初步应用

运用首次研制的安氏隐孢子虫PCR诊断试剂盒对广东省4个奶牛场和河南省1个奶牛场共234份样品,进行了安氏隐孢子虫感染的实际检测,并与常规检测方法饱和蔗糖漂浮法、改良抗酸染色法进行了比较。本试剂盒的检出率比常规检测方法提高了2%～13%,显示该试剂盒具有特异、敏感等优点,对开展隐孢子虫病的鉴别诊断和分子流行病学调查具有重要的应用价值。     

Multiattribute evaluation of two simple tests for the detection of Cryptosporidium parvum in calf faeces

There is a need for simple and inexpensive diagnostic and screening tests for the detection of Cryptosporidium parvum infection in calves. A sucrose wet mount test and a lateral immunochromatography test were evaluated for epidemiological sensitivity and specificity, cost per test, simplicity, test time and ease of batching. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the Cryptosporidium oocyst wall protein (COWP) gene locus, with gel electrophoresis, was used as a gold standard. Cohen's kappa statistic of agreement (kappa) between the Ontario Veterinary College (OVC) sucrose wet mount test and COWP PCR-RFLP was 0.82, and the sensitivity and specificity of the OVC sucrose wet mount test were 88.6% and 93.8%, respectively. The sensitivity and specificity of the lateral immunochromatography test were 78.3% and 93.3%, respectively, and agreement between this test and PCR-RFLP was good (kappa=0.73). There was substantial agreement between the OVC sucrose wet mount test and the lateral immunochromatography test (kappa=0.84). Both tests were inexpensive and easy to use; however, the lateral immunochromatography test was faster and simpler to perform than the sucrose wet mount test, and was generally more user-friendly. These tests provide practitioners and researchers with cheap, quick and accurate methods of detecting C. parvum infection in young calves.     

Comparison of viability assays for Cryptosporidium parvum oocysts after disinfection

In order to test various viability assays for Cryptosporidium parvum oocysts were used to infect HCT-8 cells in vitro or baby mice. Infected cells were either stained with fluorescent anti-Cryptosporidium-antibody or lysed and subjected to C. parvum-specific PCR after 48 h. Titrations with infective oocysts were performed and compared to oocysts disinfected with Neopredisan for 2 h at varying concentrations. Caecal smears and histological sections from infected animals were examined in parallel. The number of foci of parasite development in vitro after immunofluorescent staining correlated well with the infection dose. PCR was less quantifiable and the results were not always reproducible, especially when low infection doses were used. Disinfection resulted in a dose-dependent reduction of oocyst infectivity when compared to the controls in all three assays. The infection of cells cultured in vitro with oocysts of C. parvum provides a suitable tool for the estimation of viability after treatment with chemical disinfectants. Immunofluorescence is easy to perform and gives quantitative results, while PCR-based detection of parasite DNA, although possible, requires the use of more sophisticated tools for quantification.     

Cryptosporidium andersoni from a Danish cattle herd: identification and preliminary characterisation

In November 1997, Cryptosporidium andersoni, for the first time, was isolated from a Danish heifer. The isolate was characterised morphologically, molecularly, and furthermore inoculated into mice and one calf. Data on the distribution of cryptosporidia in the herd of origin were obtained at two separate visits in December 1997 and April 1998. C. andersoni was detected in 27 (19.0%) of 142 cattle examined at the first visit, whereas C. parvum was found in six (4.2%). At the following visit 42 (28.0%) of 150 cattle excreted C. andersoni, while 25 (16.7%) were positive for C. parvum. Oocysts of the Danish C. andersoni isolate were ovoid, 7.3(6.5-8.0) x 5.7(5.0-7.0) microm(2) (n=25), with smooth, colourless, single layer oocyst wall and distinct oocyst residuum. The length to width ratio was 1.27 (1.14-1.40, n=25). The identification was verified by sequencing of a 246bp fragment of the rDNA, which was identical to Cryptosporidium muris, the calf genotype (AF093496). The Danish C. andersoni isolate was not transmissible to mice, whereas oocysts were detected in the faeces of one experimentally infected calf from 25 days post-infection (DPI) and shed intermittently at low numbers until 165 DPI, the day of euthanasia. No macroscopic or microscopic changes that could be attributed to infection with C. andersoni were seen in the gastro-intestinal tract of the experimentally infected calf following necropsy and histological examination. This is to our knowledge the first report of C. andersoni in Scandinavia.     

河北省奶牛场犊牛安氏隐孢子虫的分离鉴定

本研究从河北省奶牛场有腹泻症状2月龄左右的犊牛粪便中分离卵囊,进行病原分离与虫株鉴定。采集腹泻犊牛的新鲜粪便24份,采用饱和蔗糖溶液漂浮法和抗酸染色法检测隐孢子虫卵囊,观察卵囊形态、大小。提取卵囊基因组DNA,进行18S rRNA基因PCR扩增及琼脂糖凝胶电泳检测。对扩增片段进行序列测定及分析,进一步确定分离虫株隐孢子虫的种类/基因型,根据18S rRNA基因核苷酸序列构建系统发育进化树,确定虫株亲缘关系。结果显示,5份样品检出隐孢子虫卵囊,感染率为20.83%。形态学观察卵囊呈长圆形或椭圆形,大小为(5.0～8.2)μm×(4.2～6.3)μm,平均大小为6.6μm×5.3μm,卵囊指数为1.24,鉴定分离虫株为安氏隐孢子虫。PCR扩增出预期大小为1 188 bp的特异性片段,序列分析和同源性分析结果表明,分离株与安氏隐孢子虫AB089285.2株、AB513856.1株、AY954885.1株的同源性为98.7%～98.8%,进一步表明分离的隐孢子虫虫株为安氏隐孢子虫。在种系进化关系上,分离株与安氏隐孢子虫AB513856.1株亲缘关系最近。本研究为揭示河北省奶牛隐孢子虫病的流行特征,实施有效防制措施提供了科学依据。     

Quantitative comparison of different purification and detection methods for Cryptosporidium parvum oocysts

Cryptosporidium parvum (C. parvum) is the causal agent of cryptosporidiosis in many animals, mainly cattle, and possesses a high zoonotic potential. It occurs worldwide and ubiquitously. Detection of C. parvum is mainly performed directly but purification of the oocysts is useful to increase sensitivity and to obtain oocyst material for further use. The study was designed to compare (a) three different direct diagnostic methods, namely modified Ziehl-Neelsen staining, carbol fuchsin staining and conventional PCR, and (b) three routine oocyst purification methods, in particular flotation with saturated sodium chloride solution, Sheather's sucrose solution and a Percoll(?) gradient. During comparison of purification methods, special regard was paid to the ability to separate morphologically intact oocysts from the morphologically degenerated fraction or viable from non-viable oocysts, respectively. Results: (a) Diagnostic methods: Most effective in C. parvum oocysts detection in calf faeces was PCR; carbol fuchsin and modified Ziehl-Neelsen stainings achieved comparable results. (b) Purification methods: Oocyst flotation using sodium chloride solution showed to be superior to Percoll(?) gradient centrifugation and sugar flotation in terms of purification quality, recovery efficacy (yield) and reduction of the proportion of degenerated or non-viable oocysts.     

Morphological and immunohistochemical features of Cryptosporidium andersoni in cattle

Light and electron microscopic features and immunohistochemical features of Cryptosporidium andersoni (C. andersoni) and host reaction in the mucosa were studied. Although the affected cattle demonstrated no apparent clinical signs, a severe infection of C. andersoni was observed in the abomasum. C. andersoni were round in shape, measured 6-8 microm in size and were mainly observed to be freely located in the gastric pits, being attached in occasional cases to the surface of the abomasum epithelium. Frequent inflammatory cells had infiltrated the lamina propria of the affected mucosa, and frequent mitotic figures were observed in epithelial cells at the dilated isthmus. To access the cell kinetics, the number of epithelial cells infected with C. andersoni were counted and compared with noninfected cattle. The number of gastric pit cells in infected cattle was significantly higher than that in the controls. The number of proliferative cells determined by the Ki-67 antigen in C. andersoni infected cattle was also significantly higher than that in the controls. Transmission electron microscopy and scanning electron microscopy revealed that the morphology of the C. andersoni organism was common to those of other Cryptosporidium spp. Immunohistochemically, several commercial antibodies against Cryptosporidium spp. showed positive reactions at the wall of these oocysts or parasitophorous vacuoles. This report is possibly the first to discuss the prominent hyperplasia of the abomasum mucosa, as well as morphologic features of C. andersoni in cattle.     

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.     

安氏隐孢子虫肌动蛋白部分编码基因的表达及免疫保护性

以筛选安氏隐孢子虫T7噬菌体展示文库获得的肌动蛋白（CA42）部分编码基因的序列设计特异性引物,扩增目的基因CA42,构建重组表达载体pET-28a-CA42,通过大肠杆菌BL21的诱导表达,产物经SDS-PAGE和Western-blotting鉴定。然后纯化重组蛋白,免疫BALB／c进行体液和细胞免疫水平的检测,并观察重组蛋白对微小隐孢子虫的交叉保护性效果。结果显示,成功构建重组原核表达质粒pET-28a-CA42,Western-blotting显示重组蛋白约为19ku,能被安氏隐孢子虫免疫小鼠的多克隆抗体识别。重组蛋白免疫BALB／c小鼠的抗体水平差异显著（P〈0．05）,CD4^＋T淋巴细胞数差异均显著,CD4^4／CD8^＋T淋巴细胞数的值与佐剂对照组和空白对照组相比差异均显著（P〈0．05）。各组小鼠经接种微小隐孢子虫卵囊后,试验组与各对照组相比卵囊减少率为32．2％,差异均显著（P〈0．05）。结果表明,成功表达了重组安氏隐孢子虫肌动蛋白,纯化的重组蛋白具有一定的免疫原性和交叉保护性。     

Cryptosporidium parvum in diarrheic calves detected by microscopy and identified by immunochromatographic and molecular methods

Cryptosporidium is an important protozoan parasite that causes diarrhea in neonates and young bovines. The objective of the present study was to determine the frequency of Cryptosporidium infection in animals of dairy farms of the Metropolitan Region (Santiago), Chile. Fecal samples of 205 newborn calves with diarrhea were studied and used for comparing the efficiency of two microscopic staining methods for diagnosis of the parasite, the auramine (AU) and a modified Ziehl-Neelsen (ZN) procedure. Out of the 205 fecal samples, we detected oocysts in 115 (56.1%) with AU and 102 (49.8%) with ZN. Comparison of results obtained with the two microscopic techniques showed significant difference (p<0.05), AU being more sensitive. On the other hand, concordance between the two methods was almost perfect (kappa value of 0.83). The results with these two operator dependent methods were confirmed using an operator independent immunochromatographic (IC) method. The IC method also enabled us to determine the identity of the parasite species as that of Cryptosporidium parvum. Identification of the parasite species was further corroborated by performing a Cryptosporidium species-specific polymerase chain reaction (PCR) test on few samples taken at random. Overall, the results showed a high number of infected animals suggesting the parasite C. parvum as a major parasitic disease agent of neonatal calves with diarrhea in dairy farms of the Metropolitan Region (Santiago) of Chile.     

应用巢式PCR-RFLP方法鉴别微小隐孢子虫、安氏隐孢子虫和火鸡隐孢子虫的研究

应用巢式聚合酶链反应（Nested PCR）-限制片段长度多态性 （restriction fragment length polymorphism, RFLP）方法对微小隐孢子虫（C．parvum）、安氏隐孢子虫（C．andersoni）和火鸡隐孢子虫（C．meleagrides）的鉴别进行了研究。结果显示C．parvum BOCC2、C．andesoni BOCC2和C．meleagrides CHCC1扩增产物片段大小分别为830bp、828bp和828bp，扩增产物分别经VspI酶切后形成3种不同的RFLP图谱，根据RFLP图谱可鉴别C．parvum、C．andersoni和C．meleagrides。本研究为我国隐孢子虫的分类和隐孢子虫病的分子流行病学研究打下了良好基础。     

Prevalence and pathogenicity of Cryptosporidium andersoni in one herd of beef cattle

Over a 35-week period from January to July 2002, a breed of Hereford beef cattle (H) and their hybrids were monitored. Five hundred and ninety-nine individual fecal samples from calves and 96 samples from their mothers were examined. First excretion of Cryptosporidium andersoni oocysts in calves was found in the 9th week after the start of calving (a calf 63-day old). The prevalence of C. andersoni in calves ranged from 11.1 to 92.9% depending on age. The mean prevalence in their mothers was found to be 43.8%. The size of oocysts was 8.48 +/- 0.78 x 6.41 +/- 0.59 microm. Infection intensity in calves ranged from 32 000 to 4 375 000 oocysts per gram (OPG) and in mothers from 78 000 to 2 552 000 OPG. Three cases of abomasal cryptosporidiosis slaughtered at the age of 81, 157 and 236 days were examined histologically and ultrastructurally [transmission electron microscopy (TEM) and scanning electron microscopy (SEM)]. Cryptosporidium infection of the abomasum was located in the upper half of the mucosal glands in the plicae spirales of the fundus, corpus and near the ostium omasoabomasicum. Cryptosporidia were not located in the glandular epithelium of the pars pylorica in the abomasum minimally 10 cm from pylorus. Histopathological changes in the site of cryptosporidial infection in the abomasum had a non-inflammatory character and included distinctive dilatation of infected parts of the glands with atrophy and metaplasia of the glandular epithelial cells, goblet cell activation and mucus hyperproduction. The TEM revealed a relatively small number of Cryptosporidium life cycle stages attached to glandular epithelial cells. In SEM the inner mucosal abomasal surface appeared swollen but was never infected by cryptosporidia.     

Assessment of three methods for multilocus fragment typing of Cryptosporidium parvum from domestic ruminants in north west Spain

The performance of three different methods, capillary electrophoresis (CE), high resolution slab-gel electrophoresis and sequencing, for PCR fragment size analysis of two Cryptosporidium parvum microsatellite regions, ML1 and ML2, was investigated by analysing 27 isolates from calves and 14 from lambs. To assess genetic variability of this protozoan in domestic ruminants in north west Spain, results were combined with sequence analysis of the 60 kDa glycoprotein (GP60) gene creating a multilocus type and analysed by farm and host species. CE showed greater overall typability (T), discriminatory power and ease of use than slab-gel electrophoresis and sequencing which were both affected by PCR stutter, especially at ML2. CE fragment sizes were consistently 4 bp longer compared to sequencing which is considered the gold standard for allele sizing but which gave the lowest typability; CE sizes were therefore adjusted. Only three alleles were identified at the ML1 locus (ML1-238, ML1-229 and ML1-226). The ML2 locus was more polymorphic and eight alleles were found (ML2-235, ML2-233, ML2-231, ML2-229, ML2-227, ML2-225, ML2-201 and ML2-176). Adjusted ML1 and ML2 CE fragment sizes were combined with GP60 subtype for 37 of the 41 C. parvum isolates which were typable at all three loci (T=0.90): nine multilocus types (MLTs) were identified. The discriminatory power of the 3-locus typing method was 0.83. Greater genetic variability was observed in calf isolates (7 MLTs) than in those from lambs (4 MLTs) although more calf isolates were studied. The most common MLT in cattle was MLT1 (ML1-238, ML2-231, GP60 subtype IIaA15G2R1), while MLT3 (ML1-238, ML2-227, GP60 IIaA16G3R1) was predominant in lambs. Our findings demonstrate that high discrimination can be achieved by means of multilocus typing. CE appears to be an economic and rapid option for performing microsatellite fragment size analysis offering good typability, discrimination and ease of use but may require calibration to sequenced standards.     

Molecular identification of Cryptosporidium parvum and Giardia duodenalis in the Italian water buffalo (Bubalus bubalis)

Livestock are commonly infected with protozoan parasites of the genera Cryptosporidium and Giardia, and some of the species and genotypes found in these animals have zoonotic significance. We characterized isolates of both parasites recovered from the Italian water buffalo (Bubalus bubalis), an economically important species whose milk is used for the production of "buffalo mozzarella" fresh cheese. Molecular analysis of the Cryptosporidium small subunit ribosomal DNA gene and of the Giardia beta-giardin gene shows the presence of both zoonotic parasites (Cryptosporidium parvum and Giardia duodenalis assemblage A) and host-specific parasites (G. duodenalis assemblage E), suggesting that water buffaloes can contribute to environmental contamination with oocysts and cysts potentially infectious to humans if their faeces are improperly disposed of. On the other hand, mozzarella cheese is probably a safe product, given that its production involves the treatment of cheese curd at 85-95 degrees C, which is likely to kill or inactivate the parasites.     

奶牛隐孢子虫病流行病学调查及初生犊牛感染试验

应用饱和糖溶液漂浮法和改良抗酸染色法调查郑州、商丘和洛阳3个地区的5个奶牛场、2个专业村的582份粪样，查出阳性样品64份，隐孢子虫总阳性率11％（64／582），发现两种不同形态的卵囊，根据其形态结构等特点鉴定为小球隐孢子虫（C．parvum）和安氏隐孢子虫（C．andersoni）。其中2个场奶牛感染安氏隐孢子虫，有1个场的奶牛感染小球隐孢子虫，且感染强度较小。犊牛感染率较育成牛、成年牛高。并进行小球隐孢子虫分离株对初生犊牛的致病性试验，其结果为潜隐期7天，排卵囊高峰出现在感染后第16天，高峰期5天。剖检后消化道黏膜经抗酸染色鉴定，仅在回肠中段发现卵囊。     

Real-time qPCR检测安氏隐孢子虫卵囊方法的建立及应用

根据GenBank公布的安氏隐孢子虫SSU rRNA基因序列设计1对引物和TaqMan探针,建立了基于TaqMan探针检测安氏隐孢子虫的实时荧光定量PCR方法,并对奶牛粪便进行了检测.结果显示,设计的探针对检测安氏隐孢子虫具有很高的特异性;粒DNA和卵囊的检测阈值分别达到5个拷贝和10个卵囊,奶牛粪便阳性率为21.15%(11/52).建立的安氏隐孢子虫TaqMan荧光定量PCR检测方法简便、快速,特异性强,敏感度高,可用于安氏隐孢子虫的快速定量检测.