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gCryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidiumpositive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

This study was conducted to determine the prevalence and risk factors for Cryptosporidium infection in calf neonates on dairy farms in Normandy. Fecal samples were randomly collected between July 2010 and September 2011 from 968 calves (7-21 days old) on 97 farms. Up to 10 calves were selected and sampled per farm, and feces examined for oocysts by microscopy. C. parvum oocyst shedding was scored semi-quantitatively (0-5). A questionnaire about calf-level care and management was completed, and mortality rates were obtained from the French national registration database (BDNI). Bivariable and multivariable analyses of potential risk factors for C. parvum oocyst shedding were conducted using generalized estimating equation (GEE) models (family=Binomial).Overall, 402 out of 968 calves (41.5%) were positive for oocysts, and 25.1% of animals had a shedding score >2. Seven of the 97 farms (7%) were negative for oocysts in all fecal samples. At the time of collection, 375 calves (39%) had diarrhea, and its prevalence strongly correlated with the score for C. parvum oocyst shedding (p<0.0001). The mortality rate at 90 days was significantly greater for calves with high combined scores of diarrhea and shedding. Factors associated with the shedding of C. parvum were the Normande breed (odds ratio=1.49; 95% confidence interval (CI): 0.93-2.37), dispensing of colostrum using a bucket (odds ratio=1.37; 95% CI: 1.00-1.89), treatment with halofuginone (odds ratio=0.46; 95% CI: 0.19-1.15) and feeding with fermented milk (odds ratio=0.32; 95% CI: 0.17-0.63). C. parvum is widespread among calves under 21 days old in dairy herds of western France. Shedding of C. parvum is associated with a high incidence of diarrhea and increased risk of mortality in young calves. This study identified some associated calf-level factors, although further investigations are necessary to determine appropriate measures that farmers and veterinary practitioners should take to reduce the prevalence of C. parvum.

a b s t r a c tCryptosporidiosis is an infection caused by protozoan parasites belonging to the genus Cryptosporidium which is responsible for a potentially severe disease in new-born ruminants. This infection is highly prevalent in small ruminants throughout the world, especially in pre-weaned animals. The clinical expression is different between goat kids and lambs, the infection being generally more severe in the former. Molecular data demonstrate geographical variations in the species of Cryptosporidium infecting small ruminants. They also support the possibility of transmission of zoonotic species from these hosts to humans. Studies are still needed on molecular epidemiology, especially in goats, and on ways to control infection.

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-The ability of the nematophagous fungus Duddingtonia flagrans to reduce the number of infective nematode larvae in coproculture was investigated in goats using different doses of chlamydospores (0, 1.25 × 10 5 , 2.5 × 10 5 , 5 × 10 5 chlamydospores/kg BW/day) given by oral administration or by voluntary consumption in feed during natural or experimental infections with nematodes. The kinetics of excretion of D. flagrans chlamydospores in the faeces was also determined using a dose of 5 × 10 5 chlamydospores/kg BW/day for five days. For all the trials, the faecal nematode egg outputs were determined by a modified McMaster method and standard coprocultures were set up (14 days, 25 °C) to determine the number of larvae emerging from culture in fungus treated and control faeces. When chlamydospores were orally administered, the number of larvae were reduced by 50 to 97% when compared to control cultures. No difference in the level of larval emergence from the culture was seen for experimental or natural infections at the different chlamydospore dose rates. In contrast, when chlamydospores were distributed in the feed, a dosedependent relationship was observed 10 days after the start of administration, the larval development being 2.0%, 14.0% and 86.9% for 5 × 10 5 , 2.5 × 10 5 and 0 spores/kg BW/day, respectively. In addition, the kinetic study showed that the larval emergence from coproculture in the fungus group was statistically lower than in the control group from the second day of administration of the chlamydospores and remained lower until the second day after the last administration (p < 0.05). The results indicate that, for goats in farm conditions, a minimum daily dose of 5 × 10 5 chlamydospores/kg BW must be used to ensure a high treatment efficacy and that daily administration is preferable for maintenance of efficacy over time.goat / biological control / Duddingtonia flagrans / nematode parasite / dose trials

Ninety-two Cryptosporidium sp.-positive fecal samples of dairy diarrheic or non-diarrheic calves from 30 cattle herds in Normandy (France) were selected. Here, the aim was to investigate the species of Cryptosporidium excreted as well as the subtypes of Cryptosporidium parvum found in 7-17-day-old dairy calves. Excretion levels were comprised between 2 × 10(4) and 4 × 10(7) oocysts per gram of feces. Here, a nested 18S SSU rRNA PCR associated with sequencing was performed for identification of Cryptosporidium species and revealed the presence of C. parvum in most cases (80/82), except for two animals which were infected with Cryptosporidium bovis. Then, C. parvum samples were submitted to gp60 PCR. For 39 samples from 24 different herds, a multilocus analysis based on four mini-microsatellites loci (MM19, MM5, MSF, and MS9-Mallon) were conducted. These results were combined with sequence analysis of the gp60 to obtain multilocus types (MLTs). Here, C. parvum gp60 genotyping identified three subtypes in the IIa zoonotic allele family: IIaA15G2R1 (88%), IIaA16G3R1 (10%), and IIaA19G2R1 (2%), and we identified 12 MLTs. The MS9-Mallon locus was reported as the most polymorphic (five alleles). The most common MLT was MLT 1 with 15 samples in 10 farms: (MS9-M: 298, MSF: 165, MM5: 264, MM19: 462, and gp60 subtype: IIaA15G2R1). When comparing diarrheic and non-diarrheic fecal samples, no difference was seen for distribution of Cryptosporidium species, C. parvum gp60 subtypes, and MLTs. Here, in a range of oocyst excretion of 10(4)-10(7) opg, both in diarrheic and non-diarrheic calves, infection was mainly due to C. parvum and to the zoonotic subtype: IIaA15G2R1.

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