C. Paraud

Molecular characterization of Cryptosporidium spp. in pre-weaned kids in a dairy goat farm in western France

A longitudinal study was undertaken to characterize the course of Cryptosporidium infection in a dairy goat farm located in western France. Two cohorts of twenty-five and fifteen animals, respectively, were sampled once a week from birth to weaning. Each individual fecal sample was screened using direct immunofluorescence (IFT) and if found positive, the Cryptosporidium species was identified using PCR analysis. Seventeen (68% [95% CI: 44-91]) animals were positive at least once during the first study and 14 (93% [95% CI: 80-100]) during the second, after IFT examination. In the first study, the age at first excretion was 17 days and the peak of excretion (mean arithmetic excretion: 22,700 oocysts per gram (opg) of feces) was recorded when kids were between 22 and 28 days old. For the second study, the age at first excretion was 10 days and the peak of excretion (mean arithmetic excretion: 3.4 × 10(6)opg) was recorded in animals aged between 10 and 14 days. Clinical signs were observed only in animals of the second cohort. DNA sequence analysis at the 18S ribosomal RNA locus was successful for 9 of the 27 IFT-positive samples in the first cohort and for 10 of the 34 positive isolates in the second cohort. All isolates were identified as Cryptosporidium xiaoi except one which was identified as Cryptosporidium parvum. Our results confirm that goat kids are hosts for C. parvum and C. xiaoi and that infection by C. xiaoi may be associated with mild clinical signs.

Evaluation of oral tilmicosin efficacy against severe cryptosporidiosis in neonatal kids under field conditions

Many compounds have been screened for their potential anti-cryptosporidial activity in ruminants but none of them has been totally efficient in controlling the disease. Macrolide antibiotics have demonstrated some activity against Cryptosporidium spp. in humans. Tilmicosin is a macrolide antibiotic, available in France in an oral form (Pulmotil AC, Lilly France). The preventive efficacy of tilmicosin was evaluated in a goat farm experiencing severe clinical cryptosporidiosis in kids. Twenty-two kids were separated from their dams just after birth and placed in a separated pen. They were divided into 3 groups: an untreated group (10 kids), group 1 (6 kids) receiving tilmicosin at 25mg/kg BW/day and group 2 (6 kids) receiving tilmicosin at 50mg/kg BW/day. Tilmicosin was individually given by oral route from day 2 of age for 10 days. Three times a week, individual faecal samples were performed to assess the oocyst output. Clinical data, i.e. diarrhea and mortality, were recorded. In control kids, the highest prevalence and intensity of excretion were observed between day 6 and day 16 of age and mortality reached 90%. Excretion dynamic and clinical consequences were similar in both treated kid groups. Finally, tilmicosin did not demonstrate any activity against severe kid cryptosporidiosis in field conditions.

Dynamics of excretion and molecular characterization of Cryptosporidium isolates in pre-weaned French beef calves.

Studies on excretion and molecular characterization of Cryptosporidium have been mostly conducted in dairy calves, both diarrhoeic and non-diarrhoeic. Little is known about Cryptosporidium in beef calves, especially in non-diarrhoeic ones. This study was conducted in a herd of Parthenais beef cattle (France) with no history of clinical cryptosporidiosis. Twenty-five calves were sampled once a week from birth to one month of age (age range: 5-34 days). At each sampling date, presence of clinical signs of cryptosporidiosis (diarrhoea) was recorded. Oocyst excretion was assessed using the Heine staining method and a direct immunofluorescence method (Merifluor(®) C/G) which allowed quantification (oocysts per gram of faeces, opg). All samples were subjected to a two-step nested PCR protocol to amplify the 18S rRNA gene and amplification products were sequenced. None of the calves presented diarrhoea. Twenty-three of them excreted oocysts at least one sampling date. Prevalence of excretion was maximal when calves were 27-34 days old, with a percentage of excretion of 85% in this age category [95% CI: 70; 100]. Mean excretion was maximal when calves were 20 to 26 days old, with a mean excretion of 7.6×10(5) opg (range: 0-8×10(6) opg). 32 isolates were successfully identified: 27 as Cryptosporidium bovis, 4 as Cryptosporidium ryanae and 1 as Cryptosporidium parvum. C. bovis was isolated from samples of calves between 11 and 33 days old. C. ryanae was isolated from samples of calves between 17 and 34 days old. C. parvum was isolated from one calf aged 13 days. This survey demonstrated the high infection rate of non-diarrhoeic beef calves by Cryptosporidium species other than C. parvum.

Cryptosporidiosis in small ruminants

Abstract Cryptosporidiosis 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.

Molecular characterization of Cryptosporidium isolates from pre-weaned calves in western France in relation to age.

Eighteen pre-weaned female calves from a single beef cattle herd in western France were sampled weekly from birth to 21/2 months of age in order to characterize Cryptosporidium oocyst output. 182 fecal samples were screened for the presence of oocysts after concentration using immunofluorescence analysis. DNA was extracted from positive samples and a PCR-RFLP protocol, with the restriction enzyme SspI and MboII, to amplify the partial SSU rRNA gene was performed. For the subtyping of Cryptosporidium parvum, a gp60 PCR was carried out. All animals excreted oocysts at at least one sampling date and 80% of the calves presented a mild diarrhea at at least one occasion, with no mortality. The prevalence of excretion reached 94% when calves were 17-23 days of age. The mean number of oocysts at the peak of excretion (10-16 days) was 5 × 10(5) oocysts per gram of feces. PCR-RFLP analysis was successful for 61 of 84 positive samples: 14 were identified as C. parvum, 15 as Cryptosporidium bovis, and 22 as Cryptosporidium ryanae. Ten mixed infections with all combinations of these species were also identified. Calves excreted the following Cryptosporidium species: C. parvum between 7 and 27 days of age, C. bovis between 11 and 38 days and C. ryanae from 19 to 72 days. The IIaA15G2R1 zoonotic subtype of C. parvum subtype was the only subtype identified. We observed the presence of different Cryptosporidium species depending on the age of the animals. This study showed that C. parvum and C. bovis can infect beef calf neonates at similar levels of oocyst excretion with or without clinical signs and that C. parvum isolates had zoonotic potential.

Control of cryptosporidiosis in neonatal goat kids: Efficacy of a product containing activated charcoal and wood vinegar liquid (Obionekk®) in field conditions

Many compounds have been screened for their potential anti-cryptosporidial activity in ruminants but none of them has been totally efficient in controlling the disease. A product containing activated charcoal and wood vinegar liquid demonstrated a good efficacy in controlling clinical signs and oocyst excretion in calves experimentally infected. This product (Obionekk(®), Obione, Charentay, France) was given to goat kids in field conditions. The product was administered in a preventive way at the dose rate of 3.75 g/day/kid of Obionekk(®) from the age of 3 days to the age of 17 days in suspension in milk replacer twice or three times a day. A significant reduction of oocyst excretion was observed when the product was given 3 times a day when compared to control group. The clinical signs (diarrhoea) were significantly reduced whatever the rhythm of distribution when compared to control group. These results demonstrated the efficacy of Obionekk(®) in preventing cryptosporidiosis in goat kids in field conditions.

High excretion of Cryptosporidium ubiquitum by peri-parturient goats in one flock in western France

Cryptosporidium spp. is an important agent of neonatal diarrhoea in goat kids. Little is known about its molecular characterization in adult goats. A longitudinal study was set up to identify the species excreted by adult goats around parturition. Individual faecal samples were collected from 20 pregnant adult goats between 1 and 5 years old in one flock. Samplings began 3 weeks before the estimated kidding date and were done weekly until kidding and for 2 weeks after kidding. Cryptosporidium oocysts were concentrated from 15 g of faeces using a caesium chloride (CsCl) method. Oocyst output was determined using a direct immunofluorescent antibody test (IFAT). Genomic DNA was extracted from each CsCl-concentrated faecal sample positive by IFAT and submitted to a nested PCR-RFLP on the SSU rDNA gene followed by sequencing to identify the isolates at species level. According to their kidding date, goats were sampled between 4 and 8 times. Sixteen goats, out of the eighteen which kidded, were found positive at least at one sampling date. Infection was asymptomatic. Prevalence of excretion was maximal 14 days before kidding with half of the goats excreting oocysts at this date. Excretion was higher before kidding than after kidding. Unexpected levels of excretion were observed with individual oocyst excretion ranging from 6 to 2.5 × 10(5) oocysts per gram of faeces. All isolates were identified as Cryptosporidium ubiquitum.

Molecular characterization of Cryptosporidium isolates from beef calves under one month of age over three successive years in one herd in western France.

Abstract Cohorts of pre-weaned calves were studied for Cryptosporidium infection over three successive years (2010–2012) in one beef cattle herd in western France. Each year 25–34 calves were sampled weekly from 3 days to one month of age in order to characterize oocyst output, Cryptosporidium species and clinical features associated with infection. Faecal samples were screened for the presence of oocysts using immunofluorescence analysis. DNA was extracted from positive samples and a PCR SSU rRNA followed by RFLP or sequencing was performed. For the subtyping of C. parvum, a gp60 PCR was carried out. Regardless of the year, 92–100% of the animals excreted oocysts on at least one sampling date. Depending on the year of observation, the age of highest prevalence varied. In contrast, the peak of excretion was systematically observed almost at the same age (2nd–3rd week of life) with excretion levels ranging from between 100 and 1.7×107 oocysts/g of faeces. Differences concerning clinical signs depending on the year of sampling were observed. Different species patterns were observed, with a predominance of C. bovis in the 1st year and a predominance of C. parvum in the last year. Moreover, two zoonotic subtypes of C. parvum, IIaA15G2R1 and IIaA18G2R1, were recorded in different years. This study shows that, in a given farm, the Cryptosporidium species and C. parvum subtypes identified as well as the prevalence of infection and level of excretion may vary greatly and show distinct patterns according to the year.

Cross-resistance to moxidectin and ivermectin on a meat sheep farm in France

Resistance to ivermectin and moxidectin was explored by a faecal egg count reduction test in two sheep flocks with suspected anthelmintic resistance. The FECRT confirmed one suspicion, with a mean percentage of reduction in egg excretion within the treated groups of 0% for ivermectin (CI 95%: -228 to 58) and 13% for moxidectin (CI 95%: -152 to 70). This was further explored by a controlled efficacy test. An experimental infection of 18 naïve lambs was set up using infective larvae isolated from this flock (5000 L3/lamb). Compared to the control group, abomasal worm burdens (Teladorsagia circumcincta) were reduced by 90% [CI 95%: 81.5-94.8] and 85% [CI 95%: 72.4-92.2] after ivermectin (p<0.05) and moxidectin (p<0.05) treatment respectively. Again, compared to the control group, there was a reduction for intestinal strongyles (Trichostrongylus colubriformis) of 100% and 99% [CI 95%: 97.5-99.7] for ivermectin and moxidectin respectively. No difference was found between the efficacy of moxidectin and ivermectin. Pharmacokinetic values indicated that the strongyles were submitted to anthelmintic concentrations usually lethal to them. This trial demonstrated the first multiple resistance of ovine strongyles in France.

Efficacy of halofuginone lactate against experimental cryptosporidiosis in goat neonates

Preliminary results obtained in calves, lambs and goat kids infected by Cryptosporidium sp. have indicated a partial prophylactic efficacy of halofuginone lactate when administered at 100 μg/kg body weight (BW). In this study, the efficacy of halofuginone lactate was evaluated in goat neonates experimentally inoculated with Cryptosporidium parvum oocysts per oral route. The trial consisted in 2 replicated experiments carried out successively at 2 months of interval. Twenty-two 2- to 4-day-old kids were experimentally inoculated once, 2-3 days after the arrival in premises, with 10(6)C. parvum oocysts per oral route and were allocated into 2 groups. Animals of group 1 acted as untreated control whereas animals of group 2 received halofuginone lactate for 10 days from the infection day to day 9 post-infection (DPI) at a daily oral dose rate of 100 μg/kg BW. Individual oocyst shedding was monitored by daily examination of faecal smears stained by carbol fuchsin and scored semi-quantitatively (0-5) until 19 DPI. Daily diarrhoea scores, weight gain and mortality were recorded. In the first experiment, oocyst excretion started 1 DPI in the control group, was highest on 4 DPI (mean score 3.6) and became undetectable from 16-19 DPI. In the treated group, oocyst shedding started 1 day later, showed lower scores compared to control on 4, 5, 6, 7 and 10 DPI and vanished from 16 to 19 DPI. No significant difference was seen for weight gains between groups. Five kids died in the control group compared to 1 kid in the treated group. In the second (replicated) experiment, oocyst excretion started 2 DPI in the control group, was highest on 4 DPI (mean score 4.5) and became undetectable 18 and 19 DPI. In the treated group, oocyst shedding started 2 days later, peaked on 13 DPI (mean score 2.3) and persisted until the end of the experiment. No significant difference was seen for weight gains between groups. Ten kids died in the control group compared to 3 kids in the treated group. The results demonstrated the efficacy of halofuginone lactate when given as a prophylactic treatment at 100 μg/kg BW during 10 days in reducing oocyst shedding, diarrhoea and mortality in goat kid cryptosporidiosis.