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

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 × 10(7)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.

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.

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.

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.

Absence of ivermectin resistance in a survey on dairy goat nematodes in France.

Anthelmintic resistance is very prevalent in ruminant strongyle populations, especially in goats. Several occurrences of multiple anthelminthic resistances have been reported in goat flocks throughout the world, including resistance to the most recent macrocyclic lactones. A faecal egg count reduction test was conducted to detect resistance to ivermectin in French goat flocks. Thirty goats per flock were randomly selected in 22 flocks and allocated into two groups of 15 animals: an untreated control group and an ivermectin-treated group (0.3 mg/kg BW per os). Individual faecal egg counts and pooled larval cultures were performed 16-17 days after anthelmintic treatment for control and treated groups. FECR's were calculated for treated group vs. control one and when <95/100, were considered as indicative of anthelmintic resistance. FECR results indicated the absence of ivermectin resistance in nematode populations from all the 22 goat farms. The nematode genera involved in control groups were of Teladorsagia/Trichostrongylus, Haemonchus and Oesophagostomum/Chabertia types.

Influence of the route of administration on efficacy and tissue distribution of ivermectin in goat.

The tissue concentration and efficacy of ivermectin after per os and subcutaneous administration were compared in goats experimentally infected with Trichostrongylus colubriformis (ivermectin-susceptible strain, INRA). Infected goats (n = 24) were treated per os (n = 9) or subcutaneously (n = 9) with ivermectin, 0.2 mg/kg, or kept as not treated controls. The faecal egg counts and small intestine worm counts were determined. Ivermectin concentration was measured in the plasma, gastrointestinal tract, lung, skin or hair, liver and adipose tissues at 0, 2, 7 and 17 days post-treatment. The efficacy of ivermectin against T. colubriformis infection in goat was 98.7 and 99.9% for subcutaneous and oral administration, respectively. Ivermectin concentration declined with time and only residual concentration was measured at 17 days post-treatment in plasma and gastrointestinal tract. Ivermectin concentration was higher after subcutaneous compared to per os injection in most of the tissue examined. In skin, hair and subcutaneous adipose tissue ivermectin persisted at significant concentrations 17 days post-treatment for both routes of administration. In our experimental conditions, ivermectin provides similar efficacy against T. colubriformis after subcutaneous or per os administration in goat. However, the lower ivermectin levels in tissues after per os administration suggest that the lasting of efficacy may be shortened after per os compared to subcutaneous administration especially in animals with poor body condition in pasture where re-infection occurs quickly after anthelmintic treatment.

Administration of Duddingtonia flagrans chlamydospores to goats to control gastro-intestinal nematodes: dose trials.

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 x 10(5), 2.5 x 10(5), 5 x 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 x 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 degrees 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 dose-dependent relationship was observed 10 days after the start of administration, the larval development being 2.0%, 14.0% and 86.9% for 5 x 10(5), 2.5 x 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 x 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.

Duration of activity of topical eprinomectin against experimental infections with Teladorsagia circumcincta and Trichostrongylus colubriformis in goats.

The immediate as well as the persistent anthelmintic efficacies of topically applied eprinomectin were evaluated in goats against induced infections with Teladorsagia circumcincta (2800 L3) and Trichostrongylus colubriformis (6000 L3). Twenty-three culled dairy goats were allocated to the following groups: control animals (group 1), animals treated 21 days prior to nematode infection (group 2), animals treated 7 days prior to nematode infection (group 3) and animals treated 21 days after nematode infection (group 4). Eprinomectin was applied at twice the cattle dose rate (1.0 mg/kg BW). According to the groups, necropsies were undertaken 28 days after nematode infection (groups 1-3) or 14 days after the anthelmintic treatment (group 4). Worm counts were determined for abomasum and small intestine. The curative anthelmintic efficacy of eprinomectin at 1.0 mg/kg BW on existing worm burdens was 100% against T. circumcincta and T. colubriformis. Quite similar worm burdens reductions were observed when eprinomectin was administered 7 days before infection whereas they were only 52.4 and 17.8% for T. circumcincta and T. colubriformis, respectively, for an administration of the drug 21 days prior to the nematode infection.

Efficacy of alpha-cyclodextrin against experimental cryptosporidiosis in neonatal goats.

The efficacy of orally administered tablets containing alpha-cyclodextrin, an excipient used in the pharmaceutical industry with demonstrated anticryptosporidial activity in vitro and in neonatal mice, was evaluated in neonatal goat kids. The formulation was evaluated for hardness and was subjected to in vitro drug release studies. Twenty goat kids were orally inoculated with 10(6) oocysts of C. parvum within the first 6 days of age. Half of the animals were treated by oral administration of four tablets of alpha-cyclodextrin/day (500 mg/kg of body weight) for six consecutive days, the treatment beginning on the day of inoculation. Infection was monitored by daily examination of faecal samples from the first day to 25 days post-inoculation. The criteria studied in evaluating efficacy were: oocyst shedding, presence of diarrhoea and weight gain at 15 and 25 days post-inoculation. alpha-cyclodextrin was effective when given at the beginning of infection: there was a longer pre-patent period, a reduction in the patent period and a decrease in the intensity of infection, these differences being statistically significant (P < 0.05) compared with untreated neonatal kids. Moreover, except in one animal, the diarrhoea was prevented in infected neonatal kids. Animals from both groups increased the body weight and no significant differences were seen between the two groups.

Effect of the nematophagous fungus, Duddingtonia flagrans, on the larval development of goat parasitic nematodes: a plot study.

Effective alternatives to anthelmintic treatment against nematode parasites of goats are required because of the high prevalence of benzimidazole resistance. Towards this objective, the nematophagous fungus, Duddingtonia flagrans (Df), was used in a plot study against two main parasitic nematode species of goats, Teladorsagia circumcincta (Tcir) and Trichostrongylus colubriformis (Tcol). Worm-free, culled goats were experimentally infected with strains of Tcir and Tcol to constitute donors. Half of the animals were periodically given Df chlamydospores at a daily dose of 2.5 x 10(5) spores/kg BW while the remaining animals were kept as controls. At 5 time periods i.e. March, May, July, September and November 2001, corresponding to the main grazing season in France for goats, faeces were collected from the 6th day of fungus administration for the following 2 days to obtain approximately 1 kg of faeces from each group of animals: Tcir/Control, Tcol/Control, Tcir/Fungus, Tcol/Fungus. For each period and each group, the faeces were deposited on a 1 m2 grass plot and the grass was cut (3 replicates) on weeks 2, 4, 6, 8,12 after deposition, for infective larval recovery. Larvae were counted and the results were expressed as a ratio of larvae/eggs deposited. On the plots with the control faeces deposited in March, July and September, the grass infectivity due to Tcir and Tcol was similar and the maximum number occurred between 2 and 4 weeks post deposition. In May, the maximum numbers of larvae were not recorded until 8 weeks after deposition, due to high daily temperatures and dryness. In November, larval development took place only for Tcir. On the plots with the fungus treated faeces, a significant reduction in grass infectivity occurred for both nematodes and ranged from 50-60% in May, July and November deposits to 80-90% in the September deposit. On the contrary to these findings, no difference was recorded between the fungus and control plots for the March deposit. In conclusion, D. flagrans is suitable for reducing the number of infective larvae in the herbage during the main part of the grazing period for the most important digestive nematodes of goats.