A physiologically based pharmacokinetic (PBPK) model exploring the blood-milk barrier in lactating species - A case study with oxytetracycline administered to dairy cows and goats.

Antibiotic excretion into milk depends on several factors such as the compound's physicochemical properties, the animal physiology, and the milk composition. The objective of this study was to develop a physiologically based pharmacokinetic (PBPK) model describing the passage of drugs into the milk of lactating species. The udder is described as a permeability limited compartment, divided into vascular, extracellular water (EW), intracellular water (IW) and milk, which was stored in alveolar and cistern compartments. The pH and ionization in each compartment and the binding to IW components and to milk fat, casein, whey protein, calcium, and magnesium were considered. Bidirectional passive diffusion across the blood-milk barrier was implemented, based on in vitro permeability studies. The model application used to predict the distribution of oxytetracycline in cow and goat milk, after different doses and routes of administration, was successful. By integrating inter-individual variability and uncertainty, the model also allowed a suitable estimation of the withdrawal periods. Further work is in progress to evaluate the predictive ability of the PBPK model for compounds with different physico-chemical properties that are potentially actively transported in order to extrapolate the excretion of xenobiotics in milk of various animal species including humans.

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.

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.