Breeding selection, farming context, and necessity to control coccidiosis
In France, the sheep-meat selection scheme is based on a breeding program supervised by a breeders’ association. It is based on the use of a core group of farms selected in the breed population. Each year, within a breed, crossbreeding is carried out between dams of sires and sires of sires, and the best male lambs resulting from these crossbreedings are collected after weaning in a common breeding center for evaluation called an “individual control station” (ICS). In an ICS, lambs are tested on criteria of interest for sheep-meat production such as average daily weight gain and fat and muscle thickness.
During 2021, two of these ICS were investigated in Berrichon du Cher (BC) and in Rouge de l’Ouest (RO) breeds, both related to the breeders’ association GEODE. Lambs were collected in the ICS at three months of age. On arrival, they received a diclazuril treatment (Vecoxan ND, MSD Santé Animale, 1 mg/kg body weight (BW) and an ivermectin treatment (Baymec ND, Norbrook Laboratories, 200 µg/kg BW) to control coccidiosis and Strongyloides papillosus infections, respectively. The lambs came from different farms of origin: in 2021, we enumerated 9 different origins for a total of 55 BC lambs entering the ICS and 12 different origins for the 52 RO lambs.
Throughout the evaluation period in the ICS, the lambs were kept exclusively indoors and fed ad libitum. During their growth phase, they were regularly weighed as part of the protocol to evaluate their growth abilities, and when signs of diarrhea appeared on several lambs, an anticoccidial treatment was carried out on all rams with diclazuril (Vecoxan ND, MSD Santé Animale, 1mg/kg BW) or sulfonamides (Sulfadimerazine 33% ND, Huvepharma, 90 mg/kg BW over three successive days).
In this very particular breeding context, where coccidiosis must be controlled as much as possible to allow the lambs to express their full genetic background, BC and RO breeders noticed the persistence of diarrhea after the anticoccidial treatment and observed that it was necessary to treat more frequently than in previous years. These observations have been recurrent for several years, despite implementation of a sanitary vacuum and disinfection of the breeding center between two years of male lamb evaluation.
A protocol to evaluate the efficacy of anticoccidial treatments was then implemented in both ICS.
Sampling and analysis
The Fecal Oocyst Count Reduction Test (FOCRT) started 43 days after the animals were recruited in the ICS. The protocol to test the efficacy of anticoccidial drugs was based largely on the protocol proposed by Odden et al. , with some changes to adapt it to our breeding context. The animals were divided into three groups of 11 to 16 lambs, with one group for each treatment that was tested. The groups were constituted so as to be homogeneous in terms of weight and farm of origin. The treatments under evaluation were: diclazuril (Vecoxan ND, MSD Santé Animale, 1 mg/kg BW orally) and toltrazuril (Baycox ND, Elanco, 20 mg/kg BW or Toltranil ND, KRKA, 20 mg/kg BW, both orally). An untreated group was added in both breeds. Individual fecal samples were taken from each lamb on the day of treatment (D0) and eight days after the treatment (D8).
Individual microscopic analyses were performed on all series of fecal samples. Eimeria spp. oocysts were counted using a modified Raynaud's method . Briefly, three grams of feces were diluted in a saturated sodium chloride solution with a density of 1.2 g/mL before being filtered three times through a tea strainer. The filtrate was then analyzed on a McMaster slide. The oocysts in both grids were counted, summed, and multiplied by 50 to obtain the number of Eimeria spp. oocysts per gram of feces. The detection limit of this technique was 50 oocysts per gram (OPG) of fecal material.
In parallel, identification of the Eimeria species was performed per treatment group and per date (D0 and D8). To do so, all individual filtrates of a group were mixed in a glass flask. After homogenization, an aliquot of the total suspension was placed in a test tube, filled to the brim, and covered with a coverslip. The flotation time lasted 15 minutes before examination of the slide. The Eimeria species were then identified by the morphological and morphometric criteria of their oocysts as defined by Eckert (Eckert, 1995) at 400x magnification. The oocyst measurements were performed with Zeiss image processing software Zen (Zen 2.6 blue edition, Carl Zeiss Microscopy GmbH, 2018).
Species identification was performed on simple and relevant criteria on non-sporulated oocysts. The identification pathway is presented in Figure 1. First, we checked the presence/absence of a polar cap on the oocyst. When a polar cap was present on the oocyst, the total length of the oocyst was measured to distinguish two sub-categories of Eimeria: species with large oocysts (> 30 µm) and species with oocysts of medium size (< 30 µm). Among the Eimeria species that have large oocysts with a polar cap, two species were readily distinguishable: Eimeria intricata, with a brown, thick wall, and Eimeria ahsata, with a very prominent polar cap. When oocysts had a medium size and a polar cap, the total length of the oocysts (Figure 2) could differentiate the two following clusters: Eimeria crandallis/Eimeria weybridgensis with smaller oocysts (total length ranging from 17 to 31 µm for a mean size of 22–24 µm) versus Eimeria granulosa/Eimeria bakuensis (from 22 to 37 µm for a mean size of 30–32 µm).
When the oocyte did not have a polar cap, the presence/absence of a micropyle was checked. Oocysts of Eimeria pallida and Eimeria parva species were readily identified as their oocysts lacked both a polar cap and a micropyle, had a round shape, and were small in size (12 to 22 µm) compared to all the other species. When a micropyle was present, the shape of the oocyst was of interest: an oval shape indicated the Eimeria ovinoidalis/Eimeria marsica cluster whereas a poultry egg shape was characteristic of Eimeria faurei.
In each group, we identified one hundred oocysts before and after treatment in order to have an accurate proportion of the species composition. However, in some groups after treatment, a smaller number of oocysts were identified due to the low number excreted after treatment. In this case, we identified as many oocysts as possible.
Calculation of the efficacy of anticoccidial treatments
The percentages reduction of the intensities of Eimeria oocyst excretion were estimated by the Kochapakdee formula :
Efficacy = (1- arithmetic mean OPG of the treated group at D8 / arithmetic mean OPG of the treated group at D0) * 100
First, this formula was used on the total number of oocysts counted on McMaster slides at D0 and D8 to obtain the overall efficacy of the drug. According to the WAAVP guidelines for anthelminthic resistance, treatment resistance occurs when the percentage of reduction is less than 95% and the lower 95% confidence interval (CI) is less than 90%. To calculate the confidence intervals, we used the following formula:
CI (95%) = (mean of efficacy in treated group ± 1.96 (Standard Variation of efficacy in treated group / √number of animals in treated group)
Secondly, we evaluated the efficacy of each drug against individual Eimeria species or clusters. Following the species identifications, the proportion of each species was calculated in each treatment group. This proportion was plotted against the total number of oocysts counted to obtain an estimate of the number of oocysts of each species in each group. Then, the same formula  was used for these estimated numbers of oocysts at D0 and D8 to obtain the efficacy of a given drug against each Eimeria species.