There are currently no guidelines available to evaluate the efficacy of anticoccidial drugs in sheep coccidiosis in the field. Some general recommendations have been made by the WAAVP 26, and protocols to evaluate the efficacy of anticoccidian drugs have been proposed 8,14,16,19,20 with substantial variations in terms of group constitution, age of the animals at the time of testing, and natural infection levels.
In our context, the need for effective anticoccidian treatment is important not only to allow the lambs to express their growth potential, but also because, after three months together in the ICS, they are sold to different breeders. If previous treatments have not been effective, then they may contaminate their new farm with resistant and potentially pathogenic Eimeria species.
In our study, male lambs from different origins were corralled in the breeding center at three months of age and were treated at the time of their arrival with diclazuril. Therefore, it was not possible to perform the FOCRT immediately upon their entry. Odden et al. 14 proposed the use of twin lambs, one in a treated group and the other one in the control group. In our study, this was not possible. However, other studies did not take this into account 8,16,19,20, but the efficacies measured were reliable because their groups were well balanced in terms of age, sex ratio, and weight. As coccidiosis is a disease that occurs in lambs, the age of the animal is important when performing the FOCRT. Compared to many studies14,16,19,20, we performed the test on older animals (four months versus one month for the above-mentioned studies) due to our experimental setting. However, this age is not necessarily a bias if the intensities of Eimeria oocyst excretions are still high at the time of the test due to a high level of contamination of the environment, as demonstrated by Alzieu et al., 8 on 2- to 3.5-month-old male lambs at the beginning of the FOCRT. Nevertheless, although evaluation of the overall efficacy of a drug is easy, estimation of the efficacy against each Eimeria species is nonetheless an onerous undertaking and the pathogen must be present at the beginning of the test and at a sufficient level in the treated groups. Indeed, progressive immunity of the animal is established for the majority of Eimeria species 2. It is then possible that FOCRT cannot be performed on all species because they may be absent at the beginning of the test or present at low levels due to the age of the animals and possible immunity.
Taking into account all these considerations, we propose a simple protocol to evaluate the efficacy of anticoccidial drugs in farms, as well as an easy-to-use key for the identification of Eimeria species by microscopy.
At the beginning of our study, the intensities of excretion were relatively close between groups within the same farm and high enough to determine treatment efficacy. The untreated groups ensured the natural dynamics of Eimeria oocyst excretion as well as the natural change in the proportion of species in the two breeding centers during the duration of the test. In our case, the intensities of Eimeria oocyst excretions were equal or even slightly higher in the two control groups of the ISC from D0 to D8.
Odden et al., 14 proposed in their protocol that the best period to evaluate the efficacy of anticoccidial treatments is when the intensity of oocyst excretion increases significantly from the beginning of the trial to the end. However, in their case, the animals were younger (“more than 14 days old”) and had never received anticoccidial drugs before the test. In our study, we performed the test on older animals that had undergone anticoccidial treatment 43 days before the beginning of the test. In our case, it seems difficult to obtain an excretion intensity that increases significantly between the beginning and the end of the FOCRT, but the maintenance of this excretion intensity between the two dates seems like an acceptable compromise that still allows interpretation of the FOCRT.
Our results show that in both ISC, the toltrazuril treatment was effective at reducing the overall intensity of Eimeria oocyst excretion. These results are very similar to those obtained in previous studies 8,16, 20–22,27. The percentages of reduction of oocyst excretions were 92.44% and 93.58% with diclazuril, which is lower than the minimum threshold of efficacy (95%) required for anthelmintic treatments 28.
In studies where the efficacy of diclazuril was not investigated, this drug exhibited an efficacy higher than 97% at four to seven days after treatment 8,18,27,29. In our case, the efficacy was slightly lower.
Regarding the efficacies of the drugs against each species, we noticed that although the general efficacy of toltrazuril was higher than 95%, some FOCRT were lower for some species. This was the case in the BC ICS, where the reduction was below 95% for the species E. crandallis/E. weybridgensis and Eimeria faurei. In this group, only a few oocysts of E. faurei were identified at D0 and D8, which likely led to inaccuracy in the calculation of the FOCRT, and the result should hence be interpreted with a degree of caution. By contrast, the number of oocysts identified as E. crandallis/E. weybridgensis was substantial at the time of the toltrazuril treatment, and we can assume that there was a real lack of reduction for this cluster of species. In the RO ICS, toltrazuril exhibited a loss of efficacy toward E. parva/E. pallida but remained effective toward the pathogenic species.
In the BC ICS, reductions of oocyst excretions below 95% were noted for the species E. ovinoidalis/E. marsica, E. crandallis/E. weybridgensis, and E. granulosa/E. bakuensis after diclazuril treatment. At D8, only a few oocysts (29 oocysts) were identified in this group because the treatment drastically reduced the total intensity of excretion. The proportions were, therefore, less accurate than if they had been estimated with 100 oocysts. Nevertheless, the majority of identified oocysts at D8 belonged to the pathogenic clusters in the diclazuril group. In the RO ICS, diclazuril no longer appeared to be fully effective against E. parva/E. pallida as well as against the pathogenic species E. ovinoidalis/E. marsica and E. crandallis/E. weybridgensis. In these situations, it is difficult to conclude that one of the pathogenic species is resistant due to the difficulty distinguishing it from a less pathogenic morphologically related species. The development of molecular tools could fill this gap. For the time being, in sheep, molecular techniques, especially real-time Polymerase Chain Reaction, are not as advanced as in poultry 30. Although some genomic sequences are available 23,31, to our knowledge, no routine technique has been developed to date.
Thus, we cannot presently draw firm conclusions regarding the actual resistance of these species of sheep Eimeria to diclazuril or toltrazuril, although it is clear that there is a lack of efficacy in these two breeding centers. Indeed, resistance is normally confirmed by experimental infections in naive lambs under coccidia-free conditions, with oocysts recovered post-treatment and submitted to a new experimental FOCRT 15. Unfortunately, we do not have the resources to validate the resistance by this method. Nevertheless, the maintenance of clinical signs after treatment observed by farmers (diarrhea and slowing of weight and mass gain) seems to indicate a significant persistence of pathogenic species after treatment.