Preliminary studies on in vitro methods for the evaluation of anticoccidial efficacy/resistance in ruminants.

Ovine Eimeria spp. infections cause increased mortality, reduced welfare and substantial economic losses, and anticocccidials are important for their control. Recent reports of anticoccidial resistance against ovine Eimeria spp. necessitate the development of in vitro methods for the detection of reduced anticoccidial efficacy, especially since the in vivo methods are both expensive, time consuming and requires the use of otherwise healthy animals. The aim of the present study was therefore to approach a preliminary standardization of in vitro assays for evaluation of the efficacy of the most commonly used anticoccidials in ruminants. For this purpose, apart from the evaluation of inhibition of oocyst sporulation, most effort was concentrated on assessment of the capacity of the different anticoccidials to inhibit both the invasion and further development (up to the first schizogony) of E. ninakohlyakimovae sporozoites in bovine colonic epithelial cells (BCEC). For this purpose, infected cultures were monitored 1, 8 and 15 days post infection to determine the infection rate, number of immature schizonts and number, size and appearance of mature schizonts, respectively. No clear inhibitory effect was found with any of the anticoccidial formulations tested, and we could not identify why there were no measurable effects from the different anticoccidials. Despite the lack of positive results, further investigations should be encouraged, as this could decrease the need for animal experiments and could be used in the initial assessment of anticoccidial efficacy of new drugs.

Excretion of Eimeria spp. oocysts in young lambs following iron supplementation.

BACKGROUND: Iron is an essential nutrient, and iron supplementation has been shown to reduce the incidence of abomasal bloat in lambs. Additionally, iron deficiency is linked to pica, which may increase uptake of Eimeria oocysts. Coccidiosis in sheep, caused by Eimeria spp., is an important infection, leading to reduced welfare and economic losses. The aims of our study were to investigate: (1) the use of iron supplementation in Norwegian sheep flocks using a questionnaire survey, and (2) whether iron supplementation reduced excretion of Eimeria oocysts and increased the growth rates of young lambs. RESULTS: A questionnaire regarding the use of iron supplementation, sent to all members of the Norwegian Sheep Recording System (n = 4993), showed that 152/1823 farmers iron-supplemented lambs, either orally (56.7%) or by injection (43.3%). The main purpose of supplementation was to prevent abomasal bloat (38.4%), coccidiosis (9.3%), or both (27.8%). In the field study, 102 twin lambs from five flocks were included: one twin (treated) received 600 mg of gleptoferron subcutaneously within 3 days of birth, whereas the control was given saline. McMaster analysis of individual faecal samples obtained at weekly intervals (n = 4 per lamb, starting at turnout) showed no significant difference in oocyst excretion between treatment groups at any sampling, except for one flock 14 days after turnout. Mean growth rates, measured at iron injection, 21 days after turnout, and in the autumn, differed significantly between treated and untreated lambs from iron injection to 21 days after turnout, however, no difference in growth rates was observed in the overall period from iron injection to autumn. Blood analysis suggested that the controls were at risk of developing iron deficiency anaemia during the housed period, but signs of anaemia were not observed. CONCLUSION: Iron supplementation of lambs was used by 8.3% of the farmers responding to the questionnaire, mainly with the intention to prevent abomasal bloat, coccidiosis, or both. The field trial results indicate that iron supplementation of young lambs do not reduce oocyst excretion and only induced a transitory increase in weight gain. However further studies, including more flocks and possibly repeated iron injections, would provide more definitive information.

Controlled efficacy trial confirming toltrazuril resistance in a field isolate of ovine Eimeria spp.

BACKGROUND: Coccidiosis due to Eimeria spp. infections in lambs causes increased mortality and substantial production losses, and anticoccidials are important for control of the infection. Anticoccidial resistance has been reported in poultry and swine, and we recently described reduced toltrazuril efficacy in ovine Eimeria spp. in some Norwegian sheep farms using a newly developed faecal oocyst count reduction test (FOCRT). The aim of the present study was to use a controlled efficacy trial to assess the efficacy of toltrazuril against a field isolate suspected of being resistant. METHODS: Twenty lambs, 17-22 days old and raised protected against exposure to coccidia, were infected with a field isolate of 100,000 Eimeria spp. oocysts. This isolate was obtained from a farm with a previously calculated drug efficacy of 56% (95% confidence interval: -433.9 to 96.6%). At day 7 post-infection, 10 of the lambs were orally treated with 20 mg/kg toltrazuril (Baycox Sheep vet., Bayer Animal Health), while the other 10 lambs (controls) were given physiological saline. Clinical examinations were conducted, and weight gains recorded. Daily faecal samples were scored for diarrhoea on a scale from 1 to 5, and oocyst excretion was determined using a modified McMaster technique. Oocysts were morphologically identified to species level. At 17-24 days post-infection, the lambs were euthanized and necropsied. RESULTS: The tested Eimeria isolate was resistant against toltrazuril, and resistance was seen in both pathogenic and non-pathogenic species. In addition, no significant differences in faecal score, growth, gross pathology or histological changes were identified between the two groups. The pathogenic E. ovinoidalis was the dominant species, and no significant difference in the individual prevalence of E. ovinoidalis post-treatment was found between treated (66.9%) and control lambs (61.9%). Other species identified included E. crandallis/weybridgensis, E. parva, E. marsica, E. faurei, E. pallida, E. ahsata and E. bakuensis. CONCLUSIONS: This study confirms toltrazuril resistance in ovine Eimeria spp.; in addition, the data support the use of FOCRT as an appropriate tool for field evaluation of anticoccidial efficacy. Due to limited anticoccidial treatment alternatives, these findings may have important implications for the sheep industry, particularly in northern Europe.

Field evaluation of anticoccidial efficacy: A novel approach demonstrates reduced efficacy of toltrazuril against ovine Eimeria spp. in Norway.

Ovine Eimeria spp. infections cause reduced welfare, increased mortality, and substantial economic losses, and anticoccidials are crucial for their control. Recent reports of toltrazuril resistance in pigs, and anecdotal reports of reduced anticoccidial efficacy in lambs, necessitate evaluation of anticoccidial efficacy. Due to the substantial lifecycle differences between nematodes and coccidia, current WAAVP methods for assessing anthelmintic efficacy are not suitable for such evaluations. Faecal samples were collected from 8 pairs of twin lambs from 36 Norwegian sheep farms 6-8 days after turnout. One twin of each pair was then treated with 20 mg/kg toltrazuril and a second faecal sample from all lambs was collected 7-11 days later. Oocyst excretion rate in all samples was determined using McMasters. Suitability of treatment timing was investigated by evaluating the increase in mean log oocyst excretion in untreated lambs. Based on comparisons between groups, a threshold of ≥0.75 (13 farms) was used to identify farms where drug efficacy could be assessed with confidence, drug efficacy on farms with increases of ≥0.5 but <0.75 (7 farms) were evaluated with caution, and drug efficacy on farms with increases of <0.5 (16 farms) was not estimated. Reduction in oocyst excretion between samples from treated lambs compared with controls from the 20 farms with a threshold of ≥0.5 were then analysed using a generalised linear mixed model. The results were classified based on 95% CI obtained using parametric bootstrapping. Among these 20 farms, two exhibited reduced drug efficacy (upper 95% CI < 95%), 13 had good efficacy (lower 95% CI > 90%), and for 5 the results were inconclusive. This is the first evidence-based report of reduced anticoccidial efficacy in ovine Eimeria spp. Additionally, we highlight the problem of sub-optimal timing of treatment (16/36 farms), which could potentially result in incorrect conclusions being reached regarding lack of drug efficacy.

Treatment against coccidiosis in Norwegian lambs and potential risk factors for development of anticoccidial resistance-a questionnaire-based study.

The objectives of this study were to investigate the use of anticoccidials in Norwegian sheep flocks and identify farms with management procedures likely to select for drug resistance. Data were obtained by a questionnaire sent to all members of the Norwegian Sheep Recording System in October 2015. The data set consisted of 1215 answers, corresponding to 8.5% of Norwegian sheep flocks. Anticoccidials were used in 82.7% of flocks. Main treatment was at turnout (38.6% of treated flocks) or 1 week after turnout (32.4%). Interestingly, clinical signs possibly related to coccidiosis were observed by almost 40% of the farmers after treatment, which might be an indication of drug resistance. Correlations between the apparently reduced anticoccidial efficacy and management conditions, such as the size of the farms, were found. From the farmers' perspective, metaphylactic treatment was used in 88.5% of treated flocks, of which approximately one third had no history of clinical coccidiosis. Even though farmers seem aware of the importance of good drenching routines based on reliable estimates of weights and calibration of drench guns, drench gun used for anticoccidial administration was never calibrated in 12.1% of the flocks. Finally, dose estimation was made by visual appraisal in 27.5% of the flocks, which can lead to incorrect dosing. Based on the present study, it cannot be determined whether the apparent treatment failure was related to management practises, incorrect administration of the drug, other infections or actual anticoccidial drug resistance.