Faecal egg count reduction tests and nemabiome analysis reveal high frequency of multi-resistant parasites on sheep farms in north-east Germany involving multiple strongyle parasite species.

Anthelmintic resistance in sheep parasitic gastrointestinal nematodes is widespread and a severe health and economic issue but prevalence of resistance and involved parasite species are unknown in Germany. Here, the faecal egg count reduction test (FECRT) was performed on eight farms using fenbendazole, ivermectin and moxidectin and on four farms using only moxidectin. A questionnaire was used to obtain data on management practices to potentially identify risk factors for presence of resistance. All requirements of the recently revised WAAVP guideline for diagnosing anthelmintic resistance using the FECRT were applied. Nematode species composition in pre- and post-treatment samples was analysed with the nemabiome approach. Using the eggCounts statistic package, resistance against fenbendazole, ivermectin and moxidectin was found on 7/8, 8/8 and 8/12 farms, respectively. No formal risk factor analysis was conducted since resistance was present on most farms. Comparison with the bayescount R package results revealed substantial agreement between methods (Cohen's κ = 0.774). In contrast, interpretation of data comparing revised and original WAAVP guidelines resulted in moderate agreement (Cohen's κ = 0.444). The FECR for moxidectin was significantly higher than for ivermectin and fenbendazole. Nemabiome data identified 4 to 12 species in pre-treatment samples and treatments caused a small but significant decrease in species diversity (inverse Simpson index). Non-metric multidimensional scaling and k-means clustering were used to identify common patterns in pre- and post-treatment samples. However, post-treatment samples were scattered among the pre-treatment samples. Resistant parasite species differed between farms. In conclusion, the revised FECRT guideline allows robust detection of anthelmintic resistance. Resistance was widespread and involved multiple parasite species. Resistance against both drug classes on the same farm was common. Further studies including additional drugs (levamisole, monepantel, closantel) should combine sensitive FECRTs with nemabiome data to comprehensively characterise the anthelmintic susceptibility status of sheep nematodes in Germany.

Efficacy of flukicides against Fasciola hepatica and first report of triclabendazole resistance on German sheep farms.

Fasciola hepatica infections lead to severe health problems and production losses in sheep farming, if not treated effectively. Triclabendazole has been used extensively over decades due to its unique efficacy range against all definitive hostfluke stages but published data about the susceptibility of F. hepatica to anthelmintics in Germany are lacking. This study aimed to identify current F. hepatica infections in German sheep flocks by coproscopic examinations and to evaluate the efficacy of anthelmintics with a focus on triclabendazole in a field study conducted from 2020 to 2022. Initial screening included 71 sheep farms, many of them with known history of fasciolosis. In this highly biased sample set, the frequency of F. hepatica infection at individual sheep and farm level were 12.8% and 35.2%, respectively. Additionally, eggs of Paramphistominae were found at frequencies of 4.8% and 15.5% at individual sheep and farm level, respectively. Due to low egg shedding intensity, faecal egg count reduction (FECR) tests could only be conducted on a few farms. The efficacy of triclabendazole was tested on 11 farms and albendazole on one farm, including 3-53 sheep/farm. Individual faecal samples were collected before and two weeks after treatment to evaluate the FECR using the sedimentation or FLUKEFINDER® or a modified FLUKEFINDER® method. On all farms a coproantigen reduction test was conducted in parallel. Lacking efficacy of triclabendazole even at double dosage was shown on one farm associated with a high number of animal losses due to acute fasciolosis. On this farm, the Fasciola miracidium development test was additionally performed, revealing a high in vitro ovicidal activity of albendazole while closantel was effective in vivo. On all other farms, sufficient efficacy of triclabendazole was observed. In conclusion, triclabendazole resistance appears not to be widespread on German sheep farms but, when present, can have serious effects on animal health.

Coproscopical diagnosis of patent Fasciola hepatica infections in sheep - A comparison between standard sedimentation, FLUKEFINDER® and a combination of both.

The liver fluke Fasciola hepatica is a highly pathogenic and zoonotic trematode with a cosmopolitan distribution. In livestock, infections may lead to significant economic losses if not diagnosed promptly and treated effectively. Particularly for small ruminants, the standard method for the detection of fluke infection is based on coproscopical methods such as the sedimentation method, which detects F. hepatica eggs in faecal samples. In this respect a recent innovative coproscopical approach to diagnose patent infections is the FLUKEFINDER® method, which relies on differential sieving before sedimentation. These two methods and a combination of both methods that allows larger amounts of faeces to be processed with the FLUKEFINDER® apparatus were compared, to assess which method is most appropriate to determine the prevalence and intensity of F. hepatica egg shedding. The methods were compared for their ability to recover eggs from ovine faecal samples containing different numbers of fluke eggs per gram (EPG) of faeces and diluting the samples further by mixing with faeces from uninfected sheep. To compare the specificity of the test procedures, positive and negative samples with a low EPG were analysed in parallel by an investigator blinded to the nature of the samples. Significant differences concerning the EPG outcome were found: The FLUKEFINDER® method demonstrated the highest EPG values (p < 0.001) in the undiluted samples as well as in all mixing levels, followed by the modified FLUKEFINDER® method. The standard sedimentation showed the lowest EPG values and the highest variability between technical replicates. The precision of the FLUKEFINDER® method and the modified FLUKEFINDER® method were significantly higher than the precision of the standard sedimentation as determined by comparison of variability between technical replicates. The highest raw egg counts were detected using the modified FLUKEFINDER® method. The FLUKEFINDER® method and the combined method showed a sensitivity of 100 % even at the lowest egg concentrations, whereas the sensitivity of the standard sedimentation was 98.1 % for the same set of samples (i.e. one false negative sample). In a separate investigation aiming to estimate the specificity no differences were found between the three methods: all protocols showed 100 % specificity and were able to correctly distinguish between truly positive and truly negative samples without any evidence of cross-contamination between positive and negative samples processed in parallel.

Field trials assessing deltamethrin (Butox®) treatments of sheep against Culicoides species.

Culicoides (Diptera: Ceratopogonidae) biting midges may transmit various diseases of economic importance, including bluetongue virus (BTV) and Schmallenberg (SV) virus, which affect ruminants. During the outbreak of BTV in central and northern Europe in 2006, and in the absence of BTV vaccines, many national veterinary services recommended the treatment of susceptible livestock with pyrethroids as a first-line defense against biting midges, although these insecticides were officially not registered and authorized for use against Culicoides midges. The efficacy of Butox® pour on (7.5 mg deltamethrin/mL) against biting midges was therefore evaluated in a double-blinded GCP field trial performed in Brandenburg, Germany. Forty female Merino sheep with an average body weight of 38 kg (±7 kg) were used for the study. Twenty randomly selected sheep were treated with 10 mL Butox® pour on. The remaining 20 sheep were left untreated and served as a control group. Midge collections took place in two separate drop traps covering two crush pens with three confined treated/untreated sheep standing inside, on weekdays at 1, 7, 14, 21, 28, and 35 days post treatment. A total of 19,057 midges were collected during this period. Midges were identified as belonging to the subgenus Avaritia, Fox (84.6%) and subgenus Culicoides, Latreille (15.4%). A total of 12,031 midges were collected inside the drop trap containing untreated sheep, in comparison to 7,026 midges collected from the vicinity of the treated sheep. Significantly, more midges had fed on control compared to treated sheep with 757 and 103 engorged midges, respectively. The results indicate that treatment of sheep with Butox® pour on provided a significant decrease in Culicoides feeding rates under field conditions for at least 35 days.

Feeding behaviour of Culicoides spp. (Diptera: Ceratopogonidae) on cattle and sheep in northeast Germany.

BACKGROUND: Culicoides spp. play an important role in the transmission of several vector-borne pathogens such as Bluetongue and Schmallenberg virus in Europe. To better understand the biology of local Culicoides species, a study divided into three parts was performed in northeast Germany to elucidate the feeding activity patterns (study A), preferential landing and feeding sites (study B) and host feeding preferences (study C) of Culicoides spp. using cattle and sheep as baits. METHODS: In study A, the activity of Culicoides spp. was monitored over a 72 h period by collecting insects at regular intervals from the interior of drop traps with cattle or sheep standing inside. In study B, Culicoides spp. were directly aspirated from the coat and fleece of cattle and sheep during the peak activity period of Culicoides. In study C, Culicoides spp. were collected using drop traps with either cattle or sheep standing inside and located 10 m apart. RESULTS: In study A, 3,545 Culicoides midges belonging to 13 species were collected, peak activity was observed at sunset. In study B, 2,024 Culicoides midges were collected. A significantly higher number of midges was collected from the belly and flank of cattle in comparison to their head region. In study C, 3,710 Culicoides midges were collected; 3,077 (83%) originated from cattle and 633 (17%) from sheep. Nearly half (46.7%) of the midges collected from cattle were engorged, significantly more than the number of engorged midges collected from sheep (7.5%). Culicoides from the Obsoletus complex (C. obsoletus and C. scoticus) were the most common Culicoides species encountered, followed by C. punctatus. Other species identified were C. dewulfi, C. chiopterus, C. pulicaris, C. lupicaris, C. pallidicornis, C. subfascipennis, C. achrayi, C. stigma, C. griseidorsum and C. subfagineus, the last two species are reported for the first time in Germany. Engorged C. chiopterus were collected in relatively high numbers from sheep, suggesting that this species may have a preference for sheep. CONCLUSIONS: An insight into the feeding behaviour of local Culicoides species under field conditions in northeast Germany was obtained, with implications for the implementation of control measures and midge-borne disease risk analysis.