Gastrointestinal nematodes in German outdoor-reared pigs based on faecal egg count and next-generation sequencing nemabiome data

Background There is a higher risk for nematode infections associated with outdoor-reared pigs. Next to Ascaris suum, Oesophagostomum dentatum and Trichuris suis, there is the potential of infections with other nodular worm species, Hyostrongylus rubidus, Stongyloides ransomi and Metastrongylus spp. lungworms. Next-generation sequencing methods describing the nemabiome have not yet been established for porcine nematodes. Methods FLOTAC was used for faecal egg counts of porcine gastrointestinal nematodes and lungworms in piglets, fatteners and adults individually. A nemabiome analyses based on ITS-2 gene region metabarcoding was used to differentiate strongyle species. Additionally, questionnaire data was analysed using mixed-effect regression to identify potential risk factors associated with parasite occurrences and egg shedding intensity. Results On 15 of 17 farms nematode eggs were detected. Ascaris suum, strongyles and T. suis were detected on 82%, 70% and 35% of the 17 farms, respectively. Lungworms were detected on one out of four farms with access to pasture. Strongyloides ransomi was not detected. 32% (CI 28–36%), 27% (24–31%), 5% (4–7%) and 3% (0.9–8%) of the samples where tested positive for strongyles, A. suum, T. suis and lungworms, respectively. The nemabiome analysis revealed three different strongyle species, with O. dentatum being the most common (mean 93.9%), followed by O. quadrispinulatum (5.9%) and the hookworm Globocephalus urosubulatus (0.1%). The bivariate and multivariate risk factor analyses showed among others that cleaning once a week compared to twice a week increased the odds significantly for being infected with A. suum (OR 78.60) and strongyles (2077.59). Access to pasture was associated with higher odds for A. suum (43.83) and strongyles (14.21). Compared to shallow litter systems, deep litter and free range systems resulted in significant higher odds for strongyles (85.74, 215.59, respectively) and T. suis (200.33, 623.08). Conclusions Infections with A. suum, O. dentatum, O. quadrispinulatum, T. suis, Metastrongylus spp. and G. urosubulatus are present in German outdoor-reared pigs. This is the first report of G. urosubulatus in domestic pigs in Europe. Metabarcoding based on the ITS-2 region is a suitable tool to analyse the porcine nemabiome. Furthermore, management practices have the potential of reducing the risk of parasite infections

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

Knockdown resistance in Stomoxys calcitrans stable fly populations on German dairy farms: kdr alleles explain susceptibility of individual flies to deltamethrin

The stable fly, Stomoxys calcitrans, is a globally important pest causing stress, economic losses and transmission of pathogens in livestock. Control on commercial farms relies predominantly on the use of insecticides, with pyrethroids being the most frequently used class of insecticides in industrialised countries. Here, laboratory isolates were obtained from four dairy farms in Brandenburg (Germany) and tested for phenotypic resistance to deltamethrin in comparison to a susceptible reference isolate using topical application. Individual flies were subsequently genotyped using allele-specific real-time PCRs. Phenotypic resistance was observed in all four field isolates with resistance ratios between 46 and 119 compared to the susceptible laboratory strain. At position 1014 of the voltage-sensitive sodium channel, allele-specific PCRs detected the wild-type, kdr-his and kdr genotypes encoding leucine, histidine and phenylalanine, respectively. In the susceptible laboratory isolate, only the wild-type was identified. On the farms with very high LD50 values, the kdr variant was most prevalent and logistic regression analysis revealed that the kdr variant increased the odds to survive exposure to deltamethrin more than the kdr-his genotype. Flies carrying two resistance alleles were less susceptible than flies that also carried one wild-type allele. In three out of four field isolates, the allele frequencies were significantly different from the expectations of the Hardy–Weinberg equilibrium suggesting ongoing selection. The data show that the phenotype can be largely explained by the kdr genotype and represent high frequencies of the L1014F kdr and L1014H kdr-his variants conferring high levels of resistance in northern Germany

Anthelmintic resistance against benzimidazoles and macrocyclic lactones in strongyle populations on cattle farms in northern Germany

Anthelmintic resistance (AR) in cattle gastrointestinal nematodes (GIN) is an increasing global concern, with low to moderate levels recently documented in Central Europe. This study reports on resistance against both macrocyclic lactones (MLs) and benzimidazoles (BZs) in northern Germany, highlighting that AR is spreading. The fecal egg count reduction test (FECRT) remains the primary tool for AR assessment, yet differing methodologies and recent guideline updates complicate resistance interpretation across studies. Statistical methods, such as Bayesian approaches used by eggCounts and bayescount, yield varying confidence intervals, further influencing results. Notably, the nemabiome analysis identified Ostertagia ostertagi and Cooperia oncophora as predominant species in the region, though unexpected diversity among farms with additional GIN species occurring sometimes even at high frequency, suggests morphological analysis of coprocultures may underestimate species prevalence. Detecting AR against both drug classes on some farms underscores the urgency of implementing sustainable strategies, such as targeted selective treatment and combinations of anthelmintics with different mode of action, to prevent scenarios of multi-drug resistance observed elsewhere. Effective resistance management requires immediate discussions with veterinarians and stakeholders to steer toward informed, preventive measures in cattle farming

Identification of Tsetse (Glossina spp.) using matrix-assisted laser desorption/ionisation time of flight mass spectrometry

Glossina (G.) spp. (Diptera: Glossinidae), known as tsetse flies, are vectors of African trypanosomes that cause sleeping sickness in humans and nagana in domestic livestock. Knowledge on tsetse distribution and accurate species identification help identify potential vector intervention sites. Morphological species identification of tsetse is challenging and sometimes not accurate. The matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF MS) technique, already standardised for microbial identification, could become a standard method for tsetse fly diagnostics. Therefore, a unique spectra reference database was created for five lab-reared species of riverine-, savannah- and forest- type tsetse flies and incorporated with the commercial Biotyper 3.0 database. The standard formic acid/acetonitrile extraction of male and female whole insects and their body parts (head, thorax, abdomen, wings and legs) was used to obtain the flies' proteins. The computed composite correlation index and cluster analysis revealed the suitability of any tsetse body part for a rapid taxonomical identification. Phyloproteomic analysis revealed that the peak patterns of G. brevipalpis differed greatly from the other tsetse. This outcome was comparable to previous theories that they might be considered as a sister group to other tsetse spp. Freshly extracted samples were found to be matched at the species level. However, sex differentiation proved to be less reliable. Similarly processed samples of the common house fly Musca domestica (Diptera: Muscidae; strain: Lei) did not yield any match with the tsetse reference database. The inclusion of additional strains of morphologically defined wild caught flies of known origin and the availability of large-scale mass spectrometry data could facilitate rapid tsetse species identification in the futur