Anthelmintic Treatment of Sheep and the Role of Parasites Refugia in a Local Context.

Gastrointestinal nematodes in grazing livestock are ubiquitous and can cause severe damage, leading to substantial losses in agricultural yields. It is undeniable that the integrated use of anthelmintics is often an essential component of successful intensive livestock management. However, anthelmintic resistance has been a major challenge for several decades, especially in pasture-based lamb production. Measures are therefore needed to reduce the risk and prevent further spread. In many countries with more extensive lamb production and pronounced resistance problems than in Sweden, the importance of keeping parasites in refugia is emphasised. To ensure that treatment is necessary, the Swedish model is based on deworming certain groups of ewes based on the parasitological results of a faecal examination and then releasing them with their lambs to safe pastures. This is intended to reduce the risk of infection, which ultimately reduces the number of subsequent treatments. Whether this preventive strategy in turn means an increased risk of resistance is debatable. In this review, we explain the importance of parasites in refugia and how they can help delay the development of resistance to anthelmintics. We also discuss how likely it is that our model contributes to an increase in resistance risk and whether there is reason to question whether it is a sustainable strategy in the long term.

Signs of multiple anthelmintic resistance in sheep gastrointestinal nematodes in Sweden.

Gastrointestinal nematodes in small ruminants are clinically and economically important parasites that often are controlled with anthelmintics. In this study, we compiled information on the anthelmintic efficacy collected on sheep farms according to routines established by Farm & Animal Health in Sweden. The efficacies of benzimidazoles (i.e. albendazole or fenbendazole, n = 30), ivermectin (n = 47), levamisole (n = 2) or moxidectin (n = 2) were examined between 2015 and 2021 in 81 treatment groups on 49 non-randomly selected farms in south-central Sweden. Drug efficacies were estimated with the faecal egg count reduction test. In addition, efficacy data were in most cases supplemented with data on the abundance of the three most common nematode genera in sheep by performing droplet digital (dd) PCR on coprocultures. Efficacies of <95% for benzimidazoles or ivermectin were identified in 37% and 77% of the tested groups, respectively. In addition, on 27 (55%) of the 49 farms where both benzimidazoles and ivermectin were tested, multiple resistance was found on 8 (30%). In contrast, on each of the two farms tested for levamisole and moxidectin both drugs proved to be 100% effective. However, because post-sampling was performed earlier than recommended in several susceptible groups (benzimidazoles = 15, and ivermectin = 10 groups), this could have underestimated the severity of the situation. Mainly larvae from the genus Haemonchus were detected in post-treatment coprocultures, in all groups with declared resistance, suggesting that this parasite was primarily associated with anthelmintic resistance. Unexpectedly, the DNA of larvae, which survived treatment, was also detected on farms declared as susceptible. Taken together, this indicates that the situation regarding the anthelmintic efficacy has deteriorated compared with the latest nationwide study on Swedish sheep farms conducted more than a decade ago. Unlike the previous study, the farm selection here was not strictly randomized but rather opportunistic i.e., only farms with a recognized parasite problem were included. Thus, there is a need for a truly randomized study to get an update on the extent of the situation of anthelmintic resistance at a national level, as well as to identify risk factors involved in the resistance selection. Research is also required to establish the optimal intervals for sampling post-treatment.

Farmers' perception on the control of gastrointestinal parasites in organic and conventional sheep production in Sweden.

A questionnaire was sent to sheep owners in Sweden to get information about anthelmintic drug use. The survey also investigated how respondents experienced problems with gastrointestinal nematode infections (GIN) focusing on Haemonchus contortus. The response rate was 31% and included both conventional and organic farms. The use of anthelmintics was low (45%), among which a majority (76%) drenched ewes on a single occasion, mostly with ivermectin (59%) followed by albendazole (19%). Other drugs were used rarely, however, unawareness of GIN risk was high (19%), especially among respondents with few animals. Anthelmintic dose calculations were done after visual appraisal by 63% and 22% calibrated the equipment before drug delivery, which is worrying since underdosing is a risk factor for the development of anthelmintic resistance. Like with anthelmintics, the perceived risk for GIN increased with herd size both by conventional and organic farmers. Faecal examination for the presence of GIN was done by 65% of the respondents and, among their sheep, H. contortus was or had been diagnosed in 41% of the herds. Irrespective of new stock had been imported from other countries or not, common problems were reported by 5% and 7% of the organic and conventional producers, respectively. Land use and grazing management strategies differed more in relation to herd size than by production form, with a majority (47%) having their sheep grazed in several paddocks, or at least the lambs were moved when separated from the ewes at weaning (25%). In contrast set stocked grazing was mainly reported on smaller farms. Co-grazing with cattle and horses were also frequently reported irrespective of production form, but with cattle to a somewhat greater degree on larger organic farms. Wild cervids, especially roe deer, were frequently observed on sheep pastures (87%). The veterinary involvement was higher on organic (65%) than on conventional farms (53%), and only 5% considered advice unimportant. Still, some conventional and organic producers treated sheeps routinely without a prior diagnosis, against the national regulations. 46% of the respondents drenched new and replacement stock. In conclusion, although some differences were observed between conventional and organic producers, the divergences were mainly due to herd size categories. Furthermore, despite a high veterinary involvement, we identified factors which can contribute to anthelmintic use, such as poor quarantine procedures, and deworming routines that can contribute to anthelmintic resistance in H. contortus.

Effects of lambing season on nematode faecal egg output in ewes.

In this study, we assessed the occurrence of a periparturient rise (PPR) in winter and spring lambing ewes in Sweden and where nematode egg excretion patterns were investigated mainly for diagnostic purposes. Gastrointestinal nematodes and Haemonchus contortus presence were monitored in parallel in all animals in each experimental group on four farms in samples that were collected mainly when the animals were stabled. Faecal examinations of the same animals were conducted on four sampling occasions between January and June 2018 Each group consisted of 12 crossbreed ewes of similar genotype. One group's peak lambing was in January or February (early), and the other in March or early May (late). The first (S1) and third samples (S3) were from approximately one to two weeks before parturition in the early (winter lambing) and late (spring lambing) group respectively, whereas the second (S2) and fourth samples (S4) were collected approximately three to five weeks post-parturition in the same groups. During the course of the study, there was a significant rise in faecal egg counts (FEC) in both groups on all farms. On three farms with a substantial amount of Haemonchus, we observed a difference in the egg excretion patterns between the two groups of ewes as revealed by a significant interaction between the sampling point and lambing period. Also, when samples corresponding to each other in relation to the number of weeks that had elapsed post-parturition (three to five weeks post-parturition, S2 for early and S4 for late) were compared, FEC were significantly lower in the early group lambing in winter than the late group lambing in spring. This indicates that besides lambing, the rise in nematode egg count is also influenced by other factors unrelated to the lambing period, such as the increased daylight in spring. Due to study limitations, we cannot provide a more detailed explanation for this, but only state that the rise appeared to be more closely linked to season than physiology as measured by day relative to parturition. Still, our results suggests that when turned out, winter lambing ewes contribute to pasture contamination to a lesser extent than those lambing in spring. These results will be used in stipulating evidence-based advice to farmers in their flock management to reduce use of anthelmintics, and at the same time efficiently produce prime lambs.

First case of monepantel resistant nematodes of sheep in Sweden.

In this study, we describe for the first time monepantel (MOP) resistance in gastrointestinal nematodes (GIN) in a Swedish sheep flock. On the farm, which had recurrent problems with Haemonchus contortus infection, the efficacy of most available anthelmintics (AH) in Sweden (i.e. ivermectin, albendazole, levamisole and monepantel), was monitored. This was done with the faecal egg count reduction test (FECRT) on three different occasions between August 2017 and April 2020. Although, MOP was used in ewes for the first time in this herd in October 2018 and then demonstrated to be highly efficacious (100% reduction), MOP-resistant worms (52% reduction) appeared in lambs already in April 2020. Resistance was detected only after two further rounds of treatment of the lambs after weaning. It is assumed that a contributing factor to this extremely rapid development was related to the fact that ewes and lambs treated during the housing period were let out on clean pasture after treatment. The ewes were treated during the housing period 2018 and grazed a clean pasture the following spring. The same ewes were treated a second time after housing 2018. The lambs were grazed with these ewes in summer 2018 and after weaning they were treated and moved to another clean pasture during the fall 2018. Anthelmintic resistance was also confirmed on two occasions to different compounds of ivermectin and once to albendazole, but not to levamisole which was tested twice. In conclusion, this is the first description of triple resistance to AH drugs in GIN of sheep in Sweden.

Detection of Haemonchus contortus on sheep farms increases using an enhanced sampling protocol combined with PCR based diagnostics.

An enhanced sampling strategy for detection of gastrointestinal parasites of sheep based on faecal sampling covering approximately 10% of the animals in the flock was evaluated with focus on the major sheep pathogen Haemonchus contortus. We also compared traditional diagnostics based on faecal eggs counts (FEC) by microscopy with DNA detection on frozen faeces samples using a droplet digital (dd)PCR assay. The investigation was carried out in 2018 in 20 conventional and 19 organic sheep flocks in Sweden with between 70 and 250 production ewes. On 76 different sampling occasions a total of 810 individual faecal samples were collected. Samples were pooled in the laboratory into 270 triplets which were examined both by microscopy and a ddPCR assay. On most farms (95%) a minimum of three triplets were investigated, first from the ewes prior to turn-out and later from the lambs after they had been grazing for at least six weeks. Extra information about the Haemonchus status was provided on 48% of the 76 sampling occasions by including more triplets compared with the old sampling strategy applied in Sweden before 2015 based on two triplets per sampling occasion irrespective of flock size. At a farm level H. contortus was identified by microscopy in 22 (56%) of the 39 flocks and by ddPCR it was found in 28 (72%) flocks with the enhanced protocol. There was a substantial agreement between the two diagnostic tests (Cohens kappa=0.70±0.087). No significant differences in infection levels were observed between the two production systems (conventional and organic) irrespective of the diagnostic method used. However, samples from the ewes were more often Haemonchus positive than those from the lambs indicating that the level of parasite control was in general acceptable. Combined, our results show that Haemonchus infection is widespread throughout Sweden. In conclusion, we have validated a practical tool for sheep producers to assess Haemonchus infection with high precision.

Detection of Haemonchus contortus on sheep farms increases using an enhanced sampling protocol combined with PCR based diagnostics.

An enhanced sampling strategy for detection of gastrointestinal parasites of sheep based on faecal sampling covering approximately 10% of the animals in the flock was evaluated with focus on the major sheep pathogen Haemonchus contortus. We also compared traditional diagnostics based on faecal eggs counts (FEC) by microscopy with DNA detection on frozen faeces samples using a droplet digital (dd)PCR assay. The investigation was carried out in 2018 in 20 conventional and 19 organic sheep flocks in Sweden with between 70 and 250 production ewes. On 76 different sampling occasions a total of 810 individual faecal samples were collected. Samples were pooled in the laboratory into 270 triplets which were examined both by microscopy and a ddPCR assay. On most farms (95%) a minimum of three triplets were investigated, first from the ewes prior to turn-out and later from the lambs after they had been grazing for at least six weeks. Extra information about the Haemonchus status was provided on 48% of the 76 sampling occasions by including more triplets compared with the old sampling strategy applied in Sweden before 2015 based on two triplets per sampling occasion irrespective of flock size. At a farm level H. contortus was identified by microscopy in 22 (56%) of the 39 flocks and by ddPCR it was found in 28 (72%) flocks with the enhanced protocol. There was a substantial agreement between the two diagnostic tests (Cohens kappa = 0.70 ± 0.087). No significant differences in infection levels were observed between the two production systems (conventional and organic) irrespective of the diagnostic method used. However, samples from the ewes were more often Haemonchus positive than those from the lambs indicating that the level of parasite control was in general acceptable. Combined, our results show that Haemonchus infection is widespread throughout Sweden. In conclusion, we have validated a practical tool for sheep producers to assess Haemonchus infection with high precision.

Detection of Tick-Borne Pathogens in Lambs Undergoing Prophylactic Treatment Against Ticks on Two Swedish Farms.

Tick-borne pathogens (TBPs), especially Anaplasma phagocytophilum, cause disease in grazing livestock. Tick prophylaxis is, therefore, a routine practice in sheep flocks in Sweden, especially in central, southern, and coastal areas of the country where ixodid ticks (Ixodes ricinus and Haemaphysalis punctata) are present. In the present study, the status of infection by A. phagocytophilum and other TBPs in lambs treated with tick prophylaxis has been assessed serologically and with polymerase chain reaction (PCR). Blood samples (n = 78) from lambs (n = 20) subjected to regular tick prophylactic treatment (flumethrin, Bayticol®) at two sites in different regions in Sweden (Östergötland, Gotland) were collected on four occasions from May until July 2013. The severity of clinical signs in Anaplasma-infected animals is known to differ between these two regions. In total, 20% of blood samples were PCR-positive for A. phagocytophilum. Serological analyses showed that 33% of all collected samples were positive for A. phagocytophilum, while 2.5% were positive for Borrelia burgdorferi s.l. and 13% for tick-borne encephalitis virus (TBEV). Percentages of lambs positive were 75 and 45% for A. phagocytophilum antibodies and DNA, respectively, while 10 and 45% were serologically positive for B. burgdorferi s.l. and TBEV, respectively. Sequencing of partial 16S rRNA genes from Anaplasma PCR positive samples revealed presence of A. phagocytophilum in all animals in Östergötland, while sequences consistent with A. phagocytophilum as well as A. capra and A. bovis were found on the island of Gotland. This is the first report of the occurrence of the latter two species in Sweden.

Failure of ivermectin treatment in Haemonchus contortus infected-Swedish sheep flocks.

Control of gastrointestinal nematodes of veterinary importance in Swedish sheep flocks is primarily based on recurring strategic anthelmintic treatments after detection of strongyle eggs in faeces samples. This study reports reduced efficacy of ivermectin (IVM) against Haemonchus contortus in naturally infected Swedish sheep flocks. Faecal egg count reduction tests (FECRT) and examinations of H. contortus-specific DNA with qPCR on larval cultures were applied to samples from 11 sheep flocks (A-K) in south-eastern Sweden between 2013 and 2014. Four of these flocks (D, E, J and K) had been in direct contact with flock H, where IVM treatment failure was first observed in October 2013, some years after the introduction of imported dairy sheep. In flock H, the resistance status to IVM was also confirmed by a larval developmental test. IVM concentrations 15-20 times higher than for susceptible strains of H. contortus were required to kill the larvae. In addition, faeces samples were obtained from 37 other Swedish sheep farms where the treatment response to IVM was screened initially in six animals using FEC and qPCR 7-10days after administration of IVM. Six farms where the majority was identified with this pre-screening test (B, C, F, G, I and K), were also investigated in more detail with FECRT as described above after the animals had been allocated to groups and treated orally or injected with a minimum of 0.2mg IVM, 0.2mg doramectin (farm F) or 0.2mg moxidectin per kg body weight (farm A and B). Four flocks (farm A, D, G and I) were also treated with 4.8mg albendazole and/or 7.5mg levamisole per kg body weight. Pre-treatment faeces samples were collected from 15 animals on the same day as deworming. Post-treatment samples were collected 7-10days later, whenever possible from 10 animals per group with the highest pre-treatment egg counts. Based on FECRT results, IVM efficacy to H. contortus was reduced on six farms (C, D, E, G, H and I) out of 11 farms studied with FECRT. This is the first report of IVM treatment failure in H. contortus-infected sheep in Sweden.

Transmission patterns of Fasciola hepatica to ruminants in Sweden.

Transmission patterns of Fasciola hepatica were investigated on beef cattle (n=3) and sheep (n=3) farms in Sweden between 2011 and 2012. The dynamics of fluke infection, particularly estimated time of infection, were screened each grazing season by ELISA detection of antibodies in lambs (n=94) and first grazing season calves (n=61). Colostral transfer of F. hepatica antibodies from seropositive ewes was detected in sheep up to 11 weeks of age. In sheep, the estimated time of infection differed significantly between herds and years. Typical 'winter infection' was observed on two sheep farms in 2012, but the most prevalent transmission pattern was found to be 'summer infection', characterised by infection of animals in late summer by F. hepatica originating from overwintered and/or spring-excreted eggs. In contrast, beef calves were infected mainly in September-October ('summer infection'). Furthermore, lymnaeid and succineid snails were collected on the pastures used by these animals both in spring and in the autumn each year. In total, 1726, 588, 138, 130, 93 and 42 specimens of Galba truncatula, Lymnaea palustris, Lymnaea glabra, Lymnaea fuscus, Radix peregra and Succinea putris, respectively, were collected and identified. These were subsequently examined for the presence of F. hepatica DNA by species-specific PCR and the findings compared against mean monthly rainfall and temperature data for each farm. The main intermediate host of the liver fluke was G. truncatula, with a prevalence range of F. hepatica infection from 0% to 82%. Only 1 out of 42 terrestrial S. putris tested positive for F. hepatica, casting doubt on the role of this species in transmission of F. hepatica in Sweden. In conclusion, two main peak periods of infection were observed: May-June (from overwintered infected snails='winter infection') and August-September (from metacercariae developed and produced by snails during summer='summer infection'). The occurrence and frequency of 'winter infection' were dependent on local environmental factors such as snail habitat availability or grazing behaviour of animals, rather than on climatic factors.

Anthelmintic resistance in Swedish sheep flocks based on a comparison of the results from the faecal egg count reduction test and resistant allele frequencies of the beta-tubulin gene.

A faecal egg count reduction test (FECRT) survey was conducted during the grazing season 2006 and 2007 to provide an updated indication of the prevalence of anthelmintic resistance in sheep flocks in Sweden. A total of 1330 faecal samples from 90 flocks on 45 farms, with a minimum of 20 ewes each, was collected by local sheep veterinarians. Per treatment group, approximately 15 lambs were dewormed either with oral suspensions of ivermectin (Ivomec vet.) or albendazole (Valbazen vet.). The efficacy on each farm was investigated either in 2006 or 2007 by faecal egg counts collected on the day of treatment and in a new sample from the same animals 7-10 days later. Third-stage larvae (L3) were initially identified morphologically from pooled cultures. These were then used as the source of genomic DNA template for two molecular tests. The first was a PCR-based test for specific identification of Haemonchus contortus, and the second was a Pyrosequencing assay for the analysis of benzimidazole (BZ) resistance targeting the P200 mutation in the parasite's beta-tubulin gene. Larval cultures indicated that Teladorsagia and Trichostrongylus were the predominant genera, but Haemonchus was diagnosed in 37% of the flocks. The PCR results revealed an almost 100% agreement with those farms that had previously been shown to have Haemonchus present, even when the % prevalence was low (approximately 3%). Only two (4%) of the surveyed farms showed evidence of BZ-resistant worm populations, with H. contortus being the species implicated according to post-treatment larval culture results. The Pyrosequencing assay detected BZ resistant allele frequencies of >40% in the Haemonchus-positive farms and 100% resistant alleles in the clinically most resistant farms. These preliminary results suggest that the FECRT is less sensitive than the molecular test at detecting BZ resistance. However, both tests need to be interpreted carefully, bearing in mind the relative proportions of species present and the starting egg and/or larval counts. Parasitological diagnosis of "clinical" resistance was also found against ivermectin in two flocks. However, both the pre-treatment FECs and the reductions in these were low, and only three lambs that had between 100 and 450 EPG after treatment were involved.

Production and secretion of interleukin-1alpha proteins by rat testis.

The present study characterizes constitutively expressed rat testicular interleukin-1alpha (IL-1alpha) proteins. IL-1 bioactivity of crude testis protein was completely neutralized by IL-1alpha antiserum, IL-1 receptor antagonist, and soluble type I IL-1 receptor. Upon non-denaturating gel permeation chromatography, bioactive IL-1 eluted at molecular sizes of 45, 31, and 17kDa and at charges of pH 5.7 and 6.0 after chromatofocusing. SDS-PAGE/Western blot analysis of proteins extracted from whole testis, seminiferous tubules, interstitial, and seminiferous tubule fluids all demonstrated IL-1alpha immunoreactivity at 45, 24, and 19kDa. Activated macrophages and tissue proteins from endotoxin treated rats showed immunoreactive 31 and 19kDa IL-1alpha. The results indicate that the testis produces three isoforms of IL-1alpha proteins that are secreted into the interstitial compartment and tubular lumen where they may exert paracrine functions. The testicular IL-1alpha isoforms may represent posttranslationally modified precursor, mature IL-1alpha, and a 24-kDa alternate splice form.