Correction to: Detection of Candidatus Neoehrlichia mikurensis in Norway up to the northern limit of Ixodes ricinus distribution using a novel real time PCR test targeting the groEL gene.

After publication of our article [1] it came to our notice that the source of the sequence for the control plasmid, pNeo (Materials and methods: Controls) was incorrectly stated as AB094461. The correct accession number is AB074461. The authors apologize for any confusion this may have caused.

Detection of Candidatus Neoehrlichia mikurensis in Norway up to the northern limit of Ixodes ricinus distribution using a novel real time PCR test targeting the groEL gene.

BACKGROUND: Candidatus Neoehrlichia mikurensis is an emerging tick-borne pathogen. It is widely distributed in Ixodes ricinus ticks in Europe, but knowledge of its distribution in Norway, where I. ricinus reaches its northern limit, is limited. In this study we have developed a real time PCR test for Ca. N. mikurensis and used it to investigate the distribution of Ca. N. mikurensis in Norway. RESULTS: Real time PCR targeting the groEL gene was developed and shown to be highly sensitive. It was used to detect Ca. N. mikurensis in 1651 I. ricinus nymphs and adults collected from twelve locations in Norway, from the eastern Oslo Fjord in the south to near the Arctic Circle in the north. The overall prevalence was 6.5% and varied locally between 0 and 16%. Prevalence in adults and nymphs was similar, suggesting that ticks acquire Ca. N. mikurensis predominantly during their first blood meal. In addition, 123 larvae were investigated; Ca. N. mikurensis was not found in larvae, suggesting that transovarial transmission is rare or absent. Sequence analysis suggests that a single variant dominates in Norway. CONCLUSIONS: Ca. N. mikurensis is widespread and common in ticks in Norway and reaches up to their northern limit near the Arctic Circle. Ticks appear to acquire Ca. N. mikurensis during their first blood meal. No evidence for transovarial transmission was found.

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.

Predicting and mapping human risk of exposure to Ixodes ricinus nymphs using climatic and environmental data, Denmark, Norway and Sweden, 2016.

BackgroundTick-borne diseases have become increasingly common in recent decades and present a health problem in many parts of Europe. Control and prevention of these diseases require a better understanding of vector distribution.AimOur aim was to create a model able to predict the distribution of Ixodes ricinus nymphs in southern Scandinavia and to assess how this relates to risk of human exposure.MethodsWe measured the presence of I. ricinus tick nymphs at 159 stratified random lowland forest and meadow sites in Denmark, Norway and Sweden by dragging 400 m transects from August to September 2016, representing a total distance of 63.6 km. Using climate and remote sensing environmental data and boosted regression tree modelling, we predicted the overall spatial distribution of I. ricinus nymphs in Scandinavia. To assess the potential public health impact, we combined the predicted tick distribution with human density maps to determine the proportion of people at risk.ResultsOur model predicted the spatial distribution of I. ricinus nymphs with a sensitivity of 91% and a specificity of 60%. Temperature was one of the main drivers in the model followed by vegetation cover. Nymphs were restricted to only 17.5% of the modelled area but, respectively, 73.5%, 67.1% and 78.8% of the human populations lived within 5 km of these areas in Denmark, Norway and Sweden.ConclusionThe model suggests that increasing temperatures in the future may expand tick distribution geographically in northern Europe, but this may only affect a small additional proportion of the human population.

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.

Detection of Anaplasma phagocytophilum in Ixodes ricinus ticks from Norway using a realtime PCR assay targeting the Anaplasma citrate synthase gene gltA.

BACKGROUND: A TaqMan real-time PCR assay targeting the Anaplasma citrate synthase gene, gltA, was developed and used for detection of Anaplasma phagocytophilum in 765 Ixodes ricinus ticks collected from dogs and cats in northern Norway (n = 669) and Telemark county in southern Norway (n = 96). RESULTS: Among the ticks from northern Norway the prevalence of A. phagocytophilum was 3.0 %, while the prevalence in southern Norway was 2.1 % (p = 0.63). The gltA PCR assay showed a high analytical sensitivity (30 genomic units) and efficiency (98.5 %), and its utility in clinical diagnostics should be evaluated in future studies. CONCLUSION: This is the first report of A. phagocytophilum occurrence in ticks collected north of the Arctic Circle in Norway. The prevalence is comparable to that found in Telemark county in southern Norway.

Occurrence of gastrointestinal nematodes in lambs in Norway, as assessed by copromicroscopy and droplet digital polymerase chain reaction.

BACKGROUND: Gastrointestinal nematodes (GINs) have a major impact on sheep production, health, and welfare worldwide. Norway is no exception, but there are only a few studies on the prevalence of GINs in Norwegian sheep. The aim of this study was to investigate the current occurrence of the most important nematodes in sheep flocks in Norway. Faecal samples were collected from flocks in 2021/2022, mainly from three geographical regions in Norway, i.e., northern, eastern, and western. In each of 134 flocks included, individual samples from 10 lambs (autumn) were pooled. Third stage larvae (L3) were cultivated and harvested (Baermann method) from the pooled samples. The DNA was then extracted and further analysed using droplet digital PCR (ddPCR). This enables assessment of the proportions of the three most important nematode species/genera, i.e., H. contortus, T. circumcincta, and Trichostrongylus. The fractional abundance/relative proportion of each species/genus was assessed by performing duplex assays with universal strongyle and species/genus-specific primers and probe sets. In addition, the occurrence of Nematodirus eggs was assessed by standard faecal egg counts (i.e., McMaster method). RESULTS: Of the 134 flocks sampled, 24 were from the northern region, 31 from eastern, and 71 from western Norway. In addition, some flocks from central (n = 7), and southern (n = 1) Norway were included. Among the sampled flocks, T. circumcincta occurred most commonly (94%), followed by H. contortus (60%) and Trichostrongylus (55%), and Nematodirus (51%). In general, mixed infections were observed, with 38% and 18% of flocks infected with three or all four genera, respectively. CONCLUSIONS: The results of this study indicate that GINs are widespread in Norway. Teladorsagia circumcincta seems to be present in most flocks based on this screening. Moreover, the results show that Nematodirus spp. infect lambs throughout the country, predominantly N. battus, and indicate that this nematode has become more abundant, which could lead to an increase in nematodirosis.

Benzimidazole-resistance associated mutation in Haemonchus contortus in Norwegian sheep, as detected by droplet digital PCR.

The aim of this study was to investigate the occurrence of benzimidazole-resistant Haemonchus contortus in Norwegian sheep flocks. Screening was based on detection of one of the resistance-conferring mutations in the ß tubulin isotype 1 gene (F200Y, TAC) in larvae (L3) cultivated from H. contortus eggs from naturally infected sheep. Faecal samples were collected in 2021/2022 from flocks in the northern (n = 34), central (n = 7), eastern (n = 40), southern (n = 1), and western (n = 87) areas of Norway. In total, samples were taken from 169 flocks (spring-ewes samples: 167, autumn-lambs samples: 134). Individual faecal samples were collected from 10 randomly selected ewes (spring) and 10 randomly selected lambs (autumn) in each flock. Faecal samples collected from each flock on each occasion were pooled (lamb and ewe samples pooled separately) and cultured for L3 development. After harvest of larvae (Baermann method), DNA was extracted and then analysed using droplet digital PCR with primer/probe sets targeting the BZ-associated F200Y (TAC) mutation. Haemonchus was found in 60% (80/134) of samples from lambs, and in 63% (106/167) from ewes. Among these, the F200Y mutation was detected in 73% (58/80) of larval samples from lambs and 69% (73/106) of larval samples from ewes, respectively. Although regional differences were evident, the mutation was detected in all areas indicating a widespread distribution and a strong potential for an increasing problem with treatment-resistant haemonchosis in Norwegian sheep flocks.

Temporal patterns of gene expression in response to inoculation with a virulent Anaplasma phagocytophilum strain in sheep.

The aim of this study was to characterize the gene expression of host immune- and cellular responses to a Norwegian virulent strain of Anaplasma phagocytophilum, the cause of tick-borne fever in sheep. Ten sheep were intravenously inoculated with a live virulent strain of A. phagocytophilum. Clinical-, observational-, hematological data as well as bacterial load, flow cytometric cell count data from peripheral blood mononuclear cells and host's gene expression post infection was analysed. The transcriptomic data were assessed for pre-set time points over the course of 22 days following the inoculation. Briefly, all inoculated sheep responded with clinical signs of infection 3 days post inoculation and onwards with maximum bacterial load observed on day 6, consistent with tick-borne fever. On days, 3-8, the innate immune responses and effector processes such as IFN1 signaling pathways and cytokine mediated signaling pathways were observed. Several pathways associated with the adaptive immune responses, namely T-cell activation, humoral immune responses, B-cell activation, and T- and B-cell differentiation dominated on the days of 8, 10 and 14. Flow-cytometric analysis of the PBMCs showed a reduction in CD4+CD25+ cells on day 10 and 14 post-inoculation and a skewed CD4:CD8 ratio indicating a reduced activation and proliferation of CD4-T-cells. The genes of important co-stimulatory molecules such as CD28 and CD40LG, important in T- and B-cell activation and proliferation, did not significantly change or experienced downregulation throughout the study. The absence of upregulation of several co-stimulatory molecules might be one possible explanation for the low activation and proliferation of CD4-T-cells during A. phagocytophilum infection, indicating a suboptimal CD4-T-cell response. The upregulation of T-BET, EOMES and IFN-γ on days 8-14 post inoculation, indicates a favoured CD4 Th1- and CD8-response. The dynamics and interaction between CD4+CD25+ and co-stimulatory molecules such as CD28, CD80, CD40 and CD40LG during infection with A. phagocytophilum in sheep needs further investigation in the future.

Prevalence of tick-borne encephalitis virus in questing Ixodes ricinus nymphs in southern Scandinavia and the possible influence of meteorological factors.

Ixodes ricinus ticks are Scandinavia's main vector for tick-borne encephalitis virus (TBEV), which infects many people annually. The aims of the present study were (i) to obtain information on the TBEV prevalence in host-seeking I. ricinus collected within the Øresund-Kattegat-Skagerrak (ØKS) region, which lies in southern Norway, southern Sweden and Denmark; (ii) to analyse whether there are potential spatial patterns in the TBEV prevalence; and (iii) to understand the relationship between TBEV prevalence and meteorological factors in southern Scandinavia. Tick nymphs were collected in 2016, in southern Scandinavia, and screened for TBEV, using pools of 10 nymphs, with RT real-time PCR, and positive samples were confirmed with pyrosequencing. Spatial autocorrelation and cluster analysis was performed with Global Moran's I and SatScan to test for spatial patterns and potential local clusters of the TBEV pool prevalence at each of the 50 sites. A climatic analysis was made to correlate parameters such as minimum, mean and maximum temperature, relative humidity and saturation deficit with TBEV pool prevalence. The climatic data were acquired from the nearest meteorological stations for 2015 and 2016. This study confirms the presence of TBEV in 12 out of 30 locations in Denmark, where six were from Jutland, three from Zealand and two from Bornholm and Falster counties. In total, five out of nine sites were positive from southern Sweden. TBEV prevalence of 0.7%, 0.5% and 0.5%, in nymphs, was found at three sites along the Oslofjord (two sites) and northern Skåne region (one site), indicating a potential concern for public health. We report an overall estimated TBEV prevalence of 0.1% in questing I. ricinus nymphs in southern Scandinavia with a region-specific prevalence of 0.1% in Denmark, 0.2% in southern Sweden and 0.1% in southeastern Norway. No evidence of a spatial pattern or local clusters was found in the study region. We found a strong correlation between TBEV prevalence in ticks and relative humidity in Sweden and Norway, which might suggest that humidity has a role in maintaining TBEV prevalence in ticks. TBEV is an emerging tick-borne pathogen in southern Scandinavia, and we recommend further studies to understand the TBEV transmission potential with changing climate in Scandinavia.

Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum.

BACKGROUND: Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain. RESULTS: Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S. CONCLUSIONS: Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.

Prevalence of anthelmintic resistance in gastrointestinal nematodes of sheep and goats in Norway.

In the period of 2008­2009, the efficacies of the benzimidazole (BZ) albendazole and the macrocyclic lactone (ML) ivermectin against gastrointestinal nematodes (GIN) of small ruminants were evaluated by means of the fecal egg count reduction (FECR) test and by post-treatment identification of surviving third stage (L3) larvae after coproculture. Sheep (n=28) and goat (n=28) flocks from three areas of Norway were randomly selected to assess the prevalence of anthelmintic resistance (AR), whereas only lambs from non-randomly selected sheep flocks (n=32) with a farm management that could select for AR were investigated the second year. Only flocks with a mean excretion of nematode eggs per gram feces (EPG) ≥ 150 at time of treatment were included in the survey. In total, 48 (80%) and 13 (46.4%) of the selected sheep and goat flocks, respectively, fulfilled the inclusion criteria. The proportions of flocks classified as resistant (i.e., FECR <95% and with a lower 95% confidence interval of <90%) for the BZ drug albendazole were 10.5% and 31.0% in the randomly and non-randomly selected sheep flocks, respectively. When restricting the area to Rogaland County, eight flocks out of ten (80%) non-randomly selected sheep flocks showed BZ resistance. The efficacy of ML was 100% in all surveyed sheep and goat flocks. In post-treatment coprocultures from the non-randomly selected flocks, the main nematode genera were Teladorsagia/Trichostrongylus in five flocks, Haemonchus in two flocks, and a mixture of these genera in the remaining two flocks. In the goat flocks, the pre-treatment infection levels of GIN were low compared to what was found in the sheep flocks. Still, in one flock, AR against BZ in Teladorsagia/Trichostrongylus was found. New strategies and recommendations to face the emerging AR situation in Rogaland County in order to limit the spread of resistant nematodes within and into other areas are urgently needed.

Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Norway: evaluation of a PCR test targeting the chromosomal flaB gene.

A consensus TaqMan real-time PCR test targeting the chromosomal flaB gene of Borrelia burgdorferi sensu lato was constructed. The test was compared with a recently published generic Light Upon eXtension (LUX) 16S rRNA real-time PCR test (Wilhelmsson et al. in J Clin Microbiol 48:4169-4176, 2010) on material consisting of 242 Ixodes ricinus ticks collected from dogs and cats in Northern Norway (n = 139) and Telemark County in Southern Norway (n = 103). Ticks positive in either test were further tested by nested PCR amplification of the 5S-23S rRNA intergenic-spacer region followed by sequencing for species identification. A tick was defined as Borrelia positive if two of three tests were positive. Thirty-four of the 242 (14 %) ticks satisfied this definition of positivity. Of these ticks 32 were positive both in the rRNA and flaB test, while two were positive only in the rRNA test. One tick was positive only in the rRNA test and was considered false positive since PCR for sequencing failed. The sensitivity of the flaB test was 94 % and the specificity 100 %. It was possible to determine the species present using Tm analysis. Among ticks from Northern Norway the prevalence of Borrelia was 13 %, whereas the prevalence in Telemark was 16 %. Among identified species (n = 33) B. afzelii was found in 16 (47 %), B. garinii in 15 (44 %) and B. valaisiana in 2 (6 %) ticks, respectively. The flaB test is a rapid, sensitive and specific test for detection and quantification of Borrelia burgdorferi s.l. in I. ricinus ticks. This is the first report on Borrelia prevalence in I. ricinus in Northern Norway.

Fasciolosis-An Increasing Challenge in the Sheep Industry.

The liver fluke Fasciola hepatica may cause severe infection in several mammalian species, including sheep and humans. Fasciolosis is a parasitic disease occurring worldwide in temperate climates and involves intermediate lymnaeid snails as vectors, in Europe the pond snail Galba truncatula in particular. In the sheep industry, the disease is a serious welfare and health problem. Fasciolosis is usually classified as acute, subacute or chronic according to the number and stage of flukes present in the liver, but with a considerable overlap. Acute disease, associated with a large number of migrating larvae, often results in sudden death due to acute and massive hemorrhage, while chronic fasciolosis is characterized by anemia, hypoalbuminaemia and weight loss. The management of fasciolosis is an increasing challenge in the sheep industry. Early diagnostic tests are limited. Protective immunity against liver flukes in sheep is low or lacking, and vaccines are not yet available. Treatment and control possibilities are challenging, and resistance to flukicide drugs is increasing. In addition, climate change with warmer and more humid weather will have a substantial effect on the establishment of both flukes and snails and will most likely increase the future distribution of F. hepatica.

Comparative Whole Genome Analysis of an Anaplasma phagocytophilum Strain Isolated from Norwegian Sheep.

Anaplasma phagocytophilum is a Gram-negative obligate intracellular tick-borne alphaproteobacteria (family Anaplasmatacea, order Rickettsiales) with a worldwide distribution. In Norway, tick borne fever (TBF), caused by A. phagocytophilum, presents a major challenge in sheep farming. Despite the abundance of its tick vector, Ixodes ricinus, and A. phagocytophilum infections in wild and domestic animals, reports of infections in humans are low compared with cases in the U.S. Although A. phagocytophilum is genetically diverse and complex infections (co-infection and superinfection) in ruminants and other animals are common, the underlying genetic basis of intra-species interactions and host-specificity remains unexplored. Here, we performed whole genome comparative analysis of a newly cultured Norwegian A. phagocytophilum isolate from sheep (ApSheep_NorV1) with 27 other A. phagocytophilum genome sequences derived from human and animal infections worldwide. Although the compared strains are syntenic, there is remarkable genetic diversity between different genomic loci including the pfam01617 superfamily that encodes the major, neutralization-sensitive, surface antigen Msp2/p44. Blast comparisons between the msp2/p44 pseudogene repertoires from all the strains showed high divergence between U. S. and European strains and even between two Norwegian strains. Based on these comparisons, we concluded that in ruminants, complex infections can be attributed to infection with strains that differ in their msp2/p44 repertoires, which has important implications for pathogen evolution and vaccine development. We also present evidence for integration of rickettsial DNA into the genome of ISE6 tick cells.

Transient Expression of Flavivirus Structural Proteins in Nicotiana benthamiana.

Flaviviruses are a threat to public health and can cause major disease outbreaks. Tick-borne encephalitis (TBE) is caused by a flavivirus, and it is one of the most important causes of viral encephalitis in Europe and is on the rise in Sweden. As there is no antiviral treatment available, vaccination remains the best protective measure against TBE. Currently available TBE vaccines are based on formalin-inactivated virus produced in cell culture. These vaccines must be delivered by intramuscular injection, have a burdensome immunization schedule, and may exhibit vaccine failure in certain populations. This project aimed to develop an edible TBE vaccine to trigger a stronger immune response through oral delivery of viral antigens to mucosal surfaces. We demonstrated successful expression and post-translational processing of flavivirus structural proteins which then self-assembled to form virus-like particles in Nicotiana benthamiana. We performed oral toxicity tests in mice using various plant species as potential bioreactors and evaluated the immunogenicity of the resulting edible vaccine candidate. Mice immunized with the edible vaccine candidate did not survive challenge with TBE virus. Interestingly, immunization of female mice with a commercial TBE vaccine can protect their offspring against TBE virus infection.

Distinct host species correlate with Anaplasma phagocytophilum ankA gene clusters.

Anaplasma phagocytophilum is a gram-negative, tick-transmitted, obligate intracellular bacterium that elicits acute febrile diseases in humans and domestic animals. In contrast to the United States, human granulocytic anaplasmosis seems to be a rare disease in Europe despite the initial recognition of A. phagocytophilum as the causative agent of tick-borne fever in European sheep and cattle. Considerable strain variation has been suggested to occur within this species, because isolates from humans and animals differed in their pathogenicity for heterologous hosts. In order to explain host preference and epidemiological diversity, molecular characterization of A. phagocytophilum strains has been undertaken. Most often the 16S rRNA gene was used, but it might be not informative enough to delineate distinct genotypes of A. phagocytophilum. Previously, we have shown that A. phagocytophilum strains infecting Ixodes ricinus ticks are highly diverse in their ankA genes. Therefore, we sequenced the 16S rRNA and ankA genes of 194 A. phagocytophilum strains from humans and several animal species. Whereas the phylogenetic analysis using 16S rRNA gene sequences was not meaningful, we showed that distinct host species correlate with A. phagocytophilum ankA gene clusters.

Anthelmintic resistance in small ruminants in the Nordic-Baltic region.

Gastrointestinal nematodes (GIN) in small ruminants result in production losses, and consequently economic losses, and are an animal welfare problem in most countries in the Nordic-Baltic region. Intensive use of anthelmintics to control helminth infections has led to anthelmintic resistance (AR), which has become a major issue in many European countries. Several studies have been performed in countries in the Nordic-Baltic region (e.g. Denmark, Sweden, Norway and Lithuania) showing increasing/emerging levels of AR. The aim of this paper is to provide an overview of the problem of AR on sheep and goat farms in the Nordic-Baltic region. This region has a limited number of registered anthelmintics. However, researchers in this area have discovered some surprising findings, such as ivermectin (IVM) resistance on a farm that had never used IVM. In Sweden there is evidence of macrocyclic lactone (ML)-resistant Haemonchus contortus being introduced with sheep imported from the Netherlands. As elsewhere in the world, the livestock trade appears to be contributing to the spread of AR in the region and isolated cases of multidrug-resistant cases have also been reported. This is surprising given that the frequency of treatments here is much lower than in other countries where sheep production is economically more important. The prevailing nematodes are Haemonchus, Teledorsagia and Trichostrongylus, while on some farms Haemonchus is dominant and clinical haemonchosis has increasingly been observed in recent decades. The reasons for this are unclear, but are probably related to this parasite's propensity to rapidly develop drug resistance and a general lack of awareness of the problem, possibly in combination with global warming and the increased livestock trade within the EU. In addition, domestic interactions through contacts with wildlife ruminants, alpacas may also be a contributing factor for transmission of AR.

Treatment against helminths in Norwegian sheep: a questionnaire-based survey.

A questionnaire was distributed to 5487 farmers throughout Norway in order to obtain information about management practices regarding helminth infections in sheep. In addition, the farmers' perceptions of helminths and anthelmintic efficacy were investigated. Most farmers (80%) treated prophylactically against nematodes, and 24% also used prophylactic treatment against Fasciola hepatica. Overall, few farmers (11%) used parasitological analysis as a tool to assess the timing of treatment, but rather based it on other factors such as previous experience (70%). In the surveyed sheep flocks, the use of benzimidazoles was reduced from 2018 (52%) to 2019 (47%) (p < 0.01), whereas the use of macrocyclic lactones increased from 2017 (23%) to 2019 (36%) (p < 0.001). Poor anthelmintic efficacy was suspected by 10% of the farmers, and 11% reported that helminths were an increasing problem in their flocks. The majority of farmers (72%) considered their veterinarian as the most important advisor for treatment of parasites, but reported a high level of uncertainty regarding which parasites were present in their flocks, with unknown status most frequently reported for Haemonchus contortus (71.5%). This is probably related to the fact that very few farmers (15%) regularly test their animals for parasites. The present study provides up-to-date information on treatment practices for helminths in Norwegian sheep flocks.

TITLE: Traitement contre les helminthes chez les moutons norvégiens : une enquête par questionnaire. ABSTRACT: Un questionnaire a été distribué à 5487 éleveurs dans l'ensemble de la Norvège afin d'obtenir des informations sur la gestion des helminthiases chez les ovins. Le questionnaire a porté, en outre, sur la perception des éleveurs concernant les helminthiases et l'efficacité des anthelminthiques. La plupart des éleveurs (80 %) réalisent des traitements prophylactiques contre les nématodes et 24 % font de même vis-à-vis de Fasciola hepatica. Globalement, peu d'éleveurs (11 %) recourent aux analyses parasitologiques pour déterminer les dates de traitement, s'appuyant plutôt sur d'autres éléments tels que leur propre expérience (70 %). Dans les troupeaux enquêtés, l'utilisation des benzimidazoles a diminué de 2018 (52 %) à 2019 (47 %) (p < 0,01) tandis que celle des lactones macrocycliques a augmenté entre 2017 (23 %) et 2019 (36 %) (p < 0,001). Une faible efficacité des anthelminthiques est suspectée par 10 % des éleveurs tandis que 11 % des éleveurs signalent que les helminthiases sont un problème croissant dans leurs troupeaux. La majorité des éleveurs (72 %) considèrent leur vétérinaire comme le conseiller le plus important en matière de traitement antiparasitaire mais rapportent un haut niveau d'incertitude concernant le type de parasites présents dans leur troupeau, en particulier en ce qui concerne la présence d'Haemonchus contortus (71,5 %). Ceci est probablement à relier au fait que très peu d'éleveurs (15 %) testent régulièrement leurs animaux vis-à-vis des parasites. La présente étude fournit des informations actualisées sur les pratiques de traitement anthelminthique des troupeaux ovins en Norvège.

Clinical and immunological responses in sheep after inoculation with Himar1-transformed Anaplasma phagocytophilum and subsequent challenge with a virulent strain of the bacterium.

In Norway, the tick-transmitted bacterium Anaplasma phagocytophilum is estimated to cause tick-borne fever (TBF) in 300 000 lambs on pastures each year, resulting in economic and animal welfare consequences. Today, prophylactic measures mainly involve the use of acaricides, but a vaccine has been requested by farmers and veterinarians for decades. Several attempts have been made to produce a vaccine against A. phagocytophilum including antigenic surface proteins, inactivated whole cell vaccines and challenge followed by treatment. In the current study, a virulent wild type strain of A. phagocytophilum named Ap.Norvar1 (16S rRNA sequence partial identical to sequence in GenBank acc.no M73220) was subject to genetic transformation with a Himar1-transposon, which resulted in three bacterial mutants, capable of propagation in a tick cell line (ISE6). In order to test the immunogenicity and pathogenicity of the live, mutated bacteria, these were clinically tested in an inoculation- and challenge study in sheep. One group was inoculated with the Ap.Norvar1 as an infection control. After inoculation, the sheep inoculated with mutated bacteria and the Ap.Norvar1 developed typical clinical signs of infection and humoral immune response. After challenge with Ap.Norvar1, 28 days later all groups inoculated with mutated bacteria showed clinical signs of tick-borne fever and bacteremia while the group initially inoculated with the Ap.Norvar1, showed protection against clinical disease. The current study shows a weak, but partial protection against infection in animals inoculated with mutated bacteria, while animals that received Ap.Norvar1 both for inoculation and challenge, responded with homologues protection.