Differences in Genotype and Antimicrobial Resistance between Campylobacter spp. Isolated from Organic and Conventionally Produced Chickens in Sweden.

Antibiotic resistance is a major challenge worldwide and increased resistance to quinolones in Campylobacter is being reported. Analysis of antibiotic resistance was performed on 157 Campylobacter strains (123 C. jejuni and 34 C. coli) from conventional and organic chickens produced in Sweden. Susceptibility for tetracycline, ciprofloxacin, erythromycin, nalidixic acid, streptomycin, and gentamycin was determined by microdilution. All 77 isolates from organic chickens were sensitive to all antibiotics, except two C. jejuni that were resistant to tetracycline. Of the 80 isolates from conventional chickens, 22.5% of C. jejuni and 11.1% of C. coli were resistant to quinolones and 5.6% of C. jejuni were resistant to tetracycline. Whole-genome sequencing resulted in 50 different sequence types of C. jejuni and six of C. coli. Nine sequence types were found in both organic and conventional chickens. Two of these (ST-19 and ST-257) included isolates from conventional broilers with different resistance phenotypes to the remaining isolates from conventional and organic broilers. There are management differences between the production systems, such as feed, breed, use of coccidiostats, and access to outdoor area. It is unlikely that quinolone resistance has arisen due to use of antimicrobials, since fluoroquinolones are not permitted in Swedish broiler production.

Droplet digital polymerase chain reaction (ddPCR) as a novel method for absolute quantification of major gastrointestinal nematodes in sheep.

In this paper, we present for the first time a new tool, based on Droplet Digital™ Polymerase Chain Reaction (ddPCR), for absolute quantification of key genera of gastrointestinal (GI) nematode parasites of grazing livestock. Four combinations of primers/probe sets targeting the internal transcribed spacer region 2 (ITS2) of the ribosomal RNA gene array were designed using the Primer3 software, following in silico analysis of nucleotide sequences from nematodes of interest downloaded from common databases. The amplified regions include both a universal region for detection of any strongylid gastrointestinal parasite and three different genus specific regions, making it possible to differentiate between the most important GI nematodes of sheep in Sweden: Haemonchus, Teladorsagia and Trichostrongylus. Analysis of samples containing serial dilutions and different mixtures of genomic DNA extracted from different species of adult worms proved useful in assessment of different threshold settings with the QuantaSoft software. Analysis of template DNA from these worms indicated that ddPCR is a viable choice for detection and absolute quantification of the different genera and also in samples with multiple species. Interpretation of the ddPCR results was straightforward and choice of analytical approach had little influence on the final results. Thus, the results obtained in the different analytical approaches seemed to be robust and the concentrations determined were uniform. Furthermore, the linear range of the Haemonchus ddPCR assay was similar to that of real-time PCR (qPCR). Taken together, our data confirm the suitability of ddPCR for detection and absolute quantification of three major sheep pathogens when tested on larval cultures from pooled ovine faeces. The results also indicate that ddPCR can be a useful complement to applications based on conventional egg counting methods such as the faecal egg reduction test (FECRT).

Genetic variants in dyf-7 validated by droplet digital PCR are not drivers for ivermectin resistance in Haemonchus contortus.

Resistance to ivermectin (IVM) in the nematode Haemonchus contortus in small ruminants is an increasing problem throughout the world. Access to molecular diagnostics will allow early detection of IVM resistance, which in turn can limit the spread of resistant isolates. One candidate gene which has recently been suggested as a marker for IVM resistance is that for dye-filling protein (dyf-7). In this study, we critically investigated the suitability of A141G and G153T single nucleotide polymorphisms (SNP) of dyf-7 as a marker in larval cultures collected from sheep farms in Sweden, involving several isolates for which resistance status had been characterised by the faecal egg count reduction test (FECRT). Initially, we designed dyf-7 primers from a worldwide collection of adult Haemonchus contortus DNA. With the sequence data, we created a haplotype network. We then optimised and used the same sets of primers and probes in a droplet digital PCR (ddPCR) assay for precise quantification of dyf-7 allele frequencies in pre- and post-anthelmintic treatment faecal larval cultures. The fractional abundance (FA) of the mutant SNP was within the range 7.8 and 31%. However, the FA was generally stable in samples collected from the same farms, even though they were obtained on different occasions up to 25 months apart. There was also no indication that the level of IVM resistance as measured by the faecal egg count reduction test was higher on farms with high FA. Furthermore, by comparing FA in samples from the same farms pre- and post-IVM treatment, we found no evidence of a correlation between dyf-7 and level of IVM resistance. Based on these results, dyf-7 is not a suitable marker for field testing of IVM resistance in H. contortus.

Gastrointestinal parasites of captive European bison Bison bonasus (L.) with a sign of reduced efficacy of Haemonchus contortus to fenbendazole.

The history of European bison Bison bonasus Linnaeus, 1758 has been stormy since its extinction in the wild after the First World War. Due to the fact that the species was restored from just 12 founders, further expansion has suffered from low genetic variability, rendering the bison vulnerable to various pathogens due to inbreeding depression. Parasites are recognised as a key biological threat to bison population. Thus, parasitological examination including monitoring of the level of anthelmintic resistance in a herd should be a routine procedure involved in management and protection of European bison. This study was conducted in a group of 27 bison kept in a European bison breeding centre in Sweden. In April 2015, a faecal egg count reduction test (FECRT) was performed in animals with ≥ 100 gastrointestinal nematode (GIN) eggs per gram faeces, to determine effectiveness of fenbendazole (FBZ) treatment. Additionally, the third stage larvae were cultured for molecular examination by a conventional PCR as well as by real-time quantitative PCR (q-PCR) for detection of the blood-sucking nematode Haemonchus contortus. Faecal sampling was conducted 1 day before and 8 days after deworming each animal. Anthelmintic treatment turned to be entirely efficient toward intestinal nematodes of genera Nematodirus and Trichuris, whereas shedding of strongylid eggs from the subfamily Ostertagiinae was reduced from 81 to 30%. Polymerase chain reaction (PCR) on cultured third-stage larvae (L3) before treatment was positive for H. contortus, Ostertagia ostertagi and Cooperia oncophora, whereas post-treatment examination revealed exclusively the DNA of H. contortus. Thus, only H. contortus was involved in post-treatment faecal egg count (FEC). FECRT showed that the reduction in strongylid FEC to FBZ in the examined bison herd was 87% (95%-confidence intervals [95% CI] = 76-93), suggesting reduced efficacy of FBZ to strongylid GIN including mainly H. contortus.

Transmission ecology of taeniid larval cestodes in rodents in Sweden, a low endemic area for Echinococcus multilocularis.

Although local prevalence of Echinococcus multilocularis may be high, this zoonotic parasite has an overall low prevalence in foxes and rodents in Sweden. To better understand opportunities for E. multilocularis transmission in the Swedish environment, the aim of this study was to investigate other taeniid cestodes and to relate observed patterns to E. multilocularis. Cestode parasites were examined in fox feces and rodents caught in different habitats from four regions of Sweden. Arvicola amphibius and Microtus agrestis were parasitized with Versteria mustelae, Hydatigera taeniaeformis s. l., and E. multilocularis, whereas Myodes glareolus and Apodemus spp. were parasitized with V. mustelae, Taenia polyacantha, H. taeniaeformis s.l., and Mesocestoides spp. Rodents caught in field habitat (Ar. amphibius, Mi. agrestis) were more likely (OR 10, 95% CI 5-19) to be parasitized than rodents caught in forest habitat (My. glareolus, Apodemus spp.). The parasite preference for each rodent species was present regardless of the type of background contamination from fox feces. These results further support the importance of both ecological barriers and individual species susceptibility in parasite transmission, and indicate that future monitoring for E. multilocularis in the Swedish environment should focus in field habitats where Mi. agrestis and Ar. amphibius are abundant.

Support for targeted sampling of red fox (Vulpes vulpes) feces in Sweden: a method to improve the probability of finding Echinococcus multilocularis.

BACKGROUND: Localized concentrations of Echinococcus multilocularis eggs from feces of infected red fox (Vulpes vulpes) can create areas of higher transmission risk for rodent hosts and possibly also for humans; therefore, identification of these areas is important. However, in a low prevalence environment, such as Sweden, these areas could be easily overlooked. As part of a project investigating the role of different rodents in the epidemiology of E. multilocularis in Sweden, fox feces were collected seasonally from rodent trapping sites in two regions with known parasite status and in two regions with unknown parasite status, 2013-2015. The aim was to evaluate background contamination in rodent trapping sites from parasite eggs in these regions. To maximize the likelihood of finding fox feces positive for the parasite, fecal collection was focused in habitats with the assumed presence of suitable rodent intermediate hosts (i.e. targeted sampling). Parasite eggs were isolated from feces through sieving-flotation, and parasite species were then confirmed using PCR and sequencing. RESULTS: Most samples were collected in the late winter/early spring and in open fields where both Arvicola amphibius and Microtus agrestis were captured. Fox feces positive for E. multilocularis (41/714) were found within 1-3 field collection sites within each of the four regions. The overall proportion of positive samples was low (≤5.4%) in three regions, but was significantly higher in one region (22.5%, P < 0.001). There was not a significant difference between seasons or years. Compared to previous national screenings, our sampling strategy identified multiple E. multilocularis positive feces in all four regions, including the two regions with previously unknown parasite status. CONCLUSIONS: These results further suggest that the distribution of E. multilocularis is highly aggregated in the environment and provide support for further development of a targeted sampling strategy. Our results show that it was possible to identify new areas of high contamination in low endemic environments. After further elaboration, such a strategy may be particularly useful for countries designing surveillance to document freedom from disease.

Assessment of flukicide efficacy against Fasciola hepatica in sheep in Sweden in the absence of a standardised test.

Anthelmintic resistance (AR) to Fasciola hepatica is emerging worldwide. Recently, AR to the adulticide compound albendazole (ABZ) was shown in Argentina and Spain. In Sweden, ABZ treatment failure against F. hepatica was first reported in sheep in 2012. The present study tested the efficacy of ABZ and triclabendazole (TCBZ) in sheep naturally infected with F. hepatica using a combination of three different diagnostic methods: faecal egg counts (FEC), coproantigen ELISA (cELISA) and Fasciola egg hatch test (FEHT). Two deworming trials, in November 2014 and January 2015, were performed on two sheep farms (farms A and B) in south-western Sweden. Except ABZ in November, treatment with ABZ or TCBZ achieved sufficient efficacy (97-100%) against adult F. hepatica on farm A. In contrast, ABZ treatment failed in the sheep flock on farm B, despite low initial faecal egg output. On farm B, ABZ efficacy based on FEC was 67% (95% CI: 35-84) and four of eight ewes tested were coproantigen-positive 21 days post-treatment. Ovicidal activity of ABZ against Fasciola eggs in isolates from both farms and one additional bovine isolate were tested by FEHT to exclude the presence of juvenile flukes and other factors such as dosing failure and poor quality of drug product. Irrespective of drug trial, data from FEHT showed significantly lower ovicidal activity of ABZ for the ovine farm B isolate than for the isolate from farm A. This confirms that the low efficacy of ABZ in sheep flock B was associated with ABZ resistance. Overall, the usefulness of three complementary methods for detection of ABZ resistance in the field was demonstrated.

First identification of Echinococcus multilocularis in rodent intermediate hosts in Sweden.

Echinococcus multilocularis is a zoonotic tapeworm with a sylvatic lifecycle and an expanding range in Europe. Monitoring efforts following its first identification in 2011 in Sweden have focused on the parasite's definitive host, the red fox (Vulpes vulpes). However, identifying rodent intermediate hosts is important to recognize opportunities for parasite transmission. During 2013-2015, livers from a total of 1566 rodents from four regions in Sweden were examined for E. multilocularis metacestode lesions. Species identity of suspect parasite lesions was confirmed by PCR and sequencing. E. multilocularis positive lesions >6 mm in diameter were also examined histologically. One Microtus agrestis out of 187 (0.5%, 95%CI: 0-2.9%), 8/439 (1.8%, 95%CI: 0.8-3.6%) Arvicola amphibius, 0/655 (0%, 95%CI: 0-0.6%) Myodes glareolus, and 0/285 (0%, 95%CI: 0-1.3%) Apodemus spp. contained E. multilocularis metacestode lesions. Presence of protoscoleces was confirmed in the infected M. agrestis and in three of eight infected A. amphibius. Six of the nine positive rodents were captured from the same field. This is the first report of E. multilocularis in intermediate hosts in Sweden. The cluster of positive rodents in one field shows that local parasite prevalence can be high in Sweden despite overall low national prevalence in foxes (<0.1%). The presence of protoscoleces in infected M. agrestis and A. amphibius indicate these species can serve as competent intermediate hosts in Sweden. However, their relative importance for E. multilocularis transmission in the Swedish environment is not yet possible to assess. In contrast, the negative findings in all M. glareolus and Apodemus spp. suggest that these species are of no importance.

Differential expression of ß-tubulin isotypes in different life stages of Parascaris spp after exposure to thiabendazole.

Anthelmintic resistance (AR) to macrocyclic lactones (ML) has been described in Parascaris of horses world-wide. In contrast, benzimidazoles (BZ) are still effective, although reduced efficacy to this drug class was recently reported. The mode of action of BZ is binding to ß-tubulin, which prevents polymerisation of microtubules. In this study, ß-tubulin gene expression of isotypes 1 and 2 was investigated at seven time points (0, 6, 24, 72, 96 and 120 h) during embryogenesis and in adult worms. In addition, an in ovo larval developmental test was developed to study ß-tubulin gene expression of both isotypes in parasacaris eggs after exposure to different concentrations of thiabendazole (TBZ) for five days at 25 °C. A strong pattern of differential expression of ß-tubulin and isotype 1 was observed in all stages, while isotype 2 expression was mainly found at an early phase of the embryogenesis. For isotype 1, a 5-fold increase was observed during the first 48 h, but gene expression gradually decreased after 72, 96 and 120 h. Isotype 2 was only expressed during the first 24h, followed by a 130-fold decrease at (time points) 72, 96 and 120 h. The in ovo larval developmental test, in which we exposed initially unembryonated eggs to increased concentrations of TBZ, did affect isotype 1 gene expression but not isotype 2. This assumes that each isotype has specific functions in different life stages. This is in agreement with the 'multi-tubulin' hypothesis, which states that different tubulin isotypes are required for specialised microtubule functions. Isotype 1 is the most likely drug target for BZs, as isotype 2 was only expressed at very low levels later in development. Increasing concentrations of TBZ altered ß-tubulin isotype 1 gene expression after exposure of the eggs for five days, but this was not seen for isotype 2.

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.

Gene expression of ABC transporters in Cooperia oncophora after field and laboratory selection with macrocyclic lactones.

The most widespread helminth parasites of grazing cattle in northern Europe are the gastrointestinal nematodes Ostertagia ostertagi and Cooperia oncophora. Heavy reliance on the use of macrocyclic lactone (ML) in cattle has led to world-wide emergence of resistance to this drug class in C. oncophora. There is evidence that members of the ATP-binding cassette (ABC) transporter family, such as P-glycoproteins (P-gp) and multidrug-resistant proteins (MRP), play a role in resistance to ML. In this study gene expression of Con-pgp9, Con-pgp11, Con-pgp12, Con-pgp16 and Con-mrp1 was examined in two isolates of C. oncophora sharing the same genetic background but exposed to ML differently. For isolate one (Laboratory-selected), adult worms were recovered before and after treatment with ML in vivo. For isolate two (Field-selected), adult worms were collected from tracer animals that had never received anthelmintics themselves. One group grazed together with untreated animals and one group grazed with animals that received suppressive prophylactic treatment with ML at monthly intervals for up to two consecutive grazing seasons. Real-time PCR data demonstrated differences in gene expression after ML selection, with the highest constitutive expression levels for Con-pgp16 and Con-mrp1. Remarkably, the same pattern of increasing expression levels of the ABC transport genes was observed in both Laboratory- and Field-selected isolates, despite the Field-selected isolate not being directly exposed to ML. The higher expression levels of ABC transporters observed in the Field-selected isolate was thus not a response to direct exposure to ML, but rather appeared to reflect a genetic characteristic inherited from worms in the previous generation which had survived exposure to ML in the co-grazing treated animals.