Effects of selected condensed tannins on Cryptosporidium parvum growth and proliferation in HCT-8 cell cultures.

Infections with Cryptosporidium spp. constitute a substantial public health burden and are responsible for widespread production losses in cattle herds. Reducing disease and shedding of Cryptosporidium spp. oocysts is an important One Health goal. There are very few therapeutic options available to treat cryptosporidiosis. Interest in plant bioactive compounds to mitigate the spread of anthelmintic resistance in ruminants has led to investigation of these phytocompounds against other parasitic taxa. Condensed tannins (CTs) are plant secondary metabolites that have shown potential against nematodes in vitro and in vivo but their applicability to Cryptosporidium spp. is comparatively under-explored. Cryptosporidium parvum infected human ileocecal colorectal adenocarcinoma (HCT)-8 cell cultures were treated with escalating doses of highly purified and well-characterized CTs from five plant species, big trefoil (Lotus pedunculatus), black currant (Ribes nigrum), sainfoin (Onobrychis viciifolia), white clover (Trifolium repens) and grapeseed (Vitis vinifera) for 44 h. Quantitative-PCR (qPCR) analysis revealed that none of the CTs examined demonstrated inhibitory potential against the parasite. Substantial inhibition of C. parvum by paromomycin was observed in positive controls in all assays (76.94-90.72% inhibition), proving the validity of the assay. Despite the lack of inhibition, these results represent an important step towards identifying alternative treatment options against this parasite.

Closed Genome Sequences of Providencia alcalifaciens Isolates from Dogs.

Eight Providencia alcalifaciens isolates from eight different dogs in Norway with acute hemorrhagic diarrhea were sequenced. Based on Illumina and Oxford Nanopore Technologies sequencing, all of the genomes were complete and closed after hybrid assembly.

Extracts of pine bark (Pinus sylvestris) inhibit Cryptosporidium parvum growth in cell culture.

The widespread apicomplexan parasite Cryptosporidium parvum is responsible for severe gastrointestinal disease in humans and animals. The treatment options are limited, and the efficacy of available drugs is low. Bark contains condensed tannins (CT), which are bioactive compounds previously shown to inhibit parasite development. Here, we examined the anti-cryptosporidial properties of bark extract of Scots pine (Pinus sylvestris) against C. parvum by means of an in vitro growth inhibition test. We hypothesised that bark extracts would have dose-dependent inhibitory effects on the development of C. parvum in cell culture.Bark extracts from Scots pine extracted with acetone, methanol, and water as solvents were investigated using human colorectal adenocarcinoma cells infected with C. parvum. Oocysts were inoculated onto the cell monolayer and bark extract was added at seven different concentrations. Parasite growth inhibition was quantified by qPCR.The acetone and methanol extracts demonstrated a sigmoid dose-dependent inhibition of C. parvum. The IC50 values were 244.6 and 279.1 µg dry matter extract/mL, and 25.4 and 24.1 µg CT/mL, for acetone and methanol extracts, respectively. The IC50 for both extracts were similar, both with regard to the dry matter concentration of each extract and to CT concentrations.Given the limited treatment options available for Cryptosporidium spp., the evidence generated in our study encourages further investigation into the in vitro and in vivo effects of pine bark extracts against C. parvum.

Who Is Dermanyssus gallinae? Genetic Structure of Populations and Critical Synthesis of the Current Knowledge.

Despite the economic and animal welfare importance of the Poultry Red Mite Dermanyssus gallinae, its genetic structure has been studied in a scattered way so far. The prophylaxis and control of such a globally distributed ectoparasite can be significantly improved by understanding its genetic population structure (composition in species and intraspecific variants). The present study aims to establish a rigorous framework for characterizing the neutral genetic structure of D. gallinae based on a literature review combined with an integrative analysis of the data available in GenBank on population-level nucleotide sequence diversity supplemented by a new dataset. The integrative analysis was conducted on sequence data extracted from GenBank coupled with new sequences of two fragments of the mitochondrial gene encoding Cytochrome Oxidase I (CO1) as well as of an intron of the nuclear gene encoding Tropomyosin (Tpm) from several PRM populations sampled from European poultry farms. Emphasis was placed on using the mitochondrial gene encoding CO1 on which the main universal region of DNA barcoding in animals is located. The species D. gallinae sensu lato is a species complex, encompassing at least two cryptic species, i.e., not distinguishable by morphological characters: D. gallinae sensu stricto and D. gallinae L1. Only D. gallinae s.s. has been recorded among the populations sampled in poultry farms worldwide. Current knowledge suggests they are structured in three mitochondrial groups (haplogroups A, B, and C). Haplogroup A is cosmopolitan, and the other two present slightly contrasted distributions (B rather in the northern part of Europe, C most frequently found in the southern part). Recent data indicate that a dynamic geographic expansion of haplogroup C is underway in Europe. Our results also show that NUMT (nuclear mitochondrial DNA) pseudogenes have generated artifactual groups (haplogroups E and F). It is important to exclude these artifact groups from future analyses to avoid confusion. We provide an operational framework that will promote consistency in the analysis of subsequent results using the CO1 fragment and recommendations for future analyses.

Complete Genome Sequences of 12 Quinolone-Resistant Escherichia coli Strains Containing qnrS1 Based on Hybrid Assemblies.

In total, 12 quinolone-resistant Escherichia coli (QREC) strains containing qnrS1 were submitted to long-read sequencing using a FLO-MIN106 flow cell on a MinION device. The long reads were assembled with short reads (Illumina) and analyzed using the MOB-suite pipeline. Six of these QREC genome sequences were closed after hybrid assembly.

Tick-borne Pathogens Detected in the Blood of Immunosuppressed Norwegian Patients Living in a Tick-endemic Area.

BACKGROUND: The knowledge regarding the occurrence and the clinical implications of tick-borne infections in immunosuppressed patients living in tick-endemic areas is limited. METHODS: Adult patients with autoimmune conditions requiring immunosuppressive treatment such as infliximab and rituximab were invited to participate in the study when they attended the hospital for treatment and/or control of the disease. Whole-blood samples were analyzed by real-time polymerase chain reaction for Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum, Rickettsia spp., Candidatus Neoehrlichia mikurensis, and Babesia spp. RESULTS: The occurrence of tick-borne pathogens in the blood of patients (n = 163) with autoimmune conditions requiring immunosuppressive treatment was evaluated. Pathogen DNA was detected in 8.6% (14/163) of the patients. The predominant pathogen was Ca. Neoehrlichia mikurensis (12/14), which was carried in the blood of infected patients for 10-59 days until treatment with doxycycline. B. burgdorferi s.l. and Rickettsia spp. were detected in 1 patient each. The B. burgdorferi-infected patient presented with fever, whereas the remaining patients were judged to have subclinical infections. B. miyamotoi, A. phagocytophilum, and Babesia spp. were not detected in any patient. CONCLUSIONS: Patients treated with biologicals and living in a tick-endemic area seem to have a high risk of contracting Ca. Neoehrlichia mikurensis infection, which, if left untreated, could result in thromboembolic complications.

Comparative morphological and transcriptomic analyses reveal chemosensory genes in the poultry red mite, Dermanyssus gallinae.

Detection of chemical cues via chemosensory receptor proteins are essential for most animals, and underlies critical behaviors, including location and discrimination of food resources, identification of sexual partners and avoidance of predators. The current knowledge of how chemical cues are detected is based primarily on data acquired from studies on insects, while our understanding of the molecular basis for chemoreception in acari, mites in particular, remains limited. The poultry red mite (PRM), Dermanyssus gallinae, is one of the most important blood-feeding ectoparasites of poultry. PRM are active at night which suck the birds' blood during periods of darkness and hide themselves in all kinds of gaps and cracks during the daytime. The diversity in habitat usage, as well as the demonstrated host finding and avoidance behaviors suggest that PRM relies on their sense of smell to orchestrate complex behavioral decisions. Comparative transcriptome analyses revealed the presence of candidate variant ionotropic receptors, odorant binding proteins, niemann-pick proteins type C2 and sensory neuron membrane proteins. Some of these proteins were highly and differentially expressed in the forelegs of PRM. Rhodopsin-like G protein-coupled receptors were also identified, while insect-specific odorant receptors and odorant co-receptors were not detected. Furthermore, using scanning electron microscopy, the tarsomeres of all leg pairs were shown to be equipped with sensilla chaetica with or without tip pores, while wall-pored olfactory sensilla chaetica were restricted to the distal-most tarsomeres of the forelegs. This study is the first to describe the presence of chemosensory genes in any Dermanyssidae family. Our findings make a significant step forward in understanding the chemosensory abilities of D. gallinae.

Assessment of differences between DNA content of cell-cultured and freely suspended oocysts of Cryptosporidium parvum and their suitability as DNA standards in qPCR.

BACKGROUND: Although more modern methods are available, quantitative PCR (qPCR) is reproducible, sensitive and specific with instruments and expertise readily available in many laboratories. As such, the use of qPCR in Cryptosporidium research is well established and still widely used by researchers globally. This method depends upon the generation of standards at different concentrations to generate standard curves subsequently used for the quantification of DNA. METHODS: We assessed four types of DNA template used to generate standard curves in drug screening studies involving Cryptosporidium spp.: (i) serially diluted Cryptosporidium parvum oocysts (106-1); (ii) diluted template DNA from pure oocysts (×10-×106 dilution of 106 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (105-1 and 5 × 104-50); and (iv) diluted DNA template (5 × 104) from cell culture incubated parasites (×10-×1000). RESULTS: Serial dilutions of both cell culture and pure oocyst suspension DNA template yielded better linearity than cell culture derived standards, with dilutions of 106 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 106 oocysts. In contrast, cell culture incubated oocysts demonstrated significantly higher DNA content than equivalent freely suspended oocysts and diluted DNA template from both cell culture derived and freely suspended oocysts across numerous concentrations. CONCLUSIONS: For many studies involving Cryptosporidium, only relative DNA content is required and as such, the superior linearity afforded by freely suspended oocysts and diluted DNA template (from either cell culture derived standards or freely suspended oocysts) will allow for more accurate relative quantification in each assay. Parasite division in the cell culture standards likely explains the higher DNA content found. These standards, therefore, have the potential to more accurately reflect DNA content in cell culture assays, and despite more modern methods available for absolute quantification, i.e. droplet digital PCR (ddPCR), the ubiquity of qPCR for the foreseeable future encourages further investigation into the reduced linearity observed in these standards such as varying oocyst seeding density, non-linear growth rates and assay efficiency.

Molecular characterization and genetic diversity of Ornithonyssus sylviarum in chickens (Gallus gallus) from Hainan Island, China.

BACKGROUND: The northern fowl mite (NFM), Ornithonyssus sylviarum, is an obligatory hematophagous ectoparasite of birds and one of the most important pests in the poultry industry on several continents. Although NFM poses a serious problem, it remains a neglected pest of poultry in China and other Asian countries. Therefore, a molecular analysis was conducted to provide baseline information on the occurrence, genetic diversity and emergence of NFM in poultry farms from China. METHODS: This study focused on morphological description and identification of adults based on electron microscopy, molecular sequencing of the mitochondrial cox1 gene and phylogenetic analysis. We have also used the DNA sequences of the cox1 gene to study the genetic diversity, population structure and demographic history. The neutrality tests were used to analyze signatures of historical demographic events. RESULTS: The mites collected were identified as the northern fowl mite Ornithonyssus sylviarum based on external morphological characterization using electron microscopy. Molecular analysis using a 756-bp long partial fragment of the cox1 gene revealed 99-100% sequence identity with NFM and phylogenetic inferences showed a bootstrap value of 99% indicating a well-supported monophyletic relationship. Molecular diversity indices showed high levels of haplotype diversity dominated by private haplotypes, but low nucleotide divergence between haplotypes. The Tajima's D test and Fu's Fs test showed negative value, indicating deviations from neutrality and both suggested recent population expansion of mite populations supported by a star-like topology of the isolates in the network analysis. Our genetic data are consistent with a single introduction of NFM infestations and the spread of NFM infestation in Hainan poultry farms and a private haplotype dominance, which suggest that infestations are recycled within the farms and transmission routes are limited between farms. CONCLUSIONS: To our knowledge, this is the first time a molecular report of NFM in chicken from China including other Asian countries using DNA barcoding. The findings have potential implications with respect to understanding the transmission patterns, emergence and populations trends of parasitic infestations of poultry farms that will help for setting the parameters for integrated pest management (IPM) tactics against mite infestations.

Darkness increases the population growth rate of the poultry red mite Dermanyssus gallinae.

BACKGROUND: The poultry red mite (PRM), Dermanyssus gallinae, is one of the most economically deleterious ectoparasites affecting egg-laying hens worldwide. It may be possible to control D. gallinae populations by manipulating lighting regimes within poultry units. However, no studies have clearly shown the effects of darkness on the population growth rate of D. gallinae. METHODS: The effect of darkness on the population growth rate of D. gallinae was investigated, together with the first description of the molecular identity of the mite from China. Mite variables under two lighting regimens (1:23 h L:D and 12:12 h L:D) were compared, including number of mites and eggs, survival and feeding rates, engorgement, oviposition, hatchability and the life-cycle of D. gallinae. RESULTS: The results showed that the number of mites (13,763 ± 956) and eggs (5424 ± 317) in the rearing system with prolonged darkness of 1:23 h L:D at 4th week were 2.4- and 3.6-fold higher than those under a conventional lighting regimen of 12:12 h L:D, respectively. The feeding rates of mites under prolonged darkness ranged from 36.7 ± 1.1% to 52.0 ± 7.0%, which were significantly higher than those under conventional lighting regimen (ranging from 22.6 ± 1.9% to 37.3 ± 1.6%). The mean weight of engorged females (0.26 ± 0.01 mg) and the mean number of eggs per female (on average 5.87 ± 0.36) under prolonged darkness were significantly higher than those under conventional lighting regimen (0.22 ± 0.01 mg and 3.62 ± 0.31, respectively). However, the survival rate ranging from 98.07 ± 0.10% to 98.93 ± 0.19%, hatchability of 97.93 ± 0.01% and the life-cycle of D. gallinae (9 days) was not affected by the lighting period. CONCLUSIONS: Our findings demonstrated that prolonged darkness significantly promoted the proliferation levels of D. gallinae, resulting in increased number of mites and eggs in the rearing system. The promoted population growth of D. gallinae was found to be related to the increased feeding rate, engorgement level and oviposition level of mites under prolonged darkness. The egg hatchability, the survival rates and the duration of life-cycle of D. gallinae were not affected by the light regimes.

Evaluation of vaccine delivery systems for inducing long-lived antibody responses to Dermanyssus gallinae antigen in laying hens.

Dermanyssus gallinae, the poultry red mite, is a global threat to the commercial egg-laying industry. Control of D. gallinae is difficult, with only a limited number of effective pesticides and non-chemical treatments available. Here, we characterize the candidate vaccine antigen D. gallinae cathepsin D-1 (Dg-CatD-1) and demonstrate that purified refolded recombinant Dg-Cat-D1 (rDg-CatD-1) is an active aspartyl proteinase which digests haemoglobin with a pH optimum of pH 4. Soluble protein extracts from D. gallinae also have haemoglobinase activity, with a pH optimum comparable to the recombinant protein, and both proteinase activities were inhibited by the aspartyl proteinase inhibitor Pepstatin A. Enzyme activity and the ubiquitous localization of Dg-CatD-1 protein in sections of adult female mites is consistent with Dg-CatD-1 being a lysosomal proteinase. Using Dg-CatD-1 as a model vaccine antigen, we compared vaccine delivery methods in laying hens via vaccination with: (i) purified rDg-CatD-1 with Montanide™ ISA 71 VG adjuvant; (ii) recombinant DNA vaccines for expression of rDg-CatD-1 and (iii) transgenic coccidial parasite Eimeria tenella expressing rDg-CatD-1. In two independent trials, only birds vaccinated with rDg-CatD-1 with Montanide™ ISA 71 VG produced a strong and long-lasting serum anti-rDg-Cat-D1 IgY response, which was significantly higher than that in control birds vaccinated with adjuvant only. Furthermore, we showed that egg-laying rates of D. gallinae mites fed on birds vaccinated with rDg-CatD-1 in Montanide™ ISA 71 VG was reduced significantly compared with mites fed on unvaccinated birds. RESEARCH HIGHLIGHTS Dermanyssus gallinae cathepsin D-1 (Dg-CatD-1) digests haemoglobin Vaccination of hens with rDg-CatD-1 in Montanide™ ISA 71 VG results in long-lasting IgY levels Serum anti-rDg-CatD-1 antibodies reduce egg laying in D. gallinae after a single blood meal.

Draft Genome Assembly of the Poultry Red Mite, Dermanyssus gallinae.

The poultry red mite, Dermanyssus gallinae, is a major worldwide concern in the egg-laying industry. Here, we report the first draft genome assembly and gene prediction of Dermanyssus gallinae, based on combined PacBio and MinION long-read de novo sequencing. The ∼959-Mb genome is predicted to encode 14,608 protein-coding genes.

Validate or falsify: Lessons learned from a microscopy method claimed to be useful for detecting Borrelia and Babesia organisms in human blood.

BACKGROUND: A modified microscopy protocol (the LM-method) was used to demonstrate what was interpreted as Borrelia spirochetes and later also Babesia sp., in peripheral blood from patients. The method gained much publicity, but was not validated prior to publication, which became the purpose of this study using appropriate scientific methodology, including a control group. METHODS: Blood from 21 patients previously interpreted as positive for Borrelia and/or Babesia infection by the LM-method and 41 healthy controls without known history of tick bite were collected, blinded and analysed for these pathogens by microscopy in two laboratories by the LM-method and conventional method, respectively, by PCR methods in five laboratories and by serology in one laboratory. RESULTS: Microscopy by the LM-method identified structures claimed to be Borrelia- and/or Babesia in 66% of the blood samples of the patient group and in 85% in the healthy control group. Microscopy by the conventional method for Babesia only did not identify Babesia in any samples. PCR analysis detected Borrelia DNA in one sample of the patient group and in eight samples of the control group; whereas Babesia DNA was not detected in any of the blood samples using molecular methods. CONCLUSIONS: The structures interpreted as Borrelia and Babesia by the LM-method could not be verified by PCR. The method was, thus, falsified. This study underlines the importance of doing proper test validation before new or modified assays are introduced.

Laboratory assessment of sensitive molecular tools for detection of low levels of Echinococcus multilocularis-eggs in fox (Vulpes vulpes) faeces.

BACKGROUND: In endemic areas with very low infection prevalence, the frequency and intensity of Echinococcus multilocularis can be extremely low. This necessitates efficient, specific and sensitive molecular tools. We wanted to compare the existing molecular tools, used in the Norwegian national surveillance programme, and compare these with new techniques for detection of this zoonotic pathogen in fox faeces. Here we present the results of screening samples containing a known level of E. multilocularis eggs with two highly sensitive DNA isolation and extraction methods combined with one conventional PCR and three real-time PCR methods for detection. METHODS: We performed a comparison of two extraction protocols; one based on sieving of faecal material and one using targeted DNA sampling. Four methods of molecular detection were tested on E. multilocularis-egg spiked fox faeces. RESULTS: There were significant differences between the multiplex PCR/egg sieving DNA extraction methods compared to the new DNA fishing method and the three real-time PCR assays. Results also indicate that replicates of the PCR-reactions improve detection sensitivity when egg numbers are low. CONCLUSIONS: The results indicate that the use of real-time PCR combined with targeted DNA extraction, improves the sensitivity of E. multilocularis detection in faecal samples containing low numbers of E. multilocularis eggs. Results also indicate the importance of replicates of the PCR-reactions when pathogen levels are low.

Illegal wildlife imports more than just animals--Baylisascaris procyonis in raccoons (Procyon lotor) in Norway.

In autumn 2011, 11 illegally imported animals were seized from a farm in southern Norway. These included four raccoon dogs (Nyctereutes procyonoides), four raccoons (Procyon lotor), and three South American coatis (Nasua nasua), all considered alien species in Norway. An additional two raccoons had escaped from the farm prior to seizure. The seized animals were euthanized and postmortem examination revealed that the four raccoons had moderate to high numbers of the zoonotic nematode Baylisascaris procyonis in their intestines, ranging from 11 to 115 nematodes per small intestine, with a mean of 53. The identity of the nematodes was confirmed using molecular analysis of ITS-1, ITS-2, cytochrome C oxidase 1, and 18S. Echinococcus multilocularis was not detected in any of the 11 animals. Toxocara and Toxascaris sp. eggs were detected in the feces of two raccoons, and two coatis had coccidia oocysts (80 and 360 oocysts per gram). Domestic dogs and other wildlife on the farm had potential access to the animal pens. Given that the eggs can remain infective for years in the environment, local veterinary and health authorities will need to remain vigilant for symptoms relating to infection with B. procyonis.

Toxocara cati larva migrans in domestic pigs--detected at slaughterhouse control in Norway.

Routine Trichinella meat inspection at the slaughterhouse detected one larva in a pooled batch of 100 pig samples. The larva was sent to the Norwegian Veterinary Institute (NVI) for species identification.Morphological examination revealed that the larva was not Trichinella spp. Molecular analysis was performed. PCR and sequencing of 5S/ITS identified the larva as Toxocara cati. A second round of digests was carried out at the meat inspection laboratory, in smaller batches to try to identify the infected animal. No further larvae were detected and it was not possible to identify which of the 100 animals the larva had come from. This is the first time that Toxocara cati has been reported in slaughterhouse pigs in Norway.Although the infected individual could not be identified, the meat originated from one of six potential farms. A small survey regarding rodent control and cats was sent to each of these farms. Cats had restricted access to food storage areas (two farms reported that cats had access) whilst none of the farms allowed cats into the production housing. Cats were, however, present on all the farms (mostly stray cats of unknown health status). Half of the farms also reported seeing rodents in the pig housing during the previous six months and half reported finding rodents in the feed and straw storage areas. We were unable to narrow down the source of infection - however contamination of food or bedding material, with cat faeces or infected rodents, in addition to the presence of infected rodents in pig housing remain potential routes of infection.

Prevalence and diversity of Babesia spp. in questing Ixodes ricinus ticks from Norway.

BACKGROUND: Ixodes ricinus ticks transmit Babesia species to vertebrate hosts. Using molecular tools we were able to detect the presence of this piroplasmid in its vector. The aims of this study were to investigate the prevalence and identity of Babesia species in questing ticks collected in various areas of Norway. METHODS: DNA from questing l. ricinus ticks were examined with a realtime PCR for the presence of Babesia. Positive samples of tick DNA were identified to species using PCR, and sequence analysis. RESULTS: From a total of 1908 questing l. ricinus ticks, 17 (0.9%) indicated the presence of Babesia spp. after realtime-PCR screening. Ixodes ricinus harbouring Babesia spp. was detected in 9 out of 22 localities. Further molecular analyses of DNA from these positive ticks indicate the presence of Babesia venatorum, B. divergens, B. capreoli and a currently undescribed Babesia in Norwegian ticks. The most prevalent was B. venatorum found in 71% of the positive ticks. CONCLUSIONS: A total of 17 out of 1908 (0.9%) ticks were positive for Babesia. Our data confirm that there are several Babesia species in ticks in Norway. Babesia venatorum was the most prevalent. This species has a zoonotic potential and may cause human babesiosis following a tick bite.

Transport of Babesia venatorum-infected Ixodes ricinus to Norway by northward migrating passerine birds.

BACKGROUND: Bovine babesiosis is regarded as a limited health problem for Norwegian cows, and the incidence has decreased markedly since the 1930s. Rare cases of babesiosis in splenectomised humans from infection with Babesia divergens and B.venatorum have been described. The objective of this study was to determine whether birds can introduce Babesia-infected ticks. There are between 30 and 85 million passerine birds that migrate to Norway every spring. METHODS: Passerine birds were examined for ticks at four bird observatories along the southern Norwegian coast during the spring migrations of 2003, 2004 and 2005. The presence of Babesia was detected in the nymphs of Ixodes ricinus by real-time PCR. Positive samples were confirmed using PCR, cloning and phylogenetic analyses. RESULTS: Of 512 ticks examined, real-time PCR revealed five to be positive (1.0%). Of these, four generated products that indicated the presence of Babesia spp.; each of these were confirmed to be from Babesia venatorum (EU1). Two of the four B. venatorum-positive ticks were caught from birds having an eastern migratory route (P< 0.001). CONCLUSIONS: Birds transport millions of ticks across the North Sea, the Skagerrak and the Kattegat every year. Thus, even with the low prevalence of Babesia-infected ticks, a substantial number of infected ticks will be transported into Norway each year. Therefore, there is a continuous risk for introduction of new Babesia spp. into areas where I. ricinus can survive.

First case of babesiosis caused by Babesia canis canis in a dog from Norway.

An Irish setter from the Oslo area was presented to the clinic with signs of babesiosis, a few days after a tick bite. Blood analysis confirmed babesiosis. Microscopic examination of thin blood film revealed large, basophilic, bodies inside erythrocytes, indicative of a large Babesia sp. Molecular analysis using PCR, indicated the presence of a Babesia spp. in the blood. Sequencing and phylogenetic analysis of the PCR fragment revealed a sequence which was 100% identical to Babesia canis canis 18S. As this dog had never been abroad, it can be concluded that this is the first report of an autochthonous infection of B. canis canis in Norway.

Echinococcus multilocularis--adaptation of a worm egg isolation procedure coupled with a multiplex PCR assay to carry out large-scale screening of red foxes (Vulpes vulpes) in Norway.

Echinococcus multilocularis, causing alveolar echinococcosis in humans, is a highly pathogenic emerging zoonotic disease in central Europe. The gold standard for the identification of this parasite in the main host, the red fox, namely identification of the adult parasite in the intestine at necropsy, is very laborious. Copro-enzyme-linked immunosorbent assay (ELISA) with confirmatory polymerase chain reaction (PCR) has been suggested as an acceptable alternative, but no commercial copro-ELISA tests are currently available and an in-house test is therefore required. Published methods for taeniid egg isolation and a multiplex PCR assay for simultaneous identification of E. multilocularis, E. granulosus and other cestodes were adapted to be carried out on pooled faecal samples from red foxes in Norway. None of the 483 fox faecal samples screened were PCR-positive for E. multilocularis, indicating an apparent prevalence of between 0% and 1.5%. The advantages and disadvantages of using the adapted method are discussed as well as the results pertaining to taeniid and non-taeniid cestodes as identified by multiplex PCR.