Bjørn Gjerde

Individual sarcocysts were isolated from fresh or alcohol-fixed muscle samples of two moose from Alberta, Canada, and examined by light (LM) and scanning electron microscopy (SEM) and molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the complete18S rRNA gene and the partial cytochrome c oxidase subunit I gene (cox1). By LM, four sarcocyst types were recognized, and the sequencing results showed that each type represented a distinct species, i.e. Sarcocystis alces, Sarcocystis alceslatrans, Sarcocystis ovalis and Sarcocystis taeniata n. sp. The finding of S. alceslatrans and S. ovalis has been reported briefly previously, but further details are provided here, including the ultrastructure of sarcoysts of S. alceslatrans as seen by SEM. The species S. alces was found for the first time in Canadian moose, whereas the finding of S. taeniata is the first record of this species in any host. The sarcocysts of S. taeniata were sac-like and about 1,000–1,100 × 60–80 μm in size. By LM, the cysts had a thin and smooth wall with no visible protrusions, whereas SEM revealed that the cyst surface had sparsely but regularly distributed, thin ribbon-like protrusions, about 2 μm long and 0.2 μm wide, lying flat against the surface and leaving most of the cyst surface naked. Similar protrusions have previously been reported from Sarcocystis grueneri in reindeer, which was found by sequence comparisons and phylogenetic analyses to be the species most closely related to S. taeniata. The phylogenetic analyses further suggested that S. taeniata, like S. alces and S. alceslatrans, use canids as definitive hosts, whereas corvid birds are known definitive hosts for S. ovalis. In contrast to the three other species found, S. taeniata displayed considerable intra-specific and intra-isolate sequence variation (substitutions, insertions/deletions) in certain regions of the 18S rRNA gene.

The arctic fox (Vulpes lagopus) is a critically endangered species in Norway, and therefore, the small population is closely monitored, and most foxes found dead are subjected to necropsy. In two deceased foxes, thin-walled muscular sarcocysts were first detected in histological sections, and numerous sarcocysts were later found in frozen and thawed muscle samples from Fox 1. These sarcocysts measured 1–12 × 0.1–0.25 mm and had closely spaced, short, knob-like protrusions, giving the cysts a serrated outline. Genomic DNA was extracted from eight isolated sarcocysts (Fox 1) and two muscle samples (Fox 2) and subjected to polymerase chain reaction amplification at four loci: the nuclear 18S and 28S ribosomal RNA genes and internal transcribed spacer 1 region and the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Both foxes were infected by the same Sarcocystis sp., which displayed little or no genetic variation at the three nuclear loci (99.9–100 % identity) and slightly more variation at cox1 (99.4–100 % identity). Sequence comparisons and phylogenetic analyses revealed that this species was distinct from other named Sarcocystis spp. but was closely related to various species using avian intermediate hosts and possibly identical to an unnamed species reported from two American dogs. The species described from the two arctic foxes was named Sarcocystis arctica n. sp.

SUMMARY: In a previous investigation, fiveSarcocystisspecies were described from Norwegian red deer and believed to be conspecific with species occurring in either reindeer or moose based on sarcocyst morphology and nucleotide sequences of the nuclear ribosomal DNA unit. The aim of the present study was to characterize numerous isolates of these sarcocyst types at the mitochondrial cytochrome c oxidase subunit I gene (cox1) in order to corroborate or refute previous species designations ofSarcocystisin red deer. TheSarcocystis tarandi- andSarcocystis rangiferi-like taxa in red deer and reindeer, respectively, were thoroughly compared by sequencing 14–27 isolates of each type. Sequence comparisons revealed four distinct sequence types, which by phylogenetic analyses were placed in four monophyletic groups according to host origin, and they were therefore considered to represent four separate species. The two taxa of this type in red deer were namedSarcocystis elongataandSarcocystis truncata, respectively. Sequencing of many isolates ofSarcocystis hjortiandSarcocystis ovalisfrom red deer and moose confirmed that these species occur in both hosts. A revised description of the two new species is given and the current knowledge concerning all sixSarcocystisspecies in red deer is reviewed.

Genomic DNA was isolated from a Linguatula serrata female expelled from a dog imported to Norway from Romania and from four Linguatula arctica females collected from semi-domesticated reindeer from northern Norway and subjected to PCR amplification of the complete nuclear 18S rRNA gene and a 1,045-bp portion of the mitochondrial cytochrome c oxidase subunit I gene (cox1). The two species differed at two of 1,830 nucleotide positions (99.9 % identity) of the complete 18S rRNA gene sequences and at 102 of 1,045 nucleotide positions (90.2 % identity) of the partial cox1 sequences. The four isolates of L. arctica showed no genetic variation in either gene. The new cox1 primers may facilitate the diagnosis of various developmental stages of L. arctica and L. serrata in their hosts. In separate phylogenetic analyses using the maximum likelihood method on sequence data from either gene, L. arctica and L. serrata clustered with members of the order Cephalobaenida rather than with members of the order Porocephalida, in which the genus Linguatula is currently placed based on morphological characters. The phylogenetic relationship of L. arctica, L. serrata and other pentastomids to other metazoan groups could not be clearly resolved, but the pentastomids did not seem to have a sister relationship to crustaceans of the subclass Branchiura as found in other studies. A more extensive taxon sampling, including molecular characterisation of more pentastomid taxa across different genera, seems to be necessary in order to estimate the true relationship of the Pentastomida to other metazoan groups.

Genomic DNA was extracted from three oocyst isolates of Hammondia triffittae from foxes and two oocyst isolates of Hammondia heydorni from dogs, as well as from cell culture-derived tachyzoites of Toxoplasma gondii (RH strain) and Neospora caninum (NC-Liverpool strain), and examined by PCR with primers targeting the cytochrome b (cytb) and the cytochrome c oxidase subunit I (cox1) genes in order to characterise both genes and, if possible, the remainder of the mitochondrial genome of these species. Several primers were designed and used in various combinations to amplify regions within and between both genes and to determine gene order. When certain forward primers targeting cytb were used in combination with certain reverse primers targeting cox1, two overlapping sequences were obtained for each species and isolate studied, which showed that a full-length copy of cytb was followed 36–37 bp downstream by a full-length copy of cox1, and these sequences are believed to represent the true mitochondrial genes and the gene order in the mitochondrial genome of the four species examined. The cytb of T. gondii, N. caninum, H. heydorni and H. triffittae comprised a total of 1,080 bp (359 amino acids) and used ATG and TAA as start and stop codon, respectively. The cox1 of these species also used TAA as stop codon, whereas the most likely start codon was ATG, resulting in a gene comprising 1,491 bp (496 amino acids). Pair-wise sequence comparisons based on either cytb or cox1 clearly separated T. gondii from N. caninum and both of these species from the two Hammondia species, whereas the latter two species were 100 % identical at cytb and shared 99.3 % identity at cox1. Phylogenetic analyses using the maximum-likelihood method confirmed these findings and placed T. gondii in a clade separate from the three other species and all four Toxoplasmatinae in a sister clade to Eimeria spp. PCR with other primers and/or primer pairs than those used to obtain the full-length mitochondrial genes yielded several types of about 1–1.5 kb long sequences, which comprised stretches of the primer-targeted genes at both ends and an intervening non-coding sequence of various length and composition. Thus, portions of cytb could be found both upstream and downstream from portions of cox1 and portions of the same gene could be found adjacent to each other (cytb→cox1; cox1→cytb; cytb→cytb; cox1→cox1). Sequence comparisons revealed that some of these gene fragments were truncated genes, whereas others included the putative start or stop codon of the full-length mitochondrial genes. From the nature of the gene fragments and/or their flanking sequences, they are assumed to be located on the chromosomes of the nuclear genome and to represent nuclear mitochondrial DNA segments (numts) or pseudogenes. In the four species examined, there were no nucleotide differences between the full-length mitochondrial copies of cytb and cox1 and their various incomplete nuclear counterparts. With a few exceptions, identical numt types and closely similar flanking sequences were obtained for all four species, which would indicate that the original transfer of these mitochondrial genes to the nuclear genome and/or the majority of any subsequent rearrangements of these gene fragments within the nuclear genome happened before the four species diverged. Yet, there were species-specific differences in the nucleotide composition of the nuclear gene fragments, identical to the differences in the mitochondrial genes, which would indicate that the incomplete nuclear copies of cytb and cox1 have been continuously updated during evolution to conform to their mitochondrial parent genes. The PCR-based findings of numts were further supported by Basic Local Alignment Search Tool (BLAST) searches against genome sequences of T. gondii and N. caninum using the concatenated mitochondrial cytb/cox1 sequences as queries. These searches revealed the presence of numerous numts of eighth distinct types in both species, with each one having a fixed starting and end point with respect to the nucleotide positions in the full-length mitochondrial genes. Four numt types were completely homologous between both species, whereas four other types differed with respect to their end point and/or the absence/presence of a 96-bp deletion. Each starting and end point was associated with a unique 100–200-bp long flanking sequence, which further revealed the presence of numts. For both species, the numt types and their various arrangements with respect to each other were identical or similar to those obtained by PCR in all four species examined. None of the identified numts covered a full-length gene, but together, the various numts covered the entire mitochondrial cytb and cox1 genes in an overlapping manner. In addition, they were fairly closely spaced on the chromosomes, and these features may explain why the nuclear copies were preferentially amplified to the exclusion of the true mitochondrial genes with most primers and primer pairs used in the present study. The possibility of a similar high prevalence of numts occurring in the nuclear genome of dinoflagellates is discussed.

Resistance against benzimidazoles (BZ) has recently been detected in Norwegian sheep flocks through a large scale prevalence survey based on the faecal egg count reduction test (FECRT). The use of this test in combination with bulk larval culture only gives an indication of which gastrointestinal nematodes genera that are involved and these results have to be confirmed by a controlled efficacy test (CET) to get accurate information about resistant nematodes populations at species level. A CET was therefore performed with larvae from two flocks where BZ resistance was previously detected through FECRT.

Sarcocysts were isolated from the muscle tissue of three roe deer from southeastern Norway and examined by light microscopy, scanning electron microscopy and/or sequencing of the small subunit ribosomal RNA (ssu rRNA) gene. By light microscopy, four sarcocyst types were found, including those of Sarcocystis gracilis and Sarcocystis oviformis, which had been characterized previously. The third cyst type had about 10 μm long, flexible, hair-like surface protrusions, consistent with those of Sarcocystis capreolicanis, and differed genetically from other known species. The name S. capreolicanis was therefore assigned to this sequence type. The fourth cyst type had densely packed, upright, finger-like surface protrusions, about 8 μm long, and was morphologically similar to an unnamed Sarcocystis sp. reported from roe deer in other countries, and identical at the ssu rRNA gene to Sarcocystis sp. Type D previously reported from moose. This species was assigned the new name Sarcocystis silva. Both S. capreolicanis and S. silva displayed considerable intraspecific variation at the ssu rRNA gene. In phylogenetic analyses based on ssu rRNA gene sequences, S. capreolicanis grouped together with other canine-transmitted Sarcocystis species, whereas S. silva was most closely related to Sarcocystis rangiferi and Sarcocystis tarandi of reindeer. Roe deer muscles containing numerous cysts of S. gracilis were fed to a silver fox (Vulpes vulpes) and a blue fox (Vulpes lagopus), both of which started shedding Sarcocystis sporocysts in their faeces 9 days later, and harboured numerous oocysts, measuring about 20 × 15 μm, in their intestinal mucosa upon euthanasia 14 days post-inoculation. DNA derived from these oocysts was amplified and sequenced at the ssu rRNA gene and belonged to S. gracilis, confirming for the first time by molecular methods that foxes are definitive hosts for this species.

In recent years, the protozoan Tritrichomonas foetus has been recognised as an important cause of chronic large-bowel diarrhoea in purebred cats in many countries, including Norway. The aim of this cross-sectional study was to determine the proportion of animals with T. foetus infection among clinically healthy cats in Norway and to assess different risk factors for T. foetus infection, such as age, sex, former history of gastrointestinal symptoms and concurrent infections with Giardia duodenalis and Cryptosporidium sp.

Anthelmintic treatment is the most common way of controlling nematode infections in ruminants. However, several countries have reported anthelmintic resistance (AR), representing a limitation for sustainable small ruminant production. The knowledge regarding worm control management represents a baseline to develop a guideline for preventing AR. The aim of the present study was therefore to improve our knowledge about the worm control practices in small ruminant flocks in Norway.