Huffmanela sp. (Nematoda: Trichosomoididae: Huffmanelinae) Encountered in the Whitecheek Shark (Carcharhinus Dussumieri) in The Arabian Gulf.

Veterinary inspection of fish caught at the western side of the Arabian Gulf off the shore of Saudi Arabia in 2017 revealed abnormal irregular zigzag black lines in the skins of 135 out of 1500 (prevalence = 9 %) whitecheek sharks (Carcharhinus dussumieri). These lesions have been noticed in the dorsal and ventral part of the head near the snout and around the eyes, as well as in the skin of the pectoral fins. The parasitological, pathological and ultramorphological aspects of the sampled fish were studied using a light microscope, as well as scanning and transmission electron microscopes. Trichosomoidid-type eggs identified as Huffmanela sp. were found in the superficial layer of the skin. The mature eggs containing larvae (n = 75) were ellipsoidal or spindle-shaped, smooth-shelled, amber-brown to dark brown or even black in colour. The width of the eggs ranged from 34 to 49µm with lengths ranging from 95 to 107µm. The polar plugs of the immature eggs were slightly protruding, whereas mature eggs had clearly protruding polar plugs. The width of the polar plugs ranged from 4.5 - 9.5µm. The shells of the eggs were smooth and there was a thin layer seen near the polar areas. The infected skin exhibited desquamated epithelium, with inflammatory cell aggregations and melanophores in the debris. The underlying musculatures of the skin were free from any eggs, but showed mild pathological changes, including congestion of blood vessels, haemorrhages and oedema. The prevalence of infection showed seasonality, peaking significantly in the spring (18.18 %), with comparable prevalence rates in winter and summer (9.43 % and 8.57 %, respectively). However, infections dropped significantly during the autumn months (3.75 %). It is believed that this is the first study reporting the presence of Huffmanela sp. in whitecheek sharks at El-Jubail Province, Saudi Arabia, Arabian Gulf.

Systematic examination of the cardiopulmonary, urogenital, muscular and gastrointestinal parasites of the Eurasian otters (Lutra lutra) in Denmark, a protected species recovering from a dramatic decline.

The Eurasian otter (Lutra lutra) is a protected species in Denmark and at present, the population is recovering due to conservation efforts. The Danish otters are mainly found in the continental part of Denmark (Jutland), but establishment in the main islands (Fyn and Zealand) has been observed. While there is a lack of systematic studies on the parasite fauna of otters in Denmark, this study aims to screen otters for their parasite fauna, especially those of zoonotic and/or veterinary importance. Thirty-three otter carcasses, road-killed (n = 30), found dead (n = 2) and shot (n = 1), were collected between June 2013 and May 2014 and examined for cardiopulmonary, urogenital, gastrointestinal, and muscle helminths by post mortem examination. Faecal samples were analysed by modified concentration McMaster technique and direct immunofluorescence test for Giardia and Cryptosporidium. At least one parasite was found in 75.8% of animals. The parasite fauna included 13 species, consisting of five nematodes: Molineus patens (30.3%), Aonchotheca putorii (27.3%), Strongyloides sp. (24.2%), Physaloptera sp. (12.1%), Eucoleus aerophilus (10.0%); one cestode: Schistocephalus solidius (6.1%); four trematodes: Metorchis bilis (33.3%), Isthimiophora melis (15.2%), Cryptocotyle sp. (3.0%), Plagiorchis sp. (3.0%); one acanthocephalan: Acanthocephalus ranae (18.2%); and two protozoans: Giardia spp. (3.1%), and Eimeria spp. (3.1%). The study showed that otters carry parasites of zoonotic and veterinary importance. Many of these parasites can also infect native carnivores and birds, and the distribution of these parasites may be affected if the otter population continue to increase in Denmark.

Echinococcus multilocularis in Denmark 2012-2015: high local prevalence in red foxes.

In Western Europe, the Echinococcus multilocularis lifecycle is predominantly sylvatic, typically involving red foxes (Vulpes vulpes) as the main definitive hosts with Microtus spp. and Arvicola spp. as intermediate hosts. During a 4-year surveillance study (2012-2015), Danish red foxes and raccoon dogs (n = 1345) were examined for E. multilocularis. Moreover, 134 insectivores and rodents collected in South Jutland during spring and summer 2016 were examined for the presence of metacestodes. The sedimentation and counting technique and molecular typing were used to identify E. multilocularis infections in the carnivores, while the rodent livers were examined macro- and microscopically for parasite lesions. Following morphological identification of E. multilocularis adult worms, the identity was verified by sequence analysis of the 12S rRNA gene in most cases (n = 13). Echinococcus multilocularis infection was demonstrated in 19 red foxes (Vulpes vulpes) originating from only two specific areas of South Jutland, namely Højer and Grindsted, and in two raccoon dogs (Nyctereutes procyonoides), originating from Højer. In Højer, 28.5% (CI 95% 11.7-45.3) of the examined red foxes were E. multilocularis positive per year. Moreover, positive red foxes were identified each year from 2012 to 2015, while E. multilocularis positive red foxes were only identified in Grindsted in 2013 (4.0%) and 2014 (6.4%). In contrast, all collected rodents were negative for E. multilocularis. We conclude that E. multilocularis is locally endemic in South Jutland with a high local prevalence in Højer.

New filtration system for efficient recovery of waterborne Cryptosporidium oocysts and Giardia cysts.

AIMS: To develop a filtration unit for efficient recovery of waterborne Cryptosporidium oocysts and Giardia cysts ((oo-)cysts) in drinking water. METHODS AND RESULTS: This unit utilizes a metallic filter and an ultrasound transducer for eluting (oo-)cysts, with a fixed retentate backwash volume; approx. 400 µl. Changes in the viability was evaluated by seeding wild type (oo-)cysts (1 × 10(4)) followed by sonication for 5, 10, 20 or 40 s (five replicates for each period). Flow cytometry analysis showed negligible increase in the mortality of (oo-)cysts exposed to 5-10 s of sonication. Recovery rate was assessed by seeding ColorSeed(™) (10 replicates) into the filter unit followed by air backwash to a glass slide and counting of (oo-)cysts by epifluorescent microscopy. High recovery rates (mean ± SD) were found: 84·9% ± 4·8 for Giardia cysts and 70% ± 6·5 for Cryptosporidium oocysts. DNA of seeded wild type (oo-)cysts (1 × 10(2); 10 replicates) was successfully amplified using real-time PCR. CONCLUSIONS: The use of a metallic filter, sonication and 'air backwash' were key factors for creating a highly efficient system for recovery of apparently undamaged protozoa. SIGNIFICANCE AND IMPACT OF THE STUDY: This reagent-less system can be used for monitoring of parasite contamination in drinking water.

Gastrointestinal parasites of cats in Denmark assessed by necropsy and concentration McMaster technique.

The large population of feral cats in Denmark may potentially transmit pathogens to household cats and zoonotic parasites to humans. A total of 99 euthanized cats; feral cats (n=92) and household cats with outdoor access (n=7), were collected from March to May 2014 from the Zealand region, Denmark. The sedimentation and counting technique (SCT) was used to isolate helminths and coproscopy was done by concentration McMaster technique (c-McMaster). Overall, 90.1% of the cats were infected and a total of 10 species were recorded by SCT: 5 nematode species: Toxocara cati (84.8%), Ollulanus tricuspis (13.1%), Aonchotheca putorii (7.1%), Paersonema spp. (3.0%), Strongyloides spp. (1.0%); 3 cestodes: Hydatigera taeniaeformis (36.4%), Mesocestoides sp. (3.0%), Dipylidium caninum (1.0%); and 2 trematodes: Cryptocotyle spp. (5.1%) and Pseudamphistomum truncatum (1.0%). O. tricuspis was the second most common gastrointestinal nematode of cats but had the highest intensity of infection. For T. cati, prevalence and worm burden were significantly higher in feral than household cats. No juvenile cats were infected with H. taeniaeformis, and age thus had a significant effect on prevalence and worm burdens of this species. Rural cats had a higher prevalence and worm burden of A. putorii than urban cats. By c-McMaster, ascarid, capillarid, strongylid or taeniid type eggs were found in 77.9% of the cats while Cystoisospora felis was found in 2.1%. The sensitivity of the c-McMaster was 82.5% for T. cati but 26.5% for taeniid eggs, using the SCT as gold standard. A positive correlation between faecal egg counts and worm burdens was seen for T. cati, but not for taeniid eggs (assumed to be H. taeniaeformis). Coprological examination also detected the eggs of extraintestinal Capillariidae species including Eucoleus aerophilus and Eucoleus boehmi, but further necropsy studies are needed to confirm these findings.

Morphological and molecular analyses of larval taeniid species in small mammals from contrasting habitats in Denmark.

Taeniid infections in intermediate hosts manifest themselves as extraintestinal larval stages which, in early development, lack species-specific characteristics. The inability to distinguish infections of zoonotic importance such as Echinococcus multilocularis from other taeniid infections that have mainly veterinary significance stimulated the development of species-specific molecular diagnostics. In this study, the prevalence of taeniid infections in potential intermediate hosts was evaluated using both morphological diagnosis and a newly described multiplex polymerase chain reaction (PCR) for species determination. Small mammals (N= 719) were trapped in three different types of habitats in north-east Zealand, Denmark. The sensitivity of the multiplex PCR (90.5%) exceeded that of morphological examination (57.9%) for identifying 95 taeniid infections. The use of the multiplex PCR resulted in higher prevalence rates due to improved detection of immature liver infections with Hydatigera taeniaeformis and Versteria mustelae, but did not affect the observed prevalence rates of peritoneal metacestodes of Taenia polyacantha. The prevalence of taeniid infections showed a significant difference according to habitat type, potentially identifying a 'sylvatic' transmission and an 'urban' transmission, with marked variation among different taeniid species. Versteria mustelae and T. polyacantha were more prevalent in rural forests, while infections with H. taeniaeformis were dominant in urban parks/forests and in residential and farm gardens. The multiplex PCR facilitated a better utilization of wildlife samples by yielding a higher number of definitive diagnoses of ambiguous taeniid infections in liver lesions, allowing for more accurate epidemiological data and, hence, a more accurate risk assessment.

The occurrence of Angiostrongylus vasorum in terrestrial slugs from forests and parks in the Copenhagen area, Denmark.

A total of 298 slugs belonging to four species, Arion lusitanicus, A. ater, A. ater rufus and Limax maximus, were collected from six different localities within a radius of 30 km from Copenhagen and examined for naturally acquired Angiostrongylus vasorum infection. Overall, 28 slugs (9%) were infected, but the prevalence varied among the studied localities: Rude Forest (26%), West Amager Forest (18%), Jaegersborg Forest and Deer Park (8%), Frederiksberg Park (4%), Assistens Cemetery Park (0%) and Frederiksberg Botanical Garden (0%). Only third-stage larvae (L3) were recovered from the slugs, in numbers ranging from 1 to 392 per slug. Overall 82% of the infected slugs harboured fewer than 10 larvae and only 14% harboured over 100 larvae.

Comparative copro-diagnosis of Echinococcus multilocularis in experimentally infected foxes.

Faecal samples from 15 foxes experimentally infected with Echinococcus multilocularis were examined until 90 days post-infection (dpi) by microscopical identification of eggs isolated by flotation/sieving, by coproantigen-enzyme-linked immunosorbent assay (cELISA), by polymerase chain reaction (PCR) on DNA, respectively, isolated directly from the faecal samples (copro-DNA PCR) and from the eggs obtained by the flotation/sieving procedure (egg-DNA PCR). Based on egg counts, three periods of the infection were defined: pre-patent (2-29 dpi), high patent (30-70 dpi) and low patent periods (71-90 dpi). Whereas all methods were highly sensitive with samples from the high patent period, cELISA was the most sensitive to detect pre-patent infections (63%). Samples from the low patent infections were positive in 77% by microscopy and in 80% by egg-DNA PCR, being significantly more sensitive than cELISA and copro-DNA PCR. The isolation of eggs from the faecal material proved to be more sensitive by the flotation/sieving procedure as compared to the classical concentration McMaster technique.