The genetic diversity of Cryptosporidium spp. and Giardia duodenalis from dairy cattle and pigs in Denmark was determined in the present study. Faecal samples from 1237 pigs and 1150 cattle ...originating from 50 sow herds and 50 dairy herds, respectively, were analysed for the presence of the two parasites by immunofluorescence microscopy. A large proportion of the (oo)cyst containing samples were selected for molecular characterization. Sequencing and phylogenetic analysis of the 18S rDNA locus and/or the HSP70 gene of 183 pig and 154 cattle isolates of Cryptosporidium revealed the presence of C. suis, pig genotype II, C. parvum (cattle genotype), C. bovis, Cryptosporidium deer-like genotype and a novel C. suis-like genotype. For both cattle and pigs, a host age-related change in distribution of species/genotypes was observed. The zoonotic C. parvum (cattle genotype) was most prevalent in young calves. For Giardia, 82 and 145 isolates from pigs and cattle, respectively, were analysed at the 18S rDNA locus and/or the gdh gene. Giardia isolates belonging to the zoonotic Assemblage A was found in both young and older calves, as well as in weaners and piglets, whereas cows seemed to be infected purely by isolates of the livestock group, Assemblage E.
Aims
To determine inactivation of Cryptosporidium parvum oocysts and reduction of Escherichia coli and enterococci in cattle slurry added aqueous ammonia.
Methods and Results
Escherichia coli, ...enterococci and nonviable C. parvum oocysts (DAPI+PI+) were enumerated every second day for 2 weeks in cattle slurry amended with 60 mmol l−1 aq. ammonia and compared with untreated slurry at three temperatures. Regardless of temperature, the proportion of nonviable C. parvum oocysts increased significantly faster over time in slurry with added ammonia than raw slurry (P = 0·021) corresponding to 62·0% higher inactivation (P = 0·001) at day 14. Additionally, 91·8% fewer E. coli and 27·3% fewer enterococci were observed in slurry added ammonia at day 14 compared to raw slurry.
Conclusion
The addition of aqueous ammonia to raw slurry significantly reduced the viability of C. parvum oocysts and numbers of bacterial indicators. Hence, ammonia is usable at lower pathogen concentrations in slurry before application to agricultural land.
Significance and Impact of the Study
Livestock waste is a valuable source of plant nutrients and organic matter, but may contain high concentrations of pathogens like E. coli and Cryptosporidium sp. that can be spread in the environment, and cause disease outbreaks. However, die‐off rates of pathogens in organic waste can increase following increasing ammonia concentrations.
Sainfoin (Onobrychis viciifolia) is a condensed tannin (CT)-containing legume and has anthelmintic potential against gastrointestinal nematodes of ruminants. This study investigated in vitro effects ...of acetone/water extracts and derived CT fractions from different types of sainfoin (i.e. accessions) against larvae of Cooperia oncophora and Ostertagia ostertagi by applying the larval feeding inhibition assay (LFIA). Seven sainfoin accessions were extracted and tested with L1 larvae at 10 and 40μg extract/ml. In addition, CT in extracts from 4 accessions were fractionated according to polymer size and tested by LFIA at two concentrations (2 and 10μg CT fraction/ml). All sainfoin extracts caused significant inhibition of L1-feeding of both C. oncophora and O. ostertagi with varying intensity compared to the control (phosphate buffered saline). For both nematode species the in vitro effect was positively correlated with CT content in the extracts, but not with any of the structural CT parameters. In contrast, the 16 CT fractions revealed significant correlations between in vitro effect and CT content, polymer size (mean degree of polymerisation, mDP) and monomeric composition (prodelphinidin percentage, % PD). These differences between crude extracts and purified fractions may stem from the fact that extracts contain complex CT mixtures, which may mask and thus suppress CT structural effects. This study provides the first indication that, apart from CT and % PD content, polymer size also contributes to anthelmintic activity of CTs. The results, therefore, suggest that the inter-accession variability in CT content and composition needs to be taken into account in future plant breeding programmes which seek to enhance the anthelmintic properties of sainfoin.
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.
•Systematic examination for internal parasites of Danish otters was performed.•75.8% of 33 otters harboured at least one parasite species.•33.3% of 30 otters were infected with the zoonotic trematode Metorchis bilis.
Molecular and biological characteristics of a new species of Cryptosporidium from the feces of pigs (Sus scrofa) is described. Oocysts are structurally indistinguishable from those of Cryptosporidium ...parvum; they are passed fully sporulated, lack sporocysts, and measure 4.9–4.4 μm (mean = 4.6 μm) × 4.0–4.3 μm (mean = 4.2 μm); length to width ratio 1.1 (n = 50). Cryptosporidium suis is not transmissible to nude mice and is poorly infectious for cattle. Molecular and phylogenetic analyses at the 18S ribosomal RNA, heat shock protein 70, and actin gene loci demonstrate C. suis to be genetically distinct from all known species and genotypes of Cryptosporidium, and thus is named as Cryptosporidium suis.
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 × 104) 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 × 102; 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.
•Overall, 90.1% of 99 Danish outdoor cats harboured gastrointestinal helminths.•Toxocara cati, the most prevalent species, was detected in 84.8% of the cats.•Ollulanus tricuspis, was found with high ...intensity in 13% (second most common).•The sensitivity of concentration McMaster was 83% for T. cati, 27% for taeniid eggs.
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.
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.
In November 1997,
Cryptosporidium andersoni, for the first time, was isolated from a Danish heifer. The isolate was characterised morphologically, molecularly, and furthermore inoculated into mice ...and one calf. Data on the distribution of cryptosporidia in the herd of origin were obtained at two separate visits in December 1997 and April 1998.
C. andersoni was detected in 27 (19.0%) of 142 cattle examined at the first visit, whereas
C. parvum was found in six (4.2%). At the following visit 42 (28.0%) of 150 cattle excreted
C. andersoni, while 25 (16.7%) were positive for
C. parvum. Oocysts of the Danish
C. andersoni isolate were ovoid,
7.3
(6.5–8.0)×5.7
(5.0–7.0)
μm
2 (
n=25), with smooth, colourless, single layer oocyst wall and distinct oocyst residuum. The length to width ratio was 1.27 (1.14–1.40,
n=25). The identification was verified by sequencing of a 246
bp fragment of the rDNA, which was identical to
Cryptosporidium muris, the calf genotype (AF093496). The Danish
C. andersoni isolate was not transmissible to mice, whereas oocysts were detected in the faeces of one experimentally infected calf from 25 days post-infection (DPI) and shed intermittently at low numbers until 165 DPI, the day of euthanasia. No macroscopic or microscopic changes that could be attributed to infection with
C. andersoni were seen in the gastro-intestinal tract of the experimentally infected calf following necropsy and histological examination. This is to our knowledge the first report of
C. andersoni in Scandinavia.
The performance of immunofluorescence microscopy (IF) and enzyme-linked immunosorbent assay (ELISA) in canine feces was evaluated. IF and
Cryptosporidium ELISA detected 10
5
oocysts/g, while the ...detection limit for
Giardia ELISA was 10
4
cysts/g. The
Cryptosporidium ELISA showed 94% specificity but only 71% sensitivity. The
Giardia ELISA correlated well with IF (sensitivity 100%, specificity 96%) and was capable of detecting animal specific
Giardia duodenalis genotypes. Visual interpretation appeared appropriate for assessment of ELISA results. The proportion of positive samples and possible zoonotic character of
Cryptosporidium and
Giardia infections in 150 asymptomatic Finnish dogs from the Helsinki area were studied. The overall proportion of dogs positive for
Cryptosporidium was 5% (7/150) and that for
Giardia 5% (8/150). In dogs ≤12 months old, the corresponding proportions were 17% and 19% (
n
=
36). Sequence analyses of the 18S rDNA gene identified the isolates as
Cryptosporidium canis and animal specific genotypes of
G. duodenalis (assemblages C–E), indicating restricted risk of zoonotic transmission.
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