Heidi L. Enemark

Molecular and phylogenetic characterization of Cryptosporidium and Giardia from pigs and cattle in Denmark

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.

Cryptosporidium andersoni from a Danish cattle herd: identification and preliminary characterisation

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) x 5.7(5.0-7.0) microm(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 246bp 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.

Molecular characterization of Danish Cryptosporidium parvum isolates.

The genetic polymorphism among 271 Danish Cryptosporidium isolates of human and animal origin was studied by partial amplification and sequencing of the Cryptosporidium oocyst wall protein (COWP) gene, the 1 8S rDNA, and a microsatellite locus. Furthermore, the microsatellite locus was studied directly using fragment analysis. A comparative analysis of DNA sequences showed the presence of 3 different subgenotypes (Cl, C2 and C3) in C. parvum isolates from Danish cattle, with prevalences of 16.7, 17.2 and 73.1% including 13 (7.0%) mixed infections. Subgenotype Cl was significantly more prevalent (P < 0.001) in the southern part of Denmark. In Cryptosporidium isolates of human origin the anthroponotic subgenotype H1 was identified, in addition to the zoonotic subgenotypes C1, C2, and C3. Of 44 human samples, 56.8% were anthroponotic, whereas 40.9% were zoonotic genotypes. One human isolate was characterized as C. meleagridis. The porcine Cryptosporidium isolates (N = 4) revealed a pattern which was genetically distinct from human and bovine isolates. Cryptosporidium in a hedgehog (Erinaceus europaeus L.) was identified for the first time. By microsatellite sequencing the hedgehog isolate showed a subgenotype distinct from the previously detected types. The assignment to subgenotype by microsatellite sequencing and fragment typing was 100% identical in samples where results were achieved by both methods. In addition, the fragment analysis proved more sensitive, easier, faster, and less expensive compared to sequencing.

A foodborne outbreak of Cryptosporidium hominis infection.

Foodborne outbreaks of cryptosporidiosis are uncommon. In Denmark human cases are generally infrequently diagnosed. In 2005 an outbreak of diarrhoea affected company employees near Copenhagen. In all 99 employees were reported ill; 13 were positive for Cryptosporidium hominis infection. Two analytical epidemiological studies were performed; an initial case-control study followed by a cohort study using an electronic questionnaire. Disease was associated with eating from the canteen salad bar on one, possibly two, specific weekdays [relative risk 4.1, 95% confidence interval (CI) 2.1-8.3]. Three separate salad bar ingredients were found to be likely sources: peeled whole carrots served in a bowl of water, grated carrots, and red peppers (in multivariate analysis, whole carrots: OR 2.1, 95% CI 1.1-4.0; grated carrots: OR 2.1, 95% CI 1.2-3.9; peppers: OR 3.3, 95% CI 1.7-6.6). We speculate that a person excreting the parasite may have contaminated the salad buffet.

Towards a standardised surveillance for Trichinella in the European Union.

Each year, more than 167 million pigs in the European Union (EU) are tested for Trichinella spp. under the current meat hygiene regulations. This imposes large economic costs on countries, yet the vast majority of these pigs test negative and the public health risk in many countries is therefore considered very low. This work reviewed the current Trichinella status across the EU as well as the national level of monitoring and reporting. It also reviewed which animal species were affected by Trichinella and in which species it should be surveyed. This information was used to design a cost-effective surveillance programme that enables a standardised monitoring approach within the EU. The proposed surveillance programme relies on identifying sub-populations of animals with a distinct risk. Low-risk pigs are finisher pigs that originate from so-called controlled housing. All other pigs are considered high-risk pigs. Controlled housing is identified by the application of a specific list of management and husbandry practices. We suggest that member states (MS) be categorised into three classes based on the confidence that Trichinella can be considered absent, in the specified sub-population of pigs above a specified design prevalence which we set to 1 per million pigs. A simple and transparent method is proposed to estimate this confidence, based on the sensitivity of the surveillance system, taking into account the sensitivity of testing and the design prevalence. The probability of detecting a positive case, if present, must be high (>95 or >99%) to ensure that there is a low or negligible risk of transmission to humans through the food chain. In MS where the probability of a positive pig is demonstrated to be negligible, testing of fattening pigs from a sub-population consisting of pigs from controlled housing can be considered unnecessary. Furthermore, reduced testing of finishers from the sub-population consisting of pigs from non-controlled housing might even be considered, if conducted in conjunction with a proportionate sampling scheme and a risk-based wildlife surveillance programme where applicable. The proposed surveillance programme specifies the required number of samples to be taken and found negative, in a MS. A MS with no data or positive findings will initially be allocated to class 1, in which all pigs should be tested. When a MS is able to demonstrate a 95% or 99% confidence that Trichinella is absent, the MS will be allocated to class 2 or 3, in which the testing requirement is lower than in class 1.

Seasonal prevalence and incidence of Cryptosporidium spp. and Giardia duodenalis and associated diarrhoea in children attending pre-school in Kafue, Zambia

Prevalence, incidence and seasonal variation of Cryptosporidium and Giardia duodenalis were studied over a 12-month period in 100 children from four pre-schools in Kafue, Zambia. Questionnaire data and a single stool sample were collected monthly from each child. Samples were processed using a commercial kit (Meridian Diagnostics Inc., USA) and oo(cysts) visualised by immunofluorescence microscopy. Cryptosporidium was detected in 30.7% (241/786; 95% CI = 27.5-33.9) while G. duodenalis was detected in 29.0% (228/786; 95% CI = 25.8-32.2). A total of 86% experienced one or more episodes of cryptosporidiosis while 75% had giardiasis. Cumulative incidence per 100 children was 75.4 for Cryptosporidium and 49.0 for G. duodenalis. Both infections were significantly more common in the wet compared to the dry season (34.8%, 162/466 vs. 24.7%, 79/320, P = 0.003 and 35.2%, 164/466 vs. 20.0%, 64/320, P < 0.001, respectively). Thus, risk ratios (RR) were 1.41 (95% CI = 1.13-1.77) and 1.76 (95% CI = 1.38-2.27) for Cryptosporidium and Giardia, respectively. Diarrhoea was significantly associated with cryptosporidiosis (RR = 1.23, 95% CI = 1.03-1.47; P = 0.029) but not with giardiasis (RR = 1.12, 95% CI = 0.91-1.53; P = 0.26). We conclude that gastro-intestinal protozoal infections are highly prevalent among children attending pre-school in peri-urban Zambia highlighting the need for further studies of risk factors.

Intestinal helminths and protozoa in children in pre-schools in Kafue district, Zambia.

Intestinal parasitic infections are among the most widespread of human infections in developing countries, and children are the most vulnerable. The aim of this study was to determine the prevalence of the protozoa Cryptosporidium and Giardia, as well as prevalence and intensity of intestinal helminths in children attending pre-school or day-care centres in Kafue District, Zambia. Single stool samples were collected from 403 children from 10 pre-schools and were subjected to duplicate Kato-Katz thick smears to identify and quantify helminths. A commercial immunofluorescence kit was used to identify Cryptosporidium- and Giardia-positive samples. The overall prevalence of helminth infection was 17.9%. Ascarislumbricoides was found in 12.0%, hookworm in 8.3%, Taenia spp. in 0.9%, Hymenolepisnana in 0.6% and Schistosomamansoni in 0.3%. The overall prevalence of Cryptosporidium and Giardia was 28.0 and 29.0%, respectively, with more girls infected with Giardia (33.8%) than boys (22.7%) (P=0.02). Significant differences in infections with A. lumbricoides and Cryptosporidium were observed between the various pre-schools (P < 0.001). These findings indicate that intestinal parasites are prevalent in children enrolled in pre-schools in Zambia. Future studies should explore local factors associated with transmission of these infections, and consequently provide the necessary health education to parents and teachers.

Pathogenicity of Cryptosporidium parvum—evaluation of an animal infection model

Abstract With the intention of developing a standardised method for assessment of pathogenicity of Cryptosporidium parvum, the CPB-0 isolate was studied by propagation in 1-day-old calves followed by inoculation into specific pathogen free (SPF) piglets. The experiment was repeated. Diarrhoea and shedding of oocysts were seen in all animals infected with the CPB-0 isolate. Clinical signs included depression, inappetence, vomiting (exclusively in the piglets), and death. Histological examination at 17 and 19 days post-infection revealed parasitic stages and microscopic changes primarily restricted to colon and rectum. The unintended presence of rotavirus in some of the experimental animals revealed an additive or synergistic effect between rotavirus and C. parvum as indicated by prolonged diarrhoea, increased oocyst shedding, decreased weight gain and elevated levels of serum haptoglobin and serum amyloid A (SAA) in piglets infected simultaneously with both pathogens. The difference in daily weight gain between infected and control animals was significant only for piglets co-infected with rotavirus. The acute phase response of haptoglobin and SAA was characterised by a large individual variation. In piglets, co-infected with rotavirus, the levels of serum haptoglobin were 3.5 and 4.6 times higher in the infected versus the controls 6 and 9dpi, respectively (mean values: 2411 μg/ml±S.D. 2023 and 1840 μg/ml±S.D. 1697). In the controls infected with rotavirus, peak haptoglobin concentration was seen 3dpi (mean: 1022 μg/ml±S.D. 425). Elevated levels of SAA were seen in 1 of 6 piglets infected with C. parvum, and in 5 of 6 piglets co-infected with rotavirus. Tumour necrosis factor alpha (TNFα) was undetectable in all serum samples from piglets. The obvious advantages of the SPF pig model are the naturally acquired intestinal microflora, the development of distinct clinical signs similar to cryptosporidiosis in humans and calves, the size of the animals, and the accessibility of individuals born within a short time span. This makes the model ideal for dose–response studies, evaluation of therapeutic agents as well as for assessment of differences in the clinical response to isolates of diverse genetic background. In conclusion, it was shown that the CPB-0 isolate was pathogenic to calves and piglets at a dose of 2.5×105 oocysts, and that the clinical signs could be replicated during separate experiments. Moreover, diarrhoea, oocyst shedding, body weight changes, histological alterations, and the acute phase response of haptoglobin and SAA were identified as useful parameters for discrimination of isolate-specific differences of pathogenicity.

Endoparasites of the raccoon dog (Nyctereutes procyonoides) and the red fox (Vulpes vulpes) in Denmark 2009–2012 – A comparative study

Graphical abstract

In vitro effects of extracts and purified tannins of sainfoin (Onobrychis viciifolia) against two cattle nematodes.

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 40 μg 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 10 μg 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.