Use of metagenomic microbial source tracking to investigate the source of a foodborne outbreak of cryptosporidiosis.

Cryptosporidium is a protozoan parasite of global public health importance that causes gastroenteritis in a variety of vertebrate hosts, with many human outbreaks reported yearly, often from ingestion of contaminated water or food. Despite the major public health implications, little is typically known about sources of contamination of disease outbreaks caused by Cryptosporidium. Here, we study a national foodborne outbreak resulted from infection with Cryptosporidium parvum via romaine lettuce, with the main goal to trace the source of the parasite. To do so, we combined traditional outbreak investigation methods with molecular detection and characterization methods (i.e. PCR based typing, amplicon and shotgun sequencing) of romaine lettuce samples collected at the same farm from which the contaminated food was produced. Using 18S rRNA typing, we detected C. parvum in two out of three lettuce samples, which was supported by detections in the metagenome analysis. Microbial source tracking analysis of the lettuce samples suggested sewage water as a likely source of the contamination, albeit with some uncertainty. In addition, the high degree of overlap in bacterial species content with a public human gut microbial database corroborated the source tracking results. The combination of traditional and molecular based methods applied here is a promising tool for future source tracking investigations of food- and waterborne outbreaks of Cryptosporidium spp. and can help to control and mitigate contamination risks.

Comparative assessment of DNA extraction procedures for Ascaris spp. eggs.

A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.

A distinct group of north European Aedes vexans as determined by mitochondrial and nuclear markers.

The floodwater mosquito Aedes (Aedimorphus) vexans (Meigen, 1830) (Diptera: Culicidae) is common in several areas of Sweden and is predicted to become more abundant in the wake of expected changes in precipitation and temperature caused by climate change. As well as being a nuisance, Ae. vexans can act as a vector of over 30 viruses. In the event of an outbreak of disease caused by a vector-borne virus, knowledge of the distribution, population structure and intermixing of populations from different locations will help direct resources to target locations to prevent spread of the pathogen. The present study analysed individual Ae. vexans from eight locations throughout Sweden. Based on the mitochondrial cytochrome oxidase I (COI) marker, a subset of the analysed mosquitoes cluster apart from the other samples. Similarly, two nuclear loci were sequenced and the same phylogenetic structure observed. These results indicate that this group represents a reproductively isolated population among Ae. vexans. Comparisons with COI sequences held in the Barcode of Life Database (BoLD) for Ae. vexans from around the world show that specimens collected in Belgium and Estonia group together with the Swedish group, suggesting that this genotype is present throughout northern Europe. These results suggest there is a cryptic taxonomic unit related to Ae. vexans in northern Europe.

Cryptosporidium infections in suckler herd beef calves.

A study was carried out to investigate how common Cryptosporidium infections are in beef calves in Swedish suckler herds and to explore which species and subtypes that occur. We further aimed at identifying factors associated with shedding of Cryptosporidium oocysts in this type of calf management. The study was conducted in two regions in Sweden and included 30 herds. Faecal samples were collected from calves younger than 3 months. A brief clinical examination was done and a questionnaire was used to collect data on management routines. Faeces were cleaned and concentrated and oocysts identified by epifuorescence microscopy. Cryptosporidium positive samples were analyzed at the 18S rRNA and GP60 genes to determine species and Cryptosporidium parvum subtype, respectively. Logistic regression was used to identify factors associated with infection. Oocysts were detected in 122 (36.7%) calves from 29 (97%) herds, at 400 to 2.4 × 107 OPG. The youngest positive calves were only 1 and 2 days old. There was no association between age and Cryptosporidium infection. Cryptosporidium bovis, Cryptosporidium ryanae, C. parvum and Cryptosporidium ubiquitum were identified, with C. bovis being the major species. Two C. parvum subtypes, IIaA16G1R1 and IIdA27G1 were identified. Routines for cleaning calf pens and number of cows in calving pens were associated with infection.

Cryptosporidium parvum infections in a cohort of veterinary students in Sweden.

In March 2013, a veterinary student tested positive for Cryptosporidium; four classmates reported similar gastrointestinal symptoms. We aimed to identify source(s) and risk factors for Cryptosporidium infection in university persons symptomatic between 21 January and 14 April 2013. Sixty-four (79%) students from a cohort of 81 fourth-year veterinary students completed questionnaires, identifying 13 cases; four were Cryptosporidium parvum GP60 subtype IIaA16G1R1b, two were IIdA24G1, seven did not submit stool samples. Thirteen cases attended the university's field clinic before symptom onset (13/37 attendees, 35%); 11 visited at least one of four farms where students recalled seeing calves with diarrhoea. C. parvum subtype IIaA16G1R1b was identified in calves at one of the farms. Entering pens of calves with diarrhoea [relative risk (RR) 7·6, 95% confidence interval (CI) 1·7-33·5] and eating in clinic cars (RR 9·1, 95% CI 1·3-65·8) were associated with being a case. Washing hands at least twice per farm visit (0 cases, P = 0·03) was protective. This outbreak investigation was notable for rapid and effective collaboration between public health, veterinary and environmental sectors, leading to swift identification of a microbiological and epidemiological link between cases, infected calves and their farms. We recommend frequent hand-washing using proper technique and dissuasion from eating in clinic cars to minimize possible exposure to contaminated surfaces.

Barcoding of biting midges in the genus Culicoides: a tool for species determination.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness virus and bluetongue virus. However, the identification of Culicoides based on morphological features is difficult. The sequencing of mitochondrial cytochrome oxidase subunit I (COI), referred to as DNA barcoding, has been proposed as a tool for rapid identification to species. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined Culicoides species in Swedish collections. In total, 237 specimens of Culicoides representing 37 morphologically distinct species were used. The barcoding generated 37 supported clusters, 31 of which were in agreement with the morphological determination. However, two pairs of closely related species could not be separated using the DNA barcode approach. Moreover, Culicoides obsoletus Meigen and Culicoides newsteadi Austen showed relatively deep intraspecific divergence (more than 10 times the average), which led to the creation of two cryptic species within each of C. obsoletus and C. newsteadi. The use of COI barcodes as a tool for the species identification of biting midges can differentiate 95% of species studied. Identification of some closely related species should employ a less conserved region, such as a ribosomal internal transcribed spacer.

Phenotypic characterization of Haemonchus contortus: a study of isolates from Sweden and Kenya in experimentally infected sheep.

The effects of cold storage of infective third-stage larvae (L3) of different isolates of the parasitic nematode Haemonchus contortus were studied with respect to infectivity, pre-patent period and propensity for larval arrestment. Two complementary experiments were conducted with 2 groups of lambs, each animal being inoculated with 2000 L3 of either Swedish or Kenyan origin. In a first experiment, L3s were cold treated at 5 degrees C for 9 months prior to infection, whereas in a second experiment larvae were newly hatched. Individual faecal egg counts (FECs), and worm burdens were determined for each experiment. The results showed that the greatest differences were associated with the pre-treatment of larvae. The pre-patent period and the FECs differed significantly between the experiments but not between the isolates used in each experiment. However, the extent of hypobiosis was significantly different between the two isolates when fresh larvae were used (36% Kenyan isolate and 70% Swedish). The storage of H. contortus at 5 degrees C had no apparent effect on the infectivity of L3s, as high establishment ranging from 43 to 74% were observed, irrespective of isolates used. This study showed that H. contortus exhibited similar phenotypic traits regardless of geographical origin. Thus, there was limited evidence for adaptations to temperate climatic conditions.

The development and overwintering survival of free-living larvae of Haemonchus contortus in Sweden.

Five complimentary studies were undertaken with the overall aim to examine the ability of free-living stages of Haemonchus contortus to over-winter and tolerate cold stress. Two studies deal with the development and long-term survival of eggs and infective larvae of two geographically different isolates (Kenya and Sweden). Eggs and larvae were monitored in climatic chambers at temperatures that fluctuated daily between -1 degrees C and 15 degrees C, or at constant temperatures of 5 degrees C and 15 degrees C. The development from egg to larvae was dependent on temperatures over 5 degrees C. The long time survival was favoured at lower temperatures. Furthermore, the overwintering capacity of the free-living stages of these isolates was estimated under Swedish field conditions. Two groups of lambs were experimentally infected with different isolates, and kept separated on previously ungrazed plots. In early May the following year, two parasite-naive tracer lambs were turned out on each of the plots to estimate the pick up of overwintered larvae. This experiment was replicated in central and southern Sweden. In addition, two experiments were performed in 2003 on pasture previously grazed by naturally infected sheep. One trial was on a pasture in southern Sweden grazed by a commercial flock, where extreme numbers of H. contortus were found towards the end of the grazing season 2002. The other study was on a pasture plot in central Sweden grazed by a hobby flock in 2002, where three of six lambs died due to haemonchiasis. Overwintered H. contortus was recorded on three of four experimental sites. Worm burdens were in all instances extremely low. No differences in development and survival were found between the isolates. Consequently, overwintering on pasture is of no practical significance in the transmission of H. contortus between grazing-seasons in Sweden.