A novel duplex ddPCR assay for detection and differential diagnosis of Ascaridia galli and Heterakis gallinarum eggs from chickens feces.

Since the EU ban on battery cages, many studies have listed Ascaridia galli and Heterakis gallinarum as the most common roundworms in the European laying hen population. A complicating factor is that the eggs of these parasites are almost identical. Thus, lack of molecular diagnostic approaches has driven epidemiological studies to take on necropsy for species discrimination, which is labor and cost intensive. Here, we describe a novel diagnostic tool based on droplet digital PCR for simultaneous identification and absolute quantification of the eggs of both of these ascarids in chickens' droppings using two different genus-specific primer-probe sets targeting the second internal transcribed spacer region (ITS-2) in the nuclear ribosomal (rRNA) gene array. No cross-reaction was observed when different combinations of DNA and species-specific primers and probes were tested. The lowest obtained frequency threshold for the detection of H. gallinarum in the presence of a constant A. galli DNA concentration was determined to be 0.8 %. After validation, we used the assay to analyze field samples collected from several Swedish laying hen farms. Out of 134 samples, 86 (64 %) were positive for A. galli while 11 (8.3 %) samples were positive for H. gallinarum. These samples were initially analyzed with flotation technique for detection of ascarid eggs. The results of the Cohen's kappa indicated substantial agreement (85.8 %) between the two tests. In conclusion, we have validated a novel molecular-based diagnostic tool for quantification and differentiation between intestinal parasites of major importance in chickens with high precision. Although this study focuses on identification of parasites of laying hens, the findings may well have a bearing on all types of chicken production systems. The present study lays the groundwork for future research into epidemiology of these two important chicken parasite species.

Public Health Follow-up of Suspected Exposure to Echinococcus multilocularis in Southwestern Ontario.

In the 3 years since the first report of canine alveolar echinococcosis (AE) in Ontario, three additional cases have been diagnosed in the province. Of the four cases reported to date, three have had no known history of travel outside the province. It is possible that this development is an indication of previously unrecognized environmental contamination with Echinococcus multilocularis eggs in some areas of the province. If so, there is the potential for an emerging threat to human health. This article describes a local public health department's investigation of the possible exposure to E. multilocularis of a number of individuals who had had contact with the latest of the four cases of canine AE, and summarizes a comprehensive decision process that can be used by public health departments to assist in the follow-up of such exposures.

The association between Anoplocephala perfoliata and colic in Swedish horses--a case control study.

A case-control study was performed to investigate the association between colic of all types in Swedish horses and infection with the equine tapeworm Anoplocephala perfoliata. Colic cases were defined by clinical signs consistent with the presence of abdominal pain, and the control horses had no signs of colic within the last year but attended a clinic for other reasons. Blood and fecal samples were collected by veterinarian from 67 horses with signs of colic and 67 control horses. The sera were analyzed using serodiagnostic assay anti-12/13 kDa IgG(T) ELISA. The fecal samples, 30 g from each horse, were analyzed with a modified sugar salt flotation method with a density of 1.280. A significant association was found between the presence of A. perfoliata eggs in feces and colic with a 16 times higher risk of colic if eggs had been observed in fecal samples. However, there was no significant association between colic and the median OD-values in the serological diagnosis, nor when recommended cut-offs were used. The study concludes that A. perfoliata is a risk factor for colic in Swedish horses and it suggests that the modified flotation method can be used as a diagnostic tool for identifying horses at risk.

Investigations and actions taken during 2011 due to the first finding of Echinococcus multilocularis in Sweden.

Echinococcus multilocularis is a parasite that can cause alveolar echinococcosis disease. After the first positive finding of E. multilocularis in Sweden in 2011, a consulting group with representatives from relevant authorities was summoned. In this group, all relevant information was shared, strategies for information dissemination and any actions to be taken due to the finding of E. multilocularis were discussed and decided. The present paper describes the actions taken during 2011 and the results thereof, including surveillance in animals, risk assessment for humans to become infected and recommendations given to the public. Further discussion about whether the parasite was introduced, and if so, how, as well as possible future development of the infection in animals and humans in Sweden and future actions are included.

First detection of Echinococcus multilocularis in Sweden, February to March 2011.

Surveillance for the fox tapeworm, Echinococcus multilocularis, has been carried out in Sweden since 2000, with about 300 red foxes analysed annually. We report the first finding of E. multilocularis in Sweden, in a fox shot in December 2010 in the south-west of the country. A second infected fox shot in the same location was detected in March 2011. This paper describes the national monitoring programme and the ongoing work to estimate the prevalence and spread of the infection.

Expulsion of small strongyle nematodes (cyathostomin spp) following deworming of horses on a stud farm in Sweden.

This study was conducted on a stud farm in Sweden to investigate the species composition of cyathostomins expelled in the faeces of horses after deworming using three different anthelmintic preparations. Twenty-seven horses excreting > or = 200 strongyle eggs per gram faeces (EPG) were divided into three comparable groups and dewormed on day 0 with either of following compounds: 0.2 mg ivermectin per kg body weight (bw), 19 mg pyrantel pamoate per kg bw or 7.5 mg fenbendazole per kg bw. For each of the 3 days following anthelmintic treatment faeces was collected from individual horses and subsamples were fixed in formalin. Four days after the anthelmintic treatment all horses were re-treated with ivermectin and faeces was collected on day 5. Individual subsamples from each of the four sampling occasions were examined for cyathostomin nematodes. Sixty-three to 270 worms per horse were identified to the species level. The majority of the worms recovered were expelled during the first day from horses treated with ivermectin or pyrantel pamoate, and during the second day from horses treated with fenbendazole. Fifteen cyathostomin species were identified and the six most prevalent were Cylicocyclus nassatus, Cyathostomum catinatum, Cylicostephanus longibursatus, Cylicocyclus leptostomus, Cylicostephanus minutus and Cylicostephanus calicatus. These species composed 91% of the total burden of cyathostomins. The number of species found per horse ranged from 6 to 13, with an average of 9. No significant differences in species composition or distribution were found between the treatment groups. On day 5, i.e. 1 day after the last ivermectin treatment, 93% of the adult worms were recovered from horses in the fenbendazole group. This study showed that it was possible to identify cyathostomins expelled in faeces of dewormed horses, and that the most prevalent species corresponded to those found in autopsy surveys performed in other countries.

A field survey on the distribution of strongyle infections of horses in Sweden and factors affecting faecal egg counts.

Faecal egg counts were performed during the first quarter of 1995 on samples from 1183 horses of varying breeds, ages and gender on 110 farms in 3 regions of Sweden. The majority of the horses had been treated with ivermectin or pyrantel when stabled the previous autumn. The risk of reinfection with strongyles between treatment and sampling was therefore considered to be minimal. Consequently, the results reflect primarily the occurrence of worms originating from inhibited strongyle larvae refractory to anthelmintic treatment. A total of 922 (78%) individuals were found to shed strongyle eggs, and 15 (14%) of the farms studied had at least one horse infected with Strongylus vulgaris. There was a significant, gradual decrease in faecal egg output in horses originating from the southern to the northern part of Sweden. Horses from studfarms showed significantly higher numbers of strongyle eggs/g faeces (EPG) as compared to those on other types of farms, such as riding-schools and boarding stables. However, there was no correlation between herd size and mean herd EPG. The output of strongyle eggs was highest in horses aged 2 and 3 years. EPG values then declined with increasing age of the horse. Horses treated with ivermectin in the previous autumn showed significantly lower EPG values than untreated horses and those treated with pyrantel pamoate.