Identification of pig-specific Cryptosporidium species in mixed infections using Illumina sequencing technology.

Nowadays molecular methods are widely used in epidemiological studies of Cryptosporidium infections in humans and animals. However to gain better understanding of parasite species or genotypes, especially when mixed infections are noticed, highly sensitive tools with adequate resolution power need to be employed. In this article, we report an application of the next generation sequencing method (NGS) for detection and characterisation of Cryptosporidium species concurrently present in pig faeces. A mixture of Cryptosporidium DNA obtained from two faecal samples was amplified at the 18 SSU rRNA gene locus and the resulting amplicons were subsequently used for MiSeq sequencing. Although initial molecular analyses indicated the possible presence of another Cryptosporidium species other than Cryptosporidium scrofarum and Cryptosporidium suis, deep sequencing only confirmed the presence of pig-specific Cryptosporidium.

Molecular studies on pig cryptosporidiosis in Poland.

Cryptosporidium intestinal parasites have been detected in farmed pigs worldwide. Infections are usually asymptomatic with a low number of oocysts shed in pig feces. This makes the recognition of infection difficult or unsuccessful when microscopic methods are used. The aim of this study was molecular identification of Cryptosporidium species in pig herds raised in Poland with regard to the occurrence of zoonotic species. In total, 166 pig fecal samples were tested. The examined pigs were aged 1 to 20 weeks. Overall, 39 pig farms were monitored for parasite presence. The detection and identification of Cryptosporidium DNA was performed on the basis of PCR-RFLP and nucleotide sequence analysis of the amplified 18 SSU rRNA and COWP gene fragments. Infected animals were housed in 21 (53.8%) of the pig farms monitored. The presence of Cryptosporidum was confirmed in 46 (27.7%) samples of pig feces. Among positive fecal samples, 34 (29.3%) were collected from healthy animals, and 12 (24%) from diarrheic pigs. Most infected animals (42.1%) were 2 to 3 months old. The following parasite species were detected: C. scrofarum, C. suis and C. parvum. Indeed, asymptomatic infections caused by C. scrofarum were observed in the majority of the herds. Mixed infections caused by C. suis and C. scrofarum were not common; however, they were observed in 8.6% of the positive animals. C. parvum DNA was found only in one sample collected from a diarrheic pig. The application of molecular diagnostic tools allowed for detection and identification of Cryptosporidium species in pigs. The sporadic findings of C. parvum are subsequent evidence for the contribution of pigs in the transmission of cryptosporidiosis from animals to humans.

Virological Quality of Irrigation Water in Leafy Green Vegetables and Berry Fruits Production Chains.

This study condenses data acquired during investigations of the virological quality of irrigation water used in production of fresh produce. One hundred and eight samples of irrigation water were collected from five berry fruit farms in Finland (1), the Czech Republic (1), Serbia (2), and Poland (1), and sixty-one samples were collected from three leafy green vegetable farms in Poland, Serbia, and Greece. Samples were analyzed for index viruses of human or animal fecal contamination (human and porcine adenoviruses, and bovine polyoma viruses), and human pathogenic viruses (hepatitis A virus, hepatitis E virus, and noroviruses GI/GII). Both index and pathogenic viruses were found in irrigation water samples from the leafy green vegetables production chain. The data on the presence of index viruses indicated that the highest percentage of fecal contamination was of human origin (28.1 %, 18/64), followed by that of porcine (15.4 %, 6/39) and bovine (5.1 %, 2/39) origins. Hepatitis E virus (5 %, 1/20) and noroviruses GII (14.3 %, 4/28) were also detected. Samples from berry fruit production were also positive for both index and pathogenic viruses. The highest percentage of fecal contamination was of human origin (8.3 %, 9/108), followed by that of porcine, 4.5 % (4/89) and bovine, 1.1 % (1/89) origins. Norovirus GII (3.6 %, 2/56) was also detected. These data demonstrate that irrigation water used in primary production is an important vehicle of viral contamination for fresh produce, and thus is a critical control point which should be integrated into food safety management systems for viruses. The recommendations of Codex Alimentarius, as well as regulations on the use of water of appropriate quality for irrigation purposes, should be followed.

An outbreak of massive mortality among farm rabbits associated with Cryptosporidium infection.

Cryptosporidium in farm rabbits is not often recognised due to a low prevalence and asymptomatic course of infection. Nonetheless, incidences of fatal diarrhoeic diseases are frequently noticed in the rabbitries. In this article, we report an outbreak where there was massive mortality among farm rabbits associated with Cryptosporidium infection. The disease was characterised by profuse diarrhoea resulting in the death of rabbits. A pooled faecal sample was screened for a presence of parasites using microscopy methods. In the tested sample no other parasites other than Cryptosporidium oocysts were found. Further identification of the parasite species was performed at a molecular level, using the 18 SSU rRNA, COWP and LIB13 PCR followed by a subtyping at the GP60 gene locus. Sequence analysis of GP60 gene fragment revealed the presence of a novel subtype VbA24 of Cryptosporidium cuniculus. In this outbreak a Cryptosporidium protozoan parasite played a major role in the etiology of the gastrointestinal disorders in rabbits resulting in massive mortality of the infected animals.

Harmonised investigation of the occurrence of human enteric viruses in the leafy green vegetable supply chain in three European countries.

Numerous outbreaks have been attributed to the consumption of raw or minimally processed leafy green vegetables contaminated with enteric viral pathogens. The aim of the present study was an integrated virological monitoring of the salad vegetables supply chain in Europe, from production, processing and point-of-sale. Samples were collected and analysed in Greece, Serbia and Poland, from 'general' and 'ad hoc' sampling points, which were perceived as critical points for virus contamination. General sampling points were identified through the analysis of background information questionnaires based on HACCP audit principles, and they were sampled during each sampling occasion where as-ad hoc sampling points were identified during food safety fact-finding visits and samples were only collected during the fact-finding visits. Human (hAdV) and porcine (pAdV) adenovirus, hepatitis A (HAV) and E (HEV) virus, norovirus GI and GII (NoV) and bovine polyomavirus (bPyV) were detected by means of real-time (RT-) PCR-based protocols. General samples were positive for hAdV, pAdV, HAV, HEV, NoV GI, NoV GII and bPyV at 20.09 % (134/667), 5.53 % (13/235), 1.32 % (4/304), 3.42 % (5/146), 2 % (6/299), 2.95 % (8/271) and 0.82 % (2/245), respectively. Ad hoc samples were positive for hAdV, pAdV, bPyV and NoV GI at 9 % (3/33), 9 % (2/22), 4.54 % (1/22) and 7.14 % (1/14), respectively. These results demonstrate the existence of viral contamination routes from human and animal sources to the salad vegetable supply chain and more specifically indicate the potential for public health risks due to the virus contamination of leafy green vegetables at primary production.

Cryptosporidium oocysts on fresh produce from areas of high livestock production in Poland.

Samples of fresh vegetables and soft fruit were collected from farmers' markets in the Lublin Area of Poland during 2006-2007; the produce was grown in areas of high to moderate livestock production. Cryptosporidium sp. oocysts were eluted from food surfaces, separated from residual food materials by IMS and identified by immunofluorescence and Nomarski differential interference contrast microscopy. Cryptosporidium sp. oocysts were detected in 6 of 128 vegetable samples (range 1-47 oocysts), but not in any of 35 fruit samples. Both empty and intact oocysts were detected. Species identity of oocyst-positive samples was performed by molecular analysis at four genetic loci. One of two 18S rRNA loci amplified DNA from 5 of the 6 oocyst-positive samples, but insufficient DNA for RFLP or sequencing analysis was available from 4 of these samples. An oocyst-positive celery sample generated an RFLP pattern consistent with C. parvum at two loci, but insufficient DNA was available for subtyping (GP60 sequencing) this isolate. Oocyst-contaminated foods originated from districts with the highest numbers of homesteads possessing cattle herds and no contaminated produce was detected from districts containing lower numbers of cattle-owning homesteads, strengthening the assumption that the origin of the contamination was livestock. The results of this study strengthen the evidence for the potential for zoonotic foodborne transmission of Cryptosporidium.