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Pig’s blood and liver are valuable edible slaughter by-products which are also the major ingredients of offal-derived foodstuffs. The aim of the study was an evaluation of the occurrence of hepatitis E virus (HEV) and porcine adenovirus (pAdV) as an index virus of faecal contamination in pig’s blood and liver for human consumption. In total, 246 samples of retail liver (n = 100) and pooled pig’s blood (n = 146) were analysed for the presence of HEV and pAdV. Blood samples were individually collected from 1432 pigs at slaughter age. Viral genomic material, including RNA of a sample process control virus was isolated from food samples using a QIAamp® Viral RNA Mini Kit. Virus-specific IAC-controlled real-time PCR methods were used for detection of target viruses. HEV RNA was found in 6 (2.4%; 95% CI: 0.9–5.2) out of 246 samples of tested foodstuffs. The virus was detected in pig’s blood (3.4%; 95% CI: 1.1–7.8) and liver (1.0%; 95% CI: 0.0–5.0) with no significant differences observed in the frequency of its occurrence between the two by-products (t = 1.33; p = 0.182 > 0.05); however PAdV was detected more frequently in pig’s blood than in liver (t = 4.65; p = 0.000 F = 0.81, p = 0.447 > 0.05 for HEV; F = 0.42, p = 0.655 > 0.05 for pAdV). Although HEV was detected in pig’s offal only sporadically, consumers cannot treat its occurrence with disregard as it demonstrates that HEV-contaminated pig tissues can enter the food chain.

Introduction The aim of the study was to estimate the diagnostic sensitivity (DSe) and specificity (DSp) of an agar gel immunodiffusion (AGID) assay for detection of myxoma virus (MYXV) in the classical form of myxomatosis and to compare its diagnostic performance to that of molecular methods (IAC-PCR, OIE PCR, and OIE real-time PCR). Material and Methods A panel of MYXV-positive samples of tissue homogenates with low (1 PCR unit – PCRU) and high (3,125 PCRU) virus levels and outbreak samples were used for method comparison studies. The validation parameters of the AGID assay were assessed using statistical methods. Results The AGID attained DSe of 0.65 (CI95%: 0.53–0.76), DSp of 1.00 (CI95%: 0.40–1.00), and accuracy of 0.67 (CI95%: 0.55–0.76). The assay confirmed its diagnostic usefulness primarily for testing samples containing ≥3,125 PCRU of MYXV DNA. However, in the assaying of samples containing Conclusions Findings from this study suggest that AGID can be used with some limitations as a screening tool for detection of MYXV infections.

Purpose Little is known about cryptosporidiosis in turtles of invasive alien species (IAS) inhabiting European bodies of fresh water. In this article, we report an occurrence of Cryptosporidium parvum in a red-eared slider turtle (Trachemys scripta elegans) captured in a rural aquatic ecosystem in eastern Poland. Methods A pair of samples consisting of feces and scrapings of intestinal mucosa (taken during necropsy) were collected from 104 animals representing the four IAS turtle species red-eared slider, yellow-bellied slider, false map and Cumberland slider. The animals were trapped in running and standing freshwater ecosystems across the Lublin province. Parasite genomic DNA was extracted from samples using a modified alkali wash and a heat-lysis method and identification of the Cryptosporidium species was performed at the 18SSU rRNA and COWP loci. Results The presence of Cryptosporidium DNA was only detected in one sample of intestinal scraping collected from a red-eared slider. A phylogenetic analysis of a 18SSU rRNA gene fragment showed 100% sequence identity between the C. parvum strain isolated from the turtle and other C. parvum strains previously detected in cattle from the Lublin province. Conclusions There was no clinical evidence that the red-eared slider turtle was truly infected rather than being merely a mechanical parasite carrier. Sporadic detection of this protozoan parasite in the studied population of IAS turtles could be associated with low natural occurrence of Cryptosporidium infections in this animal species. The results provide evidence for possible transmission of zoonotic Cryptosporidium species by IAS turtles.

European brown hare syndrome (EBHS) is lethal to several species of free-living hares worldwide. The genetic characterization of its virus (EBHSV) strains in European circulation and epidemiological knowledge of EBHSV infections is not yet complete. The study determined the nucleotide sequences of the genomes of EBHSV strains from Poland and analyzed their genetic and phylogenetic relationships to a group of hare lagoviruses. The genome of five virus strains detected in Poland between 1992 and 2004 was obtained by RT-PCR and sequencing of the obtained amplicons. The genetic relationships of the EBHSV strains were analyzed using the full genome and VP60 gene sequences. Additionally, the amino acid sequence of the VP60 gene was analyzed to identify mutations specific to recognized EBHSV subgroups. Partial amplification of the virus open reading frame (ORF)1 and ORF2 regions obtained nearly complete nucleotide genome sequences of the EBHSV strains. Phylogenetic analysis placed them in a GII.1 cluster with other European strains related to nonpathogenic hare caliciviruses. VP60 gene analysis allocated these EBHSV strains to the G1.2, G2.2–2.3 or G3 virus genetic groups. The amino acid sequence differences in the entire genome ranged from 1.1 to 2.6%. Compared to a reference French EBHSV-GD strain, 22 variable amino acid sites were identified in the VP60 region of the Polish strains, but only six were in VP10. Single amino acid changes appeared in different sequence positions among Polish and other European virus strains from different genetic groups, as well as in VP10 sequences of nonpathogenic hare caliciviruses. The results of the study showed a high genetic homogeneity of EBHSV strains from Poland despite their different location occurrence and initial detection times. These strains are also phylogenetically closely related to other EBHSV strains circulating in Europe, likely confirming the slow evolutionary dynamics of this lagovirus species.

Growing demand for fresh, unprocessed food favours the emergence of Cryptosporidium infections in humans. Mainly it is food of plant origin or unpasteurized milk which have been involved in food-borne outbreaks of cryptosporidiosis. So far consumption of shellfish contaminated with Cryptosporidium were not associated with human infections although such as possibility exists. In this study an attempt was undertaken to evaluate the analytical performance of three commonly used methods for recovery of Cryptosporidium oocysts from shellfish: i) pepsin digestion of shellfish in conjunction with immunomagnetic separation (IMS) of oocysts (method A), ii) pepsin-HCl treatment of shellfish homogenate without IMS (method B), and iii) a strainer method with direct oocyst extraction and separation from shellfish tissue using IMS (method C). Each method's performance was assessed according to the ISO standard requirements by testing shellfish homogenates seeded with different numbers of C. parvum oocysts. Two groups of parameters were compared, encompassing precision (coefficient of variation (CV)) and accuracy of measurements. These were described by linear regression models allowing calculation of the methods' limits of detection (LOD) and quantification (LOQ). In addition, oocyst recovery efficiencies from shellfish were calculated for each method. All three compared methods allowed for at least 66% recovery of Cryptosporidium oocysts from the tested samples. The best recovery (83.3–100%) in the whole range of tested suspensions was obtained for method C. The accuracy of method B was better (linearity of r2 = 0.9996 in the full measurement range) than that of method A (r2 = 0.968). Method C showed the best accuracy (r2 = 1) and precision (CV 0.2–14.1). Compared to other methods it was also characterised by the best LOD and LOQ, attaining ≅4 and ≅12 oocysts per 3 g of tested shellfish sample respectively. Despite a lack of the ability of method A to give the proportional results in oocysts recovery (non-linearity of the method) compared to the reference values, it achieved the highest LOD and LOQ values among the tested methods. As demonstrated here, the most efficient method for extraction of Cryptosporidium oocysts from shellfish tissues was method C employing sample homogenisation and separation of oocysts from tissue debris using IMS. Used alone this method does not in fact allow for identification of Cryptosporidium species but delivers quantitative results concerning the level of food contamination by parasites.

Rotaviruses of group A (RVAs) commonly occur in farm animals. In pigs, they cause acute gastrointestinal disease which is considered as significant factor of economic losses in pig farming. The aim of the study was an assessment of the prevalence of rotavirus (RV) infections in farmed pigs in Poland, genotype identification of the virus strains in conjunction with their age-related occurrence and regional (province) distribution pattern in pig herds. In total, 920 pig faecal samples were collected from pigs between the ages of one week and two years old from 131 farms. RVAs were detected using ELISA and molecular methods followed by a sequence-based identification of G (VP7) and P (VP4) virus genotypes. RV antigen was found in 377 (41%) of pig faecal samples. The correlation between pig age and frequency of RV infections was shown. In the Polish pig population, 145 RVA strains representing 33 GP genotypes were identified. Subsequent molecular analysis revealed an age-dependent and regional diversity in distribution of genotypes and virus strains. Besides typical pig RVA strains, novel strains such as G5P [34], G9P[34], and human G1P[8] were identified in this animal host. Findings from this study showed a change over time in the genotype occurrence of circulating pig RVAs in Poland. The high genetic variability of RV strains and acquisition of new virus genotypes have led to the emergence of novel, genetically distinct RVAs. The changes in the genotype occurrence of RVA strains in pigs indicate the need for their continuous epidemiological surveillance.

The red-eared slider (RES) ranavirus (RESRV) was isolated from a free-ranging RES turtle that died with evidence of respiratory disease. The RESRV genome sequence (106,878 bp) was determined, and phylogenetic analysis revealed that it is a common midwife toad virus (CMTV) strain. This study is the first report of CMTV in RES.

The most important wildlife species in the epidemiology of hepatitis E virus (HEV) infections are wild boars, which are also the main reservoir of the virus in a sylvatic environment. The aim of the study was a serological and molecular assessment of the prevalence of HEV infections in wild boars in Poland. In total, 470 pairs of samples (wild boar blood and livers) and 433 samples of faeces were tested. An ELISA (ID.vet, France) was used for serological analysis. For the detection of HEV RNA, real-time (RT)-qPCR was employed. The presence of specific anti-HEV IgG antibodies was found in 232 (49.4%; 95%CI: 44.7–54%) sera, with regional differences observed in the seroprevalence of infections. HEV RNA was detected in 57 (12.1%, 95%CI: 9.3–15.4%) livers and in 27 (6.2%, 95%CI: 4.1–8.9%) faecal samples, with the viral load ranging from 1.4 to 1.7 × 1011 G.C./g and 38 to 9.3 × 107 G.C./mL, respectively. A correlation between serological and molecular results of testing of wild boars infected with HEV was shown. HEV infections in wild boars appeared to be common in Poland.

HEV infections in animals are mainly diagnosed based on the presence of anti-HEV antibodies in sera using enzyme-linked immunosorbent assays (ELISA). In fact, commercial HEV ELISA kits are only available for selected animal species excluding rabbits. The demonstration of cross-reactivity of rabbit anti-HEV antibodies with human HEV strains makes possible diagnostic use of (human) ELISA kits for detection of specific anti-HEV antibodies in rabbit sera. The aim of the study was the application of ELISA recomWell HEV IgG (human) for identification of HEV seropositive rabbits at the time of slaughter. Native recombinant protein A was used as broadly reactive conjugate characterized by cross-species reactivity with IgG antibodies of human and other animal species. Several dilutions of protein A were employed and subsequently tested against the panel of control sera (positive, negative, and borderline) to determine its optimal concentration. Statistical analysis of OD values and their corresponding antibody titers obtained for each group of tested sera and protein A showed the most consistent performances at 1:40,000 dilution when compared to anti-human IgG (control) conjugate. Finally, the correct performance of the optimized test was verified using rabbit serum samples containing (or not) specific anti-HEV antibodies. As demonstrated here, HEV IgG (human) ELISA employing ORF2 antigens of human gt1 and gt3 HEV strains after modification and subsequent optimization can be used for the detection of anti-HEV antibodies in rabbit sera.

The aim of this study was molecular identification of Cryptosporidium species and assessment of their prevalence in different breeds of sheep and goat reared in Poland. In addition, the relationship between animal age, breed type, and the frequency of Cryptosporidium infections was determined. Fecal samples from 234 lambs and 105 goat kids aged up to 9 weeks, representing 24 breeds and their cross-breeds were collected from 71 small ruminant farms across Poland. The identification of Cryptosporidium species was performed at the 18 SSU ribosomal RNA (rRNA) and COWP loci followed by subtyping of C. parvum and C. hominis strains at GP60 gene locus. The presence of Cryptosporidium DNA at the 18 SSU rRNA locus was detected in 45/234 (19.2%) lamb feces samples and in 39/105 (37.1%) taken from goats. The following Cryptosporidium species: C. xiaoi, C. bovis, C. ubiquitum, C. parvum, and C. hominis were detected in small ruminants. Infections caused by C. xiaoi were predominant without favoring any tested animal species. Subsequent GP60 subtyping revealed the presence of C. parvum IIaA17G1R1 subtype in sheep and IIdA23G1 subtype in goats. IIdA23G1 subtype was detected in a goat host for the first time. There were no significant differences found in frequency of infections between the age groups ( 0.05) or goat kids (P = 0.06, α > 0.05). In addition, there was no correlation observed between the frequency in occurrence of particular parasite species and breed type in relation to native sheep breeds (F = 0.11; P = 0.990 > 0.05). In the case of goats, more breed-related differences in parasite occurrence were found. The results of this study improve our knowledge on the breed-related occurrence of Cryptosporidium infections in the population of small ruminants reared in Poland.

Hepatitis E virus (HEV) is a hepatovirus causing infections in humans and in many animal species. According to the current knowledge, HEV strains have been classified in the genus Orthohepevirus, family Hepeviridae, which encompasses strains belonging to one of seven virus genotypes. Genotypes 1 and 2 have only been found in humans, while genotypes 3 and 4 have been detected in humans, pigs, deer, rabbits and mongoose. The other HEV genotypes infect wild animals. However, the full spectrum of animal species being the natural reservoir of HEV has not been fully recognized. The clinical course of hepatitis in animals is asymptomatic, and infections do not cause significant losses in animal farming. Unlike in animals, infections in humans, and especially in pregnant women, can cause serious health problems. The identification of new virus strains in the animal reservoir and the possibility of transmission of some animal HEV strains to humans make the issue of public health protection and food safety even more important. This article provides an overview of data on the prevalence of HEV infections in animals and their impact on human and animal health

Cryptosporidium parvum is a zoonotic protozoan parasite occurring in a wide range of hosts. Invasions caused by this parasite have been reported in humans and in many animal species including birds. Despite its worldwide prevalence, infections have usually generated considerable losses in the livestock industry, mostly affecting calves, lambs and goat kids. It has previously been shown that ruminants are a major reservoir of zoonotic Cryptosporidium parvum and contact with an infected animal can lead to human infection. The application of molecular methods for parasitological diagnostics has increased our knowledge on the parasite hosts and its prevalence in humans and animals. They also confirmed their usefulness during epidemiological investigations and in surveillance studies of human and animal cryptosporidiosis. In this review the current state of knowledge concerning the importance of Cryptosporidium parvum invasions in farm and wild animals was presented.

Various PCR-based assays for rabbit viruses have gradually replaced traditional virologic assays, such as virus isolation, because they offer high-throughput analysis, better test sensitivity and specificity, and allow vaccine and wild-type virus strains to be fully typed and differentiated. In addition, PCR is irreplaceable in the detection of uncultivable or fastidious rabbit pathogens or those occurring in low quantity in a tested sample. We provide herein an overview of the current state of the art in the molecular detection of lagomorph viral pathogens along with details of their targeted gene or nucleic acid sequence and recommendations for their application. Apart from the nucleic acids–based methods used for identification and comprehensive typing of rabbit viruses, novel methods such as microarray, next-generation sequencing, and mass spectrometry (MALDI-TOF MS) could also be employed given that they offer greater throughput in sample screening for viral pathogens. Molecular methods should be provided with an appropriate set of controls, including an internal amplification control, to confirm the validity of the results obtained.

Hepatitis E virus (HEV) infects humans and many animal species. The rabbit HEV has been found in farmed, wild and pet rabbits as well as in human patients suggesting zoonotic transmission. Although the routes of human infection with rabbit strains are unclear a foodborne transmission is suggested especially when asymptomatically infected animals could enter the food chain. The aims of the study were an evaluation of the prevalence of HEV infections in slaughtered rabbits, identification of the virus genotype(s) and assessment of their genetic relatedness to other zoonotic HEV strains. A pair of blood and liver samples (n = 482) were collected from meat rabbits of different breeds slaughtered at the age of 2.8 to 6 months. The animals originated from 20 small-scale and 4 large-scale commercial farms operating in Poland. The presence of anti-HEV antibodies in animals was detected by the use of a recomWell HEV IgG (human) ELISA kit (Mikrogen Diagnostik) adapted to rabbit sera. The isolation of HEV and sample process control virus (feline calicivirus) RNA from homogenates of liver destined for food and virus-positive sera was performed using a QIAamp® Viral RNA Mini Kit (Qiagen). A one-step real-time reverse transcription PCR method containing a target-specific internal amplification control was used for detection of HEV. The (sub)genotype of detected rabbit HEV strains was identified based on sequence analysis of the ORF2 and ORF2/3 virus genome fragments. Anti-HEV antibodies were detected in 29 (6%) out of 482 rabbit sera samples collected from animals raised only on the small-scale rabbit farms. Four sera were also positive for HEV RNA. Viral RNA was detected in 72 (14.9%) animal livers. Analysing ELISA and PCR results using Student's t-test, there were significant differences observed in the frequency of HEV infections between rabbits from small-scale and commercial farms (t = 2.675, p = 0.015 0.05 for ELISA and t = 2.705, p = 0.014 0.05 for PCR). All detected virus strains were identified as HEV gt3 ra subtype. The results of this study provide data on the occurrence of HEV infections in rabbits entering the food chain, suggesting that a risk of foodborne HEV infection due to consumption of contaminated meat and liver exists. In this light, the presence of rabbit HEV in food animals is pertinent as an issue of food safety and the surveillance of these animals for emerging or re-emerging viruses.

In the recent years, a revival in interest in snail meat consumption has promoted a rapid development of snail farming systems to cover an increasing consumer demand and to protect the free-living snail population. Snail meat, like other types of food intended for human consumption, should be free from any microbiological hazards before being placed on the market. Snail meat may contain bacteria, but little is known about the possibility of contamination with human enteric viruses. Therefore, to test snail tissues for viruses, a suitable virus detection method is needed. Up to now, many methods for virus extraction and concentration from molluscan shellfish have been published, but only one has been adopted as an international standard procedure. In this study, a refinement of an ISO method (ISO/TS 15216–2:2013) was conducted to make it suitable for processing of edible snail tissues. The digestive glands from three edible snail species (Helix pomatia, Cornu aspersum maxima, Cornu aspersum aspersum) were used as samples, and method performance was assessed on the basis of murine norovirus RNA detection in spiked samples using a one-step real-time reverse transcription-PCR. The mean percentage of virus recovery from tissues of C. a. maxima was 5.71 %, 0.31 % for C. a. aspersum and 1.28 % for edible snail. As demonstrated here, this method was successfully applied for virus detection in snail samples, and its use can be recommended in monitoring of snails for virus contamination.

Invasiveness of alien turtles results from their impact on the functioning of the local ecosystem. It is due to predation on or competing with resident species, but also transfer of new and unknown pathogenic bacteria, viruses, parasites, or fungi. Salmonella is the most often reported microorganism, both in free-living and captive turtles. Zoonotic aspect of Salmonella spp. carriage has led to the definition of RAS (Reptile Associated Salmonellosis) acquired from domestic pet reptiles. Mycobacterium spp., Leptospira spp. and aquatic bacteria are also found in turtles. Additionally, nematode transmissions from invasive turtles to the autochthonic ones have been described. Alien turtles were less affected by parasitic invasion than animals living in a native location, but the infestation of alien parasites in native turtle species was usually more severe. Reports on viral or fungal infections in turtles are scarce. The identified knowledge gaps justify the need for research which will provide basic and systematic data on microbial threats related to alien and invasive turtles present in the natural environment of Poland. It will also give more insight in the scope and the impact of the problem on epidemiology and public health. 1. Introduction. 2. Invasive turtle species. 3. Bacteria. 3.1. Salmonella spp. 3.1.1. Reptile Associated Salmonellosis (RAS). 3.2. Mycobacteria. 3.3. Other bacteria. 4. Parasitofauna of turtles. 4.1. Invasive turtles as a source of helminth invasion of European pond turtle. 4.2. Occurrence and invasiveness of alien parasites in native and alien turtles. 5. Viral infections. 6. Mycotic infection. 7. Conclusions 1. Wstęp. 2. Inwazyjne gatunki żółwi. 3. Bakterie. 3.1. Salmonella spp. 3.1.1. Reptile Associated Salmonellosis (RAS). 3.2. Prątki. 3.3. Inne bakterie. 4. Parazytofauna żółwi. 4.1. Żółwie inwazyjne źródłem zarażenia helmintami żółwia błotnego. 4.2. Występowanie i patogenność obcych pasożytów u natywnych i obcych gatunków żółwi. 5. Infekcje wirusowe. 6. Infekcje grzybicze. 7. Podsumowanie

Recognition of myxomatosis is usually based on clinical symptoms, but amyxomatous cases of the disease require the use of laboratory methods. Nowadays PCR assays are routinely employed for detection of MYXV DNA, but none of them have had their diagnostic usefulness conclusively confirmed through validation. The aim of the study was the development and validation of a PCR with an internal amplification control (IAC) for intravital and postmortem detection of viral DNA of myxoma virus. To avoid false negative results a chimeric internal amplification control (IAC) was prepared and incorporated into the PCR reaction and amplified by the same primer set as the target DNA (M071 L). The optimal concentration of particular ingredients in the PCR mixture (including IAC concentration and volume of DNA sample) was determined. To minimize the risk of amplicon carry-over contamination, uracil N-glycosylase was added to the reaction. Before proper validation the robustness of the IAC-PCR was verified. Validation of the method encompassed the following parameters: the analytical and diagnostic specificity (ASp, DSp) and sensitivity (ASe, DSe) of the assay, repeatability, and intra-laboratory reproducibility. The assay LOD was established at 2 TCIU of the virus particles/0.2 ml tissue homogenate with a 100% capacity to detect different MYXV strains (ASp). The method was characterized by good DSp of 0.955 (0.839-0.999 CI) and DSe of 0.976 (0.914-1.00 CI). In addition, it was repeatable and reproducible and confirmed its suitability for the detection of MYXV in clinical material. The IAC-PCR developed meets OIE validation requirements for virological methods and can be used in diagnostic or epidemiological studies of rabbit myxomatosis.

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.

Numerous viruses of human or animal origin can spread in the environment and infect people via water and food, mostly through ingestion and occasionally through skin contact. These viruses are released into the environment by various routes including water run-offs and aerosols. Furthermore, zoonotic viruses may infect humans exposed to contaminated surface waters. Foodstuffs of animal origin can be contaminated, and their consumption may cause human infection if the viruses are not inactivated during food processing. Molecular epidemiology and surveillance of environmental samples are necessary to elucidate the public health hazards associated with exposure to environmental viruses. Whereas monitoring of viral nucleic acids by PCR methods is relatively straightforward and well documented, detection of infectious virus particles is technically more demanding and not always possible (e.g. human norovirus or hepatitis E virus). The human pathogenic viruses that are most relevant in this context are nonenveloped and belong to the families of the Caliciviridae, Adenoviridae, Hepeviridae, Picornaviridae and Reoviridae. Sampling methods and strategies, first-choice detection methods and evaluation criteria are reviewed., Virus hazards from food, water and the environment, their reservoirs and routes of transmission; Sampling methods and sampling strategies thereof, including the first choice test methods, and criteria for data evaluation are described.

The study was carried out in 2007, and its main aim was 1 year monitoring of surface water of the River Wieprz in Poland for the presence of human adenoviruses group F and noroviruses (NoVs). In total, 60 water samples were collected from four sampling sites situated along the river. The viruses were concentrated from water samples using glass wool, followed by elution with a glycine buffer containing skimmed milk powder. Subsequently, the viral nucleic acids were extracted and purified from water concentrates using a NucliSENS® kit and a QIAamp Viral RNA Mini Kit®. The presence of viral nucleic acids was confirmed by applying traditional PCR-based methods with incorporated internal amplification controls. Human pathogenic viruses were detected in 35% of analysed water samples. Adenoviruses were detected in 28.3% of analysed samples, and were present at all seasons of the year. 11.6% of the samples were positive for NoVs; they were present only during summer, in contrast to conventional findings. Molecular identification of norovirus strains revealed that they belong to genogroup I and II.

Myxomatosis is a highly infectious viral disease of rabbits. Myxoma virus (MYXV) is not pathogenic to other animal species except for rabbits and hares. In Poland, outbreaks of myxomatosis mainly occur on small-scale size rabbit farms in which specific immunoprophylaxis is not carried out. A typical nodular form of the disease is characterized by swelling and large myxomas over the whole body as well as by lung infection which is associated with atypical myxomatosis. However, differences in the virulence of MYXV strains are observed. They arise from a better adaptation of the virus to the rabbit host and they may also be related to the modulation of cellular response by infected animals. In fact, the changes observed in the MYXV genome do not result in the emergence of other novel virus strains than those currently circulating in the rabbit population. In addition, all previously detected strains are characterized by high phylogenetic relatedness..

The aim of this study was the application of an ultracentrifugation-based method for detection of feline calicivirus (FCV) in experimentally contaminated meat samples of roast pork chop, salami and gammon. Virus particles were liberated from food items by washing in 0.5-M glycine containing 1% bovine albumin. Food debris were removed by slow-speed centrifugation, and viruses were subsequently sedimented by ultracentrifugation. The recovery of infectious virus particles was determined by cell culture. Furthermore, RNA was extracted from virus particles and reverse-transcription polymerase chain reaction ((RT)PCR) was performed on the nucleic acid extracts. To demonstrate the efficiency of this method in removal of inhibitors from food sample extracts, an internal amplification control was constructed and incorporated into FCV (RT)PCR. Virus recovery for smoked meat, salami and cooked ham was 12.5%, 3.4% and 5.9%, respectively. The ultracentrifugation-based method is efficient in eliminating or reducing the level of inhibitory substances and can be applied for calicivirus extraction and concentration from delicatessen meat samples.

The aim of the study was to identify the Cryptosporidium parvum subtypes circulating in Polish cattle and their distribution in relation to the age and health status of tested animals. In total, 779 fecal samples were obtained from young cattle originating from 237 farms. C. parvum strains were identified at the 18 small-subunit ribosomal RNA (SSU rRNA), COWP, and LIB13 loci and were subsequently analyzed by sequencing at the 60-kDa glycoprotein (GP60) locus for subtype determination. The presence of 71 C. parvum strains belonging to IIa, IId, or IIl subtype families was shown. The strains from the IIa allele family prevailed with IIaA17G1R1, IIaA17G2R1, and IIaA15G2R1 subtypes occurring frequently. Two novel subtypes IIaA10G1R1 and IIlA19R3 were detected for the first time in a bovine host. The highest C. parvum prevalence (22.5 %, confidence interval (CI) = 2.5 %) was observed among the youngest animals up to 2 weeks of age, followed by the prevalence among those aged 2 to 4 weeks (6.6 %, CI = 2.6 %) and then among older cattle (4.9 %, CI = 2.1). The occurrence of diarrhea in animals was associated with the presence of the IIaA16G1R1b subtype, while infections caused by IIaA15G2R1 strains were more likely to be asymptomatic. The geographical distribution of subtypes revealed that strains from the IIa subtype family were detected all over the country frequently compared to the IId and IIl subtypes, the sporadic appearances of which confirmed their endemic occurrence. Subtype analysis revealed the presence of zoonotic strains indicating cattle as a reservoir for human cryptosporidiosis.

The aim of the study was to define the occurrence of human noroviruses of genogroup I and II (NoV GI and NoV GII) and hepatitis A virus (HAV) in the Baltic Sea mussels. The shellfish samples were taken at the sampling sites located on the Polish coast. In total, 120 shellfish were tested as pooled samples using RT-PCR and hybridisation with virus specific probes. NoV GI was detected in 22 (18.3 %), NoV GII in 28 (23.3 %), and HAV in 9 (7.5 %) of the shellfish. The nucleotide sequence analysis of the detected NoV GII strains showed a 97.3–99.3 % similarity to GII.4 virus strain. This is the first report describing the NoV and HAV occurrence in wild Baltic mussels and their possible role as bioindicators of seawater contamination with human enteric viruses.

The qualitative performance characteristics of a qPCR-based method to detect human adenoviruses in raspberries were determined through a collaborative trial involving 11 European laboratories. The method incorporated a sample process control (murine norovirus) and an internal amplification control. Trial sensitivity or correct identification of 25-g raspberry samples artificially contaminated with between 5 × 102 and 5 × 104 PFU was 98.5%; the accordance and concordance were 97.0%. The positive predictive value was 94.2%. The trial specificity or percentage correct identification of non-artificially contaminated samples was 69.7%; the accordance was 80.0% and the concordance was 61.7%. The negative predictive value was 100%. Application of a method for the detection of human adenoviruses in food samples could be useful for routine monitoring for food safety management. It would help to determine if a route of contamination exists from human source to food supply chain which pathogenic viruses such as norovirus and hepatitis A virus could follow.

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.

Publisher Summary Routine assays for the detection of enteric viruses in clinical specimens cannot be directly applied for food sample analysis. Therefore many laboratories have developed methodologies that allow them to detect viruses in contaminated food items. Current methodologies used for the detection of foodborne viruses consist of two main stages: extraction and concentration of viruses from food samples and detection using molecular biology techniques. There are several basic methods employing different procedures that can be used for virus extraction and detection. Each one has advantages and drawbacks. Some methods are insensitive, while others do not have the specificity allowing identification of virus type or strain. There are several methodologies that combine extraction and detection methods for noroviruses in fresh produce. In most of these methods, after initial sample treatment, viruses are concentrated and then viral RNA is extracted and purified. Other methods utilize organic solvents, which first facilitate concentration of virus particles and then allow viral RNA isolation instead of whole-virus extraction. All these procedures reduce the large volume of a food sample and allow the food extract or purified RNA solution to be used for further analysis.

The guarantee of food safety and quality along the food chain is a principal demand of the citizen. Fundamental aspects in food safety and quality, such as the presence of pathogens and their potential risk, are important consumer concerns. Other questions such as the use of genetically modified organisms (GMOs) in food production are also subjects where society demands total transparency. To guarantee food safety and quality and satisfy the requirements of the consumer, it is necessary to ensure that efficient analytical methodologies are possessed by the food industry. This review discusses current status and future challenges in two areas of analytical methodology in food safety and quality: the monitoring of microorganisms and the detection of GMOs. Current strategies for developing methods will be discussed. Lessons learnt through activities in method development and validation of leading research groups will be described. The review will also discuss the necessary requirements for the standardization of analytical tools in food safety monitoring.

A method was developed for detection of hepatitis A virus (HAV) in soft fruits (raspberries and strawberries). After washing the sample in 1 M sodium bicarbonate with added soya protein, fruits were removed by slow speed centrifugation, then particulate material and residual pectin were removed from the supernatant by flocculation and pectinase treatment during another slow speed centrifugation. Virus particles were then sedimented by ultracentrifugation. RNA was extracted from the virus particles, and nested RTPCR was performed on the nucleic acid extract. Nested RTPCR comprised an RTPCR, followed by PCR to amplify sequences within the amplicon. Internal amplification controls (IACs) were constructed for both the RTPCR and the PCR. The sensitivity of the nested RTPCR was approximately 10 RTPCRU. The overall method was shown to be able to detect 10(4) RTPCRU HAV in 90 g fresh strawberries, and 10(3) RTPCRU HAV in 60 g fresh raspberries. It is estimated that the lowest possible limit of detection of the method should be between 40 and 400 RTPCRU HAV per fruit sample. The method can be performed within one day, in suitably equipped microbiological laboratories, and is suitable for routine screening of food samples, and for analysis of suspected samples, e.g. during outbreak investigations.

Human enteric pathogenic viruses can enter the environment through discharge of waste materials from infected persons, and be transmitted back to susceptible persons to continue the cycle of disease. Contamination of food with viruses may also promote disease outbreaks. A number of studies have investigated the survival characteristics of several enteric viruses in various environments and foodstuffs, to help explain the transmissibility of these pathogens. This review deals with published work on enteric virus survival on fomites, and in waters, soil, and foods; the results of these studies have illustrated the robust survival of viruses in these environments. Much information is lacking, however, especially for foodstuffs and soils, and no detailed information is available concerning the survival of noroviruses, the most significant foodborne type.

Cryptosporidiosis is a disease reported in both humans and animals and is caused by a protozoan parasite Cryptosporidium. In most cases calves are infected by different Cryptosporidium species adapted to this animal host. Most infections are subclinical; however in some cases the watery diarrhea appears. The rapid development of new molecular diagnostic tools has provided an opportunity for better recognition of Cryptosporidium species and genotypes isolated from infected hosts. The aim of this study was an assessment of the prevalence of Cryptosporidium species in cattle herds in Poland. In total, 700 cattle fecal samples were tested. The examined cattle were at the age of 1 day to 6 years old. Overall 194 farms were monitored for parasite presence. Cryptosporidium detection in animal feces was performed using only molecular methods. Species identity of oocyst-positive samples were defined on the basis of PCR-RFLP and nucleotide sequence analysis of the amplified 18 SSU rRNA and COWP gene fragments. Cryptosporidium oocysts were detected in 119 (17%) of cattle feces. Most of the positive feces 55 (19.7%) were derived from young animals at the age of 1-4 weeks. The tested samples were positive for Cryptosporidium bovis, Cryptosporidium parvum, Cryptosporidium andersoni, and Cryptosporidium ryanae. C. parvum was not the most frequently detected parasite species, but calves below the age of 1 month were the major host for this parasite. The overall prevalence of C. parvum in Polish cattle herds was estimated at 5.1%. C. andersoni was the only species occurring in adult calves. The infected animals were housed in 67 (34.5%) of the monitored homesteads. Although C. bovis and C. ryanae were previously detected in cattle populations in many countries, this is the first report describing their presence in Polish cattle. Moreover, this is the first report on the prevalence of Cryptosporidium species in Polish cattle herds.