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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.

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 wild boar is the most important reservoir of zoonotic HEV-3 strains among different wildlife species. The aim of the study was subtype identification of wild boar HEV-3 strains circulating in Poland. Wild boar liver was used in the study in the form of homogenates prepared from 57 samples positive for HEV in a real-time RT-PCR. These samples were collected from juvenile and adult wild boars hunted in the jurisdictions of different Regional Directorates of State Forests (RDSF) across Poland. Subtype identification of detected HEV strains was based on a phylogenetic analysis of the most conserved HEV ORF2 genome fragment. Out of 57 tested samples, consensus HEV ORF2 sequences of 348 bp were obtained for 45 strains. Nineteen strains were identified and belonged to the HEV gt 3a and 3i subtypes, whereas 26 were not assigned to any virus subtype. HEV gt 3i strains prevailed in the Polish wild boar population, 16 of such were identified, and they were significantly more often observed in the RDSF Katowice area (χ2 = 28.6, p = 0.027 (2 = 48, p = 0.000 (

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.

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

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.

Rotavirus (RV) infections are a major epidemiological problem in humans and farm animals. So far, a number of human and animal RV strains have been identified. Based on the antigenic properties of the VP6 capsid protein, they have been classified into eight serogroups (A-H). The most important of them are viruses from group A (RVA), which are responsible for more than 90% of cases of rotaviral diarrhoea. The segmented structure of the virus genome and the presence of animals in human neighbourhood favour genetic reassortment between RV strains originating from different hosts. This could result in an emergence of zoonotic virus strains. The increasing number of human infections caused by virus strains having genotypes which have only been identified in animals indicates the need for epidemiological surveillance of infections. Additionally, the identification of epidemic virus strains in the outbreaks of disease in humans should be conducted. The identification of RVA strains circulating in humans and animals will allow the assessment of the impact of vaccination on the selection and emergence of zoonotic RVA strains. 1. Introduction. 2. General characteristics and classification of rotaviruses. 3. Group A rotavirus infection in humans. 4. Group A rotavirus infection in animals. 5. Genetic changes and reassortment as factors leading to the formation of zoonotic rotavirus strains. 6. Impact of human immunization on changes in genotype profile of circulating rotavirus strains. 7. Conclusions

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.