aem accepts, published online ahead of print on 16 march...
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Detection of Human Foodborne and Zoonotic Viruses on Irrigated, Field-Grown 4
Strawberries 5
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Julie Brassard,1* Marie-Josée Gagné,1 Mylène Généreux,2 and Caroline Côté2 7
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Agriculture and Agri-Food Canada, Food Research and Development Centre, 3600 Casavant 9
Boulevard West, Saint-Hyacinthe, Quebec, Canada J2S 8E31;Research and Development 10
Institute for the Agri-Environment, 3300 Sicotte Street, PO Box 480, Saint-Hyacinthe, Quebec, 11
Canada J2S 7B82 12
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* Corresponding author. Mailing address: Julie Brassard, Agriculture and Agri-Food Canada, 15
Food Research and Development Centre, 3600 Casavant Boulevard West, Saint-Hyacinthe, 16
Quebec, Canada J2S 8E3. Phone: 450-768-3233. Fax: 450-773-8461. E-mail: 17
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Running title: Pathogenic viruses on field-grown strawberries. 22
Keywords: Foodborne viruses; zoonotic viruses; strawberry; irrigation; field experiment 23
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00251-12 AEM Accepts, published online ahead of print on 16 March 2012
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This study evaluated the presence of pathogenic human and zoonotic viruses on 24
irrigated, field-grown strawberries. Norovirus genogroup I, rotavirus and swine hepatitis E virus 25
genogroup 3 were detected on strawberries and irrigation water is suspected as the 26
contamination origin. 27
28
Encouragement by public health authorities to adopt a healthy lifestyle has led to 29
increase consumer demand for fresh produce. A wide variety of fresh produce is now available 30
throughout the year in several industrialized countries, mainly because of market globalization. 31
However, vegetables and fruits, particularly berries, are increasingly associated with outbreaks 32
of foodborne illness in several parts of the world (4, 12, 21, 25) despite the perception that these 33
infections are usually related to products of animal origin (32). Produces consumed fresh or 34
after minimal processing are potential vehicles for enteric pathogens transmission. Some enteric 35
viruses, such as norovirus (NoV), rotavirus, adenovirus, astrovirus and hepatitis A virus, are 36
responsible for a large proportion of foodborne illness cases (28). In recent years, several studies 37
have focused on optimizing the concentration and detection of these viruses in fresh produce (6, 38
11, 13). In addition, many laboratory survival assays and epidemiological studies from 39
outbreaks have been conducted for certain pathogenic viruses (9, 10, 33). The resulting data can 40
now be used to provide tools to identify and, eventually, control potential sources of 41
contamination in the field. Potential sources of microbial contamination in the field include 42
irrigation water, soil, organic fertilizers and human handling (5). There is very little information 43
about the presence of viruses in irrigation water or about their persistence in the production 44
chain and on produce, although these viruses represent a large proportion of foodborne infection 45
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agents. The aim of this study was to evaluate the presence of pathogenic human and zoonotic 46
viruses on strawberries at the field scale after irrigation. 47
48
A field experiment was conducted in the Laurentides region of the province of Quebec, 49
Canada, in 2009. A split-plot factorial design was set including the method of irrigation (spray 50
and subsurface drip) as the main plot factor and the mulch (plastic or straw) as the subplot 51
factor. Plots were 7 m long and 8 m wide, and each treatment was repeated four times 52
(Supplementary Figure 1). Irrigation was performed on July 28 using water from the Chicot 53
River next to experimental site. Water samples (500 ml) were taken three times during irrigation 54
at the end of the drip line, at sprinklers, and in the river. Composite samples of 10 strawberries 55
were aseptically collected from each plot before irrigation, 1 h after irrigation, and on July 29 56
and on August 3. Fruits were cut into one-gram pieces. As a sample process controls, murine 57
calicivirus 1 (MNV-1) and feline calicivirus (FCV strain F9) were added to every water and 58
strawberry sample at concentrations of 2.5 x 102 PFU/g for MNV and 2.5 x 103 PFU/g for FCV 59
prior to extraction procedures. This provided additional quality control check throughout sample 60
processing. The procedure for virus elution and concentration from the strawberries was adapted 61
from Butot et al. (10). Briefly, 25 g of strawberries were inoculated with MNV and FCV and 62
incubated at room temperature for 30 min. Glycine-NaCl buffer (0.05 M, 0.14 M, pH 7.5) was 63
added with 200 µl pectinase, and the samples were gently shaken for 30 min. The elution buffer 64
was centrifuged at 5,000 x g for 5 min and filtered through a Whatman GD/X membrane, and 65
the filtrate was concentrated by centrifugation at 5,000 × g for 15 min on an ultrafiltration 66
device (Amicon Ultra-15, Millipore, Billerica, MA, USA). Plant RNA Isolation Aid (1:10) 67
(Applied Biosystems, Streetsvill, ON, Canada) was added to the concentrate before nucleic acid 68
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extraction. For water sample analysis, the OPFLP-04 standard method for the recovery and 69
concentration of viruses present in artificially and naturally contaminated water from Health 70
Canada’s compendium of analytical methods was used (7, 8). Negative controls for each viral 71
concentrated method from water and fruits (water, fruits and washing buffer) were included and 72
processed at the same time and tested by the detection systems. 73
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Viral RNA was extracted using the Rneasy Viral Mini Kit (Qiagen, Mississauga, ON, 75
Canada). In the first step, rotavirus, MNV, FCV, norovirus genogroup I (NoV GI) and 76
genogroup II (NoV GII), and hepatitis E virus (HEV) were detected using conventional reverse 77
transcription (RT)-PCR and nested RT-PCR assays according to previously described 78
procedures (1, 7, 14, 17, 22). All primers and probes used are shown in the Supplementary 79
Table 1. The RT-PCR amplicons were analyzed on 2% (w/v) agarose gels stained with ethidium 80
bromide. In the second step, all the positive samples obtained for HEV and rotavirus by 81
conventional RT-PCR detection were directly cloned and sequenced according to Ward et al. 82
(35) and positive samples for NoV were retested for confirmation using real-time RT-PCR 83
systems (18, 23). Nucleotide alignment was performed using the ClustalW program 84
(http://www.ebi.ac.uk/clustalw). Chi-square test was carried out for statistical analysis and the 85
level of significance was set to P-values < 0.05. 86
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On the 60 strawberry samples that were collected in this field experiment, 16 samples 88
were found positive for NoV GI, two for human rotaviruses and one for swine HEV by 89
molecular techniques (Table 1). No viruses were detected in irrigation water samples (n=9). 90
That absence could possibly be explained by a low concentration of the viruses in the water 91
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source. Spray and subsurface drip irrigation generated approximately 1400 L and 145 L of water 92
per plot respectively. If viral concentration in water is low but the amount of water received is 93
large, it may be possible that the fruits have been in contact with more viruses than what was 94
recovered in small sample. The use of a larger sample volume of water could eventually help 95
with the detection of any viruses that are potentially present. The acquisition of data on the 96
water quality status and viral contamination of the river during the weeks of growing could also 97
give indication on the possibility of viral pathogens transmission. 98
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Although no viruses could be detected in the irrigation water in this study, a significant 100
proportion (p-value < 0.05) of the fruit plots (7 out of 16, or 43%) were found positive for viral 101
contamination 1 h after irrigation only; that result contrasts with the single plot found positive 102
before irrigation. However, despite the fact that there were more contaminated plots under spray 103
irrigation 1 hour after irrigation, it was not possible to demonstrate a significant influence of 104
treatment (type of mulch and irrigation method) on strawberry viral contamination. Other 105
factors not measured in this study (soil, wind, wildlife, etc.) could have played a role in viruses 106
spreading in the plots. The fruits were not in contact with manures but only with mineral 107
fertilizers and aseptic measures (gloves, boots changes between plots, use of sterile disposable 108
material) were applied rigorously to prevent contamination during sampling. Although no direct 109
link between agricultural practices and viral contamination of small fruits was demonstrated, it 110
is undeniable that a certain proportion of the fruits were positive for viruses’ responsible of food 111
poisoning and this, directly in the field harvest time. 112
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The river used as a source of water is situated in agricultural and residential areas. This 114
could explain the presence of animal (HEV) and human (rotavirus and NoV) viruses on the 115
fruits. Human and swine Torque teno viruses have been found in 5% of fruit samples analyzed 116
(data not shown). Some enteric viruses, such as NoV and rotavirus, are able to withstand certain 117
treatments used for wastewater and their presence is frequently reported around the world in 118
surface water that contains such effluents (29). As a result, discharge from wastewater treatment 119
plants and septic tanks into environmental water can have an impact on the safety of agricultural 120
products, particularly in horticultural production, where surface water is frequently used for 121
irrigation. NoV GI, was detected at various concentrations between 3,0 x 103 and 5,0 x 101 122
particules/g (Ct values between 31 and 38) in 25% of strawberry samples analyzed in this study 123
which is consistent with other studies on fresh produces (leafy green and berries) as NoV, 124
especially GI, was found between 6.7 and 50% of analyzed samples (2). The authors also 125
suspected irrigation water as a potential source of contamination because NoV GI is frequently 126
reported in surface water, and appears to be more resistant in the environment than NoV GII (2, 127
24). NoV GI seems to be also linked to cases of recreational (contact with contaminated water) 128
or sporadic foodborne illness and is less often reported as a cause of foodborne illness outbreaks 129
affecting a large number of people. NoV GI seems to be transmitted more effectively via food 130
and the environment than by person-to-person contact. Involvement of NoV GI in cases of 131
foodborne gastroenteritis is probably underestimated because of the virus low contagiousness, 132
but its resistance in the environment makes it a pathogen that should be monitored, mainly in 133
horticultural and seafood products. 134
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For rotaviruses detected and sequenced in this study, both isolates showed an identity of 136
98% to 99%, suggesting that they belong to the same virus strain. That strain is a member of 137
rotavirus A G1P[8], the most common human rotavirus strain in Canada and the United States 138
(15, 30). Rotavirus is the first cause of young children’s hospitalization due to gastroenteritis in 139
industrialized countries and is responsible of hidden societal costs (19, 26). Despite the 140
introduction of vaccination against rotavirus, it is important to know which strains are in 141
circulation in the population and the environment because rotavirus is also widespread in wild 142
and domestic animal species, and it has been suggested that zoonotic transmission plays a 143
substantial role in the introduction of novel strains into the human population (3). In addition, it 144
seems that this transmission could be achieved via fresh products. 145
146
Sequencing determined that the HEV detected by nested RT-PCR 1 h after irrigation was 147
the swine HEV genotype 3 strain. The phylogenetic analysis of the amplified genomic fragment 148
presented in Figure 1 showed a nucleotide sequence identity of 99% with another swine HEV 149
strain detected on a swine farm in Quebec (35). That study was conducted between May 2003 150
and January 2004, and the identified strain was still in circulation at the time of the present 151
experiment in the summer of 2009. Discharge from farms into the environment and certain 152
agricultural practices such as manure spreading can also have an impact on the safety of 153
horticultural products (36). Because enteric and hepatic viruses are resistant in the environment, 154
they can be found in surrounding rivers following rain and runoff events and eventually, end up 155
on fresh produce via irrigation (34). The circulation of swine hepatitis E virus (HEV) genotype 3 156
on Quebec farms has already been demonstrated in a previous study. However, this is the first 157
time that swine HEV, considered a zoonotic virus primarily because of its transmission via 158
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contaminated pork (27), has been found on fruits. However, it is not possible to evaluate the 159
survival of the virus or its ability to infect the host, because HEV replication in the laboratory is 160
only moderately successful. The detection of HEV on produce nevertheless paves the way for 161
epidemiological investigations into other possible sources of contamination that could explain 162
the increase in autochthonous cases of hepatitis caused by HEV genotype 3 in non-endemic 163
countries (20). Recently in Quebec, a research team in gastroenterology demonstrated a high 164
prevalence of HEV found in paediatric organ recipients developing chronic hepatitis caused by 165
HEV genogroup 3 genetically related to animal strains. The authors also suggest a potential 166
zoonotic transmission of the virus via food in those immunocompromised children (16). It was 167
also reported that individuals with an impaired immune system, including children, the elderly, 168
pregnant women and people with HIV/AIDS are more susceptible to such infections even at low 169
levels of enteric viruses present in the environment or on food (31). 170
171
In conclusion, human and zoonotic pathogens, norovirus genogroup I, rotavirus and 172
swine hepatitis E virus genogroup 3 responsible for food poisoning and highly resistant in 173
environment, were detected in small proportion on strawberries in the field. In this study, it was 174
not possible to establish a clear link between irrigation water and the contamination origin. The 175
impact of other contamination factors such as agricultural practices and soil should be evaluated 176
in the future. 177
178
Nucleotides sequence accession numbers: The nucleotide sequences of HEV and rotavirus 179
determined in this study were deposited in GenBank under accession numbers HQ415969, 180
JF748713-JF748715. 181
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182
This research was supported by Agriculture and Agri-Food Canada Research Branch Peer 183
Reviewed Research Projects F.1401.EP, and by the “Programme de Solution à l’Innovation 184
Horticole” of the Québec Ministry of Agriculture and Fisheries and Food. 185
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Table 1: Molecular detection of foodborne viruses on strawberry samples before and after
irrigation.
nd: not detected ns: no sample
Plots
Viruses detected at different times before and after irrigation
Before
1 hour
1 day
6 days
1
nd
nd
nd
nd
2
nd
nd
nd
nd
3
nd
nd
nd
nd
4
nd
NoV GI
ns
NoV GI
5
nd
NoV GI
NoV GI Human RV
NoV GI
6
nd
nd
NoV GI
NoV GI
7
nd
NoV GI
ns
nd
8
nd
nd
NoV GI
nd
9
nd
NoV GI
nd
NoV GI
10
nd
nd
nd
NoV GI
11
nd
nd
nd
NoV GI
Human RV
12
nd
NoV GI
ns
nd
13
nd
Swine HEV
nd
nd
14
nd
nd
nd
nd
15
nd
nd
nd
nd
16
NoV GI
NoV GI
nd
nd
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