Introduction

Produce contamination with enteric pathogens, including viruses such as norovirus, is a major contributor to foodborne illness (Hall et al. 2014; Painter et al. 2013). Fecal bacteria and viruses, including coliphages, are used as indicators in food safety studies, as they have similar molecular and ecological characteristics to enteric pathogens and are relatively easier to detect (Allwood et al. 2004; Dawson et al. 2005; Endley et al. 2003; Espinosa et al. 2009; Love and Sobsey 2007; Smartt and Ripp 2011).

Although both F+ and somatic coliphages have been suggested as indicators of enteric viruses (Aw and Gin 2010; Mocé-Llivina et al. 2005; Wu et al. 2011), somatic coliphages (Garcia et al. 2015; Xu and Warriner 2005), compared to F+ coliphages (Allwood et al. 2004; Endley et al. 2003; Oron et al. 2001; Ravva et al. 2015; Shahrampour et al. 2015), have been less studied on produce in the agricultural environment. Few studies have attempted to quantify and compare the coliphage presence or concentrations among different types of fruits and vegetables, or various components of the farm environment. No studies have compared the coliphage presence or concentrations across the various production steps from farm to fork.

Increased understanding of coliphage ecology in the farm environment is needed to inform best practices to mitigate the risk of viral pathogen contamination of produce. Thus, the goal of the study was to compare amounts of somatic coliphages among fresh produce (cantaloupes, jalapeños, and tomatoes) and environmental samples (hand rinses, soil, source water, and irrigation) across the production steps on farms.

Materials and Methods

The study area included eleven farms and three packing facilities in Nuevo León and Coahuila México. Five farms produced cantaloupes (Cucumis melo var. cantalupensis), five produced jalapeño peppers (Capsicum annuum), and five produced tomatoes (Solanum lycopersicum) (four of which were also included as jalapeño farms). All participating farms used drip irrigation systems and well water to grow produce. Data on participating farm characteristics and food safety practices were collected via a farmer interview and observational survey collected by study staff from each farm (Heredia et al. 2016). Ethical approval for the study was conferred by Emory University (IRB00035460).

Samples were collected during May 2011–December 2012 as previously described (Fabiszewski de Aceituno et al. 2016; Heredia et al. 2015; Heredia et al. 2016). Samples of produce rinses were collected during various production steps on the farm: before harvest, during harvest, during transport away from the field, and at the packing facility, if present. For each sampling date and location on the farm, triplicate random subsamples were collected and composited. Produce was collected in Whirl-Pak bags (Nasco, Fort Atkinson, WI) previously filled with 0.15 % sterile peptone water (PW), shaken for 30 s, massaged for 30 s, and then shaken once more for 30 s. Composite produce samples represented 54 tomatoes, 42 jalapeños, or 6 cantaloupes in 1500 ml of PW. The numbers of tomatoes, jalapeños, and cantaloupes used in each rinse were so chosen to provide an equivalent surface area per sample across produce types (approximately 4500 cm2 of fruit/composite sample or 3 cm2 of fruit/ml PW).

Samples were also collected of matched hand rinses (3 workers’ hands/2250 ml PW) during the three latter production steps. Matched samples of soil (25 g/75–225 ml PW), and water- (4.5 L collected) from the source well pumps (source water) and from irrigation hoses in the field (irrigation water) were collected before harvest. Thus, on each sampling date, 8–10 samples were collected per farm (depending on whether a packing facility was present), and each farm was visited up to three times per growing season.

Somatic coliphages were quantified with FastPhage MPN Quanti-tray (Charm Sciences, Inc., Lawrence, MA). For each test, 100 ml sample was used except for highly turbid samples, for which 10 ml of sample was diluted with 90 ml PW. Quanti-trays were incubated for 6 h at 37 °C. On each day of sample collection, a negative control (PW) and a positive control (provided with FastPhage MPN), were processed. Sample data were not included if controls did not perform as anticipated. A sample was considered positive if fluorescence was observed (using an ultraviolet light). The most probable number (MPN) of coliphages was calculated using an IDEXX Quanti-Tray®/2000 MPN Table (IDEXX Laboratories, Westbrook, ME). The theoretical limits of detection and quantitation were 1–2419.6 MPN/100 ml.

Data from 459 samples were analyzed using Statistical Analysis Systems software 9.3. Concentration data were adjusted to MPN/fruit (produce), MPN/hand (hands), MPN/g (soil), and MPN/100 ml (water), and log10 transformed prior to analyses. Differences between produce types were tested by Pearson’s χ 2 test with Bonferonni adjustment (proportion of samples positive for coliphages) and the Kruskal–Wallis and Steel–Dwass All Pairs tests (mean rank coliphage concentrations). Logistic models identified differences in the proportion of produce and hand rinse samples that were positive for coliphages between production steps on produce farms, adjusted for year of sample collection; Firth corrections were employed when there were less than five positive or negative observations in any comparison group. Linear models identified differences in the concentrations of coliphages on produce and farm workers' hands between production steps in produce farms, adjusted for year of sample collection. P values ≤0.05 were considered significant for all statistical tests.

Results

The FastPhage MPN Quanti-tray method was relatively rapid, and required little reagent preparation time; however, the method required maintenance of viable positive control during shipping, and was sensitive to deviations in incubation time. Coliphages were detected on all sample types and during all production steps at concentrations ranging from the lower limit of detection to the upper limit of quantitation (Tables 1, 2, and 3; Fig. 1).

Table 1 Somatic coliphages on produce—percents of positive samples and concentrationsa from various production steps on farms
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Table 2 Somatic coliphages on hands—percents of positive samples and concentrationsa from various production steps on farms
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Table 3 Somatic coliphages on soil and water—percents of positive samples and concentrationsa before harvest on farms
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Fig. 1
figure 1

Somatic coliphage concentrations on produce and environmental samples collected from farms producing cantaloupe, jalapeño, and tomato. Boxes represent the 25–75 % interquartile range (IQR). Midlines represent the median. Whiskers extend from the median ± 1.58 × IQR. The superscripta indicates statistically significant difference compared to jalapeño; the superscript b indicates statistically significant difference compared to tomato (by Steel–Dwass multiple comparison, α = 0.05)

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Coliphages were found on 83 % of produce samples (range across produce types, 78–89 %) at concentrations ranging from <1 MPN/fruit to >4.78 log10 MPN/fruit (geometric mean range across produce types 0.91–3.64 log10 MPN/fruit) (Table 1; Fig. 1). When comparing the percent of samples that tested positive for coliphages among produce types, coliphage percentages were similar on produce from different produce farms (cantaloupe compared to jalapeño p = 0.38, cantaloupe compared to tomato p = 0.51, jalapeño compared to tomato p = 1.00). However, coliphage concentrations on produce from cantaloupe farms (geometric mean 3.64 log10 MPN/fruit) were significantly higher (p < 0.01) compared to both jalapeño (1.22 log10 MPN/fruit) and tomato (0.91 log10 MPN/fruit) farm samples.

When comparing across the various production steps, no significant differences in the percent of samples positive for coliphages were detected, except in the case of jalapeño (Table 1). Jalapeño was 50 % more likely to contain coliphages when sampled in the field before harvest than in the packing facility (p = 0.03). No significant differences in coliphage concentrations across production steps were detected except in the case of tomato. Geometric mean coliphage concentrations on packing shed tomato samples was 1.76 log10 MPN/fruit, 0.85–1.08 higher than samples from each of the preceding production steps (p value range <0.01–0.02) (Table 1). In summary, coliphage prevalence and concentrations were similar among produce from all production steps on farms except in two cases: jalapeño had higher coliphage percentages in the field than in the packing facility, and tomato had higher coliphage concentrations in the packing facility compared to all preceding production steps.

Coliphages were found on 65 % of hand samples at <0.57 to >3.96 log10 MPN/hand; (Table 2; Fig. 1). The percent of hand rinse samples that tested positive for coliphages were similar across farms producing different produce types (p = 1.00 for all comparisons). However, coliphage concentrations on hands from cantaloupe farms (geometric mean 3.01 log10 MPN/hand) were significantly higher (p < 0.01) compared to both jalapeño (1.62 log10 MPN/hand) and tomato (1.53 log10 MPN/hand) farm samples. When comparing across the various production steps, no significant differences were detected in the percent of hand rinse samples positive for coliphages (p value range 0.06–0.90) or in coliphage concentrations on hands (p value range 0.10–0.97).

Coliphages were found on 34 % of soil samples (at <1 MPN/g to >3.34 log10 MPN/g), 46 % of source water samples, and 45 % of irrigation water samples (at concentrations of <1 MPN/100 ml to >3.38 log10 MPN/100 ml) (Table 3, Fig. 1). Source water had no significant differences in coliphage percentage (p = 0.86–1.00) or concentrations (p = 0.83) among produce types. However, irrigation water from cantaloupe farms had significantly lower percentages of coliphages than those from tomato farms (p = 0.03), and soil from cantaloupe farms had significantly lower percentages of coliphages than those from jalapeño or tomato farms (both p < 0.01). Interestingly, coliphage concentrations were significantly higher (p < 0.01) on soil from cantaloupe farms (−0.18 log10 MPN/g) compared to tomato farms (−0.71 log10 MPN/g).

In summary, coliphages were prolific in the produce farm environment. Coliphage profiles of produce and environmental samples varied across produce types. In general, samples from cantaloupe farms seemed to exhibit significant differences in coliphage percentages and concentrations when compared to those of tomato and jalapeño farms. Coliphage profiles on produce also varied across production steps; jalapeños had more coliphages before harvest than during packing, while tomatoes had more coliphages at packing than all preceding production steps.

Conclusions

This study found that coliphages are prevalent in the pre-harvest and post-harvest farm environment on produce, hands, soil, and water. This enabled collection of the robust quantitative data needed to compare virus profiles across produce types and production steps. Somatic coliphages were included in this study as a proxy for viral enteric pathogens. It would be of interest in future studies to quantify male specific coliphage in addition, and to compare coliphage contamination to foodborne viral pathogens such as hepatitis and norovirus.

We found that the presence and concentrations of coliphages on produce and in the farm environment depend on produce types and production steps, similar to the relationships of produce type and production step with bacterial indicators (Heredia et al. 2016). These findings support the use of targeted produce-specific interventions at multiple points in the process of growing and packing produce to reduce the risk of enteric virus contamination and improve food safety during fruit and vegetable productions (U.S. FDA 2015).