Contamination of fresh produce sold on the Italian market with Cyclospora cayetanensis and Echinococcus multilocularis.

To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced 'ready-to-eat' (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination.

Detection and monitoring of Drosophila suzukii in raspberry and cherry orchards with volatile organic compounds in the USA and Europe.

Spotted wing drosophila (SWD) causes significant economic loss in fruit crops to growers worldwide. There is immediate need for efficacious and selective monitoring tools that can detect infestations early. Previously, volatile organic compounds derived from apple were studied and a quinary chemical component blend (QB) was identified as the key SWD attractant in a blueberry orchard in the United States. This study's aim was to determine whether previously observed QB efficacy, selectivity, and early detection levels could be attained within raspberry and cherry fields in the USA and Europe. Results demonstrated that sticky trap baited QB dispenser provided earlier SWD detection potential than the usually adopted apple cider vinegar (ACV) trap. The number of SWD captured/trap by QB baited trapping systems was significantly lower than that of the ACV trap. However, percent SWD/trap of QB baited traps was same within cherry. Lower non-target capture will save farmer/grower's labor and time allocated to traps installation and drosophila species identification. Within the USA, SWD selectivity of QB baited liquid traps was consistently greater than sticky trap in raspberry field, suggesting that the QB dispenser can be an alternative to the standard ACV lure and that trap design could improve selectivity further.

(7Z)-Tricosene Improves Pheromone Trap Catch of Raspberry Bud Moth, Heterocrossa rubophaga.

Heterocrossa rubophaga, the raspberry bud moth, is a pest of commercial Rubus berry crops and wild Rubus species. Its pheromone was initially identified as a single component, (7Z)-nonadecen-11-one. Here we report further investigation of this carposinid pheromone including volatile collection, gland extraction, mass spectrometry, microchemical analysis, electrophysiology and field testing. The alkene (7Z)-tricosene was identified from female gland extracts and the synthetic compound gave antennal responses from the male moth. Field testing of (7Z)-tricosene showed that it was unattractive alone but in combination with (7Z)-nonadecen-11-one it more than doubled male moth trap catch for all doses tested compared to that of (7Z)-nonadecen-11-one alone. We recommend a blend of 300 µg of (7Z)-nonadecen-11-one and 300 µg of (7Z)-tricosene as an improved monitoring tool for raspberry bud moth for use by berry growers. This is the second report of the Z-alkene as a pheromone component in the Carposinidae family among the three carposisnid pheromones identified to date.

Evaluation of the U.S. Food and Drug Administration validated molecular method for detection of Cyclospora cayetanensis oocysts on fresh and frozen berries.

Outbreaks and sporadic cases of Cyclospora cayetanensis have been linked to consumption of berries. The efficacy of the U.S. Food and Drug Administration (FDA) method for detection of C. cayetanensis was evaluated in fresh berries (blackberries, strawberries, blueberries and mixed berries) and in frozen mixed berries. The protocol included seeding with C. cayetanensis oocysts, produce washing, DNA extraction and a dual TaqMan assay. As few as five oocysts were detected in every type of fresh berry analyzed. All berry samples seeded with 200 oocysts were positive and all unseeded berry samples were negative. No significant differences were observed among any of the berry types analyzed in detection rates, CT values and estimated oocyst recovery percentages. Mixed berries were seeded and frozen for up to seven weeks. As few as five oocysts were also detected. No significant differences were observed in C. cayetanensis CT values between fresh and frozen mixed berries at any seeding level. In conclusion, the FDA BAM Chapter 19B method for the detection of Cyclospora was robust, consistent, and showed high sensitivity in all types of berries analyzed. Evaluation of the FDA detection method in berries will provide reliable laboratory support for surveillance programs and for outbreak investigations.

Yield Response to Orange Cane Blotch of Blackberry Grown in the Georgia Coastal Plain.

Orange cane blotch (OCB), an algal disease on commercial blackberry plants in the southeastern United States, has been an increasing concern among producers. The causal agent, Cephaleuros virescens, produces brightly colored green to orange lesions on blackberry stems, but proof of actual damage and impact on crop yield has not been documented. Naturally infected stem sections were viewed using transmission and scanning electron microscopy to evaluate cane damage. Surface abrasions, intercellular growth, and occasional intracellular growth were observed on the surface and epidermal layers. Field studies at four commercial sites over 2 years were conducted to assess the impact of OCB on yield in 'Ouachita' blackberry plants not treated with algicidal chemicals. Neither cane diameter nor berry size was impacted by severity of OCB; however, berry number decreased with increasing OCB intensity in a nonlinear manner, thereby resulting in reduced yields.

A novel multiplex real-time PCR for the detection of Echinococcus multilocularis, Toxoplasma gondii, and Cyclospora cayetanensis on berries.

Foodborne parasites (FBP) are of major public health importance and warrant appropriate detection and control strategies. Most of the FBP considered for risk-ranking by a panel of experts are potentially transmitted via consumption of contaminated fresh produce, including berries. In this study we focused on the potential of three FBP, namely Echinococcus multilocularis, Toxoplamsa gondii, and Cyclospora cayetanensis, as contaminants of berries. Surveys to assess these parasites as contaminants of fresh produce in general, and berries in particular, are scanty or non-existent mainly due to the lack of optimized laboratory methods for detection. The aim of the present study was to develop and evaluate a novel multiplex qPCR for the simultaneous detection of E. multilocularis, T. gondii, and C. cayetanensis from berry fruits. The efficiency and linearity of each channel in the multiplex qPCR were within the acceptable limits for the range of concentrations tested. Furthermore, the method was shown to have good repeatability (standard deviation ≤0.2 Cq) and intermediate precision (pooled standard deviation of 0.3-0.6 Cq). The limit of detection was estimated to 10 oocysts for Toxoplasma and Cyclospora, and 5 eggs for Echinococcus per 30 g of raspberries or blueberries. In conclusion, evaluation of the present method showed that the newly developed multiplex qPCR is highly specific, precise, and robust method that has potential for application in food-testing laboratories.

Interlaboratory validation of an improved method for detection of Cyclospora cayetanensis in produce using a real-time PCR assay.

A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are recovered from produce using an enhanced produce washing solution containing 0.1% Alconox and a commercially available method to disrupt the C. cayetanensis oocysts and extract DNA. A real-time PCR assay targeting the C. cayetanensis 18S rDNA gene with an internal amplification control to monitor PCR inhibition provides species-specific identification. Five laboratories blindly analyzed a total of 319 samples consisting of 25 g of cilantro or 50 g of raspberries which were either uninoculated or artificially contaminated with C. cayetanensis oocysts. Detection rates for cilantro inoculated with 200, 10, and 5 oocysts, were 100%, 80%, and 31%, respectively. For raspberries, the detection rates for samples inoculated with 200, 10, and 5 oocysts were 100%, 90% and 50%, respectively. All uninoculated samples, DNA blank extracts, and no-template PCR controls were negative. Reproducibility between laboratories and analysts was high and the method was shown to be an effective analytical tool for detection of C. cayetanensis in produce.

Improved capture of Drosophila suzukii by a trap baited with two attractants in the same device.

The improvement of trap-lure combinations is an important part of integrated pest management programs that involve monitoring pests for timely insecticide applications, or for their use in control strategies such as mass trapping or bait stations. In this study improvements in the capture of Drosophila suzukii were not observed following the inclusion of different color stimuli with respect to a red-black stripe cup trap. This red-black stripe trap with a hemispherical dome-shaped lid had a significantly improved physical retention of flies compared to traps fitted with a flat lid. Retention was further improved when an additional tube device, which could be baited with a supplemental attractant, was introduced through the dome-shaped lid. Under laboratory conditions, this trap, in which apple cider vinegar + 10% ethanol was present as the drowning solution and the additional tube device was baited with a fermenting mixture of sugar and yeast, was significantly more effective in catching D. suzukii flies than other conventional attractants or a commercial lure. The capture rate of this trap-lure combination remained higher than that of a commercial lure, even after 20 days of use under laboratory conditions. In a guava orchard this trap was 15-fold more effective in catching D. suzukii flies than similar traps baited with apple cider vinegar alone, 4 to 7 fold more effective than similar traps baited with a commercial lure, and 1.7-fold more effective than a fermenting mixture of yeasts and wheat flour. In commercial blackberry orchards, this trap was 6-fold more effective in trapping D. suzukii flies than the clear trap baited with apple cider vinegar used by growers. The efficacy of this trap presents a promising line of future research for monitoring and control of D. suzukii and likely other drosophilid pests.

Economic analysis of revenue losses and control costs associated with the spotted wing drosophila, Drosophila suzukii (Matsumura), in the California raspberry industry.

BACKGROUND: The spotted wing drosophila (SWD), Drosophila suzukii (Matsumura), is an invasive vinegar fly with a preference for infesting commercially viable berries and stone fruits. SWD infestations can reduce yields significantly, necessitating additional management activities. This analysis estimates economic losses in the California raspberry industry that have resulted from the SWD invasion. RESULTS: California raspberry producers experienced considerable revenue losses and management costs in the first years following SWD's invasion of North America. Conventional producers have since developed effective chemical management programs, virtually eliminating revenue losses due to SWD and reducing the cost of management to that of purchasing and applying insecticides more often. Organic raspberry producers, who do not have access to the same chemical controls, continue to confront substantial SWD-related revenue losses. These losses can be mitigated only by applying expensive insecticides registered for organic use and by performing labor-intensive field sanitation. CONCLUSION: SWD's invasion into North America has caused extensive crop losses to berry and cherry crops in California and elsewhere. Agricultural producers and researchers have responded quickly to this pest by developing management programs that significantly reduce revenue losses. Economic losses are expected to continue to fall as producers learn to manage SWD more efficiently and as new control tactics become available. © 2016 Society of Chemical Industry.

Simultaneous detection of the protozoan parasites Toxoplasma, Cryptosporidium and Giardia in food matrices and their persistence on basil leaves.

Toxoplasma gondii, Cryptosporidium spp. and Giardia intestinalis are emerging pathogen parasites in the food domain. However, without standardized methods for their detection in food matrices, parasitic foodborne outbreaks remain neglected. In this study, a new immunomagnetic separation assay (IMS Toxo) targeting the oocyst's wall of T. gondii was developed using a specific purified monoclonal antibody. Performance of this IMS Toxo coupled to microscopic and qPCR analyses was evaluated in terms of limit of detection (LOD) and recovery rate (RR) on: i) simple matrix (LOD = 5 oocysts; RR between 5 and 56%); ii) raspberries and basil (LOD = 33 oocysts/g; RR between 0.2 and 35%). Finally, to simultaneously extract the three protozoa from these food matrices, T. gondii oocysts were directly concentrated (without IMS Toxo) from the supernatant of the IMS of Cryptosporidium and Giardia (oo)cysts. This strategy associated to qPCR detection led to LOD <1 to 3 (oo)cysts/g and RR between 2 and 35%. This procedure was coupled to RT-qPCR analyses and showed that the three protozoa persisted on the leaves of basil and remained viable following storage at 4 °C for 8 days. These data strengthen the need to consider these protozoa in food safety.

Repellent Activity of the Essential Oil from the Heartwood of Pilgerodendron uviferum (D. Don) Florin against Aegorhinus superciliosus (Coleoptera: Curculionidae).

The weevil Aegorhinus superciliosus Guérin (Coleoptera: Curculionidae), which is endemic to Central-Southern Chile and Argentina, is one of the major berry pests in Chile and the most important pest in the La Araucanía Region (38°44'9″S, 72°35'25″W). Due to the poor effectiveness and problems surrounding the implementation of the traditional control methods using organophosphate and carbamate insecticides, new strategies for controlling this pest are needed. In this communication, we evaluated the behavioral responses of male and female A. superciliosus to volatile compounds released from the essential oil (EO) obtained from the heartwood of Pilgerodendron uviferum (D. Don) Florin using olfactometric bioassays. The composition of the EO was analyzed using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). According to these analyses, δ-cadinol (24.16%), cubenol (22.64%), 15-copaenol (15.46%) and δ-cadinene (10.81%) were the principal components of the EO. The Pilgerodendron uviferum EO, which is almost exclusively composed of sesquiterpenes (99.5%), exhibited a repellent effect against A. superciliosus adults, regardless of the sex or concentration used (56.6 mg/cm³ and 1.58 × 10(-2) mg/cm³). The EO has low volatility and greater persistence than the EOs composed of monoterpenes and is considered a good model in the search for raspberry weevil repellents.

Pilot-Scale Pulsed UV Light Irradiation of Experimentally Infected Raspberries Suppresses Cryptosporidium parvum Infectivity in Immunocompetent Suckling Mice.

Cryptosporidium spp., a significant cause of foodborne infection, have been shown to be resistant to most chemical food disinfectant agents and infective for weeks in irrigation waters and stored fresh vegetal produce. Pulsed UV light (PL) has the potential to inactivate Cryptosporidium spp. on surfaces of raw or minimally processed foods or both. The present study aimed to evaluate the efficacy of PL on viability and in vivo infectivity of Cryptosporidium parvum oocysts present on raspberries, a known source of transmission to humans of oocyst-forming apicomplexan pathogens. The skin of each of 20 raspberries was experimentally inoculated with five 10-µl spots of an oocyst suspension containing 6 × 10(7) oocysts per ml (Nouzilly isolate). Raspberries were irradiated by PL flashes (4 J/cm(2) of total fluence). This dose did not affect colorimetric or organoleptic characteristics of fruits. After immunomagnetic separation from raspberries, oocysts were bleached and administered orally to neonatal suckling mice. Seven days after infection, mice were euthanized, and the number of oocysts in the entire small intestine was individually assessed by immunofluorescence flow cytometry. Three of 12 and 12 of 12 inoculated mice that received 10 and 100 oocysts isolated from nonirradiated raspberries, respectively, were found infected. Four of 12 and 2 of 12 inoculated mice that received 10(3) and 10(4) oocysts from irradiated raspberries, respectively, were found infected. Oocyst counts were lower in animals inoculated with 10(3) and 10(4) oocysts from irradiated raspberries (92 ± 144 and 38 ± 82, respectively) than in animals infected with 100 oocysts from nonirradiated raspberries (35,785 ± 66,221, P = 0.008). PL irradiation achieved oocyst reductions of 2 and 3 log for an inoculum of 10(3) and 10(4) oocysts, respectively. The present pilot-scale evaluation suggests that PL is an effective mode of decontamination for raspberries and prompts further applicability studies in industrial contexts.

Phylogenetic analysis of the complete mitogenome sequence of the raspberry weevil, Aegorhinus superciliosus (Coleoptera: Curculionidae), supports monophyly of the tribe Aterpini.

The superfamily Curculionoidea is one of the most diverse groups of insects in the world, including many species which are crop pests. Within this group, the native raspberry weevil, Aegorhinus superciliosus (Guérin, 1830), is an important pest in blueberry and raspberry fields in southern South America. Using a 454 sequencing approach, we sequenced and annotated the mitogenome of A. superciliosus, it, providing the first such information for any species in the tribe Aterpini, subfamily Cyclominae. The assembled mitogenome is a circular DNA molecule 15,121bp in length containing all 37 genes normally found in metazoans. Mitogenome organization and transcriptional orientation in A. superciliosus showed the same pattern that characterizes the suborder Polyphaga. Bayesian and Maximum Likelihood phylogenetic analyses supported the monophyly of the tribe Aterpini and the subfamily Cyclominae, recovering this clade in a sister group relationship with Entiminae and Hyperinae. The monophyly of these three subfamilies defines a critical transition to an ectophagous lifestyle in the larvae, from an ancestrally endophagous larval lifestyle in all other lineages. The sequenced mitogenome of A. superciliosus can provide basic data for future studies investigating population history, molecular systematics, stress ecophysiology and phylogeography.

Seasonal phenology of Amphorophora agathonica (Hemiptera: Aphididae) and spread of viruses in red raspberry in Washington.

Amphorophora agathonica (Hottes) is the primary vector of aphid-transmitted viruses in red raspberry in the Pacific Northwest region of the United States. To better understand the biology of the aphid, we estimated the lower developmental threshold and studied the seasonal activity of A. agathonica in commercial fields in northern Washington state. In addition, we monitored the spread of raspberry viruses (raspberry latent virus and raspberry leaf mottle virus, RLMV) to determine how rapidly fields became infected and whether there was a relationship between aphid presence and infection. The lower developmental threshold of A. agathonica was estimated to be 2.7°C. In the field, apterous and alate aphid populations began rapidly increasing at ≍800 growing degree-days and peaked at 1,050 growing degree-days. RLMV spread rapidly, with 30-60% of plants in four different commercial fields testing positive after three growing seasons. There was no discernible relationship between the presence or abundance of aphids based on 10 leaves sampled per plant location, and the odds of that plant becoming infected with RLMV.