Quantification of viable protozoan parasites on leafy greens using molecular methods.

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.

Establishment of a Novel Molecular Detection Method for Sarcocystis in Venison.

Recently, horse meat (basashi) contaminated with Sarcocystis spp. caused food poisoning in Japan. An official detection method provided by the Ministry of Health, Labour and Welfare (MHLW), Japan, was designed to detect Sarcocystis fayeri to diagnose and control outbreaks of basashi food poisoning. In 2011, Sarcocystis-contaminated venison also caused food poisoning. However, the official MHLW detection method was not adequate for detecting Sarcocystis spp. in venison. In this study, we established a novel PCR-based detection method that amplifies 18S rRNA gene based on the conserved region of the sequence in 32 species of Sarcocystis for screening and quantification. Fifty venison samples from three areas in Hokkaido were examined by the MHLW method and the novel detection method. All samples were Sarcocystis spp.-positive. A sequence analysis indicated the presence of a species of Sarcocystis specific to sika deer (Cervus nippon), and not to horses. Another primer pair was designed for a quantitative real-time PCR assay to determine the copy number of the Sarcocystis-18S rRNA gene in parasitized venison. The melting curve analysis revealed high specificity of this assay. The calculated curve demonstrated that this quantitative PCR assay showed R2 value of 0.993 with 10-106 copies. Using this quantitative real-time PCR assay, the gene copy numbers were determined in 50 venison samples. The copy numbers of each sample ranged from 104 to 107 per gram. The copy numbers differed according to the area in Hokkaido. This indicates that the density of Sarcocystis spp. that infect Sika deer in Hokkaido is affected by the area. The novel screening and quantitative PCR method for Sarcocystis in venison was useful for collecting epidemiological information on Sarcocystis in wild Japanese sika deer, which will contribute to improve the safety of venison products in Japan.

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.

Simultaneous detection of four protozoan parasites on leafy greens using a novel multiplex PCR assay.

Pathogen contamination of fresh produce presents a health risk for consumers; however, the produce industry still lacks adequate tools for simultaneous detection of protozoan parasites. Here, a simple multiplex PCR (mPCR) assay was developed for detection of protozoan (oo)cysts and compared with previously published real-time PCR assays and microscopy methods. The assay was evaluated for simultaneous detection of Cryptosporidium, Giardia, Cyclospora cayetanensis, and Toxoplasma gondii followed by parasite differentiation via either a nested specific PCR or a restriction fragment length polymorphism (RFLP) assay. Spiking experiments using spinach as a model leafy green were performed for assay validation. Leaf-washing yielded higher recoveries and more consistent detection of parasites as compared with stomacher processing. Lowest limits of detection using the nested mPCR assay were 1-10 (oo)cysts/g spinach (in 10 g samples processed), and this method proved more sensitive than qPCR for parasite detection. Microscopy methods were more reliable for visual detection of parasites in lower spiking concentrations, but are more costly and laborious, require additional expertise, and lack molecular confirmation essential for accurate risk assessment. Overall, the nested mPCR assay provides a rapid (<24 h), inexpensive ($10 USD/sample), and simple approach for simultaneous detection of protozoan pathogens on fresh produce.

Evaluation of the U.S. Food and Drug Administration validated method for detection of Cyclospora cayetanensis in high-risk fresh produce matrices and a method modification for a prepared dish.

The performance of the U.S. Food and Drug Administration (FDA) validated method for regulatory detection of Cyclospora cayetanensis in leafy greens and berries was evaluated in additional high-risk fresh produce items and in a dish prepared with these produce commodities. The method was robust and reproducible in basil, parsley, shredded carrots, shredded cabbage and carrot mix, and could detect as few as 5 oocysts in 25 g samples. Some differences in C. cayetanensis detection were found among the fresh produce analyzed. Significantly lower target gene copy numbers per reaction were obtained with shredded carrots, and shredded cabbage and carrot mix compared to leafy greens, which highlights the importance of evaluating the performance characteristics of validated methods in different food matrices. In the prepared dish, coleslaw with dressing, the method was optimized to detect 5 oocysts in a 25 g sample by using 1.0% Alconox® in the washing solution instead of 0.1% as originally described. These data are important to assess the prevalence of C. cayetanensis in different produce items and to support outbreak investigations.

An overview of methods/techniques for the detection of Cryptosporidium in food samples.

Cryptosporidium is one of the most important parasitic protozoa of concern within the food production industry, worldwide. This review describes the evolution and its development, and it monitors the methodology that has been used for Cryptosporidium in food material since 1984, when the first publication appeared regarding the detection of Cryptosporidium parvum in food materials. The methods that are currently being used for the detection of Cryptosporidium oocysts in food material (mainly vegetables) and all of the other available published methods are discussed in this review. Generating more consistent and reliable data should lead to a better understanding of the occurrence, transport and fate of the oocysts in food material. Improvements in monitoring and developing effective methodology, along with food security, offer more practical possibilities for both the developed and developing worlds.

Use of Tunisian flavored olive oil as anisakicidal agent in industrial anchovy marinating process.

BACKGROUND: Natural compounds are more frequently used against Anisakis, responsible for the important fish-borne disease anisakidosis. The aim of the study was to evaluate the effectiveness of enriched Tunisian olive oil with different spices (cumin, turmeric, clove, thyme, and lemon) against Anisakis larvae type 1. RESULTS: In vitro experiment: larvae were submerged separately in the aforementioned oils and then examined to check viability. For each oil, LT50 and LT100 were calculated. Turmeric and cumin oils are the most effective against the parasites; followed by lemon, thyme and clove oils. For the in vivo experiment, turmeric and cumin oils were tested in anchovy fillets previously artificially parasitized with L3 larvae. Cumin was the most effective against parasites (dead after 5 days) compared with turmeric (8 days). For the two oils, the resulting odor was pleasant, as was the taste, while changes in color were much more evident in turmeric fillets. CONCLUSION: All the flavored oils demonstrated a good nematodical action against Anisakis. Cumin oil was the most effective against encysted larvae. Turmeric oil showed the best activity in the in vitro experiment. The use of flavored oils in the marinating process could represent an efficient strategy to devitalize Anisakis. © 2017 Society of Chemical Industry.

Human pathogens in plant biofilms: Formation, physiology, and detection.

Fresh produce, viewed as an essential part of a healthy life style is usually consumed in the form of raw or minimally processed fruits and vegetables, and is a potentially important source of food-borne human pathogenic bacteria and viruses. These are passed on to the consumer since the bacteria can form biofilms or otherwise populate plant tissues, thereby using plants as vectors to infect animal hosts. The life cycle of the bacteria in plants differs from those in animals or humans and results in altered physiochemical and biological properties (e.g., physiology, immunity, native microflora, physical barriers, mobility, and temperature). Mechanisms by which healthy plants may become contaminated by microorganisms, develop biofilms, and then pass on their pathogenic burden to people are explored in the context of hollow fiber microfiltration by which plant-derived microorganisms may be recovered and rapidly concentrated to facilitate study of their properties. Enzymes, when added to macerated plant tissues, hydrolyze or alter macromolecules that would otherwise foul hollow-fiber microfiltration membranes. Hence, microfiltration may be used to quickly increase the concentration of microorganisms to detectable levels. This review discusses microbial colonization of vegetables, formation and properties of biofilms, and how hollow fiber microfiltration may be used to concentrate microbial targets to detectable levels. The use of added enzymes helps to disintegrate biofilms and minimize hollow fiber membrane fouling, thereby providing a new tool for more time effectively elucidating mechanisms by which biofilms develop and plant tissue becomes contaminated with human pathogens. Biotechnol. Bioeng. 2017;114: 1403-1418. © 2017 Wiley Periodicals, Inc.

Non-Destructive and rapid evaluation of staple foods quality by using spectroscopic techniques: A review.

Staple foods, including cereals, legumes, and root/tuber crops, dominate the daily diet of humans by providing valuable proteins, starch, oils, minerals, and vitamins. Quality evaluation of staple foods is primarily carried out on sensory (e.g. external defect, color), adulteration (e.g. species, origin), chemical (e.g. starch, proteins), mycotoxin (e.g. Fusarium toxin, aflatoxin), parasitic infection (e.g. weevil, beetle), and internal physiological (e.g. hollow heart, black heart) aspects. Conventional methods for the quality assessment of staple foods are always laborious, destructive, and time-consuming. Requirements for online monitoring of staple foods have been proposed to encourage the development of rapid, reagentless, and noninvasive techniques. Spectroscopic techniques, such as visible-infrared spectroscopy, Raman spectroscopy, nuclear magnetic resonance spectroscopy, and spectral imaging, have been introduced as promising analytical tools and applied for the quality evaluation of staple foods. This review summarizes the recent applications and progress of such spectroscopic techniques in determining various qualities of staple foods. Besides, challenges and future trends of these spectroscopic techniques are also presented.

[(COMPARATIvE EVALUATION OF THE EFFICIENCY OF MANUAL AND AUTOMATIC SCANNING MICROSCOPY IN THE PARASITOLOGICAL STUDIES OF FOODSTUFFS)].

The paper gives the results of experimental studies, as well as sanitary parasitological ones investigating meat and fish, which have been performed using manual microscopy and Mecos-C2 automated digital microscopy and scanning micro- scopy. It shows the effectiveness of automated versus manual microscopy in performing parasitological studies of foods and the possibility of rationalizing production labor. The investigation has been conducted to develop the reform of labora- tory services of the healthcare system in the Russian Federation and proposes to introduce a hardware technology into a group of mass methods for parasitological analyses in sanitary-epidemiological, veterinary and environmental areas.

An alternative method to recover Toxoplasma gondii from greenery and fruits.

Toxoplasma gondii oocysts are an important form of contamination with a high dispersion in the environment, but their detection is still a challenge. This study evaluated the recovery of oocysts from strawberries and crisphead lettuce. Samples (250 g of strawberries or one head of lettuce) were experimentally inoculated with 10, 10(2), 10(3) and 10(4) T. gondii oocysts, by two separate processes, spot dripping and immersion. Then, 50 g of each sample was washed, filtered through a cellulose ester membrane, and concentrated by centrifugation. Three aliquots were taken for DNA extraction in a direct way, after freeze-thaw (FT) cycles or ultrasound (US), followed by PCR (B22-B23 and Tox4-Tox5 primers). The T. gondii DNA was amplified with the primers B22-B23 in all samples contaminated by dripping and when DNA extraction was carried out after FT or US. These techniques may be useful in epidemiological surveillance in the control of this zoonosis.

Enhancing the Detection of Giardia duodenalis Cysts in Foods by Inertial Microfluidic Separation.

The sensitivity and specificity of current Giardia cyst detection methods for foods are largely determined by the effectiveness of the elution, separation, and concentration methods used. The aim of these methods is to produce a final suspension with an adequate concentration of Giardia cysts for detection and a low concentration of interfering food debris. In the present study, a microfluidic device, which makes use of inertial separation, was designed and fabricated for the separation of Giardia cysts. A cyclical pumping platform and protocol was developed to concentrate 10-ml suspensions down to less than 1 ml. Tests involving Giardia duodenalis cysts and 1.90-µm microbeads in pure suspensions demonstrated the specificity of the microfluidic chip for cysts over smaller nonspecific particles. As the suspension cycled through the chip, a large number of beads were removed (70%) and the majority of the cysts were concentrated (82%). Subsequently, the microfluidic inertial separation chip was integrated into a method for the detection of G. duodenalis cysts from lettuce samples. The method greatly reduced the concentration of background debris in the final suspensions (10-fold reduction) in comparison to that obtained by a conventional method. The method also recovered an average of 68.4% of cysts from 25-g lettuce samples and had a limit of detection (LOD) of 38 cysts. While the recovery of cysts by inertial separation was slightly lower, and the LOD slightly higher, than with the conventional method, the sample analysis time was greatly reduced, as there were far fewer background food particles interfering with the detection of cysts by immunofluorescence microscopy.

A PCR procedure for the detection of Giardia intestinalis cysts and Escherichia coli in lettuce.

Giardia intestinalis is a pathogen associated with foodborne outbreaks and Escherichia coli is commonly used as a marker of faecal contamination. Implementation of routine identification methods of G. intestinalis is difficult for the analysis of vegetables and the microbiological detection of E. coli requires several days. This study proposes a PCR-based assay for the detection of E. coli and G. intestinalis cysts using crude DNA isolated from artificially contaminated lettuce. The G. intestinalis and E. coli PCR assays targeted the ß-giardin and uidA genes, respectively, and were 100% specific. Forty lettuces from local markets were analysed by both PCR and light microscopy and no cysts were detected, the calculated detection limit was 20 cysts per gram of lettuce; however, by PCR, E. coli was detected in eight of ten randomly selected samples of lettuce. These data highlight the need to validate procedures for routine quality assurance. These PCR-based assays can be employed as alternative methods for the detection of G. intestinalis and E. coli and have the potential to allow for the automation and simultaneous detection of protozoa and bacterial pathogens in multiple samples. Significance and impact of the study: There are few studies for Giardia intestinalis detection in food because methods for its identification are difficult for routine implementation. Here, we developed a PCR-based method as an alternative to the direct observation of cysts in lettuce by light microscopy. Additionally, Escherichia coli was detected by PCR and the sanitary quality of lettuce was evaluated using molecular and standard microbiological methods. Using PCR, the detection probability of Giardia cysts inoculated onto samples of lettuce was improved compared to light microscopy, with the advantage of easy automation. These methods may be employed to perform timely and affordable detection of foodborne pathogens.

Detection of Adult Beetles Inside the Stored Wheat Mass Based on Their Acoustic Emissions.

The efficacy of bioacoustics in detecting the presence of adult beetles inside the grain mass was evaluated in the laboratory. A piezoelectric sensor and a portable acoustic emission amplifier connected with a computer were used. Adults of the most common beetle pests of stored wheat have been detected in varying population densities (0.1, 0.5, 1, and 2 adults per kilogram of wheat). The verification of the presence of the insect individuals was achieved through automated signal parameterization and classification. We tried out two different ways to detect impulses: 1) by applying a Hilbert transform on the audio recording and 2) by subtracting a noise estimation of the recording from the spectral content of the recording, thus allowing the frequency content of possible impulses to emerge. Prediction for infestation was rated falsely negative in 60-74%, 48-60%, 0-28%, and 0-4% of the cases when actual population density was 0.1, 0.5, 1, and 2 adults per kilogram, respectively, irrespective of pest species. No significant differences were recorded in positive predictions among different species in almost all cases. The system was very accurate (72-100%) in detecting 1 or 2 insects per kilogram of hard wheat grain, which is the standard threshold for classifying a grain mass "clean" or "infested." Our findings are discussed on the basis of enhancing the use of bioacoustics in stored-product IPM framework.

An inexpensive feeding bioassay technique for stored-product insects.

ABSTRACT We used the red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), to compare three feeding bioassay techniques using flour disks. The area (scanner or digital photographs) and mass (sensitive balance) of the same flour disks were measured daily for 1 or 2 wk to assess feeding by insects. The loss in mass and area over 4 h was measured, as some variation over time was noticed in the disks with no insects feeding on them. The gravimetric method correlated well with both measurements of the area for the disks held in a growth chamber: scanner (R2 = 0.96), digital photography (R2 = 0.96). There was also a high correlation (R2 = 0.86) between the disk weight and area scanned at normal lab conditions. There were differences in the percentage of the disks remaining over time depending on the temperature and whether they were weighed or scanned. Measuring the mass of the disks resulted in a relatively larger percent of disk remaining compared with the scanned area. Mass measurements required a sensitive balance, handling of the disks and the insects, and appeared slightly more sensitive to humidity and temperature changes over time. Scanning the disks requires flat bed scanner access but less handling of both insects and disks. Digital photographs could be taken quickly, requiring less equipment, although photographs had to be further processed to determine area Scanning or taking digital photographs of flour disk area was an effective technique for measuring insect feeding.

A review of current statistical methodologies for in-storage sampling and surveillance in the grains industry.

Effective, statistically robust sampling and surveillance strategies form an integral component of large agricultural industries such as the grains industry. Intensive in-storage sampling is essential for pest detection, integrated pest management (IPM), to determine grain quality and to satisfy importing nation's biosecurity concerns, while surveillance over broad geographic regions ensures that biosecurity risks can be excluded, monitored, eradicated or contained within an area. In the grains industry, a number of qualitative and quantitative methodologies for surveillance and in-storage sampling have been considered. Primarily, research has focussed on developing statistical methodologies for in-storage sampling strategies concentrating on detection of pest insects within a grain bulk; however, the need for effective and statistically defensible surveillance strategies has also been recognised. Interestingly, although surveillance and in-storage sampling have typically been considered independently, many techniques and concepts are common between the two fields of research. This review aims to consider the development of statistically based in-storage sampling and surveillance strategies and to identify methods that may be useful for both surveillance and in-storage sampling. We discuss the utility of new quantitative and qualitative approaches, such as Bayesian statistics, fault trees and more traditional probabilistic methods and show how these methods may be used in both surveillance and in-storage sampling systems.

Assessing the applicability of currently available methods for attributing foodborne disease to sources, including food and food commodities.

A variety of approaches to attribute foodborne diseases to specific sources are available, including hazard occurrence analysis, epidemiological methods, intervention studies, and expert elicitations. The usefulness of each method to attribute disease caused by a foodborne hazard depends on the public health question being addressed, on the data requirements, on advantages and limitations of the method, and on the data availability of the country or region in question. Previous articles have described available methods for source attribution, but have focused only on foodborne microbiological hazards. These articles have described strengths and weaknesses of each method, but no guidance on how to choose the most appropriate tool to address different public health questions has thus far been provided. We reviewed available source attribution methods; assessed their applicability to attribute illness caused by enteric, parasitic, and chemical foodborne hazards to the responsible sources; and renamed some of the approaches. The main objective was to make recommendations on the most appropriate method(s) to attribute human disease caused by different foodborne hazards. We concluded that the proportion of disease that can be attributed to specific foods items or transmission routes may be estimated for the majority of the evaluated hazards by applying one or more of the source attribution methods assessed. It was also recognized that the use of source attribution methods may be limited to specific countries, reflecting the data availability.

Seroprevalence of Toxoplasma gondii in beef cattle and dairy cattle in northeast China.

The seroprevalence of Toxoplasma gondii in beef cattle and dairy cattle in Heilongjiang Province, northeast China, was surveyed between April 2009 and May 2011. A total of 1803 (693 beef cattle and 1110 dairy cattle) serum samples were collected from 10 administrative regions rearing beef cattle and dairy cattle, and antibodies to T. gondii were examined by indirect hemagglutination (IHA) test. The overall seroprevalence of T. gondii in beef cattle and dairy cattle was 2.6% (46/1803), and the prevalence in beef cattle (3.0%) was slightly higher than that in dairy cattle (2.3%). The prevalence of antibodies in adult animals was higher than that in calves, but the differences among the age groups were not significant (p>0.05). The seroprevalence in female (3.4%) and male (2.5%) beef cattle was not statistically significant (p>0.05). Though the prevalence in intensively reared beef cattle and dairy cattle was lower than that in semi-intensively reared animals, the difference was not statistically significant (p>0.05). The results of this survey indicated the presence of T. gondii infection in beef cattle and dairy cattle in Heilongjiang Province, the coldest province in China, which may cause economic losses to the local livestock industry, and may be a source of T. gondii infection for humans in this region.

PCR test for detecting Taenia solium cysticercosis in pig carcasses.

Polymerase chain reaction (PCR) test was employed to detect Taenia solium DNA in muscle lesions for validation of the meat inspection results of slaughtered pigs. Two sets of oligonucleotide primers, one targeted against the large subunit rRNA gene (TBR primers) and the other targeted against cytochrome c oxidase subunit 1 gene (Cox1 primers) of T. solium were used in this study. On reactivity in PCR test, the TBR primers and the Cox1 primers yielded products of 286 and 984 bp, respectively, in cysticercosis positive cases. Both the sets of primers were found to be highly specific, since they did not yield any PCR product in negative controls. A total of 225 pig carcasses were screened for cysticercosis by meat inspection, out of which 25 carcasses with visible cysts (16 viable and 9 degenerated cysts) were also confirmed to be positive for cysticercosis in PCR test. However, out of the 35 carcasses with suspected lesions on meat inspection, only two were found to be positive for cysticercosis in PCR test. The detection limits for both the primer sets were analyzed. The TBR primer set could detect up to 10 pg of cysticercus DNA, whereas the Cox1 primer set could detect only up to 1 ng. It is evident from the study that PCR test is an efficient tool for validation of meat inspection results and also to rule out ambiguity in carcass judgment of suspected cases of porcine cysticercosis.

Sensitivity of candling as routine method for the detection and recovery of ascaridoids in commercial fish fillets.

Ascaridoids are one of the main parasitic hazards in commercial fish. Candling is the current industrial screening method whereby visible ascaridoid larvae are detected on a light table and manually removed. The aim of this study was to assess the sensitivity (Se) and negative predictive value (NPV) of this method. To make targeted recommendations to the fish industry, the Se was calculated per fish part, larval genus, and fish species. All fish parts (n = 615) were first candled, and larvae were collected, followed by enzymatic digestion to recover the remaining larvae. A fish part was considered positive if at least one larva was detected using candling and/or enzymatic digestion, with both methods combined as reference standard. The overall Se of candling was 31% (95% CI 23-41%) and NPV was 87% (95% CI 85-90%). The Se increased with higher numbers of larvae/100 g infected muscle. A low NPV was found for the belly flaps, therefore we either advise the removal or proper freezing of this part. Lastly, the Se and larval recovery was the highest for the darker and larger Pseudoterranova spp. larvae. Due to the low overall efficacy of candling, further assessment of its cost-benefit and impact on consumers' health risk should be conducted.