The proposed research addresses the 2016 Agriculture and Food Research Initiative (AFRI) Foundational Program, area priority code A1331. This research aims to validate the effectiveness of loop-mediated isothermal amplification (LAMP) method coupled with bioluminescence assay in real-time (BART) of Campylobacter jejuni in uncooked poultry. C. jejuni, in addition to C. coli, is the estimated third-leading cause of bacterial foodborne illness in the United States, with over 800,000 reported cases each year (Scallan et al., 2011). The predominant C. jejuni and C. coli causative agents in humans include major symptoms of infection being diarrhea, fever, abdominal cramps, and vomiting (FDA, 2012). Although human infections can arise from a variety of sources, the most frequent being undercooked or improperly handled poultry, due to contaminated poultry carcasses containing ~100-100,000 Campylobacter cells; therefore, handling raw or undercooked poultry is a significant risk factor for illness. In order to reduce the number of illnesses from Campylobacter, rapid detection of contaminated food items is essential. Rapid detection can assist outbreak investigation efforts, leading to prompt removal of contaminated food from supermarket shelves. Identification of Campylobacter in foods using conventional methods is laborious and time-consuming due to the need for enrichment, plating, and confirmation tests (Hunt et al., 2001). Rapid methods using nucleic acid amplification, such as polymerase chain reaction (PCR) and real-time PCR, have been successfully used to detect Campylobacter in food products for the purpose of screening (USDA, 2016). Although PCR-based methods are well-established and are known to be rapid and reliable, they require the use of sophisticated equipment and are susceptible to inhibition from food matrices (Notomi et al., 2000; Yang et al., 2014). Loop-mediated isothermal amplification (LAMP) is an emerging method for pathogen detection that presents several distinct advantages over PCR-based systems (Notomi et al., 2000; Zhang et al., 2014). LAMP is carried out at a constant temperature and does not require the complex equipment used for thermal cycling. One technique that has emerged recently as a promising detection method is called the bioluminescent assay in real-time (BART), which allows for real-time detection of the target pathogen and has shown strong quantification capability in food samples (Gandelman et al., 2010, Yang et al., 2016).
Similar to the results from a recent study conducted by Yang et al. (2016) that used of LAMP-BART to detect Salmonella in food samples, the overall goal of this project is to demonstrate the effectiveness of a loop-mediated isothermal amplification method coupled with bioluminescence for the rapid detection of Campylobacter jejuni in uncooked poultry. In the study using Salmonella, the method was reported to be specific, robust, sensitive and rapid, which is what is also expected with C. jejuni and uncooked poultry (Yang et al., 2016).
The project consists of two specific objectives, which are to 1) carry out initial testing and optimization of a loop-mediated isothermal amplification (LAMP) coupled with bioluminescent assay in real-time (BART) for the detection of Campylobacter jejuni in pure culture and 2) test the effectiveness of the LAMP-BART assay for detection of C. jejuni in artificially inoculated samples of uncooked poultry and compare the results to culture-based methods. LAMP-BART presents a simple, specific and sensitive method with high potential to be used for on-site, quantitative detection of Campylobacter in food products. This method will promote food safety by facilitating pathogen detection and thereby enhancing routine surveillance efforts and outbreak investigations.
2. Review of Literature
2.1. An examination of foodborne illness
Foodborne illness is commonly referred as “foodborne disease, foodborne infection, or food poisoning,” and is a result of consuming contaminated foods or drinks (CDC 2016). Although it can be commonly prevented, foodborne illness is a result of foods and beverages contaminated by a variety of bacteria, viruses, microbes, toxins, metals, prions, and parasites, including Botulism, Campylobacter, Clostridium perfringens, Cyclospora, Listeria, Norovirus, Salmonella, Shigella, Vibrio, and Escherichia coli (Mead 1999, CDC 2016). As many different microbes can contaminate food, many different foodborne diseases with varying severities and symptoms may arise. In conjunction with about 21 other pathogens, the ten commonly associated with foodborne illness are responsible for about 48 million cases of foodborne illness annually, of which an estimated 128,000 result in hospitalization and 3,000 result in death (CDC 2016, FDA 2016). Of all bacterial causes of foodborne infection in the United States, Campylobacter jejuni has been the most commonly reported bacteria associated with food poisoning, with a 14% increase of incidence from 2012 to 2013 (Epps et al. 2013). The table below provides an in-depth summary of the most common pathogens that cause foodborne disease across the United States.
In the United States, a recent study ranked Campylobacter in poultry as the highest pathogen-food combination with the largest burden on public health considering the number of cases, hospitalization, death, economic cost, and health-related quality of life
TABLE OF MOST COMMON FOODBORNE ILLNESS PATHOGENS??
2.1.1. Reporting of outbreaks in the United States
Despite knowing that pathogens cause an estimated 9.4 million foodborne illnesses annually in the United States, the data provided by the CDC suggests there is under-reporting of foodborne illness outbreaks by consumers (Scallan et al. 2011, CDC 2013). An outbreak is described as the occurrence of two or more similar illnesses resulting from ingestion of a common food (CDC 2013). Resources for reporting foodborne disease outbreaks are readily available on the CDC website for the general public, health department, and healthcare professionals. Data recorded from the general public, health department, and healthcare professionals from each outbreak include the number of illnesses, hospitalizations, and deaths; the etiologic agent (confirmed or suspected); the implicated food vehicle; factors contributing to food contamination; and the settings of food preparation and consumption (CDC 2013).
The general public is asked to contact the local health department (county or city health department) and speak with an environmental health specialist or sanitarian about a possible food problem. Additional contact information facilitating the process of reporting a foodborne illness for the general public is also available on the CDC page. Health departments should report any suspected cases of foodborne illness to the state health department, as required by each state (CDC 2015). From there, the state health departments report to the CDC via the National Notifiable Diseases Surveillance System (NNDSS) (CDC 2015). Minor adjustments to the procedure are (such as forms and lab results) should be followed depending on the severity and source of the disease (CDC 2015). Healthcare professionals should follow particular guidelines available through healthcare agencies and the CDC (CDC 2015). A typical process healthcare professionals should abide by is the following: recognizing foodborne illness, diagnosing foodborne illness/clinical microbiology testing, treating of foodborne illness, and surveillance and reporting of foodborne illness (MMWR 2004).
2.1.2. Recognizing and diagnosing the pathogenicity of Campylobacter jejuni
Campylobacter consist of gram-negative, spiral-shaped, thermophilic bacteria that tend to be 0.2 to 0.9 microns wide and 0.5 to 5 microns long (Epps et al. 2013). Campylobacter are motile and consist of polar flagellum at one or both ends and can grow optimally aerobically or anaerobically at 37 OC – 42 OC, but cannot tolerate drying and oxygen (CDC 2016, Epps et al. 2013). The infectious dose of Campylobacter was estimated to be between 500-800 organisms for humans, as discovered in 1981 (Epps at el. 2013).
Campylobacter jejuni is the most common species that affects about 14 cases for each 100,000 persons in the population (CDC 2016). Foodborne disease from campylobacter jejuni is referred to as Campylobacteriosis and results in diarrhea, cramps, abdominal pain, fever, and vomiting, typically lasting from 2-10 days (FDA 2016). Diagnosis of foodborne illness can be difficult, as many symptoms may overlap and be slightly different that patients with chromic diarrhea, severe abdominal pain, viral syndrome, or gastrointestinal tract disease. Dependent upon the parasite, the symptoms will vary slightly, but all have the same underlying symptoms: bloody diarrhea, weight loss, diarrhea leading to dehydration, fever, severe abdominal pain, and sudden onset of nausea and vomiting (MMWR 2004). The best way to obtain important diagnostic clues is laboratory testing.
2.1.3. Treatment of foodborne illness from Campylobacter jejuni
Typically, those infected with Campylobacter can recover without any specific treatment; however the Center for Disease Control and Prevention (CDC) recommends patients to drink extra fluids while the symptoms last (CDC 2016). Only those with a severely weakened immune system or sever disease may receive antimicrobial therapy to treat the infection and aid with the symptoms (CDC 2016). Although antibiotics are commonly used to treat, control, and prevent infections, Campylobacter jejuni is almost intrinsically resistant to penicillians, cephalosporins, trimethoprim, sulfamethoxazole, rifampicin and vancomycin (Epps et al. 2013).
2.1.4. Causes and transmission of foodborne illness from Campylobacter
In developing countries Campylobacter is hyperendemic and major sources of infection are environmental and food contamination (Epps et al. 2013). However, in developed countries, such as the United States, food production animals are considered to be the primary source infection (Epps et al. 2013). Although beef and pork constitute part of the reservoirs for foodborne illness, most infections of Campylobacter are a result of handling and consumption of poultry meat (Butzler 2004). Poultry includes broilers, laying hens, turkeys, ducks, and ostriches, according to Epps et al. (2013). A reason as to why Campylobacter are predominantly in poultry is due to the higher metabolic temperatures of poultry, allowing for predisposition for the thermotolerant Campylobacter (Epps et al. 2013). Transmission can also easily occur while traveling. Alternatively, rodents and flies can act as transmission vectors for Campylobacter (Sahin et al. 2015). In addition, nearby livestock, farm personnel, and equipment can act as potential risk factors of transmission due to lack of separation and improper hygiene and sanitation (Sahin et al. 2015). Rarely does contamination occur through feed, fresh litter, and water (Epps et al. 2013, Sahin et al. 2015). Vertical transmission from breeder flocks via eggs is seldom the reason for contamination among birds (Sahin et al. 2015). Since transmission of C. jejuni can occur in a variety of ways and through several different reservoirs, it is advised to take many precautionary measures to reduce the risk of getting food poisoning.
2.1.5. Poultry as a food vehicle of C. jejuni
Domestic poultry (chickens, turkeys, ducks, geese) and wild birds are commonly infected with Campylobacter due to the thermophilic bacteria growing optimally in avian hosts. Campylobacter is a commensal bacterium, frequently found in the intestinal tract of birds, and is insignificant for poultry health (Sahin et al. 2015). Although it is not a poultry pathogen, Campylobacter is the leading cause of foodborne gastroenteritis in humans, and contaminated poultry is recognized as the main source for human exposure (Sahin et al. 2015). The most frequent species found in birds are Campylobacter jejuni and Campylobacter coli, but neither produce little or overt disease in birds (Sahin et al. 2015). Although Campylobacter is normal microbial flora of poultry, prevalence of the bacteria varies by season, region, and production type (Sahin et al. 2015). Horizontal transmission between the food poisoning bacteria and the environment is the most common way of contamination. Birds may show no sign of illness, despite being infected with Campylobacter, creating unknown spread of the disease-causing bacteria (CDC 2013). Since Campylobacter can contaminate poultry in a variety of ways, implementation of strict measures to prevent contamination and illness are crucial.
2.1.6. Prevention of foodborne illness from Campylobacter
Some food handling practices can prevent contamination in the kitchen and can prevent foodborne diseases as a result. CDC suggests doing the following to prevent Camylobacter infection: cook all poultry products thoroughly (all poultry should be coked at a minimum internal temperature of 165 OC), practice thorough hand washing technique before preparing food, after handling raw foods of animal origin and before touching anything else, and after touching pet feces, prevent cross-contamination in the kitchen using separate cutting boards for foods of animal origin, properly clean and sanitized cutting boards, countertops, and utensils, and avoid drinking unpasteurized milk or untreated surface water (CDC 2016). Also, educating travelers on the risk involved while traveling, especially when traveling to undeveloped countries, as well as giving them advice on the how to avoid Campylobacter infections while staying in different countries (Butzler 2004). Additional preventative measures include: strict biosecurity and good hygiene practices, treatment of water, litter treatment, and feed additives (Sahin et el. 2015). Also, there is potential research that Camplylobacter is reduced through application of bacteriophages (Sahin et al. 2015).
Despite efforts from the food industries to prevent Campylobacter infections, outbreaks may still occur due to the high demand of poultry and milk. However, when two or more cases of similar illness resulting from ingestion of a common food occur, it is imperative to find the source of the outbreak and take the product out of commerce to prevent further cases. Rapid detection tests are vital when conducting epidemiological tests and environmental trace back.
2.2. Rapid detection methods for diagnosing foodborne illness
Methods can be based on immunological, biochemical, and microbiological and molecular methods. Although rapid detection methods can be used for enumeration and characterization, methods can be used for isolation, detection, and identification of Campylobacter in foods. As for rapid methods based on biochemistry, Analytical Profile Index (API) strips can be used for identification of bacteria and yeast, while a VITEK test can be used for identification of an isolated organism. Immunological tests include a Latex Agglutination Test, and Enzyme-linked immunosorbent assay (ELISA) for identification of specific pathogens. Lastly, nucleic acid-based methods include targeted screening/confirmation methods and DNA typing methods. Polymerase chain reaction (PCR), real time-PCR, multiplex PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), and oligonucleotide DNA microarray are detection methods, while DNA sequencing, Pulsed-Field Gel Electrophoresis and whole-genome sequencing are DNA typing methods (Law et al. 2015). Rapid detection methods are typically automated, time-efficient, sensitive, and specific to the prevention and treatment of foodborne infections.
2.2.1. Loop-mediated isothermal amplification (LAMP)
Loop-mediated isothermal amplification (LAMP) is an emerging method for pathogen detection that presents several distinct advantages over PCR-based systems (Notomi et al., 2000; Zhang et al., 2014). LAMP is carried out at a constant temperature and therefore does not require the complex equipment used for thermal cycling or a thermostable DNA polymerase. Also, in comparison to PCR, LAMP has shown increased tolerance to reaction inhibitors, resulting in increased sensitivity and potentially simplified procedures for food sample preparation (Yang et al., 2014; Zhang et al., 2014). LAMP utilizes four to six specific primers combined with a strand-displacing DNA polymerase and is capable of amplifying a few copies of target DNA into 109 copies within one hour (Nagamine et al., 2002; Notomi et al., 2000). Various detection systems for LAMP products have been proposed. These include naked eye monitoring, turbidity, fluorescent dyes and gel electrophoresis (Zhang et al., 2014). A recently emerged and promising detection method is called the bioluminescent assay in real-time (BART) (Gandelman et al., 2010). This method allows for real-time detection of the target pathogen and has shown strong quantification capability in food samples (Yang et al., 2016).
2.2.2. Bioluminescent assay in real-time (BART)
BART continuously reports the exponential increase of inorganic pyrophosphate (PPi) produced during isothermal amplification of a particular targeted nucleotide through bioluminescent output (Gandelman et al. 2010). During nucleic acid synthesis to ATP (through ATP sulfurylase), inorganic pyrophosphate (PPi) is produced, which is monitored through bioluminescence generated by a thermostable firefly luciferase (Gandelman et al. 2010). The enzyme luciferase emits varying light intensities, according to different nucleic acid amplification in various animal species tested.
Figure 1. Equations
2.2.3. Advantages and disadvantages for rapid detection methods of pathogens
Since conventional methods typically require long incubation times, conventional methods consist of culturing microorganisms on agar plates and are selective, not ideal for rapid detection of pathogens, and delay removal of the food from commerce (Law et al. 2014, Mandal et al. 2011). Therefore, a variety of rapid detection methods have been developed. Several rapid detection methods have been discussed above- all of which overcome the limitations of conventional detection methods (Law et al. 2014). The nucleic acid-based methods described above have high sensitivity and are widely used for the detection of foodborne pathogens
2.2.4. Limitations of LAMP-BART as a rapid detection method
A major limitation of LAMP-BART is that it does not allow for the discrimination of viable and non-viable cells. Despite this, it is expected to be a valuable on-site screening tool for the quantitative detection of pathogens in foods.
Although LAMP-BART has proven successful in the detection of other pathogens, it is possible that it will not perform well with C. jejuni in food samples. If this is the case, additional optimization of the assay may be necessary. Furthermore, cultural isolation and identification of C. jejuni is known to be challenging as compared to other common bacterial foodborne pathogens.
2.3. Rationale and significance
In order to reduce the number of foodborne illnesses from Campylobacter, rapid detection of contaminated food items is vital. Rapid detection methods can aid outbreak investigations, leading to prompt removal of the food vehicle out of commerce. Conventional methods for identifying Campylobacter are laborious and time-consuming; therefore, rapid methods are emerging. The goal of this research is to successfully analyze and compare the results from an emerging rapid detection method to culture-based methods in various artificially inoculated samples of uncooked poultry, which will be fulfilled through the following aims:
I. Carry out initial testing and optimization of a loop-mediated isothermal amplification (LAMP) coupled with bioluminescent assay in real-time (BART) for the detection of Campylobacter jejuni in pure culture. The working hypothesis for this aim is that LAMP-BART will be capable of detecting Campylobacter at levels of 100 – 101 CFU/reaction in pure culture and 103 -104 CFU/g in food samples without enrichment (Yamazaki et al., 2009; Yang et al., 2016).
II. Test the effectiveness of the LAMP-BART assay for detection of C. jejuni in artificially inoculated samples of uncooked poultry and compare the results to culture-based methods. The working hypothesis for this aim is that the LAMP-BART method is expected to show a strong correlation between the number of cells per reaction in pure culture and the Tmax values, resulting in potential for quantification capability (Yamazaki et al., 2009; Yang et al., 2016).
Upon completion of the research, results will highlight processed samples that are either successes or failures, and give further insight to the effect of various processing methods on fish. In addition, the research will indicate the success and/or the failure of the full-length and mini-barcodes. In the case that mini-barcodes perform as well or better than full barcodes, setting quality standards may be beneficial for further research on processed fish samples.