Project Report on

Emerging food pathogen and advanced methods for their detection

SUBMITTED TO:
DR.Ravinder Nagpal
DEPARTMENT OF BIOTECHNOLOGY LOVELY PROFESSIONAL UNIVERSITY PHAGWARA

SUBMITTEDBY:
MANDEEP KAUR ROLLNO.R292A20 REGD NO: 10802086 MSC (HONS) MICROBIO M.PHI

ACKNOWLEDMENT
I am Mandeep Kaur of Msc. (hons) Microbiology 1st year 1st semester in Lovely Professional University Phagwara feels highly indebted to the person who co-operated with me during the project work. The various persons to whom I am thankful for their expert guidance are my friends, classmates for the completion of my project. I am really thankful to my teacher DR.Ravinder Nagpal for his expert guidance. I cannot express my feelings in words for their love, affection, inspiration and blessings rendered by my parents. Above all I am highly indebted to GOD without whose grace this little work could have never been possible. Mandeep kaur

Abstract
Food safety and food security now a day have become the major issue of considerable health concern in both the developed and developing countries. The cross border inflow of processed foods such as dairy products requires stringent food laws and regulatory measures to ensure the quality and safety of such foods specifically against high risk dairy pathogens and microbial contaminants during their export and import. Hence, to comply with these food laws and regulatory standards, the need for developing innovative, rapid and reliable analytical techniques has been a very important step for monitoring the quality and safety of such perishable foods. Since dairy products are extremely vulnerable to microbial contamination during their processing in countries like India due to unhygienic conditions and mishandling of food, high risk food pathogens like Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella sp. etc, can have access into these foods and hence can pose threat to the health of the consumers. Hence, it is now mandatory to constantly monitor these foods for the presence of target pathogens to completely rule out the possibility of food poisoning outbreaks associated with such food pathogens. To achieve this target, appropriate sensitive and reliable analytical tools are required to quickly detect and enumerate these pathogens in such foods to ensure their safety and correct risk assessment. The conventional methods of identification and confirmation involving growth on selective medium, biochemical tests and immunological assays are extremely laborious, cumbersome, and results are delayed considerably to make them virtually impractical for any follow up action. Extremely powerful and sensitive molecular techniques based on the genetic architect of these pathogens could be extremely valuable tool for assessing the microbiological quality and safety of the dairy foods not only qualitatively but also in quantitative terms. One such molecular diagnostic technique having immense potential in determining food safety and quality is Real-Time PCR, which is currently being explored by the dairy industry in the developed countries. However, a lot of research is in vogue to further improve the capability of this powerful technique vis--vis above mentioned high risk pathogens, resulting into generation of very useful information and knowledge in this particular area. Molecular techniques such as the polymerase chain reaction (PCR), multiplex PCR, and Real-time PCR are very useful tools used frequently in many research laboratories throughout the world. Use of PCR-based techniques has facilitated the discovery of more effective methods for the detection of food borne pathogens associated with food-producing animal environments and food borne pathogens causing disease in humans. These DNA-based detection methods offer improvements in detection and characterization of food borne pathogens beyond classical plating and phenotypic methods. The past few years have yielded advancements in development of very sensitive, rapid, automated, and increasingly easy-to-use molecular detection methods for a variety of different food borne pathogens.

INTRODUCTION
The first person to suggest the role of microorganisms in spoiling foods was A.Kircher, amonk, who as early as 1658 examined decaying bodies, meat, milk and other substances and saw what he referred to as worms invisible to naked eye. Primary sources of microorganisms found in foods are of mainly eight types. 1. Soil and water 2. Plant and plant products 3. Food utensils 4. Intestinal tract of humans and animals. 5. Food handlers 6. Animal feeds 7. Animal hides 8. Air and dust The emergence of pathogens is the result of a number of impacts in all parts of the food chain. The emerging technologies in food production explain how new pathogens can establish themselves in the food chain and compromise food safety. The various common food borne bacteria are:Acinetobacter,Aeromonas,Alcaligenes,Alteromonas,Bacillus,Brochothrix,Campyl obacter,Carnobacterium,Citrobacter,Clostridium,Corynebacterium,Enterobacter, Enterococcus,Erwinia,Escherichia,Lactobacillus,Listeria,Salmonella,Shigella,Sta phylococcus,Yersini

Emerging Food borne Pathogens

Emerging Foodborne Pathogens: There are a number of factors involved in the emergence or re-emergence of pathogens associated with foodborne illness in the United States and other developed countries. These include environmentallyrelated factors, such climate changes and deforestation, food-related factors, such as changes in food production and distribution practices, consumer-related factors, such as increased international travel and changes in eating habits, and pathogen-related factors, such as genetic changes in microorganisms as a result of exposure to environmental stresses. One major factor is the increased globalization of the food supply, resulting in transfer of pathogenic agents between countries. The use of antimicrobials for prophylaxis in animals has contributed to the emergence of bacterial strains resistant to multiple antibiotics. These include: Arcobacter; Campylobacter; Trematodes and helminths; emerging strains of E. coli; Hepatitis viruses; Prion diseases; Vibrios; Yersinia;

Listeria; Helicobacter pylori; Enterobacteriaceae; Mycobacterium paratuberculosis; and enterocci.

Campylobacter;

Mycobacterium paratuberculosis:- Mycobacterium are fastidious and

slow growing organisms, and this has made their culture very difficult. Most strains of M. paratuberculosis require the iron-binding siderophore mycobactin in culture media and they grow very slowly even in its presence. In addition, mycobacterium form cell walldeficient forms, spheroplasts, and this changes their staining properties (no longer acid-fast) and makes it extremely difficult to culture them. Mycobacterium paratuberculosis was first associated with an intestinal disease of cattle about a hundred years ago. At first it was difficult to distinguish this bacterium from tubercle bacilli but eventually they were isolated and characterized and Johnes disease was distinguished from intestinal tuberculosis. Although a number of diagnostic tests are available, it is not always possible to detect these bacteria in sick cattle. The bacteria penetrate the intestinal mucosa, are phagocytized by macrophages, and continue to multiply inside the macrophages. Granulomas, compact foci of inflammatory cells, form at these sites and the animals develop a chronic no responsive diarrhea which causes progressive weight loss, debilitation, and death. A number of granulomatous infections occurring in humans are known to be caused by mycobacteria. These include tuberculosis, leprosy, some skin ulcers, and some opportunistic infections in AIDS patients . It is suspected, but by no means proven, that mycobacteria are involved in some other granulomatous diseases, including Crohns and sarcoidosis. Sarcoidosis is a disease primarily affecting the lungs whereas crohn disease affects the ileum (part of the small intestine) and the colon. In diseases, lymph nodes and other organs are sometimes involved. . When M. paratuberculosis was first isolated from patients with Crohns disease, it took up to 18 months for the primary isolation . Later attempts to isolate M. paratuberculosis from clinical specimens of Crohns disease patients have not been very successful, with only six strains of DNA probeconfirmed M. paratuberculosis isolated from patients with Crohns disease. Mycobacterium paratuberculosis is a causative factor in human granulomatous diseases such as Crohns disease, or (ii) what vehicle might transmit these bacteria to humans.. It is likely that if M. paratuberculosis is involved in this disease it will turn out to be one of several etiological cofactors. Genetic predisposition, other environmental factors, and the measles virus have all been suggested to have a role in the development of these disease symptoms. A potential foodborne pathogen, has been linked to outbreaks of illness in new-born and premature babies and contamination of infant formulae has been suggested as a source of infection. E.

Enterobacter sakazakii:-

sakazakii is a gram-negative straight rod belonging to the Enterobacteriaciae family and qualifies as a coliform bacterium. Having dimensions of 3 m in length and 1 m in width, the cells are motile by peritrichous flagellaand do not form spores. Prior to 1980, E. sakazakii was referred to as a yellow-pigmented Enterobactercloacae but it was reclassified based on differences in DNA-DNA hybridization, biochemical reactions, antibioticsusceptibility, and production of yellow-pigmented colonies.

Clinical Symptoms of E. sakazakii Infections: These symptoms include poor feeding response, irritability, jaundice, grunting respirations, and instability of body temperature. In a la rge number of the neonatal cases, infection progressed to meningitis (an acute inflammation of the membranes of the brain and spinal cord), with survivors suffering from severe neurological impairment. Ventriculitis (inflammation in the ventricles of the brain), brain cysts and abscesses, cerebral infarction, and late development of hydrocephalus (abnormal increase in the amount of cerebrospinal fluid within the cranial cavity) are characteristic of central nervous system infections. Other medical conditions have also been associated with E. sakazakii infections, including bacteremia (bacteria in the blood) as well as necrotizing enterocolitis (localized death of small and large intestine tissues). After the first signs of sepsis appear, death may occur within a few hours to several days. In survivors, continued colonization by E. sakazakii varies from 2 to 8 weeks. Source of E. sakazakii Infections:- of E. sakazakii but no reservoir was found in surface water, soil, mud, rotting wood, grain, bird dung, rodents, domestic environments, cattle, or untreated cow's milk. The bacterium was however found in cheese, minced beef, sausage meat, and vegetables Future Research on E. sakazakii: - More research is needed to determine virulence factors, genetic diversity, appropriate recovery and subspeciation methods, environmental niches, and methods for control of this emerging pathogen in foods and the environment. It is the most commonly reported bacterial cause of foodborne infection in the United States. Adding to the human and economic costs are chronic sequelae associated with C. jejuni infection Guillian-Barr syndrome and reactive arthritis. In addition, an increasing proportion of human infections caused by C. jejuni are resistant to antimicrobial therapy. Mishandling of raw poultry and consumption of undercooked poultry are the major risk factors for human campylobacteriosis. Efforts to prevent human illness are needed throughout each link in the food chain. Campylobacter jejuni infections are now the leading cause of bacterial gastroenteritis.

Campylobacter jejuni: -

Scanning electron microscope image of Campylobacter jejuni, illustrating its corkscrew appearance and bipolar flagella.

Transmission to Humans : Most cases of human campylobacteriosis are sporadic. Outbreaks have different epidemiologic characteristics from sporadic infections. In contrast, sporadic Campylobacter isolates peak during the summer months. A series of case-control studies identified some risk factors for sporadic campylobacteriosis, particularly handling raw poultry and eating undercooked poultry. Other risk factors accounting for a smaller proportion of sporadic illnesses include drinking untreated water; traveling abroad; eating barbequed pork or sausage; drinking raw milk or milk from bird-pecked bottles; and contact with dogs and cats, particularly juvenile pets or pets with diarrhea. Person-to-person transmission is uncommon. Overlap is reported between serotypes of C. jejuni found in humans, poultry, and cattle, indicating that foods of animal origin may play a major role in transmitting C. jejuni to humans Reservoirs: The ecology of C. jejuni involves wildlife reservoirs, particularly wild birds. Species that carry C. jejuni include migratory birdsranes, ducks, geese , and seagulls . The organism is also found in other wild and domestic bird species, as well as in rodents . Insects can carry the organism on their exoskeleton. Sequelae to Infection: Guillain-Barr syndrome (GBS), a demy elating disorder resulting in acute neuromuscular paralysis, is a serious sequel of Campylobacter infection. An estimated one case of GBS occurs for every 1,000 cases of campylobacteriosis. Up to 40% of patients with the syndrome have evidence of recent Campylobacter infection. Campylobacteriosis is also associated with Reiter syndrome, a reactive arthropathy. In approximately 1% of patients with campylobacteriosis, the sterile post infection process occurs 7 to 10 days after onset of diarrhea. Multiple joints can be affected, particularly the knee joint. Pain and incapacitation can last for months or become chronic. Both GBS and Reiter syndrome are thought to be autoimmune responses stimulated by infection. Many patients with Reiter syndrome carry the HLA B27 antigenic marker. The pathogenesis of GBS and Reiter syndrome is not completely understood.

Treatment of C. jejuni Infections: Supportive measures, particularly fluid and electrolyte replacement are the principal therapies for most patients with campylobacteriosis. Severely dehydrated patients should receive rapid volume expansion with intravenous fluids. For most other patients, oral rehydration is indicated. Although Campylobacter infections are usually self limiting, antibiotic therapy may be prudent for patients who have high fever, bloody diarrhea, or more than eight stools in 24 hours; immunosuppressed patients, patients with bloodstream infections, and those whose symptoms worsen or persist for more than 1 week from the time of diagnosis. When indicated, antimicrobial therapy soon after the onset of symptoms can reduce the median duration of illness from approximately 10 days to 5 days. When treatment is delayed infection is therapy may not be successful. Ease of administration, lack of serious toxicity, and high degree of efficacy make erythromycin the drug of choice for C. jejuni infection; however, other antimicrobial agents, particularly the quinolones and newer macrolides including azithromycin, are also used

Cyclospora cayetanensis: Cyclospora cayetanensis is a protozoan that

causes disease in humans, and perhaps other primates. It has been linked in the United States from fecally-contaminated imported raspberries and was virtually unknown before about 1990, but has been on the rise since. The health risk associated with the disease is usually confined to adult foreigners visiting endemic regions and acquiring the infection: this is why C. cayetanensis has been labeled as causing travelers diarrhea. Cyclospora cayetanensis is an apicomplexan, cyst-forming coccidian protozoan that causes a self-limiting diarrhea. Morphologically speaking, C. cayetanensis has spherical oocysts that are between 7.5 and 10 micrometers in diameter that also have a 50 nanometer thick wall with an outer threadlike coat that has been called a wrinkle by some researchers. This species was placed in the Cyclospora genus because of the spherical shape of its sporocysts.

Cyclospora cayetanensis oocysts

Characterization: The only hosts C. cayetanensis uses are humans. The protozoan lives out its lifecycle intracellularly within the hosts epithelial cells

and gastrointestinal tract. Infection is transmitted through the oral-fecal route, and begins when a person ingests oocysts in fecally contaminated food or water. Various chemicals in the hosts gastrointestinal tract cause the oocysts to excyst and release sporozoites; generally, two are observed per oocyst. Sporulation can take anywhere from one to several weeks, meaning that person-to-person transmission is not a likely problem. Symptoms: C. cayetanensis causes gastroenteritis, with the extent of the illness varying based on age, condition of the host, and size of the infectious dose. Symptoms include "watery diarrhea, loss of appetite, weight loss, abdominal bloating and cramping, increased flatulence, nausea, fatigue, and low-grade fever," though this can be augmented in more severe cases by vomiting, substantial weight loss, explosive diarrhea, and muscle aches. Typically, patients who come in with a persistent watery diarrhea lasting over several days may be suspected of harboring the disease, especially if they have traveled to a region where the protozoan is endemic. The incubation period in the host is typically around a week, and illness can last six weeks before self-limiting. Unless treated, illness may relapse. It is important to note here that the more severe forms of the disease can occur in immunocompromised patients such as those with AIDS. Detection method: Due to its small size, intracellular habitat, and inability to properly uptake many histological stains, diagnosis of Cyclospora cayetanensis can be very difficult. Four methods have thus far been established for positive diagnosis of the protozoan: microscopic detection in stool samples of oocysts; recovering oocysts in intestinal fluid/small bowel biopsy specimens; demonstration of oocyst sporulation; and amplification by polymerase chain reaction (PCR) of C. cayetanensis DNA. Since detection is so hard, one negative result should not discount the possibility of C. cayetanensis: tests involving fresh stool samples over the next few days should also be considered.

Cryptosporidium parvum: An emerging food pathogen in food industry.

Cryptosporidium parvum is an emerging pathogen linked to water and food borne illness with recent epidemiological data reporting C. parvum as the second most common enteric pathogen causing diarrhoeal illness after Campylobacter. C. parvum is a parasite found in the intestinal tract of animals including cattle and sheep. Many animal carriers of Cryptosporidium are asymptomatic, but infected animals can shed up to 107 C. parvum oocysts per gram of faeces. Cryptosporidium species are transmitted by ingestion of oocysts excreted in the faeces of infected humans or animals and can therefore be spread from animal to person, person-to-person, through ingestion of faecally contaminated water or food, or by direct contact with contaminated environmental surfaces. Cryptosporidium is a coccidian protozoan parasite that has gained much attention in the last 20 years as a clinically important human pathogen.

Cryptosporidial oocysts

The increasing population of immunocompromised persons and the various outbreaks of cryptosporidiosis through infection by water-borne Cryptosporidium oocysts (often in drinking water) have placed an even greater emphasis on this pathogen. Little is known about the pathogenesis of the parasite, and no safe and effective treatment has been successfully developed to combat cryptosporidiosis. Unlike other intestinal pathogens, Cryptosporidium can infect several different hosts, can survive most environments for long periods of time due to its "hardy cyst" (Keusch, et al., 1995), and inhabits all climates and locales. Clinical symptoms: The various symptoms of cryptosporidiosis differ greatly between immunocompetent and immunocompromised individuals. In immunocompetent patients, cryptosporidiosis is an acute, yet self-limiting diarrheal illness (1-2 week duration), and symptoms include Frequent, watery diarrhea, Nausea, Vomiting Abdominal cramps, Low-grade fever For immunocompromised persons, the illness is much more severe Debilitating, cholera-like diarrhea (up to 20 liters/day), severe abdominal cramps, Malaise Low-grade fever, Weight loss, Anorexia. Due to lack of tissue specificity, C. parvum infection has also been identified in the biliary tract (causing thickening of the gallbladder wall) and the respiratory system . Detection and Diagnosis: When C. parvum was first identified as a human pathogen, diagnosis was made by a biopsy of intestinal tissue. However, this method of testing can give false negatives due the "patchy" nature of the intestinal parasitic infection Staining methods were then developed to detect and identify the oocysts directly from stool samples. The modified acid-fast stain is traditionally used to most reliably and specifically detect the presence of cryptosporidial oocysts. Immunologically, anti-cryptosporidial IgM, IgG, and IgA can be detected by the enzyme-linked immunoabsorbent assay (ELISA) or by the antibody immunofluorescence assay (IFA), but neither of these assays can provide a direct diagnosis of cryptosporidiosis.

Recently, new genetic methods of detecting C. parvum have been developed, using PCR (Polymerase Chain Reaction) or other DNA-based detection methods. For more information about these improvements in the specificity and sensitivity of cryptosporidial detection.

Arcobacter butzleri:

Chronic diarrhea emerging pathogen. The genus Arcobacter, belonging to the family Campylobacteraceae, includes polar flagellated, curved or spiral Gram negative bacteria described first in 1977 as Vibrio/Spirillum organisms and later as aerotolerant Campylobacter species. A. butzleri have been associated with abortion and enteritis in animals and with diarrhea and bacteremia in adults and children. The latter is also considered an emerging food pathogen and it seems to be most frequently isolated from human beings than the other species. However, little is known about its clinical significance. Thus, case-control and virulence studies need to be conducted in order to establish pathogenicity factors and clinical features of A. butzleri. Although Arcobacter intestinal infections in humans have been reported since 1987, there is no standardized method for the isolation of these bacteria. Therefore, there is a need of optimized primary isolation procedures. This represents an important step for the definition of a laboratory identification protocol and their clinical and epidemiological significance.

Listeria monocytogene : Old Enemy, New Syndrome: The major increases in

the understanding of Listeria disease as a foodborne pathogen have occurred only over the last 15 years. Because Listeria grows at refrigerator temperature, it has an unusual survival advantage. Of the six major species of Listeria, only Listeria monocytogenes is a human pathogen. It has several serotypes, of which 4b is the major epidemic problem. Diseases caused: Listeriosis: it is not characterized by unique symptom since the course of the disease depends on the stste of the host. however the following conditions are known as predispose to adult listeriosis and to significant in mortality rate:neoplasm,AIDS,alcoholism,diabetes,cardiovascular disease, renal transplant, and corticosteroid therapy.when susceptible adults contract the disease,meningitis andsepis are the most common recognized symptoms. . Listeria traditionally affects pregnant women, their fetuses and newborns, the elderly and immunocompromised hosts disproportionately. Listeria syndrome, "febrile gastroenteritis," which occurs in previously healthy individuals. There have been three large outbreaks that put this syndrome on the map. Disease in these instances was characterized by fever, diarrhea, headache, and abdominal pain

Helicobacter pylori: Not Just Ulcers Physicians have been dragged to the
recognition that Helicobacter pylori is the major cause of peptic ulcer disease and

gastric ulcerations in patients who do not have alcohol or aspirin abuse as risk factors. This presentation highlighted the fact that we may have to recognize Helicobacter as a cause of several other clinical syndromes. In addition to upper gastrointestinal tract disease, there is accumulating evidence that Helicobacter species may be associated with lower bowel colonization. Helicobacter hepaticus has been found colonized in the liver, cecum, and colon of mice, in which it is associated with inflammatory bowel disease-like syndromes and rectal prolapse. A major question is whether Helicobacter may be related to inflammatory bowel disease and even to cholecystitis, hepatitis, and cancer of the liver in humans. Evidence is collecting that Helicobacter heilmannii can be associated with gastric cancer and mucosal-associated lymphoid tissue (MALT) lymphoma. The reservoir of H heilmannii appears to be dogs, cats, and their owners. Other Helicobacter species that may be related to disease in humans include Helicobacter fennelliae and Helicobacter cinadei, which had been associated with acute proctocolitis, bacteremia, and cellulitis or arthritis in immunocompromised human hosts. Helicobacter rappini has been associated with diarrhea and bacteremia in humans.

Saccharomyces cerevisiae: It is an opportunistic human pathogen, used

for centuries for food and production of alcoholic beverages, calls for research in molecular tools to distinguish between probiotic and clinical strains.

Hepatitis E virus: It is a potential emerging food borne pathogen.and swine

may serve as a source for infection in humans, the most challenging issue in greater part of the world raising pigs. Tick-borne encephalitis virus infection, either thick borne or caused by consumption of raw milk, is an increasing trend in the industrialized part of the world.

Salmonella hadar: An Emerging Food-Borne Gastroenteritis Pathogen in

Europe. S. hadar becoming in the third commonest Salmonella serotype isolate in Spain, displacing S. virchow, with an annual increase of 63% respect previous years. Salmonellosis is the name for the acute lower gastrointestinal tract disease caused by infection with the bacteria Salmonella. The infection is usually contracted by ingestion of Salmonella-contaminated foodstuffs but can be acquired by inhalation or by exposure of mucous membranes, such as by splashing of contaminated urine or the use of a contaminated rectal thermometer. Salmonellosis is commonly manifested clinically in both man and animals by sudden onset, usually 12 to 72 hours after ingestion of the organism, of one or more signs of gastrointestinal infection including high fever, abdominal pain, diarrhea and sometimes vomiting. . When severe, the diarrhea may contain mucous, blood and shreds of intestinal lining Advanced methods in detection of food pathogen

A major problem in maintaining food safety is the need to rapidly detect microorganisms in order to curb outbreaks that can affect large populations. This is especially important because of wide scale distribution of perishable foods. Traditional methods of identification of food-borne pathogens, which cause disease in humans, are time-consuming and laborious, so there is a need for the development of innovative methods for the rapid identification of food-borne pathogens. Recent advances in molecular cloning and recombinant DNA techniques have revolutionized the detection of pathogens in foods. A variety of methods are now in use for the purpose of characterizing or fingerprinting species and strains of organisms of interest in foods: serotyping Phage typing nucleic acid probes Polymerase chain reaction multilocus enzyme electrophoresis restriction enzyme analysis Random amplication of polymorphic DNA Pulsed field gel electrophoresis Restriction fragment length polymorphism Ribotyping

Serotyping: Serotyping is most widely applied to gramnegativeenteric bacterial

pathogens such as Salmonelal and Escherichia. Among gram positives, serotyping is very important for the genus Listeria.The gist of a typical serotyping scheme is the use of specific antibodies (antiserum) to identify homologous antigens. In the case of many food borne pathogens, the antigens are particulate, and agglutination methods are employed. For soluble antigens such as toxins, methods such as gel diffusion may be used. The O and H antigens of enteric bacteria this typing scheme results in the recognition of three antigenic sites somatic (O, Ger. ohne), capsular (K, Ger. kapsel), and flagellar (H, Ger. hauch). The O antigens consist of the O polysaccharide side chains that are exposed on the surface these are heterogeneous structures, and antigenic specificity is determined by the composition and linkage of the O group sugars.

Bacteriophage Typing: Phage typing is based on the specificity of a given

phage for its host bacterium, and this relationship allows one to use known phages to identify their specific hosts. All food borne pathogens can be phage typed, but the practice is applied more to some than others.

Nucleic Acid (DNA) Probes: A DNA probe consists of the DNA sequence
from an organism of interest that can be used to detect homologous DNA or RNA sequences. In effect, the probe DNA must hybridize with that of the strain to be sought. Ideally, the probe contains sequences that code for a specific product. The probe DNA must be labeled in some way in order to be able to assess whether hybridization has occurred. Radioisotopes are the most widely used labels, and they include 32P, 3H, 125I, and 14C, with 32P being the most widely used.

Reporter groups such as alkaline phosphatase, peroxidase, fluorescein, happens, digoxigenin, and biotin have been developed and used.47 One of the drawbacks to the use of some of the reporter methods is the limited number of probes. When biotin issued, its presence is detected with avid in-enzyme. Conjugates, antibiotic, or photobiotic. Chromosomal DNA is often the source of target nucleicacid, but it contains only one target per cell. Multiple targets are provided by mRNA, rRNA, and plasmid DNA, thus making for a more sensitive detection system. Synthetic oligonucleotideprobes may be constructed of 20-50 bases, and under proper conditions, hybridization times of 30-60 minutes are possible. After separating fragment strands by electrophoresis, they are transferred to cellulose nitrate filters and hybridized to the radio labeled probe. After gentle washing to remove unreacted probe DNA, the presence of the radiolabel is assessed by autoradiography.

Multilocus Enzyme Electrophoresis Typing: This technique may be

employed to estimate the overall genomic relationships among strains of organisms within species by determining the relative electrophoretic mobilities of a set of water-soluble cellular enzymes. The variation inelectrophoretic mobility can be related to allelicvariation and to levels of genetic variation within populations of a species. Typically, from 15 to25 enzymes are tested for on starch gels. Because some of the enzymes may have different mobilities (be polymorphic) among strains of the same species, multilocus enzyme electrophoresis(MEE) typing can be used to characterize strains for epidemiologic purposes in the same general way as serotyping or phage type.

Restriction Enzyme Analysis: By this method, chromosomal DNA of test

A strain is digested by use of an appropriate restrictionendonuclease. The latter class of enzymes makes double-stranded breaks in DNA at specific nucleotide sequences. One of the most widely used restriction end nucleases is EcoRI (obtained from Escherichia coli), which recognizes the DNA base sequence GAATTC andcleaves between GA. Another endonuclease isHhal (obtained from Haemophilus influenzae), and it recognizes the sequence GTPyPuAC (Py= any primidine, Pu = any purine base). The cleavage site for Hhal is between PyPu, and ithas been found to be of value in studying the epidemiology of L. monocytogenes.202After some L. monocytogenes serovars 4bstrains associated with three food-associated outbreaks were subjected to restriction enzymeanalysis (REA) using Hhal, the method was found to be valuable as both a taxonomic too land an epidemiologic tracer.20

Random Amplification of Polymorphic DNA: In essence, random

amplification of polymorphicDNA (RAPD) employs the use of PCR to obtain randomly amplified polymorphic DNAelectrophoretic profiles. Briefly, cells are harvested, suspended in water, and lysed for theirDNA. The DNA along with a specific primer such as a 10-mer (10-bp section) is mixed withDNA and Taq polymerase. PCR is carried out at varying temperatures for 40 or more cycles,

followed by electrophoresis of the products on anagarose gel. Following staining of gel (typically with ethidium bromide), the bands are photographed and analyzed. Purified genomic DNAis not needed for RAPD, nor is there a need forprior sequence data. By using a capillary air thermalcycler, which was able to complete 30 cyclesin <1.0 hour, RAPD results could be obtained in3 hours

starting with colony growth.

Pulsed Field Gel Electrophoresis: This method entails the digestion of

genomicDNA by one or more restriction enzymes, separation of the restriction fragments by field inversion electrophoresis, and resolution of fragmentsin agarose gels. In contrast to conventional electrophoresis where a gel is run in one direction,PFGE is carried out with pulse times ramped from 1 to 100 seconds over varying periods of time, which is determined by the sizes of molecules.The alternating electrical fields force molecules to change directions, and the electrophoreticprofiles are designated pulsovars. It has been used to fingerprint food borne outbreak strains of several pathogens.

Restriction Fragment Length Polymorphism: DNA restriction

fragment length polymorphisms (RFLP) are heritable differences in the Lengths of DNA fragments that arise when DNAis digested by a restriction endonuclease. In brief, cellular DNA is digested with a restriction enzyme, Separated by electrophoresis, followed by Southern blot hybridization with a DNA probe from a given gene library of the organism in question. Along with MEE, it was used to demonstrate the recurrence of strains of L.monocytogenes in raw milk and nondairy foods.

POLYMERASE CHAIN REACTION: This technique, first outlined in

1971 by Kleppe et al., 102 is applicable more to the identification of food borne organisms than to their enumeration. The currently used methodologyis that further developed by scientists at the Perkin Elmer-Cetus Corp., 165179 among others. For his efforts in the development of PCR, K. B.Mullis was Co-winner of the Nobel Prize in chemistry in 1993. In brief, by employing thermostableDNA polymerases and 5'- and 3'-specific oligonucleotideprimers, a single molecule of DNA can be amplified to 107 molecules after a series of amplification cycles, typically from 20 to 50.The amplified DNA is then detected by use of either agarose gels or Southern hybridization employing either radio labeled or

nonradiolabeledprobes.

Electro immunoassay Technology: Electro immunoassays couple specific antibody-antigen binding to the production of an electrical signal. The technology is comprised of a circuit with a capture antibody attached to the solid surface in the area of the electrode gap .Upon addition of sample; the target antigen binds to the capture antibody. In the next step, a colloidal goldlabeled detection antibody is bound, creating a capture-target-detector sandwich. The final step is the deposition of silver ions onto the colloidal gold, which produces a conductive silver bridge, closing the circuit and resulting in a measured drop in resistance. This detection technology can also be applied to nucleic acid hybridization assays.

Electro immunoassay technology is composed of a circuit with a capture antibody attached to the solid surface in the area of the electrode gap

The Detex system is an electro immunoassay biosensor system consisting of the MC-18 immunoassay instrument and disposable pathogen-detection kits for various target pathogens the disposable components include cartridges containing the circuit with capture antibodies attached, and reagent packs containing all of the necessary reagents for conducting the assays. The MC-18 can perform 27 tests per run, divided into nine sectors of three tests each. The system is multiparametric, meaning that any combination of pathogen tests can be performed simultaneously. For example, one sector can be loaded for E. coli O157 testing and the next sector for Salmonella testing. The system is also capable of duplex testing, meaning that it can conduct two tests in one cartridge. For example, one sample can simultaneously be tested for both Salmonella and E. coli O157. This is achieved by the addition of a second circuit to the cartridge. Dual enrichment media formulations compatible with the duplex tests are also being developed by the company. The system is fully automated, enabling the user to complete the following steps.
1. Enrich the food product in the appropriate broth(s). 2. Heat-inactivate a sample of the enriched broth. 3. Load the appropriate reagent packs and cartridges into the carousel. 4. Add a 150 l aliquot to the cartridge. The Detex electro immunoassay biosensor system

Future Applications of Electro immunoassay Technology: As an immunoassay platform, the Detex system menu can be expanded to include tests for other

antigenic targets for various markets. Alternatively, the electrical detection concept can be combined with molecular probes for carrying out hybridization assays, thus expanding the utility of the current platform with some modest changes such as temperature control. Technology research is under way to employ the described electrical detection technology in an ultra sensitive quantitative biosensor device that would have broad applications in industrial and clinical diagnostics. There are reports of optical biosensor developments for food-borne pathogen detection. The ideal device would be an ultra sensitive in-line sensor that could quantitate total bacterial load as well as specific pathogen levels.

Real-time PCR: Real-time PCR

Real Time PCR is the DNA-based technique that monitors amplification of target DNA in real-time by monitoring florescence. Real-time PCR can be used to quantify bacteria from various samples including milk, faeces, food and water, and can be used for processing, detecting and confirming pathogens in multiple samples at one time. Real Time modifies the technique in a way that reduces the chance of false positives observed in traditional PCR by 99.9%. Even a single copy of target DNA can be detected due to high dynamic range. Real-time PCR using double-stranded DNA dyes A DNA-binding dye binds to all double-stranded (ds)DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified. However, dsDNa dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as "primer dimers"). This can potentially interfere with or prevent accurate quantification of the intended target sequence. 1. The reaction is prepared as usual, with the addition of fluorescent dsDNA dye. 2. The reaction is run in a thermocycler, and after each cycle, the levels of fluorescence are measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). With reference to a standard dilution, the dsDNA concentration in the PCR can be determined.

Fluorescent reporter probe method

Using fluorescent reporter probes is the most accurate and most reliable of the methods, but also the most expensive. It uses a sequence-specific RNA or DNAbased probe to quantify only the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and allows quantification even in the presence of some non-specific DNA amplification. This potentially allows for multiplexing - assaying for several genes in the same reaction by using specific probes with different-coloured labels, provided that all genes are amplified with similar efficiency. It is commonly carried out with an RNA-based probe with a fluorescent reporter at one end and a quencherof fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonucleaseactivity of the taq polymerasebreaker the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter. 1. The PCR is prepared as usual , and the reporter probe is added. 2. As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target. 3. Polymerisationof a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-3-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence. 4. Fluorescence is detected and measured in the real-time PCR thermocycler, and its geometric increase corresponding to exponential increase of the product is used to determine the threshold cycle (CT) in each reaction.

Future prospects: DNA Chips: DNA chips or DNA arrays, which are
nothing but ordered arrays of oligonucleotides immobilized on an organic substrate, provide new opportunities for assessing genetic diversity among the microorganisms. These arrays rely on hybridization of isolated microbial DNA to large sets of oligonucleotides or DNA fragments present at a precise location on a miniaturized inorganic substrate (e.g. glass slide), and are now being investigated for bacteriological testing by various groups. The number of oligonucleotides spotted on a chip can vary from several hundred to almost a million. Oligonucleotides can be derived from every region of the genome, and point mutations (single nucleotide polymorphisms) are targeted as well. Such DNA chips will certainly be commercially available in the near future for large scale testing. The only difficulty that this method may face is the cost of a chip, which can only be used once, and the need to purchase the expensive equipment for hybridization and analysis. Nevertheless, this technique is going to be the method of choice for identification of dairy organism in the near future because of its high degree of reliability in terms of specificity and sensitivity. REFRENCES 1.world.org/ProjectDetails.aspx?ProjectId=eebaab25dbae428594ac39569895c95c 2. books.google.co.in/books?isbn= 3. linkinghub.elsevier.com/retrieve/pii 4. jmm.sgmjournals.org/cgi/content/full/54/1/51 5. www.ist-world.org/ProjectDetails.aspx?ProjectId 6. www.zampbioworld.org/bionews/index.php/2008/10/16/9098 7. linkinghub.elsevier.com/retrieve/pii/ 8. www.pathogencombat.com/about/relevance.aspx - 42k 9.www.ars.usda.gov/research/projects/projects.htm 10. www.springerlink.com/index/j0x65l1827068854 11. www.who.int/mediacentre/factsheets/fs124/en/ - 23k 12. www.ugacfs.org/faculty/Erickson/EBWhitePaper.mpd.PDF 13.www.med-chem.com/Para/New/cc-intro.htm 14. www.horizonpress.com/gateway/foodborne-emerging-pathogens.html 15. gateway.nlm.nih.gov/MeetingAbstracts/102246291.html - 12k 16. cat.inist.fr/?aModele=afficheN&cpsidt=1909138 17. linkinghub.elsevier.com/retrieve/pii/S0168160507004199 18. jmm.sgmjournals.org/cgi/content/full/54/1/