{SOMEWHERE OVER THE RAINBOW.}

Saturday, July 21, 2007

title:{Marine biotoxins-detection methods}

Methods of analysis

In general

Ciguatoxins are odourless, tasteless and generally undetectable by any simple test. Therefore, bioassays have traditionally been used to monitor suspected fish. Many native tests for toxicity of fish have been examined including the discolouration of silver coins or copper wire, or the repulsion of flies and ants, but all of these were rejected as invalid (Park, 1994).
Feeding tests to cat or mongoose are simple and relatively sensitive but they are cumbersome and non-quantitative. The mouse bioassay requires purification of fish extracts since the mouse is not very sensitive to ciguatoxin. The alternative mosquito bioassay correlates well with cat and mouse bioassay. Other bioassays that have been developed have used chicken, brine shrimp and guinea pig atrium. All traditional bioassays have one common disadvantage, the lack of specificity for individual toxins. Recent studies have also focused on the development of chemical methods, such as TLC and LC for the detection and quantification of ciguatera-related toxins. Alternative assays based on immunochemical technology have been developed and show greatest promise for use in seafood safety monitoring programmes .

Bioassays

All the mentioned bioassays have the limited chemical specificity for individual toxins in common ,although for a broad screening this property can be advantageous detecting a poisoning. The bioassays are semi-quantitative and sensitive. Ciguatoxin induces characteristic signs of toxicity but the use of some animal species can be problematic in terms of cost and ethical difficulties.
in vivo assays

Mouse bioassay

The mouse bioassay, based on the method described by Banner et al. (1960) is presently the most widely used assay for the detection of ciguatoxins in fish. The method consists of injecting i.p. (intraperitoneal) serially diluted semi-purified or crude toxic extracts into mice and observing the symptoms for 24 hours. This assay has been described for the detection of ciguatoxins in up to 20 mg of ether extract from the flesh of fish. Mice are observed continuously for the first two hours, after that regularly checks are performed. Two mice are tested for each fraction. Mice are housed at 23 ± 2 oC and observed over seven days and signs and times to death recorded. Rectal body temperature is intermittently measured. The relationship between dose and time to death is used to quantify each fraction. Total lethality is expressed in mouse units (MU). For the mix of ciguatoxins found in carnivorous fish (Lewis and Sellin, 1992; Lewis et al. 1991) this relationship is approximated by log MU = 2.3 log (1 + T-1), where MU is the number of mouse units of ciguatoxin injected and T is time to death in hours (see also Table 7.2). One MU is the LD50 dose for a 20 g mouse which is equivalent to 5 ng, 48 ng and 18 ng of CTX-1, CTX-2 en CTX-3, respectively. (Lewis and Sellin, 1992; Lewis et al. 1991). It is recommended that additional purification is undertaken to separate the various toxins, especially the maitotoxins (see Chapter 7.3.1) from ciguatoxins since maitotoxins induce effects in mice, often mistaken for effects of ciguatoxins despite the clear differences (see Table 7.2). Therefore modified extraction procedures have been reported that may improve separation of these two types of toxins (Yokayama et al., 1988; Holmes et al., 1991; Holmes and Lewis, 1994; Legrand et al., 1992).
The mouse assay has been traditionally used but it is unsuitable as a market test. There are other disadvantages such as the variation in mouse weight, that must be limited involving a large breeding colony of the mice, and the death time relationship to dose is non-linear.

Chicken assay

This assay provides a rapid means of assaying the toxicity of fish liver by administering small portions of liver directly into the crop of young chickens at 10 percent of their body weight. Administration of fish flesh is physically more difficult but can be accomplished (Vernoux et al., 1985).

Mongoose and cat assay

For the mongoose (Banner et al., 1960) and cat assay (Lewis, 1987; Bagnis et al., 1985) the same procedure is followed as with chicken, only flesh of fish is fed and also in large quantity (5 to 15 percent of the test animal weight was fed). The cat is less satisfactory as test model because it often regurgitates part of the test meal. Test animals are observed for 48 hours. Although the tests are simple in screening fish for toxicity, they are cumbersome and not quantitative (Bagnis et al., 1987).

Brine shrimp assay

The brine shrimp assay was the first non-vertebrate assay developed. However, false positive results were caused by the toxic effects on brine shrimp of the Tween 80 recommended to emulsify the extract and no toxic effect attributable to ciguatoxin could be detected (Granade et al., 1976; Hungerford, 1993).

Mosquito assay

A bioassay using mosquitoes has also been developed. Only a few laboratories perform this assay, perhaps because of difficulties in obtaining and housing mosquitoes and a lack of familiarity in handling and recognising signs characteristic of intoxication by ciguatoxins. This procedure involves intrathoracic injection of the mosquitoes of serially diluted fish extract, and the toxicity is expressed in mosquito LD50.. It is a rapid assay, depending on a simple extraction requiring a small amount of fish. However, the assay is non-specific and non-quantitative (Bagnis et al., 1985, 1987).

Biochemical assays

Immunoassays

An ideal assay for the detection of marine toxins should be simple, highly sensitive and specific. Therefore the evaluation of marine toxin detection assays has moved in the direction of immunologic analysis (Hokama and Smith, 1990). Immunochemical methods such as a radioimmunoassay (RIA) (Hokama et al., 1977), a competitive enzyme immunoassay (EIA) (Hokama et al., 1983, 1984, 1986), and a rapid enzyme immunoassay stick test (Hokama, 1985; Hokama et al., 1985, 1987) have been developed. Problems with these immunochemical methods are their cross-reactivity with other polyether compounds and the limited antibody supply.

Radioimmunoassay

In 1977, a radioimmunoassay (RIA) was developed for the detection of ciguatoxin directly in contaminated fish (Hokama et al., 1977). In this assay, CTX conjugated to human serum albumin was injected into sheep and rabbits, thereby producing antibodies. The sheep antibody to CTX was used in the RIA after being purified and coupled to 125I as a label. In practice, some false positives were reported. This method could not be used for analyses of large numbers of fishes.
enzyme-linked immunosorbent assay (ELISA)
The practicality of detection improved when Hokama et al. (1983) developed an enzyme immunoassay (EIA) for the detection of CTX. The procedure incorporated a sheep anti-ciguatoxin horseradish peroxidase conjugate and colorimetric determination of absorbance following the enzymatic reaction. The assay was shown to be similar in efficacy to the earlier RIA developed, but less expensive and more practical. However, it was still tedious and therefore abandoned as detection method.

11:27 AM;

Wednesday, July 11, 2007

title:{Purpose of genetic modification}

The purpose of genetic modification is to

- give the organism/food desirable traits such ashigher yields
- increased muscle bulk (animals)
- ability to resist disease, pests or chemicals
- prolonged shelf-life
- higher nutritional value
- reduced allergens
- ability to grow in environments that are usually too hostile.

Some of these properties are especially important to developing countries where food supplies are scarce and conditions are often unsuitable for successful agriculture. e.g. drought, poor food storage or transport systems. People in developing nations may also benefit from increasing the levels of nutrients in foods as they are more likely to suffer from nutrient deficiencies.
Other uses of GM crops manufacture of medical drugs and vaccines (known as 'neutraceuticals') on a large scale from GM crops such as oilseed rape.

Development of renewable sources of valuable materials like plastics in engineered crops such as oilseed rape potato and maize.

Bioremediation, or the removal of toxic substances in the environment such as industrial waste.

10:27 PM;

Sunday, July 1, 2007

title:{How the transfer of genes are done}

First the desired gene is identified and isolated. It is broken up into fragments using restriction enzymes (‘genetic scissors’).

Fragments of DNA are sequenced and trimmed.

It is then associated with start and stop genes, a promotor and a genetic marker e.g. conferring antibiotic resistance forming a ‘construct’.

This construct is incorporated using DNA ligases (‘genetic glue’) into plasmids (extrachromosomal circular pieces of DNA) from organsims such as a soil bacterium called Agrobacterium Tumefaciens. The plasmid must be cut open using the same restrictases used on the DNA so that complementary base pairing can occur.

The recombinant plasmid containing foreign genes or transgenes is taken up by a bacterial cell which is then cultured , producing more copies of the gene so that it is amplified.
The colonies produced are screened for the presence of the genes. A colony containing the gene will be grown further (subculturing).

The gene produced can be integrated into the DNA in the nucleus of the cultured cells from the plant or animal that is to be modified.
a non-virulent bacterial vector is used for the transfer or laboratories use a technique called biolistics where a gun is used to fire genes carried on fragments of gold into plant cells 1, 14
Plant cells it their cell walls removed than (protoplasts) may be used to facilitate the entry of new genes.

If the appropriate DNA signals/ promoters have been attached to the gene prior to insertion into the host organism, the new gene will work because the genetic code is universal i.e.the genes of all organisms are made up of DNA.

If the plant cells are grown on selective media e.g. containing an antibiotic, those expressing the transgenes with an ab- resistant marker can be identified.

All methods rely on the fact that once genetically modified single cells or small pieces of plant tissue have been produced, they can be multiplied very rapidly (i.e plant cells are totipotent) using plant hormones so that whole GM Plants are produced.

It is possible for expression of the inserted gene to be localised to certain parts of the plant such as the leaves or stem, by using approptiate promotors. This allows pest resistance genes to be restricted to parts of a plant suscetible to attack by the pest, for example.
Animals can be genetically modified by inserting numerous copies of the desired gene into the nucleus of a fertilised egg. In about 1% cases, the host DNA will be altered and this transgene will be passed onto the embryo through cell division.

Another type of GM technology is the antisense approach where a resident gene in the organismis switched off or turned down (underexpressed). The gene is identified, cloned then attached to a more powerful promotor in reverse. Consequently, the gene is transcribed to produce antisense RNA in higher concentrations than the normal sense RNA, preventing protein synthesis.19 Alternatively, only a part of the normal gene is inserted. This antisense method is used in increasing the shelf- life of foods such as the FlavrSavr tomato, and in reducing the allergenicity of foods by inducing underexpression of the gene controlling the allergen.
Co-suppression has similar applications to antisense technolgy but works differently; in some cases. by inserting several copies of the undesired gene into a plant, silencing of both the endogenous and the introduced gene occurs so that the corresponding protein is not synthesised

10:30 PM;

Wednesday, June 20, 2007

title:{GM Products: Benefits and Controversies}

Benefits

Crops
- Enhanced taste and quality
- Reduced maturation time
- Increased nutrients, yields, and stress tolerance
- Improved resistance to disease, pests, and herbicides
- New products and growing techniques

Animals
- Increased resistance, productivity, hardiness, and feed efficiency
- Better yields of meat, eggs, and milk
- Improved animal health and diagnostic methods

Environment
- "Friendly" bioherbicides and bioinsecticides
- Conservation of soil, water, and energy
- Bioprocessing for forestry products
- Better natural waste management
- More efficient processing

Society
- Increased food security for growing populations

Controversies

Safety
- Potential human health impact: allergens, transfer of antibiotic resistance markers, unknown effects Potential environmental impact: unintended transfer of transgenes through cross-pollination, unknown effects on other organisms (e.g., soil microbes), and loss of flora and fauna biodiversity
- Access and Intellectual Property
- Domination of world food production by a few companies
- Increasing dependence on Industralized nations by developing countries
- Biopiracy—foreign exploitation of natural resources

Ethics
- Violation of natural organisms' intrinsic values
- Tampering with nature by mixing genes among species
- Objections to consuming animal genes in plants and vice versa
- Stress for animal

Labeling
- Not mandatory in some countries (e.g., United States)
- Mixing GM crops with non-GM confounds labeling attempts

Society
- New advances may be skewed to interests of rich countries

10:51 PM;

Monday, June 11, 2007

title:{Introduction to Genetically modified food}

Genetically Modified (GM) foods are produced from genetically modified organisms(GMO) which have had their genome altered through genetic engineering techniques. The general principle of producing a GMO is to insert DNA that has been taken from another organism and modified in the laboratory into an organism's genome to produce both new and useful traits or phenotypes. Typically this is done using DNA from certain types of bacteria. GM Foods have been available since the 1990s, with the principal ones being derived from plants; soybean, corn, canola and cotton seed oil.

The first commercially grown genetically modified food crop was the Flavr Savr tomato which was made more resistant to rotting by Californian company Calgene. Calgene was allowed to release it into the market in 1994 without any special labeling, where it was welcomed by consumers who purchased the fruit at two to five times the price of standard tomatoes. However, production problems and competition from a conventionally bred, longer shelf-life variety prevented the product from becoming profitable.

A variant of the Flavr Savr was used by Zeneca to produce tomato paste which was sold in Europe during the summer of 1996. Its labeling and pricing were designed as a marketing experiment which proved that, at the time, European consumers would accept genetically engineered foods. This attitude would be drastically changed after outbreaks of Mad Cow Disease weakened consumer trust in government regulators, and protesters rallied against the introduction of Monsanto's "Roundup-Ready" soybeans. The next GM crops included insect-protected cotton and herbicide-tolerant soybeans both of which were commercially released in 1996. These crops have been widely adopted in the United States. They have also been extensively planted in several other countries (Argentina, Brazil, South Africa, India, and China) where agriculture is a major part of the total economy. Other GM crops include insect-protected maize and herbicide-tolerant maize, cotton, and rapeseed varieties.

In the USA regulation of a genetically modified food is determined by the objective characteristics of the food and the intended use of the food, irrespective of the way it was developed. FDA policy states that a formal pre-market review by the FDA is to be taken when the objective characteristics of any substance added to the food raises safety issues.

Prior to marketing a new GM food product, manufacturers are required to submit documentation to the FDA to demonstrate its safety and then await approval before selling it to consumers. The context for assessing safety of novel foods is the fact that existing foods often contain toxic components but are still able to be consumed safely. For instance, potatoes and tomatoes can contain toxic levels of solanine and alpha-tomatine alkaloids respectively, and this situation is recognised in the concept of "Substantial Equivalence" that was developed by the OECD in 1993 as a criterion for identifying whether a novel food is at least as safe as the equivalent existing food. The US FDA takes a safety assessment approach that is consistent with this OECD concept in their regulation of novel foods (including those made by recombinant DNA methods). This policy is outlined in an FDA statement. Critics of GM food believe this regulatory model fails to sufficiently protect consumers and claim that the FDA is subject to pressure and influence by industry. One concern voiced is that a novel crop may have unintended changes created during the insertion of new genetic material.

On the other hand, plant scientists, backed by results of modern comprehensive profiling of crop composition, point out that crops modified using GM techniques are less likely to have unintended changes than are conventionally bred crops.

7:43 PM;

Sunday, April 29, 2007

title:{foodborne illnesses in seafood}

Here are the foodborne illness associated with seafood. I have elaborate about the more common ones and those associated with shrimps(shellfish).

These are the bacteria pathogens which can be found in raw and processed seafood.

Listeria monocytogenes

L. monocytogenes has been associated with such foods as raw milk, supposedly pasteurized fluid milk, cheeses (particularly soft-ripened varieties), ice cream, raw vegetables, fermented raw-meat sausages, raw and cooked poultry, raw meats (all types), and raw and smoked fish. Its ability to grow at temperatures as low as 3°C permits multiplication in refrigerated foods.

Salmonella

Raw meats, poultry, eggs, milk and dairy products, fish, shrimp, frog legs, yeast, coconut, sauces and salad dressing, cake mixes, cream-filled desserts and toppings, dried gelatin, peanut butter, cocoa, and chocolate.
Various Salmonella species have long been isolated from the outside of egg shells. The present situation with S. enteritidis is complicated by the presence of the organism inside the egg, in the yolk. This and other information strongly suggest vertical transmission, i.e., deposition of the organism in the yolk by an infected layer hen prior to shell deposition. Foods other than eggs have also caused outbreaks of S. enteritidis disease

Clostridium botulinum

The types of foods involved in botulism vary according to food preservation and eating habits in different regions. Any food that is conducive to outgrowth and toxin production, that when processed allows spore survival, and is not subsequently heated before consumption can be associated with botulism. Almost any type of food that is not very acidic (pH above 4.6) can support growth and toxin production by C. botulinum. Botulinal toxin has been demonstrated in a considerable variety of foods, such as canned corn, peppers, green beans, soups, beets, asparagus, mushrooms, ripe olives, spinach, tuna fish, chicken and chicken livers and liver pate, and luncheon meats, ham, sausage, stuffed eggplant, lobster, and smoked and salted fish.

Vibrio cholerae Serogroup O1

Cholera is generally a disease spread by poor sanitation, resulting in contaminated water supplies. This is clearly the main mechanism for the spread of cholera in poor communities in South America. The excellent sanitation facilities in the U.S. are responsible for the near eradication of epidemic cholera. Sporadic cases occur when shellfish harvested from fecally polluted coastal waters are consumed raw. Cholera may also be transmitted by shellfish harvested from nonpolluted waters since V. cholerae O1 is part of the autochthonous microbiota of these waters.

Vibrio cholerae Serogroup Non-O1

Shellfish harvested from U.S. coastal waters frequently contain V. cholerae serogroup non-Ol. Consumption of raw, improperly cooked or cooked, re-contaminated shellfish may lead to infection.

Vibrio parahaemolyticus

Infections with this organism have been associated with the consumption of raw, improperly cooked, or cooked, recontaminated fish and shellfish. A correlation exists between the probability of infection and warmer months of the year. Improper refrigeration of seafoods contaminated with this organism will allow its proliferation, which increases the possibility of infection.

Vibrio vulnificus

This organism has been isolated from oysters, clams, and crabs. Consumption of these products raw or recontaminated may result in illness.

These are the parasites that can be found in raw seafood

Anisakis simplex and related worms

Seafoods are the principal sources of human infections with these larval worms. The adults of A. simplex are found in the stomachs of whales and dolphins. Fertilized eggs from the female parasite pass out of the host with the host's feces. In seawater, the eggs embryonate, developing into larvae that hatch in sea water. These larvae are infective to copepods (minute crustaceans related to shrimp) and other small invertebrates. The larvae grow in the invertebrate and become infective for the next host, a fish or larger invertebrate host such as a squid. The larvae may penetrate through the digestive tract into the muscle of the second host. Some evidence exists that the nematode larvae move from the viscera to the flesh if the fish hosts are not gutted promptly after catching. The life cycles of all the other anisakid genera implicated in human infections are similar. These parasites are known to occur frequently in the flesh of cod, haddock, fluke, pacific salmon, herring, flounder, and monkfish.

Diphyllobothrium spp.

The larvae of these parasites are sometimes found in the flesh of fish.

Eustrongylides sp.

Fish from fresh, brackish or salt water.

Ascaris lumbricoides and Trichuris trichiura

The eggs of these worms are found in insufficiently treated sewage-fertilizer and in soils where they embryonate (i.e., larvae develop in fertilized eggs). The eggs may contaminate crops grown in soil or fertilized with sewage that has received nonlethal treatment; humans are infected when such produce is consumed raw. Infected foodhandlers may contaminate a wide variety of foods.

Other parasites

· Eustrongylides sp.
· Acanthamoeba and other free-living amoebae

These are the virus that sometimes contaminate seafood

Hepatitis A Virus

HAV is excreted in feces of infected people and can produce clinical disease when susceptible individuals consume contaminated water or foods. Cold cuts and sandwiches, fruits and fruit juices, milk and milk products, vegetables, salads, shellfish, and iced drinks are commonly implicated in outbreaks. Water, shellfish, and salads are the most frequent sources. Contamination of foods by infected workers in food processing plants and restaurants is common.

The Norwalk virus family

Norwalk gastroenteritis is transmitted by the fecal-oral route via contaminated water and foods. Secondary person-to-person transmission has been documented. Water is the most common source of outbreaks and may include water from municipal supplies, well, recreational lakes, swiming pools, and water stored aboard cruise ships.
Shellfish and salad ingredients are the foods most often implicated in Norwalk outbreaks. Ingestion of raw or insufficiently steamed clams and oysters poses a high risk for infection with Norwalk virus. Foods other than shellfish are contaminated by ill food handlers.

Other Gastroenteritis Viruses

Viral gastroenteritis is transmitted by the fecal-oral route via person-to-person contact or ingestion of contaminated foods and water. Ill food handlers may contaminate foods that are not further cooked before consumption. Enteric adenovirus may also be transmitted by the respiratory route. Shellfish have been implicated in illness caused by a parvo-like virus.

Other virus that are associated with seafood
· Hepatitis E virus
· Rotavirus

These are the nature toxins that are found in seafood

Various Shellfish-Associated Toxins

Shellfish poisoning is caused by a group of toxins elaborated by planktonic algae (dinoflagellates, in most cases) upon which the shellfish feed. The toxins are accumulated and sometimes metabolized by the shellfish.

Types pf shellfish poisoning
-Paralytic Shellfish Poisoning
-Diarrheic Shellfish Poisoning
-Neurotoxic Shellfish Poisoning
-Amnesic Shellfish Poisoning

All shellfish (filter-feeding molluscs) are potentially toxic. However, PSP is generally associated with mussels, clams, cockles, and scallops; NSP with shellfish harvested along the Florida coast and the Gulf of Mexico; DSP with mussels, oysters, and scallops, and ASP with mussels.

Other toxins

· Ciguatera poisoning
· Scombroid
· Tetrodotoxin (Pufferfish)

12:23 AM;

Saturday, April 14, 2007

title:{Recall plan 2}

Today I shall touch on how to develop a recall plan

Recall is a voluntary action taken by manufacturers and distributors to remove food which is in violation of laws administered by the FDA. FDA may request a recall, but cannot order one without a court order. Product recovery is only classified as a recall when the product is violative.

Recall is time consuming and costly. It may destroy a company’s reputation. Careful control over production and processing is an absolute necessity to prevent the need for a recall. In the event a recall is necessary, use the plan, paying close attention to the notification step, the use of designated spokespersons, and good communications to all involved. Careful planning will allow the processor to implement the recall in a timely, organized fashion with a minimum of confusion. This will help to minimize the public health consequences and losses to the company.

Product identification
Each batch or production lot of the product will be properly coded as described in. This code will allow the product lot to be identified as to date, batch product personnel production records, and ingredient. Without records, the whole product line is suspect. All of the production procedures are suspect.

Notification
Persons to be notified in the event of a recall include FDA, key company personnel, and distributors. A list of phone numbers and names have to be prepared.
In the event of a recall, the media may also need to be notified. Only one employee should be assigned the duty of spokesperson. Questions should be refered to him.The notification shall include the product, container size, and code of affected lots. The extent of the hazard and the level of the recall will be as determined by FDA. FDA will approve the recall strategy. The notification will include instructions for consumers and distributors for product recovery and information feedback. The contact person should

Product Recovery
Plans for recovery include procedures for segregation of affected lots, storage, warehousing, and control. Procedures in place shall allow determination of the effectiveness of the recall. The recall is concluded when FDA determines that recovery is adequate and there is no longer any threat to the public.

Things to include in the recall plan
A. Product coding
B. Raw materials records
C. Processing & production records
D. Departures from scheduled processes
E. Records of initial distribution
F. Key contacts and phone numbers
G. Sample notification form
H. Plans for recovery of recalled product and evaluation of effectiveness.

Many thanks to John E. Rushing, Ph.D. Department of food science.

11:11 PM;