DNA sequencing - applications, limitations & other methods

 

Applications of DNA sequencing

Used for the identification of genes or mutations responsible for hereditary disorders.

Used for parental verification, criminal investigation and identification of individuals using available samples such as hair, nail, blood or tissue.

Identification of GMO species and any minor variations in the plant genome.

Used to construct whole chromosomal maps, restriction digestion maps, and genome maps.

Open reading frames, non-open reading frames and protein-coding DNA sequences can be identified.

Used in exon/ intron, repeat sequence and tandem repeat identification and detection.

Used in gene manipulation and gene editing

New variations in nature can be determined through sequencing.

Used in metagenomic studies

For Microbial identification and study of the new bacterial species.

For evolutionary studies and for generating evolutionary map

For studying asymptomatic high-risk population, prior to the occurrence of disease

Limitations of DNA sequencing

DNA sequencing is performed using computer algorithm-based assistive techniques and so for such computational data processing high-speed supercomputer is required.

It is difficult to sequence sequences like tandem repeats, repetitive DNA, fragmented genes, other duplicated regions, etc. 

There are chances of errors in the pre-sample processing which will result in economic losses.

Automated sequencing

The identification of sequence from the electrophoretic pattern through manual Sanger method was tedious. Recent advances have enabled the semi-automated Sanger sequencing method which is Sanger’s method with some minor variations.

Here, instead of 4 different reaction tubes, a single tube is used and thus during electrophoresis the DNA runs in a single lane in gel. Fluorescent-labeled ddNTPs are used.  Capillary electrophoresis is used to separate DNA molecules on the basis of size.  It is powerful enough to separate single base pair fragment. The chromatogram generated after Capillary electrophoresis will give output as fluorescent peaks, each colour representing a particular ddNTP.

In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths.

Pyrosequencing:

This was described in 1993 by Bertil Pettersson, Mathias Uhlen and Pål Nyren.  Principle of the method is the detection of the pyrophosphate released during the chain reaction of nucleotide addition. The order of the nucleotide is determined by the PPi released during the joining of two adjacent nucleotides.

Three enzymes are required in the pyrosequencing method which work in a sequential manner for the detection of the PPi. The three enzymes are:

·                  DNA polymerase (without exonuclease activity)

·                  Luciferase

·                  Sulfurylase

The real-time polymerase activity monitoring is done for the detection of the released pyrophosphate

 
Enzyme polymerase add dNTPs to single-stranded DNA. If the correct complementary base is added, pyrophosphate is released.

Enzyme sulfurylase converts PPi into ATP (energy) with the help of the APS (adenosine 5´ phosphosulfate).

In the presence of ATP and oxygen, luciferase  converts luciferin into oxyluciferin  and a photon of light is released.  

So, once the correct nucleotide is added, light will be released by the enzymatic reaction which is detected by a photodiode or a photomultiplier tube.

Based on the substrate used, two types of pyrosequencing methods are there, solid-phase pyroseq and liquid phase pyroseq.

The pyrosequencing method is more accurate and faster than Sanger sequencing.

But this method involves more chemical steps and thus is more complex. 

Whole-genome shotgun sequencing:

This technique is also a modification of Sanger’s chain termination method. Shotgun sequencing concept was originally discovered by Sanger F and his colleagues for sequencing the whole genome. This technique can be used to sequence the entire genome of an organism.

The principle is the same as Sanger’s method.  There is an additional step of DNA fragmentation to generate multiple fragments. The entire genome of an organism is fragmented with the help of endonuclease enzymes or by mechanically, and the smaller fragments are sequenced individually.  

The computer-based software analyses each and every overlapping fragment and reassemble it to generate the complete sequence of entire genome.

Steps involved:

1.               Fragmentation of DNA to about 2 -20kb.

2.        Formation of libraries of subfragments, fragments are ligated in vectors and an entire library is generated  

3.               Sequencing the subfragments  

4.               Generation and reading of overlapping fragments (contigs) by using computer.  

 The technique is faster and cheaper, and can be used to sequence whole genome of an organism. This technique depends on computational analysis and a huge, powerful, supercomputer is required.

In 1981, for sequencing cauliflower mosaic virus genome shotgun sequencing method was used.

 

Clone by clone sequencing:

For sequencing the whole genome, Clone by Clone Method can be used.  In 1980 and 1990 the genomes of C. elegans and S. cerevisiae were sequenced using the clone by clone sequencing, respectively and this technique was used during the human genome project.

This method is similar to shot gun sequencing method, but have additional steps.

1.      In the first step, instead of smaller fragments, large clumps of DNA fragments are constructed and the location of each fragment is noted through gene mapping.  Using bacterial artificial chromosome, multiple copies of each fragment are generated. 

2.      In the next step, all these copied fragments are further fragmented into smaller pieces and inserted into vectors.

3.      Now sequencing of these short fragments are performed as per shotgun technique and overlapping fragments are assembled by using computer.

4.      In the last step, the data obtained during gene mapping is used to assemble the complete sequence. So the sequences can be arranged on each chromosome based on their location.

Sequencing of whole chromosomes can be done without any gaps.

   More tedious, time-consuming and costly since more procedures like mapping, cloning, and restriction digestion are involved. 

Next-generation sequencing (NGS) or High-throughput sequencing

The most recent set of DNA sequencing technologies are collectively referred to as next-generation sequencing.  Next-generation sequencing involves amplification of millions of copies of a particular fragment and sequences are analyzed by computational program.  There are a variety of next-generation sequencing techniques that use different technologies.  Examples are Polony sequencing, Massively parallel signature sequencing (MPSS), 454 pyrosequencing, Illumina (Solexa) sequencing, Combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Nanopore DNA sequencing, etc.

These varieties of next-generation sequencing techniques use different technologies, however, most share a common set of features,

·                  Highly parallel: many sequencing reactions take place at the same time

·                  Micro scale: reactions are tiny and many can be done at once on a chip

·                  Fast: because reactions are done in parallel, results are ready much faster

·                  Low-cost: sequencing a genome is cheaper than with Sanger sequencing

Next-generation sequencing is kind of like running a very large number of tiny Sanger sequencing reactions in parallel thus allowing, large quantities of DNA to be sequenced much more quickly and cheaply. The NGS is the most advanced, fast, accurate and 100% effective technique for DNA sequencing.

 There are several platforms for NGS, they are

·                  Single-molecule real-time (RNAP) sequencing

·                  Illumina (Solexa) sequencing

·                  Polony sequencing

·                  DNA nano ball sequencing

·                  SOLiD sequencing

·                  Single-molecule SMRT(TM) sequencing

·                  Massively parallel signature sequencing (MPSS)

·                  High throughput sequencing

·                  Helioscope (TM) single-molecule sequencing

 The NGS process can be divided into 4 different steps:

1.               Library preparation

2.               Cluster generation

3.               DNA sequencing

4.               Data analysis

These processes are common to all next-generation DNA sequencing techniques:

1. Library Preparation: DNA is randomly fragmented to generate libraries, which are then ligated together using specific linkers/adapters.

2. Cluster generation or Amplification: PCR and clonal amplification techniques are used to amplify the library.

3. Sequencing: DNA is sequenced using one of several methods.

4. Data Analysis: Bioinformatics techniques are used to process the generated data in order to align the reads, find deviations, and assemble the entire genome.

Library preparation

Firstly, DNA is fragmented either enzymatically or by sonication to create smaller strands. Short and double stranded pieces of synthetic DNA called Adaptors are then ligated to these fragments using DNA ligase enzyme. The adaptors enable the sequence to become bound to a complementary counterpart. 

Cluster generation/Amplification 

The DNA library is amplified so as to generate enough signal from the sequencer in the next stage, which is accurate and can be reliably detected. PCR amplification methods produce many DNA clusters in enormous quantities.

 Sequencing & Data Analysis 

Various companies have developed multiple types of Next Generation Sequencing competitive techniques (Pyrosequencing, Sequencing by ligation (SOLiD), Reversible terminator sequencing (Illumina)

 Applications of NGS: 

 Researchers have been able to gather enormous amounts of genomic sequencing data through next-generation sequencing. This technology has a wide range of applications including the diagnosis and understanding of complex diseases, whole-genome sequencing, epigenetic analysis, mitochondrial sequencing, transcriptome sequencing, understanding how altered expression of genetic variants affects an organism, and exome sequencing (sequence of all the exons in a genome, which is the protein-coding portion of a genome. In humans, the exome is about 1.5% of the genome)– mutations in the exome are thought to contain up to 90% of mutations in the human genome, which results in disease.

 Gene therapy in cancer treatment focuses on methods like antisense RNA, which prevents the synthesis of targeted proteins, and suicide gene therapy, which introduces genes to selectively kill cancer cells. The challenge lies in delivering these genes precisely to avoid harming healthy cells. Sequencing tumor genomes enables tailored chemotherapy and personalized medicine, revolutionizing diagnostics and treatment planning.

 The decreasing cost of DNA sequencing leads to its wider adoption, but it also presents challenges. Processing and storing the vast amounts of sequencing data pose computational hurdles. Ethical concerns arise regarding ownership and security of individuals’ DNA data, as it can be misused by insurance companies, mortgage brokers, or employers.

 Additionally, while sequencing can identify disease risks, issues remain regarding patient awareness and the availability of effective treatments.

 

 

 

Preparation of Acetate Buffer

 Aim: To prepare 0.1 M acetate buffer of pH 5.0 

Procedure

Acetate buffer has a pH range of 3.6 to 5.6. To obtain a buffer of respective pH, mix A (acetic acid) and B (sodium acetate) in the proportions indicated below and adjust the final volume to 100 ml using distilled water The pH can be checked using a sensitive pH meter.

Stock solution A- 0.2 M solution of acetic acid

Stock solution B- 0.2 M solution of sodium acetate

 Mixed 14.8 ml of A and 35.2 ml of B diluted to 100 ml using distilled water to obtain 0.1 M acetate buffer of pH 5.0

 

Solution A (ml) (Acetic acid)	Solution B (ml) (Sodium acetate)	pH
46.3	3.7	3.6
44	6	3.8
41	9	4
36.8	13.2	4.2
30.5	19.5	4.4
25.5	24.5	4.6
20	30	4.8
14.8	35.2	5.0
10.5	39.2	5.2
8.8	41.2	5.4
4.8	45.2	5.6

 Result

The buffer prepared is 0.1 M acetate buffer with pH 5. It is confirmed using a pH meter.

 

Food borne infections - Bacterial: Staphylococcal, Escherichia, Salmonella

 Salmonellosis

Salmonellosis is the most frequently occurring bacterial food-borne illness.

Salmonellae are gram-negative non-spore-forming rods that ferment glucose, usually with gas, but usually do not ferment lactose or sucrose. They grow over a wider range of temperature, pH, and water activity. They grow well at room temperatures, optimum is about 37^oC. The pH range for growth is 4.1 to 9.0. The lowest aw for growth varies with the food but is about 0.93 to 0.95.

The likelihood of infection by consumption of a food containing salmonellae depends on the resistance of the consumer, the infectiveness of the particular strain of Salmonella. and the number of organisms ingested. Salmonellae can attain considerable numbers in foods without causing detectable alterations in appearance, odor, or even taste. Human beings and animals are directly or indirectly the source of the contamination of foods with salmonellae. The organisms may come from cases of the disease or from carriers.

Most frequently isolated serovars, such as S. typhimurium cause human gastroenteritis.

A large variety of foods are involved in causing outbreaks of Salmonella infections. Most common are various kinds of meats, poultry and products from them, especially if they are held unrefrigerated for long periods. Fresh meats may carry Salmonella bacteria that caused disease in the slaughtered animals or may be contaminated by handlers. Meat products, such as meat pies, hash, sausages, cured meats (ham, bacon, and tongue), sandwiches, and chili, often are allowed to stand at room temperatures, permitting the growth of salmonellae. Milk and milk products, including fresh milk, fermented milks, ice cream, and cheese, have caused infections. Since eggs may carry the salmonellae, foods made with eggs and not sufficiently cooked or pasteurized may carry live organisms, e.g., pastries filled with cream or custard, cream cakes, etc.

The organisms also may come from cats, dogs, swine, and cattle, but more important sources for foods are poultry and their eggs and rodents. About one-third of all the food products involved in Salmonella outbreaks are meat and poultry products, eggs etc. Infected rodents, rats and mice, may contaminate unprotected foods with their faeces and thus spread Salmonella bacteria. Flies may play an important role in the spread of Salmonella, especially from contaminated fecal matter to foods.

Changes in processing, packaging, and compounding of foods and feeds in recent years have resulted in an apparent increase in salmonellosis from these products. Salmonellae have been introduced by the incorporation of cracked and dried eggs in baked goods, candy, ice cream, and convenience foods such as cake and cookie mixes.

Large-scale handling of foods, increase the spread of trouble, precooked foods and food vending machines add to the risk. Feeds, especially those from meat or fish by-products, may carry Salmonellae to poultry or meat animals.

As with other infectious diseases, individuals differ in their susceptibility to Salmonella infections, but in general morbidity is high in any outbreak. The susceptibility of humans varies with the species and strain of the organism and the total numbers of bacteria ingested

Salmonellosis has a longer incubation period - usually 12 to 36 hr. The principal symptoms of a Salmonella gastrointestinal infection are nausea, vomiting, abdominal pain, and diarrhea that usually appear suddenly. This may be preceded by a headache and chills. Other evidences of the disease are watery, greenish foul-smelling stools, prostration, muscular weakness, faintness, usually a moderate fever, restlessness, twitching, and drowsiness. The mortality is low, being less than 1 percent. The severity and duration vary not only with the amount of food eaten and hence the numbers of Salmonella bacteria ingested and with the individual.

Intensity may vary from slight discomfort and diarrhea to death in 2 to 6 days. Usually, the symptoms persist for 2 to 3 days, followed by uncomplicated recovery, but they may linger for weeks or months. About 0.2 to 5 percent of the patients may become carriers of the Salmonella organism.

The laboratory diagnosis of the disease is difficult unless Salmonella can be isolated from the suspected food and from the stools of individuals.

For the prevention of outbreaks of food-borne Salmonella infections:

(I) avoid contamination of the food with salmonellae from sources such as diseased human beings and animals and carriers and ingredients carrying the organisms, e.g., contaminated eggs

(2) destroy the organisms in foods by heat (or other means) when possible, as by cooking or pasteurization, paying special attention to held-over foods

(3) prevent the growth of Salmonella in foods by adequate refrigeration or by other means.

In the prevention of contamination, care and cleanliness in food handling and preparation are important. The food handlers should be healthy (and not be carriers) and clean. Rats and other vermin and insects should be kept away from the food. Ingredients used in foods should be free of salmonellae, if possible.

 Foods should not be allowed to stand at room temperature for any length of time, but if this happens, thorough cooking will destroy the Salmonella organisms unlike Staphylococcus enterotoxin.

Warmed-over leftovers, held without refrigeration, often support the growth of Salmonella, as may canned foods that have been contaminated and held after the cans were opened. Inspection of animals and meats at packing houses may remove some Salmonella-infected meats but is not in itself a successful method for the prevention of human salmonellosis.

Staphylococcus Food Intoxication

One of the most commonly occurring food poisonings is caused by the ingestion of the enterotoxin formed in food during growth of certain strains of Staphylococcus aureus. The toxin is termed an enterotoxin because it causes gastro- enteritis or inflammation of the lining of the intestinal tract.

Staphylococcus, typically appear as clusters of grapes or in pairs and short chains. Growth on solid media usually is golden or yellow but may be unpigmented in some strains. Most enterotoxin-producing S. aureus cultures are coagulase-positive (coagulating blood plasma), produce a thermal stable nuclease, and are facultative in their oxygen requirements in a complex glucose medium but grow better aerobically than anaerobically. Some of the toxigenic cocci are very salt-tolerant (10 to 20 percent NaCl), and also tolerate nitrites fairly well and therefore can grow in curing solutions and on curing and cured meats if other environmental conditions are favourable. They also are fairly tolerant of dissolved sugars (50 to 60 percent sucrose). They are fermentative and proteolytic but usually do not produce obnoxious odors in most foods or make them appear unattractive.

S. aureus produces six enterotoxins (A, B, Cl , C2 , D, and E) that differ in toxicity; most food poisoning is from type A. The range of conditions permitting growth of the staphylococcus, and hence toxin production, varies with the food involved. The better medium the food is for the coccus, the wider the range of temperature, pH, or aw over which growth can take place. The temperature range for growth and toxin production is about 4 to 46^oC, depending on the food. S. aureus grows most rapidly between 20 and 45^oC. A and D are more often associated with food-poisoning outbreaks. Toxin production and growth of the Staphylococcus is best at 40^oC.

The sources from which the food-poisoning staphylococci enter foods are human or animal. The nasal passages of many persons are laden with these organisms, which are a common cause of sinus infections. Also, boils and infected wounds may be sources. Staphylococci are becoming increasingly important in causing mastitis in cows, and some of these cocci can form enterotoxin in milk or milk products.

Production of enterotoxin by the Staphylococci is more likely when competing microorganisms are absent, few, or inhibited for some reason. Therefore, a food that had been contaminated with the Staphylococci after a heat process would be favourable for toxin production. The type of food has an influence on the amount of enterotoxin produced; much is produced in meat products and custard-filled bakery goods. The presence of starch and protein in considerable amounts enhance toxin production by the staphylococci. Type B enterotoxin is the most heat-resistant. The normal cooking of foods will not destroy the toxin formed therein before the heat process. Such foods might cause poisoning, although no live staphylococci could be demonstrated.

About 75 percent of all staphylococcal food-poisoning outbreaks occur because of inadequate cooling of foods. Other foods incriminated include other meats and meat products, fish and fish products, milk and milk products, cream sauces, salads, puddings, custards, pies, and salad dressings. The fillings in bakery goods usually are good culture media in which the staphylococci can grow during the time that these foods are held at room temperatures. Toxin production has even been reported in imitation cream filling. The contaminated leftover turkey, or other fowl, along with the gravy and dressing, is kept out of the refrigerator, it may cause poisoning.

Foods that ordinarily are too acid for good growth of the staphylococci may have this acidity reduced by added ingredients, such as eggs or cream, and then become dangerous. Growth and toxin production by staphylococci may take place in the steam tables in cafeterias and restaurants and in food-vending machines that keep foods heated for extended periods if temperatures and times are not properly controlled.

Individuals differ in their susceptibility to staphylococcus poisoning, so that of a group of people eating food containing toxin some may become very ill and few may be affected little or not at all. The incubation period for this kind of poisoning usually is brief, 2 or 4 hr unlike the other common food poisonings and infections, which usually have longer incubation periods.

The most common human symptoms are nausea, vomiting, retching, abdominal cramping of varying severity, and diarrhea. Blood and mucus may be found in stools and vomitus in severe cases. Headache, muscular cramping, sweating, chills, prostration, weak pulse, shock, and shallow respiration may occur. Usually a subnormal body temperature is found rather than fever. The duration is brief, usually only a day or two, and recovery ordinarily is uneventful and complete. The mortality is extremely low.

For the most part no treatment is given, except in extreme cases, when saline solutions may be given parenterally to restore the salt balance and counteract dehydration. Diagnosis of the poisoning would depend, on isolation of staphylococci and demonstration that this produce enterotoxin or isolation and detection of the enterotoxin.

The means of prevention of outbreaks of staphylococcus food poisoning include

(1) prevention of contamination of the food with the staphylococci  (2) prevention of the growth of the staphylococci and (3) killing staphylococci in foods.

Contamination of foods can be reduced by general methods of sanitation, by using ingredients free from the cocci, e.g., pasteurized rather than raw milk, and by keeping employees away from foods when these workers have staphylococcal infections in the form of colds, boils, carbuncles, etc. Growth of the cocci can be prevented by adequate refrigeration of foods and, in some instances, by adjustment to a more acid pH. Also the addition of a bacteriostatic substance, such as serine or an antibiotic, has been suggested. Some foods may be pasteurized to kill the staphylococci before exposure of the foods to ordinary temperatures, e.g., pasteurization of custard filled puffs and eclairs for 30 min at 190.6 to 218.3 C oven temperature.

Enteropathogenic Escherichia Coli

E. coli is generally regarded as part of the normal flora of the human intestinal tract and that of many animals. Serotypes of E. coli have been implicated in human diarrheal diseases or foodpoisoning outbreaks are designated as enteropathogenic E. coli (EEC).

The human disease syndromes resulting from the ingestion of EEC have been divided into two main groups. The first group consists of strains which produce an enterotoxin and result in a choleralike or enterotoxigenic illness in humans. These enterotoxigenic strains usually produce two enterotoxins, a heat-stable (ST) and a heat-labile (LT) toxin, and are thought to be responsible for infantile diarrheal diseases and traveller’s diarrhea. EEC serotypes capable of elaborating the enterotoxins if ingested, are colonized in the upper small intestine and produce the enterotoxins. The enterotoxins cause fluid accumulation in the intestinal lumen.

The second major group consists of invasive strains which produce a cytotoxin and result in the invasive illness, colitis, or dysentery like syndrome. These serotypes are non-enterotoxigenic, grow in the colon, and invade or penetrate the epithelial cells of colonic mucosa, resulting in fever, chills, headache, abdominal cramps etc

A large infective dose of EEC is required for either the enterotoxigenic or invasive illness to occur. Therefore, foods must be highly contaminated or inadequately preserved or refrigerated to allow for prolific growth. The optimal temperature for growth is 37^oC, with a temperature range for growth of 10 to 40^oC. The optimal pH for growth is 7.0 to 7.5, with the minimum at pH 4.0 and the maximum at pH 8.5. The organism is relatively heat sensitive and can readily be destroyed at pasteurization temperatures and by the proper cooking of foods.

In addition, the hemorrhagic E. coli (EHEC) strains can result in illness in humans as manifested by bloody diarrhea and severe abdominal pain.

Thorough and sanitary methods of cooking, chilling foods after use, maintaining personal hygiene, treating water and ensuring sanitary disposal of sewage can control E. coli infections

Mycotoxins

 Fungal toxins

Mycotoxins are fungal metabolites which may contaminate foods, animal feeds, and are toxic to humans or their domestic animals. They are proposed to have carcinogenic properties. As common adulterants of foods or animal feeds, they are important food contaminants. Mycotoxicosis results from the ingestion of toxin in a mold-contaminated food.

            The fungi include the molds, yeasts, mildews, blights, rusts, and mushrooms. Many fungi are useful. Some are edible, e.g., mushrooms and single-cell protein from yeast. Others are widely used in industrial and food fermentations; e.g., Aspergillus oryzae is used in the production of soy sauce, miso, and sake, and molds take part in the ripening of certain cheese. The metabolite of Penicillium chrysogenum, penicillin, has contributed immensely to human well-being. Some mushrooms are harmful or poisonous to humans, but in contrast, molds have generally been regarded as harmless.

The two predominant genera of fungi in stored products are probably Penicillium and Aspergillus, members of which produce mycotoxins.

 Ergot

The first documented case of mycotoxicosis was that of rye ergot. Claviceps purpurea parasitizes rye and other grains and produces many lysergic acid derivatives which are responsible for the syndrome. Consumption of the infested grain or flour made from it over a period of time can result in gangrenous ergotism.

Outbreaks of ergotism were quite common during the Middle Ages. More recent outbreaks of ergotism have been reported in the Soviet Union (1926-1927), England (1928), and France (1951).

Claviceps purpurea is a parasite of grasses including cereals. As part of its life cycle, the tissues of infected grains are replaced by sclerotium which is fungal mycelium. The sclerotium helps to survive the adverse conditions of the winter and germinate later. It is also known as an ergot.

Ergotism is the name for severe pathological syndromes affecting humans or other animals that have ingested plant material containing ergot alkaloid, such as ergot-contaminated grains. The common name for ergotism is "St. Anthony's fire", in reference to the severe burning sensations in the limbs.

Ergotism, is due to ergot alkaloids which is toxic and cause a constriction of the peripheral blood capillaries leading, to fingers and toes becoming gangrenous and necrotic.Ergots contain alkaloid metabolites which may be incorporated into the flour, and the bread, made from the harvested grain. The ergot sclerotium contains high concentrations (up to 2% of dry mass) of the alkaloid ergotamine, and ergoline group. Ergot alkaloids have a wide range of biological activities including effects on circulation and neurotransmission. Ergot contains lysergic acid which is a precursor for the synthesis of LSD which is a potent synthetic hallucinogenic drug.

There are two types of ergotism. The first is characterized by muscle spasms, fever and hallucinations and the victims may appear dazed, be unable to speak, or have other forms of paralysis or tremors, and suffer from hallucinations. This is caused by stimulation of the central nervous system by some of the alkaloids. The second type of ergotism is marked by violent burning, and shooting pain of the poorly vascularized distal organs, such as the fingers and toes. It is caused by effects of ergot alkaloids on the vascular system due to vasoconstriction, sometimes leading to gangrene and loss of limbs due to severely restricted blood circulation.

Ergot metabolites also have profound effects on the central nervous system stimulating smooth muscle activity. The neurotropic activities of the ergot alkaloids may also cause hallucinations, convulsions, and even death. Other symptoms include strong uterine contractions, nausea, seizures, high fever, vomiting, loss of muscle strength and unconsciousness.

Ergot alkaloids has been used in pharmaceutical preparations, to treat migraine headaches, and to induce uterine contractions and control bleeding after childbirth.  Since the Middle Ages, controlled doses of ergot were used to induce abortions and to stop maternal bleeding after childbirth.

The causative agents of most ergot poisonings are the ergot alkaloid class of fungal metabolites. The fungi of the genera Penicillium and Aspergillus also produce ergot alkaloids, particularly some isolates of the human pathogen Aspergillus fumigatus.

Aflatoxin

Aflatoxicosis is a fungal toxicosis that may affect all species of animals. Aflatoxins are produced by certain strains of Aspergillus flavus and A. parasiticus. These fungi grow on carbohydrate-rich feeds such as peanuts, cottonseed, corn, sorghum and cereal grains when they are stored in hot conditions without adequate drying and aeration. Optimal conditions for the production of aflatoxin would be an aw of 0.85 and a temperature of 25 to 40^oC.

The number and types of aflatoxins produced vary with the strain. For example, A. flavus strains produce B1 and its related metabolities, while A. parasiticus produces both B1 and G1 and the related metabolites.

In 1959 there was the deaths of several thousand turkey poults and other poultry on farms in East Anglia due to poisoning of the groundnut meal used as a protein supplement in the pelleted feed. The contaminant, which was called aflatoxin, fluoresces intensely under ultra-violet light and was shown to be produced by the mould Aspergillus flavus growing on the groundnuts.

Acute aflatoxicosis can be caused by ingestion of high doses of aflatoxin over a short period of time. Aflatoxin toxicity may result in nausea, vomiting, abdominal pain, convulsions, and other signs of acute liver injury. Long-term exposure also leads to various complications like growth retardation, cirrhosis, and hepatocellular carcinoma.

The two major aflatoxins have been designated B1 and G1 because they fluoresce blue (B1) and green (G1) when exposed to long-wave ultraviolet light. Aflatoxins B2 and G2 are the dihydroderivatives of B1 and G1. Aflatoxins M1, M2, and P1 are the hydroxylated derivatives of B1 and B2 which are excreted in the urine, faeces, and milk as metabolic products of B1 and B2 following their consumption by mammals.  

Aflatoxin B1, the most toxic of the aflatoxins, is toxic to various animals. Many of the other aflatoxins have been shown to be toxic or carcinogenic to different species of fish, mammals, and poultry. When cows eat feed containing aflatoxin, aflatoxin M1 and M2 is excreted in the milk. Although M1 and M2 are less toxic than the parent compounds B1 and B2, M1 retains its toxic and carcinogenic ability in many animals. M1 has also been detected in the urine of Philippine women who had consumed peanut butter containing aflatoxin.

Many commodities will support the growth of toxigenic strains, including various dairy products, bakery products, fruit juices, cereals, and forage crops.  Aflatoxins have been reported from a wide range of foods and animal feeds. Initially, it was considered that aflatoxin contamination was a problem of poor storage of commodities after harvest allowing the growth of storage fungi such as Aspergilli with consequent formation of mycotoxins. High humidity and warm temperatures can give rise to the highest levels of aflatoxin in food.

Aflatoxins can be produced in the growing crop before harvest also. Aflatoxigenic species of Aspergillus can establish an endophytic relationship with the healthy plant and produce low, but significant, amounts of aflatoxin when the plant is stressed, such as occurs during a drought.

It is assumed that a correlation is there between aflatoxin and liver cancer and liver damage in different parts of the world. Very young children may be exposed to aflatoxins even before they are weaned because mothers, consuming aflatoxin in their food, may secrete aflatoxin M1 in their milk.

.