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Infection and Immunity

Infection and Immunity
Infection and immunity involve interaction between the host (man) and the infecting microorganism. Based on their relationship to their hosts, microorganisms can be classified as saprophytes and Parasites.
Saprophytes are free-living microbes that subsist on dead or decaying organic matter. They are found in soil and water and play an important role in the degradation of organic materials in nature.
Parasite: Parasite may be defined as a microorganism, which lives on a living host and derives nutrition from the host, without any benefit to the host.
Commensal: Commensals (con, with; mensa, table, i.e. living together) live in complete harmony with the host body without causing any harm to it. The commensals constitute the normal bacterial flora of the body such as Staphylococcus epidermidis of skin and Escherichia coli of gastrointestinal tract. The commensals subsist on secretions, food residues or waste products of the body.
Opportunistic pathogens: Some commensals or saprophytes can produce disease when the body resistance is lowered; such organisms are termed as opportunistic pathogens.
Pathogens: A microorganism capable of producing disease is called pathogen. Majority of the pathogenic bacteria are heterotrophs.
Pathogenicity: Pathogenicity refers to the ability of a class of bacteria to produce disease.
Virulence: Virulence is a measure of the degree of pathogenicity and depends on invasiveness and toxigenecity of the organism.
The term infection and disease are often used synonymously when discussing hosp-parasite relationships. The difference between these terms is helpful to understand the development of the normal microflora, which represents a successful host-microorganism relationship.
Colonizer: A microorganism that makes contact with the host but does not cause the host to produce an immune response that would destroy it or cause an allergic response is called a Colonizer.
Infection: Microorganisms that make contact with the host and elicit an immune response (antibody production) are said to cause infection.
Disease: If the microorganism brings about an abnormal condition in the host, in addition to elicting antibodies, it is said to cause a Disease.
Infection: Infection may be defined as lodgement and multiplication of an infectious agent in the body. All infections do not invariably results in disease. Some infections may remain asymptomatic and others may lead to development of signs and symptoms after breakdown of host-parasite relationship in favour of the parasite. It is necessary to distinguish between the term infection and infectious disease. Infection does not always results in disease. In fact disease is but a rare consequence of infection, which is a common natural event.
Infection may be classified in various ways such as:
Primary infection: Initial infection with a parasite in a host is termed as primary infection.
Reinfection: Subsequent infections by the same parasite in the host are termed as reinfections.
Secondary infection: When a new parasite sets up an infection in a host whose resistance is lowered by a preexisting infectious disease, is termed as secondary infection.
Focal infection (focal sepsis): A localized site of infection from which bacteria and their products are spread to other parts of the body. Or. Indicates a condition where, due to infection or sepsis at localized sites such as appendix or tonsils, generalized effects are produced.
Cross infection: when in a patient already suffering from a disease a new infection is set up from an other host or another external source, it is termed as cross infection.
Nosocomial infections: Cross infections occurring in hospitals are called nosocomial infections.
Iatrogenic infection: It refers to physician induced infections resulting from investigative, therapeutic or other procedures.
Depending on whether the source of infection is from the host’s own body or form external sources, infections are classified as endogenous or exogenous, respectively. Based on the clinical effects of infections, they may be classified into different varieties.

Inapparent infection: Is one where clinical effects are not apparent.
Sub-clinical infection: it is a synonoym of inapparent infection.
Atypical infection: Is one in which the typical or characteristic clinical manifestations of the particular infectious disease are not present.
Latent infections: Some parasites, following infections, may remain in the tissues in a latent or hidden form proliferating and producing clinical disease when the host resistance is lowered. This is termed latent infections.
Sources of infection: Infection may be acquired endogenously or exogenously.
Endogenous sources: This occurs with microorganisms of the host’s normal flora which behave as pathogens outside their habitat.
Exogenous sources:
Man:
The commonest source of infection for human beings is the human beings themselves. The parasite may originate from a patient or a carrier. A Carrier is a person who harbors the pathogenic microorganism without suffering form any ill effect because of it. Several types of carriers have been identified. A healthy carrier is one who harbors the pathogen but has never suffered form the disease caused by the pathogen. While a convalescent carrier is one who has recovered form the disease and continues to harbor the pathogen in his body. Depending on the duration of carriage, carriers are classified as temporary and chronic carriers. The temporary carrier state lasts less than six months, while chronic carriage may last for several years and some times even for the rest of ones life. The term contact carrier is applied to a person who acquires the pathogen from a patient, while the term paradoxical carrier refers to a carrier who acquires the pathogen form another carrier.
Animals:
Many pathogens can infect both man and animals. Zoonoses are infections between vertebrate- animals and man. Many of those infections affect agridultural workers and veterinary surgeons. General public acquires infection through contaminated meat and milk. The infected animals serve as reservoir hosts. An epidemic occurring in animal is called epixootic (plague in rats), where as the term enzootic refers to endemic occurring in animals (Plague in rats and brucellosis in cattels).
Insects:
Blood sucking insects may transmit pathogens to human beings. The diseases so caused are called arthropod borne diseases. Insects such as mosquitoes, ticks, mites, flies, fleas and lice that transmit infections are called vectors. Transmission may be mechanical (transmission of dysentery or typhoid bacilli by the domestic fly). Such vectors are called mechanical vectors. In other instances, the pathogen multiplies in the body of the vector, often undergoing part of its developmental cucle in it. Such vectors are termed as Biological vectors (Aedes aegypti mosquito in yellow fever, Anopheles mosquito in malaria). Biological vectors transmit infection only after the pathogen has undergone a developmental cycle. The interval between the time of entry of the pathogen into the vector and the vector becoming infective is called the extrinsic incubation period.
Beside acting as a vector, some insects may also act as reservoir hosts. Infection is maintained in such insects by transovarial or transstadial passage.
Soil and water:
Some pathogens can survive in the soil for very long periods. Spores of tetanus bacilli may remain viable in the soil for several decades and serve as the source of infection. Fungi are also parasites such as roundworm and hookworm survive in the soil and cause human infections.
Water may act as the source of infection either due to contamination with pathogenic microorganisms (cholera vibrio, infective hepatitis virus) or due to the presence of aquatic vectors (Cyclops in guinea worm infection).
Food: Contaminated food may act as a source of infection. The presence of pathogens in food may be due to external contamination (food poisoning by staphylococcus) or due to pre-existent infection in meat or other animal products (salmonellosis).
Methods of transmission of infection
The routes of transmission may be by way of contact, vehicle, vector, air and transplacental. The infective agent has to find out a portal of entry for successful parasitism.
Contact: Infection may be acquired by contact, which may be direct or indirect. Direct contact refers to transmission of microorganisms from person to person by close personal association. Handshaking, kissing, sneezing, coughing and sexual contact represent the most usual ways that microorganisms are transferred by direct means. Sexually transmitted diseases such as syphilis and gonorrhea illustrate spread by direct contact. The term contagious disease had been used for diseases transmitted by direct contact. Where as infectious diseases are those signifying all other modes of transmission. This distinction is now not generally employed.
Sneezing and coughing may also be considered a method of direct spread, provided the individuals are within a few feet of each other. The microorganisms are expelled in droplets that are carried only a few feet and then drop to horizontal surface. Direct spread of this kind is characteristic of measles, a viral disease.
Indirect contact may be through the agency of fomites, which are inanimate objects such as clothing, pencils or toys that may be contaminated by a pathogen from one person and act as a vehicle for its transmission to another. Pencils shared by school children may act as fomites in the transmission of diphtheria and face towels in trachoma.
Inhalation:
Respiratory infections such as influenza and tuberculosis are transmitted by inhalation of the pathogen. Such microbes are shed by the patients into the environment, in secretions from the nose or throat during sneezing, speaking or coughing. Large drops of such secretions fall to the ground and dry there. Pathogens resistant to drying may remain viable in the dust and act as sources of infection. Small droplets, under 0.1 mm in diameter, evaporate immediately to become minute particles or droplet nuclei (usually 1-10 µm in diameter) which remain suspended in the air for long periods, acting as sources of infection.
Ingestion:
Intestinal infections are generally acquired by the ingestion of food or drink contaminated by pathogens. Infection transmitted by ingestion may be waterborne (cholera), foodborne (food poisoning), or handborne (dysentery). The importance of fingerborne transmission is being increasingly recognized, not only in the case of pathogens entering through the mouth, but also those that enter through the nose and eyes.
Inoculation:
pathogens, in some instances, may be inoculated directly into the tissues of the host. Tetanus spores implanted in deep wounds, rabies virus deposited subcutaneously by dog bite and arboviruses injected by insect vectors are examples. Infection by inoculation may be iatrogenic when unsterile syringes and surgical equipment are employed. Hepatitis B and the Human Immunodeficiency Virus (HIV) may be transmitted through transfusion of infected blood, or the use of contaminated syringes and needles, particularly among addicts of injectable drugs.
Insects:
Insects may act as mechanical or biological vectors of infectious diseases.
Congenital:
Some pathogens are able to cross the placental barrier and infect the ferus in utero. This is known as vertical transmission. This may result in abortion, miscarriage or stillbirth. Live infants may be born with manifestations of a disease, as in congenital syphilis. Intrauterine infection with the rubella virus, especially in the first trimester of pregnancy, may interfere with organogenesis and lead to congenital malformation. Such infections are known as teratogenic infections.
Iatrogenic and laboratory infections:
Infection may sometimes be transmitted during administration of injections, lumbar puncture and catheterization, if meticulous care in asepsis is lacking. Modern methods of treatment such as exchange transfusion, dialysis, and heart and transplant surgery have increased the possibilities for iatrogenic infections. Laboratory personnel handling infectious material are at risk and special care should be taken to prevent laboratory infection.
The outcome of an infection will depend on the interaction between microbial factors, which predispose to pathogenicity and host factors that contribute to resistance.
Factors predisposing to microbial pathogenicity:
The term pathogenicity and virulence refer to the ability of a microbe to produce disease or tissue injury but it is convenient to make a fine distinction between them. Pathogenicity is generally employed to refer to the ability of a microbial species to produce disease while the term virulence is applied to the same property in a strain of microorganism. Thus the species M. tuberculosis or polio virus is referred to as being pathogenic. The pathogenic species M. tuberculosis and the polio virus contain strains of varying degrees of virulence including those which are avirulent, such as the vaccine strains.
Enhancement of virulence is known as exaltation and can be demonstrated experimentally by serial passage in susceptible hosts. Reduction of virulence is known as attenuation and can be achieved by passage through unfavourable hosts, repeated cultures in artificial media, growth under high temperature or in the presence of weak antiseptic, desiccation or prolonged storage in culture. Virulence is the sum total of several determinants, as follows:
Adhesion: The initial event in the pathogenesis of many infections is the attachment of the bacteria to body surfaces. This attachment is not a chance event but a specific reaction between surface receptors on host cells and adhesive structures on the surface of bacteria. These adhesive structures are called adhesins. Adhesins may occur as organized structures, such as fimbriae or fibrillae and pili, or as colonization factors. This specific adhesin may account for the tissue tropisms and host specificity exhibited by many pathogens. Adhesins serve as virulence factors and loss of adhesins often renders the strain avirulent. Adhesins aer usually made of protein and are antigenic in nature. Specific immunization with adhesins has been attempted as a method of prophylaxis in some infections, as for instance against E. coli diarrhea in calves and piglets and gonorrhea in human beings.
Invasiveness:Invasiveness refers to the ability of microorganisms to penetrate tissue. Highly invasive pathogens characteristically produce spreading or generalized lesions (e.g. streptococcal septicemia following wound infecton), while less invasive pathogens cause more localized lesions. Invasiveness is not always associated with virulence or vice versa. e.g. Clostridium tetani, the causative agent of tetanus, is not an invasive organism but is extremely virulent once it has infected the host because of the release of a potent toxin. Many viruses are capable of invading tissue but are not pathogenic to humans.
Toxigenicity: Bacteria produce two types of toxins namely exotoxins and endotoxins.
Exotoxins:
Exotoxins are heat labile proteins which are secreted by certain species of bacteria and diffuse readily into the surrounding medium. They are highly potent in minute amounts and constitute some of the most poisonous substances known. One mg of tetanus or botulinum toxin is sufficient to kill more than one million guinea pigs and it has been estimated that 3 kg of botulinum toxin can kill all the inhabitants of the world. Most exotoxins are highly antigenic that is they can stimulate the formation of antibodies (antitoxins) when injected into appropriate hosts. For this reasons exotoxins can be used to induce active immunity against toxin caused diseases such as diphtheria and tetanus. However, because many toxins are potent and lethal poisons, they cannot be injected into the human body without some modification of the toxin molecule. They can be modified chemically by phenol, formaldehyde and various acids. In its modified form the toxin is called a tosoid. When injected into the body toxoid have the ability to stimulate the formation of antibodies that neutralize the specific exotoxins from which they were derived and toxoid are not toxic to the host. Toxoids or incactivated virulent microorganisms use in immunization are referred to as vaccines.
Endotoxins:
Endotoxins are heat stable lipopolysaccharides (LPS) which form an integral part of the cell wall of Gram negative bacteria. Their toxicity depends on the lipid component (Lipid A). they are not secreated out side the bacterial cell and are released only by the disintegration of the cell wall. They cannot be toxoided. They are poor antigens and their toxicity is not completely neutralized by the homologous antibodies. They are active only in relatively large doses. They do not exhibit specific pharmacological activities. All endotoxins, whether isolatd from pathogenic or nonpathogenic bacteria, produce similar effects. Administration of small quantities of endotoxin in susceptible animals causes an elevation of body temperature manifestd within 15 minutes and lasting for several hours. The pyrogenic effect of fluids used for intravenous administration is usually dur to the presence of endotoxins from contaminant bacteria. Intravenous injections of large doses of endotoxin and massive gram negative septicemias cause endotoxic shock marked by fever, leucopenia, thrombocytopenia, significant fall in blood pressure, circulatory collapse and bloody diarrhea leading to death.
Communicability: The ability of a parasite to spread form one host to another is known as communicability. This property does not influence the production of disease in an individual host but determines the survival and distribution of a parasite in a community. A correlation need not exist between virulence and communicability. In fact a highly virulent parasite may not exhibit a high degree of communicability due to its rapidly lethal effect on the host. In general infections in which the pathogen is shed in secreations, as in respiratory or intestinal diseases, are highly communicable. In some instances, as in hydrophobia, human infection represents a dead end. There being an interruption in the spread of the pathogen to other hosts. Development of epidemic and pandemic diseases requires the strain of pathogen to possess high degrees of virulence and communicability.
Other bacterial products:
Some bacterial products other than toxins, though devoid of intrinsic toxicity, may contribute to virulence by ingibiting the mechanisms of host resistance. Pathogenic staphylococci produce a thrombin-like enzyme coagulase, which prevents phagocytosis by forming a fibrin barrier around the bacteria and walling off the lesion.
Fibrinolysins promote the spread of infections by breaking down the fibrin barrier in tissues. Hyaluronidases split hyaluronic acid which, is a component of intercellular connective tissue and thus facilitate the spread of infection along tissue spaces. Leucocidins damage polymorphonuclear leucocytes. Many pathogens produce hemolysins capable of destroying erythrocytes but their significance in pathogenicity is not clearly understood.
Bacterial appendages:
Capsulated bacteria are not readily phagocytosed. Some bacterial surface antigens such as the Vi and K antigens help the bacteria to withstand phagocytosis and the lytic activity of complement.
Characteristics differentiating Exotoxins from Endotoxins
Exotoxins Endotoxins
Generally produced by Gram positive bacteria Produced by Gram negative bacteria
They are protein in nature Lipopolysaccharide in nature
They are heat labile Heat stable
Actively secreated by cells; do no diffuse into surrounding medium

diffuse into surrounding medium Form part of cell wall;
Often are high molecular weight Low molecular weight components
Action of exotoxin is often enzymic No enzymic action
Shows specific pharmacological effect Effect is non-specific; action common to all endotoxins.

for each exotoxins

Exotoxins have specific tissue affinities
No specific tissue affinity
Active in very minute doses
Active only in very large doses
Highly antigenic in nature
They are weakly antigenic
Exotoxins can be toxioded
Cannot be toxioded
Action specifically neutralized by antibody
Neutralization by antibody is ineffective
Infecting dose: Successful infections require that an adequate number of bacteria should gain entry into the host. The dosage may be estimated as the minimum infecting dose (MID) or minimum lethal dose (MLD).
MID: The minimum number of bacteria required to produce clinical evidence of infection in susceptible animal under standard conditions.
MLD: The minimum number of bacteria required to cause death of susceptible animal under standard conditions.
As animals exhibit considerable individual variation in susceptibility, these doses are more correctly estimated as statistical expressions, ID50 and LD50, as the dose required for infecting or killing 50 per cent of the animals tested under standard conditions.
Route of infection: Some bacteria, such as streptococci, can initiate infection whatever be the mode of entry. Others can survive and multiply only when introduced by the optimal routes. Cholera vibrio are infective orally bur are unable to cause infection when introduced subcutaneously. This difference is probably related to modes by which different bacteria are able to initiate tissue damage and establish themselves. Bacteria also differ in their in their sites of election in to the host body after introduction into tissues. They also differ in their ability to produce damage of different organs in different species of animals. e.g. Tubercle bacilli injected into rabbits cause lesions mainly in the kidney and infrequently in the liver and spleen, but in guinea pigs the lesions are mainly in the liver and spleen, the kidneys being spared.
The reasons for such selective multiplication in tissues are largely obscure, though they may be related to the presence in tissues of substances that may selectively hinder or favour their multiplication.
Types of infectious diseases: Infectious diseases may be localized or generalized. Localised infections may be superficial or deep seated, Generalized infection involve the spread of the infecting agent from the site of entry by contiguity, through the bloodstream. Circulation of bacteria in the blood is known as bacteremia. Transient bacteremia is a frequent event even healthy individuals and may occur during chewing, brushing or teeth or straining at stools. The bacteria are immediately mopped up by phagocytic cells and are unable to initiate infection. Bacteremia of greater severity and longer duration is seen during generalized infections as in typhoid fever.
Septicemia is the condition where bacteria circulate and multiply in the blood, form toxic products and cause high, swinging type of fever.
Pyemia is a condition where pyogenic bacteria produce septicemia with multiple abscesses in the internal organs such as the spleen, liver and kidney.
Depending on their spread in the community infectious diseases may be classified into different types:
Endemic diseases are those, which are constantly present in a particular area. Typhoid fever is endemic in most parts of India.
Epidemic deseases are those which spreads rapidly, involving many persons in the area at the same time.
Pandemic is an epidemic that spreads through many areas of the world involving very large numbers of people within a short period.
Prosodemic diseases are those which spread by direct person to person contact, hence evolve very slowly, such creeping or smouldering epidemics are refered as prosodemic diseases (e.g. cerebrospinal fever).
IMMUNITY
Def: Immunity refers to the resistance exhibited by the host towards injury caused by microorganisms and their products. Protection against infectious diseases is only one of the consequences of the immune response, which in its entirety is concerned with the reaction of the body against any foreign antigen.
Classification of Immunity: Immunity against infectious diseases if of different types. It can be classified as follows:
Immunity can be broadly of two types (i) Innate or Native immunity and (ii) Acquired immunity.
Innate immunity: it is the resistance to infections that an individual possess by virtue of his genetic and constitutional make up. It is not affected by prior contact with microbes or immunization. It may be nonspecific (when it indicates a degree of resistance to infections in general) or specific (when the resistance to a particular pathogen is concerned).
Innate immunity may be considered at three different levels (i) species, (ii) race and (iii) individual. i.e. Innate immunity is species specific, race specific and individual specific.
(i) Species immunity refers to the total or relative refractoriness to a pathogen, shown by all members of a species. i.e. Animals of same species exhibit uniform pattern of susceptibility to infections, (e.g. B. anthracis infects human but not chicken, birds are immune to tetanus).
The mechanism of species immunity is not clearly understood but may be due ot the physiological and biochemical differences between tissues of different species may be responsible for species-specific immunity. This has been proved by pasteur’s experiment on anthrax in frogs, which are naturally resistant to the disease but become susceptible when their body temperature is raised is raised from 25°C to 35°C.
(ii) Racial immunity: With in a species, different races may show differences in the susceptibility to infections. This is known as racial immunity. e.g. High resistance of Algerian sheep to anthrax. Such racial differences are known to be genetic in origin and by selection and inbreeding, it is possible to develop at will race that possess high degrees of resistance or susceptibility to various pathogens. It has been reported that the people of Negroid origin in the USA are more susceptible than the Jews to tuberculosis. An interesting instance of genetic resistance to plasmodium falciparum malaria is seen in some part of Africa and the Mediterranean coast. A hereditary abnormality of red cells (sickling), prevalent in the area, confers immunity to infection by the malarial parasite and may have evolved from the survival advantage conferred by it in a malarial environment.
(iii) Individual immunity: Resistance to infection varies with different individuals of the same species and the race. Thus, certain individuals may be found within a highly susceptible population, who unaccountably cannot be infected by some microorganisms even though they have no previous contact with the same. The individual immunity is commonly observed in endemic outbreaks.
Factors influencing innate immunity:
There are several factors, which influences the level of innate immunity such as age, hormonal influence, nutrition etc.
IMMUNITY
Innate (natural) Acquired (Specific)
i. Species: Birds immune to tetanus
ii. Racial: Algerian sheep immune to anthrax
iii. Individual: Seen in endemic area
Active

Passive
Natural Artificial Natural Artificial
Follows clinical Induced by vaccination Tranplacental passage Injection
or subclinical IgG of preformed infections antibody
Age: Two extreme of life, i.e. fetus and old person carry high susceptibility of infectious diseases. In the former, the immune apparatus is immature whereas in the latter there is gradual waning of their immune response. The fetus in uterus is protected by the placental barrier form maternal infection. Certain pathogens, e.g. Rubella, Cytomegaloviruses and Toxoplasma gondii cross the placental barrier leading to congential malformation.
Hormonal influence: Certain hormonal disorders such as diabetes mellitus, hypothyroidism and adrenal dysfunction influence susceptibility to infection. In diabetes there is high incidence of staphylococcal sepsis partly due to altered metabolism and elevated level of carbohydrates in tissues. The susceptibility to infection is increased in both hypo and hyper adrenal states. Corticosteroids depress the host resistance by suppression of the inflammatory response and inhibiting antibody formation.
Nutrition: In general, malnutrition predisposes to bacterial infection such as gram-negative bacterial septicemia. Tuberculosis, herpes virus infection, measles and candidiasis. Cell mediated immunity and antibody response to T lymphocyte dependent antigens are primarily reduced in malnutrition.
ACQUIRED IMMUNITY
Resistance acquired by an individual during lifetime is called as acquired immunity. Acquired immunity is of two types: active and passive.
Active acquired immunity:
Active immunity is the resistance induced in an individual after effective contact with an antigen. It follows either natural infection or vaccination. Here the immune system actively participates in producing antibodies and often cell mediated immunity also. Active immunity develops slowly over a period of days or weeks but persists for a long time, usually for years.
Types of Active Immunity:
Active immunity may be acquired either naturally or artificially.
(i) Natural active acquired immunity: It is acquired by natural infection by the organisms. In large majority of the cases this occurs by sub clinical infections after repeated exposure to small doses of the infecting organism, which pass unnoticed. Such immunity is usually long lasting. Sub clinical attacks by pathogenic microbes play important role in preventing epidemics, e.g. poliomyelitis, tuberculosis. Some of these follow overt infections, for example person recovering from an attack of small pox develops natural active immunity.
(ii) Artificial active acquired immunity: It is the resistance produced by vaccination. The vaccines are preparations of live, attenuated or killed microorganisms, or their antigens or active materials derived from them (toxoids). The commonly employed vaccines are as follows:
Bacterial vaccines: (i) Live BCG, anthrax, plague and brucella vaccines. (ii) Killed TAB for enteric fever, autovaccine.
Viral vaccines: (i) Live- small pox, measles, influenza, mumps and Sabin poliomyelitis vaccine. (ii) Killed- Salk vaccine for poliomyelitis.
Bacterial products: Toxoids for tetanus and diphtheria.
Attenuation of live vaccine is done by ageing of culture, cultivation at high temperature (anthrax bacilli grown at 42°C), passage through animals of different species (variola virus through rabbit and calf), drying (rabies), by continued cultivation in presence of antagonistic substance (BCG) and by repeated subculture in artificial media (streptococci).
In killed vaccine the organism are killed by heat, formalin, phenol, alcohol, ultraviolet light and photodynamic inactivation. These are preserved in phenol, alcohol, N-merthiolate. Toxoid are preparations of bacterial exotoxins inactivated by formaline (formal toxoid) or by alum (APT). they retain immunogenecity but not toxigenecity.
Passive acquired immunity:
The resistance that is induced in the recipient by transfer of antibodies preformed against infective agent or toxin in another host, is called passive immunity. The immune system plays no active role and the protective mechanism comes into force immediately after transfer of antibodies (immune serum). Passive protection is short lasting only for days or weeks. It is useful when instant immunity is required. Passive immunity is of two types: Natural and Artificial.
Natural passive acquired immunity:
It is the resistance passively transferred from the mother to foetus and infant, e.g. transfer of maternal antibody to foetus transplacentally and to infant through milk (colostrum).
Artificial passive acquired immunity
It is the resistance passively transferred to a recipient by the parenteral administration of antibodies. Passive administration of antibody is very useful in some clinical conditions.
Differences between active and passive immunity
Active immunity Passive immunity
Produced actively by the immune system of host Received passively by the host and the hosts immune system does not participate
Induced by infection or by contacts with immunogen i.e. vaccines Conferred by introduction of ready made antibodies
Immune response is durable and effective Immune response is short lived and less effective
Immunity develops only after a period of lag period Immunity effective immediately. No lag period Immunological memory is present
No immunological memory.

Subsequent challenge with booster dose is more effective Subsequent administration of a ntibody is less effective due to immune elimination.
After antigenic stimulus negative phase may occur No negative phase
Serves no purpose in immunodeficient hosts Applicable in immunodeficient hosts.
Used for prophylaxis to increase body resistance Used for treatment of acute infections.

STOCK CULTURE AND ITS MAINTENANCE

STOCK CULTURE AND ITS MAINTENANCE

INTRODUCTION:All practicing microbiologists have felt the need to preserve the viability of microorganisms with which they work. In addition, all the cultural characteristics of a culture, as they were at the time of preservation, must be conserved. The nature of work being done will determine whether the preservation requirement is only very short-term or for an unlimited time period. Long-term preservation of a culture is required if a culture is to be deposited in one of the service culture collections with a view to preserving something of scientific value “for perpetuity”. Many methods of preservation for microorganisms have been developed. Here, it is to be noted that there exist different types of microorganisms (bacteria, viruses, algae, protozoa, yeasts and mould). Therefore, there are two criteria for selecting a method of preservation for a given culture. They are:1. The period of preservation desired, and 2. The nature of a culture to be preserved. Definition: - Stock cultures are those cultures of microorganisms that are stored or maintained for future use in such a fashion that their growth and productive capacities remains unaltered.There are two types of stock cultures: (i) working stocks and (ii) Primary stocks.· The working stock cultures are those which are used frequently and they must be maintained in a vigorous and uncontaminated condition. These cultures are maintained as agar slants, agar stabs, spore preparations or broth cultures and they are held under refrigeration. They must be checked constantly for possible changes in growth characteristics, nutrition, productive capacity and contamination.· Primary stocks are cultures that are held in reserve for practical or new fermentations, for comparative purposes, for biological assays or for possible later screening programs. These cultures are not maintained in a state of high physiological activity and they are delved into only rarely. Transfers from these cultures are made only when a new working-stock is required, or when the primary stock culture must be sub-cultured to avoid death of the cells. Thus, primary stock cultures are stored in such a manner as to require the least possible numbers of transfers over a period of time. Death of a high percent of cells in a primary stock culture is not particularly serious, if viable cells can still be recovered for subculture to fresh medium. Primary stock cultures stored at room temperature are maintained in sterile soil or in agar or broth that is provided with an overlay of sterile mineral oil. Agar and broth cultures without mineral oil also are refrigerated and cultures in milk or agar are maintained frozen at low temperatures. Finally, primary stock cultures are lyophilized or freeze-dried and stored at low temperatures. Often mote than one of these procedure is employed to insure against loss of the cultures or changes in the cells.There are three basic aims in maintaining and preserving the microorganisms. They are:- i. To keep culture alive ii. Uncontaminated and iii. As healthy as possible, both physically and physiologically, preserving their original properties until they are deposited in any major collections. iv. To have adequate stocks and appropriate sustems for replenishing stocks when necessary. Serial subculture:-This is the simplest and most common method of maintaining microbial cultures. Microbes are grown on agar slants and are transferred to fresh media before they exhaust all the nutrients or dry out. An exception to this is aerobic Streptomyces spp. Where drying up of the medium has been found successful, provided the initial growth showed the production of serial hyphae. The drying of medium appeared to encourage good sporulation and the preserved specimen became simply a dried out strand of agar coated with spores, which remained viable for a few years at room temperature. For some microbial cultures no other methods have been found satisfactory, but for the majority of species other methods are available.The maintenance of refrigerated stock cultures on agar or in broth is the least desirable of these procedures. Although the cultures may survive six months or more under refrigeration, usually they are transferred more frequently. These frequent culture transfers and the many cell generations accompanying these transfers allow the possible occurrence of and selection for undesired genetic changes in the organisms. Also the potential for contamination is markedly increased with frequent transfer of the cultures. Certain microorganisms, such as Blakeslea trispora used in b-carotene production, cannot be stored at refrigeration temperatures, because they die out relatively quickly at these temperatures. However, such cultures can be held as agar slants at room temperature with transfers being made to fresh medium when the culture have become nearly dried out. Overlaying cultures with mineral oil: Agar slant and stab cultures of many microorganisms will survive several years at room temperature if the growth is submerged under sterile mineral oil. The oil overlay provides dissolved oxygen, prevents drying of the agar and apparently decreases the metabolic activity of the cells to an almost negligible rate. However, genetic changes do occur in cultures stored in this manner.The steps involved in this method are:· Inoculation of agar salnt/stab with the culture to be maintained.· Inoculated agar slant/stab is subjected to incubation until good growth appears.· Using sterile technique, a healthy agar slant/stab culture is covered with sterile mineral oil to a depth of about 1 cm above the top of the agar slant. If a short slant is used, less oil is required.· Finally, oiled cultures can be stored at room temperature. But better viability is obtained when stored at lower temperatures (15 °C).Note: The depth of oil of 1 cm is fairly critical, as the oxygen transmission by layers of mineral oil in excess of 1 cm becomes less favorable. If less oil is used, strands of mycelium may be exposed which allows the cultures to dry out. If the bottles or screw capped tubes are used, the rubber liners should be removed form the caps as the oil tends to dissolve the rubber and this can be toxic to the culture. This method has following advantages: i. Practically all bacterial species or strains tested live longer under oul than in the control tubes without oil. Some bacterial species have been preserved satisfactorily for 15-20 years. ii. Transplants may be prepared when desired without affecting the preservatio of the stock cultures. iii. The method is especially advantageous when working with unstable variants where occasional transfers to fresh media or growth in mass cultures results in changes in the developmental stages of the strains. iv. This method also appears to be an ideal method of storage for a busy laboratory with limited funds and a relatively small collection. Soil culture (Soil stock):Sterile soil has found wide use for the stock culture maintenance of microorganisms that form spores. This method is particularly applied for the preservation of sporing microbes specially fungi. In fact, microorganisms that do not form spores also will survive in sterile soil, bur they may die out unexpectedly after a period of time. Soil stocks are prepared as follows: i. A mixture of soil (20 %), sand (78%) and calcium carbonate (2%) is prepared and distributed into tubes (a few grams per tube). They are sterilezed for 8-15 hours at 130°C and then cooled. ii. A small volume of thick suspension of spores or of an actively growing culture is then added to the sterile soil and incubated till good growth. iii. The inoculated tubes are kept in desiccators under vacuum. The reason behind this is to evaporate the excess water. Then the tubes are sealed. iv. Soil stocks, thus prepared are stored at room temperatures with cotton plugs or screw caps protected from dust. These cultures can be stored in refrigerators at about 5-8 °C temperature. Lyophilization or freeze-drying: Lyophilization is the most satisfactory method of long-term preservation of microorganisms. It is universally used for the preservation of bacteria, viruses, fungi, sera, toxins, enzymes and other biological materials. Lyophilization is the most popular form of suspended metabolism. It consists of drying of cultures or a spore suspension from the frozen state under reduced pressure. This can be accomplished in several ways. The major steps involve in this techniques are: i. A thick cell or spore suspension is prepared in a suitable protective medium (10% skimmed milk or bovine serum, 5% inositol in distilled water). ii. Using sterile techniques, this thick suspension is distributed in small quantities into glass ampoules. iii. These ampoules are subjected to deep-freezing by keeping the cultures at lower temperature (-20°C). iv. Then the chilled ampoules are connected with a high vacuum system usually incorporating a desiccant (e.g. phosphorous pentoxide, silica gel or a freezing trap), and immersed into a freezing mixture of dry ice and alcohol (-78°C). v. The vacuum pump is turned on and the ampoules are evacuated till drying is complete. vi. Freeze dried ampoules are then immediately sealed off and stored under refrigeration. If properly prepared and stored, most lyophilized cultures will remain viable for long periods (> 20 yrs.), without the occurrence of genetic changes. When needed, the cultures are recovered from the ampoules by suspending the lyophilized cells in a minimal amount of growth medium and then incubating. Advantages of lyophilization: i. As the ampoules are sealed there is no risk of contamination of infection with mites. ii. The prepared ampoules are easily stored, they are not readily broken and most species remain viable for many years. iii. There is less opportunity for the cultures to undergo changes in characteristics (i.e. they remain unchanged during storage period). iv. Owing to the small size of glass ampoules, hundreds of lyophilized cultures can be stored in a small storage space. In addition to this, the ampoules size makes them ideal for postage. v. Lyophilization cuts down the number of transfers. Liquid nitrogen storage: This method is also called as Cryogenic storage. It is like lyophilization, a satisfactory method for the long-term preservation of microorganisms. It has also been successful with many specimens which cannot be preserved by lyophilization. The maintenance of microbes is done by suspended metabolism. Life is regarded as “Stand still” at –130°C and below, so at the temperature of liquid nitrogen (-196°C), provided the cultures survive the treatment, the period of preservation should be indefinite. Thus, long-term preservation without any change in the cultural characteristics is now attainable. Major steps involved in the methods are: i. The culture to be maintained is suspended (thick suspension) as a cell or spore suspension in 10% glycerol. ii. This thick suspension in glycerol is distributed into ampoules (resistant to cold-shock). iii. Ampoules filled with a culture suspension are frozen (at the rate of about 1°C per minute to –20 to –35°C) and are sealed. iv. The frozen ampoules are then clipped on metal (aluminium) canes, one above the other and six to each cane. The canes in turn are packed in metal boxes or canisters (aluminium), which holds about 20 canes. These are perforated to allow the free running of the liquid nitrogen. v. The cultures are revived by removing form the container rapidly thawing and culturing them in the usual manner. Advantages: i. It is effective method of preservation ii. No subculturing is required iii. The cultural characteristics remain unchanged. iv. The ampoules are not open to contamination or infection by mites, since they are sealed. v. The living material of a type, which would not normally grow in a culture and would not be preserved in a culture collection, can be retained in a viable state. Regardless of the method or methods chosen for preservation of primary stock cultures, it is of utmost importance that good, descriptive records be kept on these cultures and that the cultures be well labeled. If little is known or recorded about a newly isolated microbial strain, we cannot hope to be able to recognize changes that may have occurred in that culture after prolonged storage period.

INDUSTRIAL MICROBIOLOGY AND SCREENING

INDUSTRIAL MICROBIOLOGY
Definition:
Industrial Microbiology deals with all forms of microbiology that have an economic aspect. It deals with those areas of microbiology on which, in some manner, a monetary value can be placed, regardless of whether the microbiology involves a fermentation product or some form of deterioration, disease or waste disposal.
OR
Use of Microbes to obtain a product or service of economic value constitutes Industrial Microbiology.

Fermentation: Any process mediated by or involving Microorganisms in which a product of economic value is obtained is called Fermentation
Industrial microbiology is an important branch of microbiology dealing with those areas of microbiology involving economic aspects, where valuable products are prepared from cheaper and often disposable substrates.
· Hence it has become possible for the industrial microbiologist to compare with the industrial chemist. e.g. fermentative production costs of all antibiotics, except one or two are appreciably less than the synthetic production costs of the same.
Scope of Industrial Microbiology:
Industrial microbiology is a very broad area for study. In fact, many nonindustrial areas of microbiology are important to industrial microbiology and should be taken into consideration in understanding the concepts and practice of Industrial Microbiology. These areas include: soil and Agricultural Microbiology, Medical Microbiology, Microbial Physiology, Cytology and Morphology, Virology, Genetics, Marine Microbiology, Food and Dairy Microbiology and Immunology.
Disciplines not normally considered to be included in microbiology are also important to industrial microbiology and include organic, inorganic and physical chemistry, biochemistry, engineering, medicine, economics, sales and law, particularly patent law and labor law, governmental regulations on the use of certain substrates and the sale of certain products also are relevant to industrial microbiology, as a consideration of space and marine exploration.
Further more, areas not presently considered to have any relationship to industrial microbiology, under the proper conditions easily can become a matter for consideration e.g. An industrial concern producing fiberglass may have no apparent need for a knowledge of industrial microbiology. But, if a change or improvement in some processing step suddenly allows microbial growth on the sizing applied to the glass fibers, then industrial microbiology immediately assumes importance. That is to say that many different branches of microbiology and non-microbiological fields are directly or indirectly involved in the study of industrial microbiology.
The industrial microbiologist is always dependent upon a biochemical engineer and vice versa. Apart form the cultural responsibilities; the remaining bioparameters of the fermentation process are to be controlled by the biochemical engineer. Thus economization of a fermentation process requires both a biochemical engineer and a microbiologist.
FERMENTOR: DESIGN AND ROLE OF DIFFERENT PARTS OF FERMENTOR
In fermentation industries, microbes are to be grown in specially designed vessels loaded with particular type of nutritive media. These vessels are referred to as Fermentor or Bioreactors.
Bioreactors or fermentors are complicated in design, because they must provide for the control and observation of many facts of microbial growth and biosynthesis. The design of fermentor depends upon the purpose for which it is to be utilized. Industrial fermentors are designed to provide the best possible growth and biosynthesis conditions for industrially important microorganisms and allows ease of manipulation for all operations associated with the use of the fermentors. The fermentor used for a particular process should possess following characters:

Characteristics of and Ideal Fermentor or bioreactor:
There cannot be a fermentor ideal for all most all fermentation processes, but if there is then it should following characteristics:
1. Material used in the fabrication of fermentor should be strong enough to withstand the interior pressure due to the fermentation media, it should be resistant to corrosion and free form any toxic effect for the microbial culture and the product formed by the microbial culture.
2. A fermentor should permit easy control of contaminating microbes.
3. It should be provided with the inoculation point for aseptic transfer of inoculum.
4. Should be equipped with the aerating device (Spargers).
5. Should be equipped with a stirring device for uniform distribution of air, nutrients and microbes (Impellers).
6. There should be provision of baffles to avoid vortex formation.
7. Fermentor should be provided with a sampling valve for aseptic withdrawing of sample for different laboratory tests.
8. Fermentor should possess a device for controlling temperature (Temperature sensor and water jacket internally fitted with heating coil).
9. Fermentor should be provided with pH controlling device for monitoring and maintaining pH of media during fermentation process (pH probe and Acid base reservior).
10. Should be provided with a facility for intermittent addition of antifoam agents for controlling foam formation (Reservior of sterile Antifoming agents or mechanical foam breakers).
11. There should be provision fro feeding certain media components during the progress of fermentation (Precursors).
12. A drain at the bottom is essential for the removal of the completed fermentation broth for further processing.
13. A man hole should be provided at the top of fermentor for acess inside the fermentor for different purposes like repairing and thorough cleaning of feermentors between runs.
14. A exit valve should be provided at the top for the exit of metabolic gases produced during fermentation processes.
TYPES OF FERMENTORS:
Batch fermentors are used to carry out microbiological processes on batch basis. They are available with varying capacities. The capacity of the fermentor may range form a few hundred to several thousand gallons. The capacity of the fermentor is usrally stated on the basis of the total volume capacity of the same. Thus, based on total volume capacity the fermentors are of following types:
i) Small Laboratory fermentors (ii) Pilot plant fermentors (iii) Large industrial fermentors (iv) Horton spheres.
The small laboratory fermentor ranges from 1-2 liters with a maximum up to of 12 –15 liters.
Pilot plant fermentors have a total volume of 25 –100 gallons upto 2000 gallons total volume.
Larger fermentors range form 5,000 or 10,000 gallons total volume to approximately 1,00,000 gallons.
Horton spheres are rarely employed with a size range of 2,50,000 to 5,00,000 gallons total capacity.
Actually the working volume in a fermentor is always less than that of the total volume. In other words, a ‘head space’ is left at the top of the fermentor above the level of fermentation media. The reason for keeping a head space is to allow aeration, splashing and foaming of the aqueous medium. This head space usually occupies a fifth to a quarter or more of the volume of the fermentor.
pH Control:
pH control is achieved by acid or alkali addition, which is controlled by an auto- titrator. The autotitrator in turn is connected to a pH probe.
Temperature control:
Temperature control is achieved by a water jacket around the vessel. This is often supplemented by the use of internal coils, in order to provide sufficient heat-transfer surface.
Agitation:
The agitating device consists of a strong and straight shaft to which impellors are fitted. An impeller, in turn consists of a circular disc to which blades are fitted with bolts. Different types of blades are available and are used according to the requirements. The shaft passes through a bearing in the lid of the fermentation tank. It is rotated with the help of an electric motor mounted externally at the top of the tank. The liquid medium is thrown up towards the walls of the fermentor while rotating the impeller blades at a high speed. This results in the formation of a vortex, which is eliminated, usually by four equally spaced baffles attached to the walls of the fermentor.
Aeration:
Usually, the aerating device consists of a pipe with minute holes, through which pressurized air escapes into the aqueous medium in the form of tiny air bubbles. This aeration device is called a “SPARGER”. The size of the holes in a sparger ranges from 1/64 to 1/32 of an inch or larger. Holes smaller than this requires too high air pressure for economical bubble formation. One should always remember that the smaller the air bubbles, the greater is the bubble surface area. It is desirable to adjust the size of the air bubbles to give the greatest possible aeration without greatly increasing the overall cost of the fermentation process. The reason for this is that sterile air is a costly item for large-scale fermentation. The cheapest means of sterilization of air is to pass it through a sterile filter composed of glass wool, carbon particles or some other finely divided material that will trap microorganisms present in the air.
Spargers in fermentors for growth of mycelium forming organisms often utilize 1/4 inch holes to prevent plugging of the holes by hyphal growth. Pipes crimped at the end or with a single small hole to produce a stream of air bubbles also are employed in some instances. The air bubbles from the sparger are picked up and dispersed through the medium by the action of the impeller blade mounted above the sparger.
In some very large fermentation tanks, an impeller is not utilized. The medium is stirred by the directed rush of air bubbles from a sparger at the bottom of the tank. These tanks are specially designed and usually do not contain baffles.
Foam Control:
Aeration and agitation of a liquid medium can cause the production of foam. This is particularly true for the media containing high levels of proteins or peptides. If the foam is not controlled, it will rise in the head space of the tank and be forced from the tank along with the exit valve. This condition often causes contamination of the fermentation from organisms picked up by breaking of some of the foam which then drains back into the tank. Excessive foaming also causes other problems for fermentation.
The usual procedure for controlling foam is to add an antifoaming agent, although a supplementary impeller blade mounted high in the tank may at times be effective. An antifoam agent lowers surface tension and in the process decreases the stability of the foam bubbles so that they burst. The antifoam may be added at media makeup or may be added after sterilization or as called for during the fermentation process.
There are two types of antifoam agents:
(i) Inert Antifoam agents
(ii) Antifoam agents made from crude organic materials.

e.g. Animal and vegetable oils , lard oil, corn and soybean oil, long chain alcohols such as octadecanol. In addition mixtures of oils and alcohols are effective in controlling foam. Silicone compounds are ideal inert antifoam agents but are too expensive.
Antifoam agents are often difficult to sterilize, particularly if they are of an oily nature, because of poor heat penetration and transport through the oil.
The use of inert antifoam agents, such as various silicone compounds is the ideal way to control foam, but these agents generally are too expensive for use in large scale industrial fermentations.
When antifoam is required in a tank, it is added either manually or electrically. Obviously, manually addition requires that some one continuously observe the tank so that the antifoam can be added as required. Electrical addition of antifoam is usually preferred. To accomplish this automatic addition, a sensing mechanism is employed to determine when the foam has risen into the head space of bioreactor. Such a device is provided with two electrodes mounted in the top of the fermentor. These electrodes are connected to a pump associated with a reservoir of sterile antifoam and as the foam rises in the reactor it touches the two electrodes in the process allowing current to flow between them so as to activate the pump for addition of antifoam. The foam then collapses away from the electrode thus breaking the electrical connection between them and stopping further addition of antifoam agent.
SCREENING
In Microbial Technology Microorganisms holds the key to the success or failure of a fermentation process. It is therefore important to select the most suitable microorganisms to carry out the desired industrial process.
The most important factor for the success of any fermentation industry is of a production strain. It is highly desirable to use a production strain possessing the following four characteristics:
It should be high-yielding strain.
It should have stable biochemical/ genetical characteristics.
It should not produce undesirable substances.
It should be easily cultivated on large-scale.
Def:
Detection and isolation of high-yielding species form the natural sources material, such as soil, containing a heterogeneous microbial population is called Screening
OR
Screening may be defined as the use of highly selective procedures to allow the detection and isolation of only those microorganisms of interest from among a large microbial population.

Thus to be effective, screening must, in one or a few steps allow the discarding of many valueless microorganisms, while at the same time allowing the easy detection of the small percentage of useful microorganisms that are present in the population.
The concept of screening will be illustrated by citing specific examples of screening procedures that are or have been commonly employed in industrial research programs.
During screening programs except crowded plate technique a natural source such as soil is diluted to provide a cell concentration such that aliquots spread, sprayed or applied in some manner to the surface of the agar plates will yield well isolated colonies (30-300).
Primary screening of Organic acid/ amine producer:-
· For primary screening of organic acid or organic amine producers, soil sample is taken as a source of microorganism.
· It is diluted serially to an extent to get well-isolated colonies on the plate when spread or applied in some form.
· After preparation of dilution these dilutions are applied on a media incorporated with a pH indicating dye such as Neutral red (Pink to yellow)or Bromothymol blue (Yellow -blue), into a poorly buffered agar nutrient medium. The production of these compounds is indicated by a change in the color of the indicating dye in the close vicinity of the colony to a color representing an acidic or alkaline reaction.
· The usefulness of this procedure is increased if media of greater buffer capacity are utilized so that only those microorganisms that produce considerable quantities of the acid or amine can induce changes in the color of the dye.
An alternative procedure for detecting organic acid production involves the incorporation of calcium carbonate (1-2 %) in the medium so that organic acid production is indicated by a cleared zone of dissolved calcium carbonate around the colony. These procedures are not foolproof, however, since inorganic acids or bases also are potential products of microbial growth. For instance, if the nitrogen source of the medium is the nitrogen of ammonium sulfate the organism may utilize the ammonium ion, leaving behind the sulfate ion as sulfuric acid, a condition indistinguishable form organic acid production. Thus cultures yielding positive reactions require further testing to be sure that an organic acid or base actually has been produced.
Primary screening of antibiotic producer (Crowded plate technique):
· The crowded plate technique is the simplest screening technique employed in detecting and isolating antibiotic producers.
· It consists of preparing a series of dilution of the source material for the antibiotic producing microorganisms, followed by spreading the dilution on the agar plates.
· The agar plates having 300- 400 or more colonies per plate after incubation for 2-4 days are observed since they are helpful in locating the colonies producing antibiotic activity.
· Colonies showing antibiotic activity is indicated by the presence of a zone of inhibition (arrow in fig) surrounding the colony.
· Such a colony is sub- cultured to a similar medium and purified.
· It is necessary to carry on further testing to confirm the antibiotic activity associated with a microorganism since zone of inhibition surrounding the colony may sometimes be due to other causes. Notable among these are a marked change in the pH value of the medium resulting from the metabolism of the colony, or rapid utilization of critical nutrients in the immediate vicinity of the colony.
· Thus, further testing again is required to prove that the inhibitory activity associated with a microorganism can really be attributed to the presence of an antibiotic.
The crowded plate technique has limited application, since usually we are interested in finding a microorganism producing antibiotic activity against specific microorganism and not against the unknown microorganism that were by chance on the plate in the vicinity of an antibiotic producing organism. Antibiotic screening is improved, therefore by the incorporation into the procedure of a “Test organism” that is an organism used as an indicator for the presence of specific antibiotic activity.
Dilutions of soil or of other microbial sources are applied to the surface of agar plates so that well isolated colonies will develop. The plates are incubated until the colonies are a few millimeters in diameter and so that antibiotic production will have occurred for those organisms having this potential. A suspension of test organism is then sprayed or applied in some manner to the surface of the agar and the plates are further incubated to allow growth of the test organism. Antibiotic activity is indicated by zones of inhibited growth of the organism around antibiotic producing colonies. In addition a rough approximation of the relative amount of antibiotic produced by barious colonies can be gained by measuring in mm the diameters of the zones of inhibited test organism growth. Antibiotic producing colonies again must be isolated and purified before further testing.
Primary screening of growth factor (Amino acid/ Vit) producer (Auxanography):
This technique is largely employed for detecting microorganisms able to produce growth factors (eg. Amino acid and Vitamins) extracellularly. The two major steps are as follows:
Step I
A filter paper strip is kept across the bottom of a petri dish in such a way that the two ends pass over the edge of the dish.
A filter paper disc of petri dish size is placed over paper strip on the bottom of the plate.
The nutrient agar is poured on the paper disc in the dish and allowed to solidify.
Microbial source material such as soil, is subjected to dilution such that aliquots on plating will produce well isolated colonies.
Plating of aliquots of properly diluted soil sample is done.
Step II
A minimal medium lacking the growth factor under consideration is seeded with the test organism.
The seeded medium is poured on the surface of a fresh petri dish and allowed to solidify.
The agar in the first plate as prepared in step- I is carefully and aseptically lifted out with the help of tweezers and a spatula and placed without inverting on the surface of the second plate as prepared in the second step.
The growth factor(s) produced by colonies present on the surface of the first layer of agar can diffuse into the lower layer of agar containing the test organism. The zone of stimulated growth of the test organism around the colonies is an indication that they produce growth factor(s) extracellularly. Productive colonies are sub cultured and are further tested.
OR
A similar screening approach can be used to find microorganisms capable of synthesizing extracellular vitamins, amino acids or other metabolites. However, the medium at makeup must be totally lacking in the metabolite under consideration. Again the microbial source is diluted and plated to provide well-isolated colonies and the test organism is applied to the plates before further incubation. The choice of the particular test organism to be used is critical. It must possess a definite growth requirement for the particular metabolite and for that metabolite only, so that production of this compound will be indicated by zones of growth or at least increased growth of the test organism adjacent to colonies that have produced the metabolite.
Enrichment culture technique:
This technique was designed by a soul microbiologist, Beijerinck, to isolate the desired microorganisms form a heterogeneous microbial population present in soil. Either medium or incubation conditions are adjusted so as to favour the growth of the desired microorganism. On the other hand, unwanted microbes are eliminated or develop poorly since they do not find suitable growth conditions in the newly created environment. Today this technique has become a valuable tool in many screening program for isolating industrially important strains.
Secondary screening
Secondary screening is strictly essential in any systematic screening programme intended to isolate industrially useful microorganisms, since primary screening merely allows the detection and isolation of microbes that possess potentially interesting industrial applications. Moreover, primary screening does not provide much information needed in setting up a new fermentation process. Secondary screening helps in detecting really useful microorganisms in fermentation processes. This can be realized by a careful understanding of the following points associated with secondary screening:
1. It is very useful in sorting our microorganisms that have real commercial value from many isolates obtained during primary screening. At the same time, microbes that have poor applicability in a fermentation process are discarded. It is advisable to discard poor cultures as soon as possible since such studies involve much labour and high expense.
2. It provides information whether the product produced by a microorganism is a new one or not. This may be accomplished by paper, thin layer or other chromatographic techniques.
3. It gives an idea about the economic position of the fermentation process involving the use of a newly discovered culture. Thus one may have a comparative study of this process with processes that are already known, so far as the economic status picture is concerned.
4. It helps in providing information regarding the product yield potentials of different isolates. Thus this is useful in selecting efficient cultures for the fermentation processes.
5. It determines the optimum conditions for growth or accumulation of a product associated with a particular culture.
6. It provides information pertaining to the effect of different components of a medium. This is valuable in designing the medium that may be attractive so far as economic consideration is concerned.
7. It detects gross genetic instability in microbial cultures. This type of information is very important, since microorganisms tending to undergo mutation or alteration is some way may lose their capability for maximum accumulation of the fermentation products.
8. It gives information about the number of products produced in a single fermentation. Additional major or minor products are of distinct value, since their recovery and sale as by-products can markedly improve the economic status of the prime fermentation.
9. Information about the solubility of the product in various organic solvents is made available. (useful in product recovery operation and purification).
10. Chemical, physical and biological properties of a product are also determined during secondary screening. Moreover, it reveals whether a product produced in the culture broth occurs in more than one chemical form.
11. It reveals whether the culture is homofermentative or heterofermentative.
12. Determination of the structure of product is done. The product may have a simple, complex or even a macromolecular structure.
13. With certain types of products (e.g. antibiotics) determination of the toxicity for animals, plants or man are made if they are to be used for therapeutic purpose.
14. It reveals whether microorganisms are capable of chemical change or of even destroying their own fermentation products. E.g. microorganism that produce the adaptive enzyme, decarboxylase can remove carbon dioxide from amino acid, leaving behind an organic amine.
15. It tells us something about the chemical stability of the fermentation product.

Thus, secondary screening gives answers to many questions that arise during final sorting out of industrially useful microorganisms. This is accomplished by performing experiments on agar plates, in flasks or small bioreactors containing liquid media, or a combination of these approaches. A specific example of antibiotic producing Streptomyces species may be taken for an understanding of the sequence of events during a screening programme.
Those streptomycetes able to produce antibiotics are detected and isolated in a primary screening programme. These streptomycetes exhibiting antimicrobial activity are subjected to an initial secondary screening where their inhibition spectra are determined. A simple “Giant – Colony technique” is used to do this. Each of the streptomycal isolates is streaked in a narrow band across the centers of the nutritious agar plates. Then, these plates are incubated until growth of a streptomycete occurs. Now, the test organisms are streaked from the edges of the plates upto bur not touching the streptomycete growth. Again, the plates are incubated. At the end of incubation, growth inhibitory zones for each test organism are measured in millimeters. Thus, the microbial inhibition spectrum study extensively helps in discarding poor cultures. Ultimately, streptomycete isolates that have exhibited interesting microbial inhibition spectra need further testing. With streptomycetes suspected to produce antibiotics with poor solubility in water, the initial secondary screening is done in some different way.
Further screening is carried our employing liquid media in flask, since such studies give more information than that which can be obtained on agar media. At the same time, it is advisable to use accurate assay technique (e.g. paper disc agar diffusion assay) to exactly determine the amounts of antibiotic present in samples of culture fluids. Thus , each of the streptomycete isolates is studied by using several different liquid media in Erlenmeyer flasks provided with baffles. These streptomycete cultures are inoculated into sterilized liquid media. Then , such seeded flasks are incubated at a constant temperature. Usually such cultures are incubated at near room temperature. Moreover, such flasks are aerated by keeping them on mechanical shaker, since the growth of streptomycetes and production of antibiotics occur better in aerated flasks than in stationary ones. Samples are withdrawn at regular intervals under aseptic conditions and are tested in a quality control laboratory. Important tests to be carried out include:
i. Checking for contamination,
ii. Checking of pH
iii. Estimation of critical nutrients
iv. Assaying of the antibiotic, and
v. Other determinations, if necessary

The result of the above test, points out the best medium for antibiotic formation and the stage at which the antibiotic yields are greatest during the growth of culture on different media. After performing all necessary routine tests in the screening of an actually useful streptomycete for the fermentation process, other additional determinations are made. They are:
i. Screening of fermentation media through the exploitation of which the highest antibiotic yields may be obtained.
ii. Determination of whether the antibiotic is new.
iii. Determination of the number of antibiotics accumulated in the culture broth is made.
iv. Effect of different bioparameters on the growth of streptomycete culture, fermentation process and accumulation of antibiotic.
v. Solubility picture of antibiotic in various organic solvents. Also, it is to be determined whether antibiotic is adsorbed by adsorbent materials.
vi. Toxicity tests are conducted on mice or other laboratory animals. An antibiotic is also tested for the adverse effects if any, on man, animal or plant.
vii. The streptomycete culture is characterized and is classified upto species.
viii. Further studies are made on a selected individual streptomycete culture. For example mutation and other genetic studies for strain improvement are carried out.
In conclusion, tests are designed and conducted in such a way that production streptomycete strains may be obtained with least expenses. Similar screening and analytical techniques could be employed for the isolation of microbial isolates important in the production of other industrial chemical substances.

Candidiasis

Oropharyngeal Candidiasis, Esophageal Candidiasis (OPC)
What is OPC?
Candidiasis of the mouth and throat, also known as a "thrush" or oropharyngeal candidiasis (OPC), is a fungal infection that occurs when there is overgrowth of fungus called Candida. Candida is normally found on skin or mucous membranes. However, if the environment inside the mouth or throat becomes imbalanced, Candida can multiply. When this happens, symptoms of thrush appear. Candida overgrowth can also develop in the esophagus, and is called Candida esophagitis, or esophageal candidiasis.
OPC can affect normal newborns, persons with dentures, and people who use inhaled corticosteroids. It occurs more frequently and more severely in people with weakened immune systems, particularly in persons with AIDS and people undergoing treatment for cancer. Candida esophagitis usually occurs in people with weakened immune systems. It is very unusual in otherwise healthy people.
Most cases of OPC are caused by the person’s own Candida organisms which normally live in the mouth or digestive tract. A person has symptoms when overgrowth of Candida organisms occurs.
Symptoms of OPC
People with OPC infection usually have painless, white patches in the mouth. Others may have redness and soreness of the inside of the mouth. Cracking at the corners of the mouth, known as angular cheilitis, may occur. Symptoms of Candida esophagitis may include pain and difficulty swallowing. Other conditions can cause similar symptoms, so it is important to see your doctor.
How is OPC diagnosed?
OPC is often diagnosed based on the clinical appearance of the mouth and by taking a scraping of the white patches and looking at it under a microscope. A culture may also be performed. Because Candida organisms are normal inhabitants of the human mouth, a positive culture by itself does not make the diagnosis.
How is OPC treated?
Prescription treatments include clotrimazole troches or lozenges and nystatin suspension (nystatin “swish and swallow”). Another commonly prescribed treatment is oral fluconazole. For infection which does not respond to these treatments, there are a number of other antifungal drugs that are available.

What will happen if a person does not seek treatment for a OPC?
Symptoms, which may be uncomfortable, may persist. In rare cases, invasive candidiasis may occur.
Can OPC become resistant to treatment?
Yes, OPC and Candida esophagitis can become resistant to antifungal treatment over time. Therefore, it is important to see your doctor for evaluation if you think you have OPC or Candida esophagitis.
Genital / Volvovaginal Candidiasis (VVC)
What is genital candidiasis /VVC?
Candidiasis, also known as a "yeast infection" or VVC, is a common fungal infection that occurs when there is overgrowth of the fungus called Candida. Candida is always present in the body in small amounts. However, when an imbalance occurs, such as when the normal acidity of the vagina changes or when hormonal balance changes, Candida can multiply. When that happens, symptoms of candidiasis appear.
Symptoms of genital candidiasis /VVC:
Women with VVC usually experience genital itching or burning, with or without a "cottage cheese-like" vaginal discharge. Males with genital candidiasis may experience an itchy rash on the penis.
How is genital candidiasis/VVC transmitted?
Nearly 75% of all adult women have had at least one genital "yeast infection" in their lifetime. On rare occasions, men may also experience genital candidiasis. VVC occurs more frequently and more severely in people with weakened immune systems. There are some other conditions that may put a woman at risk for genital candidiasis:
Pregnancy
Diabetes mellitus
Use of broad-spectrum antibiotics
Use of corticosteroid medications
What are the symptoms of OPC?
Most cases of Candida infection are caused by the person’s own Candida organisms. Candida yeasts usually live in the mouth, gastrointestinal tract, and vagina without causing symptoms. Symptoms develop only when Candida becomes overgrown in these sites. Rarely, Candida can be passed from person to person, such as through sexual intercourse.
How is genital candidiasis/VVC diagnosed?
The symptoms of genital candidiasis are similar to those of many other genital infections. Usually the diagnosis is made by taking a sample of the vaginal secretions and looking at it under a microscope to see if Candida organisms are present.
How is genital candidiasis/VVC treated?
Several antifungal drugs are available to treat genital candidiasis/VVC. Antifungal vaginal suppositories or creams are commonly used. The duration of the treatment course of suppositories and creams ranges from single dose therapy to 7 days of therapy. Uncomplicated VVC may also be treated with single-dose, oral fluconazole. Oral fluconazole should be avoided in pregnancy. These drugs usually work to cure the infection (80%-90% success rate), but some people will have recurrent or resistant infections. Short-course treatments should be avoided in recurrent or resistant infection.
Are over-the-counter (OTC) treatments for genital candidiasis/VVC safe to use?
Over-the-counter treatments for VVC are available. As a result, more women are diagnosing themselves with VVC and using one of a family of drugs called "azoles" for therapy. However, misdiagnosis is common, and studies have shown that as many as two-thirds of all OTC drugs sold to treat VVC were used by women without the disease. Using these drugs when they are not needed may lead to a resistant infection. Resistant infections are very difficult to treat with the currently available medications for VVC.
Can Candida infections become resistant to treatment?
Overuse of these antifungal medications can increase the chance that they will eventually not work (the fungus develops resistance to medications). Therefore, it is important to be sure of the diagnosis before treating with over-the-counter or other antifungal medications.
What will happen if a person does not seek treatment for genital candidiasis/VVC?
Symptoms, which may be very uncomfortable, may persist. There is a chance that the infection may be passed between sex partners.

How can someone tell the difference between genital candidiasis/VVC and a urinary tract infection?
Because VVC and urinary tract infections share similar symptoms, such as a burning sensation when urinating, it is important to see a doctor and obtain laboratory testing to determine the cause of the symptoms and to treat effectively.
Invasive Candidiasis (VVC)
What is invasive candidiasis?
Invasive candidiasis is a fungal infection that occurs when Candida species enter the blood, causing bloodstream infection and then spreading throughout the body.
How common is invasive candidiasis and who can get it?
One form of invasive candidiasis, candidemia (a bloodstream infection with Candida), is the fourth most common bloodstream infection among hospitalized patients in the United States. A survey conducted at CDC found that candidemia occurs in 8 of every 100,000 persons per year. Persons at high risk for candidemia include very-low-birth-weight babies, surgical patients, hospitalized patients or patients with a central venous catheter, and those whose immune systems are weakened.
What are the symptoms of invasive candidiasis?
The symptoms of invasive candidiasis are not specific. Fever and chills that do not improve after antibiotic therapy are the most common symptoms. If the infection spreads to deep organs such as kidneys, liver, bones, muscles, joints, spleen, or eyes, additional specific symptoms may develop, which vary depending on the site of infection. If the infection does not respond to treatment, the patient’s organs may fail and cause death.
How is invasive candidiasis transmitted?
Invasive candidiasis is extremely rare in persons without risk factors. In persons at risk, invasive candidiasis may result when a person’s own Candida organisms, normally found in the digestive tract, enter the bloodstream. On rare occasions, it can also occur when medical equipment or devices become contaminated with Candida. In either case, the infection may spread throughout the body.
How is invasive candidiasis diagnosed?
Invasive candidiasis is usually diagnosed by either culture of blood or tissue or by examining samples of infected tissue under the microscope.
How is invasive candidiasis treated?
There are a number of antifungal drugs that are now available to treat invasive candidiasis. Fluconazole is a drug that can be taken by mouth or given intravenously (IV) to treat invasive candidiasis. Another class of antifungal drugs, the echinocandins, are also commonly used to treat invasive candidiasis. There are three echinocandins, all IV only: caspofungin, micafungin, and anidulafungin. All threse are effective in treating invasive candidiasis. Other drugs that are sometimes used to treat invasive candidiasis include voriconazole (by mouth or IV) and amphotericin B formulations (IV only).

Cholera

CHOLERA
In January 1991, epidemic cholera appeared in South America and quickly spread to several countries. A few cases have occurred in the United States among persons who traveled to South America or ate contaminated food brought back by travelers.
Cholera has been very rare in industrialized nations for the last 100 years; however, the disease is still common today in other parts of the world, including the Indian subcontinent and sub-Saharan Africa.
Although cholera can be life-threatening, it is easily prevented and treated. In the United States, because of advanced water and sanitation systems, cholera is not a major threat; however, everyone, especially travelers, should be aware of how the disease is transmitted and what can be done to prevent it.
What is cholera ?
Cholera is an acute, diarrheal illness caused by infection of the intestine with the bacterium Vibrio cholerae. The infection is often mild or without symptoms, but sometimes it can be severe. Approximately one in 20 infected persons has severe disease characterized by profuse watery diarrhea, vomiting, and leg cramps. In these persons, rapid loss of body fluids leads to dehydration and shock. Without treatment, death can occur within hours.
How does a person get cholera?
A person may get cholera by drinking water or eating food contaminated with the cholera bacterium. In an epidemic, the source of the contamination is usually the feces of an infected person. The disease can spread rapidly in areas with inadequate treatment of sewage and drinking water. The cholera bacterium may also live in the environment in brackish rivers and coastal waters. Shellfish eaten raw have been a source of cholera, and a few persons in the United States have contracted cholera after eating raw or undercooked shellfish from the Gulf of Mexico. The disease is not likely to spread directly from one person to another; therefore, casual contact with an infected person is not a risk for becoming ill.
What is the risk for cholera in the United States?
In the United States, cholera was prevalent in the 1800s but has been virtually eliminated by modern sewage and water treatment systems. However, as a result of improved transportation, more persons from the United States travel to parts of Africa, Asia, or Latin America where epidemic cholera is occurring. U.S. travelers to areas with epidemic cholera may be exposed to the cholera bacterium. In addition, travelers may bring contaminated seafood back to the United States; food borne outbreaks have been caused by contaminated seafood brought into this country by travelers.
What should travelers do to avoid getting cholera?
The risk for cholera is very low for U.S. travelers visiting areas with epidemic cholera. When simple precautions are observed, contracting the disease is unlikely. All travelers to areas where cholera has occured should observe the following recommendations:
Drink only water that you have boiled or treated with chlorine or iodine. Other safe beverages include tea and coffee made with boiled water and carbonated, bottled beverages with no ice.
Eat only foods that have been thoroughly cooked and are still hot, or fruit that you have peeled yourself.
Avoid undercooked or raw fish or shellfish, including ceviche.
Make sure all vegetables are cooked avoid salads.
Avoid foods and beverages from street vendors.
Do not bring perishable seafood back to the United States.
A simple rule of thumb is "Boil it, cook it, peel it, or forget it. "
Is a vaccine available to prevent cholera?
A recently developed oral vaccine for cholera is licensed and available in other countries (Dukoral from SBL Vaccines). The vaccine appears to provide somewhat better immunity and have fewer adverse effects than the previously available vaccine. However, CDC does not recommend cholera vaccines for most travelers, nor is the vaccine available in the United States . Further information about Dukoral can be obtained from the manufacturers
Can cholera be treated?
Cholera can be simply and successfully treated by immediate replacement of the fluid and salts lost through diarrhea. Patients can be treated with oral rehydration solution, a prepackaged mixture of sugar and salts to be mixed with water and drunk in large amounts. This solution is used throughout the world to treat diarrhea. Severe cases also require intravenous fluid replacement. With prompt rehydration, fewer than 1% of cholera patients die.
Antibiotics shorten the course and diminish the severity of the illness, but they are not as important as rehydration. Persons who develop severe diarrhea and vomiting in countries where cholera occurs should seek medical attention promptly.

How long will the current epidemic last?
Predicting how long a Cholera epidemic will last is difficult. The cholera epidemic in Africa has lasted more than 30 years. In areas with inadequate sanitation, a cholera epidemic cannot be stopped immediately, and, although far fewer cases have been reported from Latin America and Asia in recent years, there are no signs that the global Cholera pandemic will end soon. Major improvements in sewage and water treatment systems are needed in many countries to prevent future epidemic cholera.
What is the U.S. government doing to combat cholera?
U.S. and international public health authorities are working to enhance surveillance for cholera, investigate cholera outbreaks, and design and implement preventive measures. The Centers for Disease Control and Prevention investigates epidemic cholera wherever it occurs and trains laboratory workers in proper techniques for identification of V. cholerae. In addition, the Centers for Disease Control and Prevention provides information on diagnosis, treatment, and prevention of cholera to public health officials and educates the public about effective preventive measures.
The U.S. Agency for International Development is sponsoring some of the international government activities and is providing medical supplies to affected countries. The Environmental Protection Agency is working with water and sewage treatment operators in the United States to prevent contamination of water with the cholera bacterium. The Food and Drug Administration is testing imported and domestic shellfish for V. cholerae and monitoring the safety of U.S. shellfish beds through the shellfish sanitation program. With cooperation at the state and local, national, and international levels, assistance will be provided to countries where cholera is present, and the risk to U.S. residents will remain small.
Where can a traveler get information about cholera?
The global picture of cholera changes periodically, so travelers should seek updated information on countries of interest. The Centers for Disease Control and Prevention maintains a travelers' information telephone line on which callers can receive recent information on cholera and other diseases of concern to travelers. Data for this service are obtained from the World Health Organization. The number is 877-FYI-TRIP (394-8747) or check out http://www.cdc.gov/travel.
Cholera
Cholera is an acute diarrhoeal infection caused by ingestion of the bacterium Vibrio cholerae. Transmission occurs through direct faecal-oral contamination or through ingestion of contaminated water and food. The disease is characterized in its most severe form by a sudden onset of acute watery diarrhoea that can lead to death by severe dehydration and kidney failure. The extremely short incubation period - two hours to five days - enhances the potentially explosive pattern of outbreaks, as the number of cases can rise very quickly. About 75% of people infected with cholera do not develop any symptoms. However, the pathogens stay in their faeces for 7 to 14 days and are shed back into the environment, potentially infecting other individuals. Cholera is an extremely virulent disease that affects both children and adults. Unlike other diarrhoeal diseases, it can kill healthy adults within hours. Individuals with lower immunity, such as malnourished children or people living with HIV, are at greater risk of death if infected by cholera.
Background
During the 19th century, cholera spread repeatedly from its original reservoir or source in the Ganges delta in India to the rest of the world, before receding to South Asia. Six pandemics were recorded that killed millions of people across Europe, Africa and the Americas. The seventh pandemic, which is still ongoing, started in 1961 in South Asia, reached Africa in 1971 and the Americas in 1991. The disease is now considered to be endemic in many countries and the pathogen causing cholera cannot currently be eliminated from the environment.
Two serogroups of V. cholerae - O1 and O139 - can cause outbreaks. The main reservoirs are human beings and aquatic sources such as brackish water and estuaries, often associated with algal blooms (plankton). Recent studies indicate that global warming might create a favourable environment for V. cholerae and increase the incidence of the disease in vulnerable areas. V. cholerae O1 causes the majority of outbreaks worldwide. The serogroup O139, first identified in Bangladesh in 1992, possesses the same virulence factors as O1, and creates a similar clinical picture. Currently, the presence of O139 has been detected only in South-East and East Asia, but it is still unclear whether V. cholerae O139 will extend to other regions. Careful epidemiological monitoring of the situation is recommended and should be reinforced. Other strains of V. cholerae apart from O1 and O139 can cause mild diarrhoea but do not develop into epidemics.
Risk factors and vulnerable populations
Cholera is mainly transmitted through contaminated water and food and is closely linked to inadequate environmental management. The absence or shortage of safe water and sufficient sanitation combined with a generally poor environmental status are the main causes of spread of the disease. Typical at-risk areas include peri-urban slums, where basic infrastructure is not available, as well as camps for internally displaced people or refugees, where minimum requirements of clean water and sanitation are not met. However, it is important to stress that the belief that cholera epidemics are caused by dead bodies after disasters, whether natural or man-made, is false. Nonetheless, rumours and panic are often rife in the aftermath of a disaster. On the other hand, the consequences of a disaster -- such as disruption of water and sanitation systems or massive displacement of population to inadequate and overcrowded camps -- can increase the risk of transmission, should the pathogen be present or introduced.
Since 2005, the re-emergence of cholera has been noted in parallel with the ever-increasing size of vulnerable populations living in unsanitary conditions. Cholera remains a global threat to public health and one of the key indicators of social development. While the disease is no longer an issue in countries where minimum hygiene standards are met, it remains a threat in almost every developing country. The number of cholera cases reported to WHO during 2006 rose dramatically, reaching the level of the late 1990s. A total of 236 896 cases were notified from 52 countries, including 6311 deaths, an overall increase of 79% compared with the number of cases reported in 2005. This increased number of cases is the result of several major outbreaks that occurred in countries where cases have not been reported for several years. It is estimated that only a small proportion of cases - less than 10% - are reported to WHO. The true burden of disease is therefore grossly underestimated.
Prevention and control of Cholera outbreaks
Among people developing symptoms, 80% of episodes are of mild or moderate severity. Among the remaining cases, 10%-20% develop severe watery diarrhoea with signs of dehydration. If untreated, as many as one in two people may die. With proper treatment, the fatality rate should stay below 1%.
Measures for the prevention of cholera have not changed much in recent decades, and mostly consist of providing clean water and proper sanitation to populations potentially affected. Health education and good food hygiene are equally important. In particular, systematic hand washing should be taught. Once an outbreak is detected, the usual intervention strategy is to reduce mortality by ensuring prompt access to treatment and controlling the spread of the disease.
The majority of patients - up to 80% - can be treated adequately through the administration of oral rehydration salts (WHO/UNICEF ORS standard sachet). Very severely dehydrated patients are treated through the administration of intravenous fluids, preferably Ringer lactate. Appropriate antibiotics can be given to severe cases to diminish the duration of diarrhoea, reduce the volume of rehydration fluids needed and shorten the duration of vibrio excretion. Routine treatment of a community with antibiotics, or "mass chemoprophylaxis", has no effect on the spread of cholera and can have adverse effects by increasing antimicrobial resistance. In order to ensure timely access to treatment, cholera treatment centres should be set up among the affected populations whenever feasible.
The provision of safe water and sanitation is a formidable challenge but remains the critical factor in reducing the impact of cholera outbreaks. Recommended control methods, including standardized case management, have proven effective in reducing the case-fatality rate. Comprehensive surveillance data are of paramount importance to guide the interventions and adapt them to each specific situation. In addition, cholera prevention and control is not an issue to be dealt by the health sector alone. Water, sanitation, education and communication are among the other sectors usually involved. A comprehensive multidisciplinary approach should be adopted for dealing with a potential cholera outbreak.
Oral cholera vaccines
The use of the parenteral cholera vaccine has never been recommended by WHO due to its low protective efficacy and the high occurrence of severe adverse reactions. An internationally licensed oral cholera vaccine (OCV) is currently available on the market and is suitable for travellers. This vaccine was proven safe and effective (85–90% after six months in all age groups, declining to 62% at one year among adults) and is available for individuals aged two years and above. It is administered in two doses 10-15 days apart and given in 150 ml of safe water. Its public health use in mass vaccination campaigns is relatively recent. Within the past few years several immunization campaigns were carried out with WHO support. In 2006, WHO published official recommendations for OCV use in complex emergencies.
Travel and trade
Today, no country requires proof of cholera vaccination as a condition for entry and the International Certificate of Vaccination no longer provides a specific space for recording cholera vaccinations. Past experience clearly showed that quarantine measures and embargoes on movements of people and goods - especially food products - are unnecessary. At present, WHO has no information that food commercially imported from affected countries has been implicated in outbreaks of cholera in importing countries. The isolated cases of cholera that have been related to imported food have been associated with food which had been in the possession of individual travellers. Therefore, it may be concluded that food produced under good manufacturing practices poses only a negligible risk for cholera transmission. Consequently, WHO believes that food import restrictions, based on the sole fact that cholera is epidemic or endemic in a country, are not justified.