IMMUNOTECHNOLOGY

An antigen is a substance/molecule that when introduced into the body triggers the production of an antibody by the immune system which will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as pollen or cells such as bacteria. Originally the term came from antibody generator and was a molecule that binds specifically to an antibody, but the term now also refers to any molecule or molecular fragment that can be bound by a major histocompatibility complex (MHC) and presented to a T-cell receptor"Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system. Autoimmune disorders arise from the immune system reacting to its own antigens. Antigen

Each antibody binds to a specific antigen; an interaction similar to a lock and key. Similarly, an immunogen is a specific type of antigen. An immunogen is defined as a substance that is able to provoke an adaptive immune response if injected on its own.Said another way, an immunogen is able to induce an immune response, while an antigen is able to combine with the products of an immune response once they are made. The overlapping concepts of immunogenicity and antigenicity are thereby subtly different. According to a current text book:

Immunogenicity is the ability to induce a humoral and/or cell-mediated immune response

Antigenicity is the ability to combine specifically with the final products of the [immune response] (i.e. secreted antibodies and/or surface receptors on T-cells). Although all molecules that have the property of immunogenicity also have the property of antigenicity, the reverse is not true."

At the molecular level, an antigen is characterized by its ability to be "bound" at the antigen-binding site of an antibody. Note also that antibodies tend to discriminate between the specific molecular structures presented on the surface of the antigen (as illustrated in the Figure).

UNIT I ANTIGENS

Antigens are usually proteins or polysaccharides. This includes parts (coats, capsules, cell walls, flagella, fimbrae, and toxins) of bacteria, viruses, and other microorganisms. Lipids and nucleic acids are antigenic only when combined with proteins and polysaccharides. Non-microbial exogenous (non-self) antigens can include pollen, egg white, and proteins from transplanted tissues and organs or on the surface of transfused blood cells. Vaccines are examples of immunogenic antigens intentionally administered to induce acquired immunity in the recipient.

Cells present their immunogenic-antigens to the immune system via a histocompatibility molecule. Depending on the antigen presented and the type of the histocompatibility molecule, several types of immune cells can become activated.

Related concepts

• Epitope - The distinct molecular surface features of an antigen capable of being bound by an antibody (a.k.a. antigenic determinant). Antigenic molecules, normally being "large" biological polymers, usually present several surface features that can act as points of interaction for specific antibodies. Any such distinct molecular feature constitutes an epitope. Most antigens therefore have the potential to be bound by several distinct antibodies, each of which is specific to a particular epitope. Using the "lock and key" metaphor, the antigen itself can be seen as a string of keys - any epitope being a "key" - each of which can match a different lock. Different antibody idiotypes, each having distinctly formed complementarity determining regions, correspond to the various "locks" that can match "the keys" (epitopes) presented on the antigen molecule.

• Allergen - A substance capable of causing an allergic reaction. The (detrimental) reaction may result after exposure via ingestion, inhalation, injection, or contact with skin.

• Superantigen - A class of antigens which cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release.

• Tolerogen - A substance that invokes a specific immune non-responsiveness due to its molecular form. If its molecular form is changed, a tolerogen can become an immunogen.

• Immunoglobulin binding protein - These proteins are capable of binding to antibodies at positions outside of the antigen-binding site. That is, whereas antigens are the "target" of antibodies, immunoglobulin binding proteins "attack" antibodies. Protein A, protein G and protein L are examples of proteins that strongly bind to various antibody isotypes.

Origin of the term antigen

In 1899 Ladislas Deutsch (Laszlo Detre) (1874–1939) named the hypothetical substances halfway between bacterial constituents and antibodies "substances immunogenes ou antigenes". He originally believed those substances to be precursors of antibodies, just like zymogen is a precursor of zymase. But by 1903 he understood that an antigen induces the production of immune bodies (antibodies) and wrote that the word antigen was a contraction of "Antisomatogen = Immunkörperbildner". The Oxford English Dictionary indicates that the logical construction should be "anti(body)-gen"[6].

Classification of antigens

Antigens can be classified in order of their class.

Exogenous antigens Exogenous antigens are antigens that have entered the body from the outside, for example by inhalation, ingestion, or injection. The immune system's response to exogenous antigens is often subclinical. By endocytosis or phagocytosis, exogenous antigens are taken into the antigen-presenting cells (APCs) and processed into fragments. APCs then present the fragments to T helper cells (CD4+) by the use of class II histocompatibility molecules on their surface. Some T cells are specific for the peptide:MHC complex. They become activated and start to secrete cytokines. Cytokines are substances that can activate cytotoxic T lymphocytes (CTL), antibody-secreting B cells, macrophages, and other particles.

Some antigens start out as exogenous antigens, and later become endogenous (for example, intracellular viruses). Intracellular antigens can be released back into circulation upon the destruction of the infected cell, again. Endogenous antigens Endogenous antigens are antigens that have been generated within previously normal cells as a result of normal cell metabolism, or because of viral or intracellular bacterial infection. The fragments are then presented on the cell surface in the complex with MHC class I molecules. If activated cytotoxic CD8+ T cells recognize them, the T cells begin to secrete various toxins that cause the lysis or apoptosis of the infected cell. In order to keep the cytotoxic cells from killing cells just for presenting self-proteins, self-reactive T cells are deleted from the repertoire as a result of tolerance (also known as negative selection). Endogenous antigens include xenogenic (heterologous), autologous and idiotypic or allogenic (homologous) antigens. Autoantigens An autoantigen is usually a normal protein or complex of proteins (and sometimes DNA or RNA) that is recognized by the immune system of patients suffering from a specific autoimmune disease. These antigens should, under normal conditions, not be the target of the immune system, but, due to mainly genetic and environmental factors, the normal immunological tolerance for such an antigen has been lost in these patients.

Tumor antigens

Tumor antigens or neoantigens are[citation needed] those antigens that are presented by MHC I or MHC II molecules on the surface of tumor cells. These antigens can sometimes be presented by tumor cells and never by the normal ones. In this case, they are called tumor-specific antigens (TSAs) and, in general, result from a tumor-specific mutation. More common are antigens that are presented by tumor cells and normal cells, and they are called tumor-associated antigens (TAAs). Cytotoxic T lymphocytes that recognize these antigens may be able to destroy the tumor cells before they proliferate or metastasize.

Tumor antigens can also be on the surface of the tumor in the form of, for example, a mutated receptor, in which case they will be recognized by B cells.

Nativity

A native antigen is an antigen that is not yet processed by an APC to smaller parts. T cells cannot bind native antigens, but require that they be processed by APCs, whereas B cells can be activated by native ones.

Antigenic specificity

Antigen(ic) specificity is the ability of the host cells to recognize an antigen specifically as a unique molecular entity and distinguish it from another with exquisite precision. Antigen specificity is due primarily to the side-chain conformations of the antigen. It is a measurement, although the degree of specificity may not be easy to measure, and need not be linear or of the nature of a rate-limited step or equation.

Structure of antigen

Preparation Of Antigens For Raising Antibodies Polyclonal antibodies (or antisera) are antibodies that are obtained from different B cell resources. They are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope.

Production

These antibodies are typically produced by immunization of a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is purified from the mammal’s serum.By contrast, monoclonal antibodies are derived from a single cell line.

Many methodologies exist for polyclonal antibody production in laboratory animals. Institutional guidelines governing animal use and procedures relating to these methodologies are generally oriented around humane considerations and appropriate conduct for adjuvant (agents which modify the effect of other agents while having few if any direct effects when given by

themselves) use. This includes adjuvant selection, routes and sites of administration, injection volumes per site and number of sites per animal. Institutional policies generally include allowable volumes of blood per collection and safety precautions including appropriate restraint and sedation or anesthesia of animals for injury prevention to animals or personnel.

The primary goal of antibody production in laboratory animals is to obtain high titer, high affinity antisera for use in experimentation or diagnostic tests. Adjuvants are used to improve or enhance an immune response to antigens. Most adjuvants provide for an injection site, antigen depot which allows for a slow release of antigen into draining lymph nodes.

Many adjuvants also contain or act directly as:

1. surfactants which promote concentration of protein antigens molecules over a large surface area, and

2. immunostimulatory molecules or properties. Adjuvants are generally used with soluble protein antigens to increase antibody titers and induce a prolonged response with accompanying memory.

Such antigens by themselves are generally poor immunogens. Most complex protein antigens induce multiple B-cell clones during the immune response, thus, the response is polyclonal. Immune responses to non-protein antigens are generally poorly or enhanced by adjuvants and there is no system memory.

Antibodies are currently also being produced from isolation of human B-lymphocytes to produce specific recombinant polyclonal antibodies. The biotechnology company, Symphogen, produces this type of antibody for therapeutic applications. They are the first research company to develop recombinant polyclonal antibody drugs to reach phase two trials. This production prevents viral and prion transmission.

Animal selection

Animals frequently used for polyclonal antibody production include chickens, goats, guinea pigs, hamsters, horses, mice, rats, and sheep. However, the rabbit is the most commonly used laboratory animal for this purpose. Animal selection should be based upon:

1. the amount of antibody needed,

2. the relationship between the donor of the antigen and the recipient antibody producer (generally the more distant the phylogenetic relationship, the greater the potential for high titer antibody response) and

3. the necessary characteristics [e.g., class, subclass (isotype), complement fixing nature] of the antibodies to be made. Immunization and phlebotomies are stress associated and, at least when using rabbits and rodents, specific pathogen free (SPF) animals are preferred. Use of such animals can dramatically reduce morbidity and mortality due to pathogenic organisms, especially Pasteurella multocida in rabbits.

Goats or horses are generally used when large quantities of antisera are required. Many investigators favor chickens because of their phylogenetic distance from mammals. Chickens transfer high quantities of IgY (IgG) into the egg yolk and harvesting antibodies from eggs eliminates the need for the invasive bleeding procedure. One week’s eggs can contain 10 times more antibodies than the volume of rabbit blood obtained from one weekly bleeding. However, there are some disadvantages when using certain chicken derived antibodies in immunoassays.

Chicken IgY does not fix mammalian complement component C1 and it does not perform as a precipitating antibody using standard solutions.

Although mice are used most frequently for monoclonal antibody production, their small size usually prevents their use for sufficient quantities of polyclonal, serum antibodies. However, polyclonal antibodies in mice can be collected from ascites fluid using any one of a number of ascites producing methodologies.

When using rabbits, young adult animals (2.5–3.0 kg or 5.5-6.5lbs) should be used for primary immunization because of the vigorous antibody response. Immune function peaks at puberty and primary responses to new antigens decline with age. Female rabbits are generally preferred because they are more docile and are reported to mount a more vigorous immune response than males. At least two animals per antigen should be used when using outbred animals. This principle reduces potential total failure resulting from non-responsiveness to antigens of individual animals.

Antigen preparation

The size, extent of aggregation and relative nativity of protein antigens can all dramatically affect the quality and quantity of antibody produced. Small polypeptides (<10 ku) and non-protein antigens generally need to be conjugated or crosslinked to larger, immunogenic, carrier proteins to increase immunogenicity and provide T cell epitopes. Generally, the larger the immunogenic protein the better. Larger proteins, even in smaller amounts, usually result in better engagement of antigen presenting antigen processing cells for a satisfactory immune response. Injection of soluble, non-aggregated proteins has a higher probability of inducing tolerance rather than a satisfactory antibody response.

Keyhole limpet hemocyanin (KLH) and bovine serum albumin are two widely used carrier proteins. Poly-L-lysine has also been used successfully as a backbone for peptides. Although the use of Poly-L-lysine reduces or eliminates production of antibodies to foreign proteins, it may result in failure of peptide-induced antibody production. Recently, liposomes have also been successfully used for delivery of small peptides and this technique is an alternative to delivery with oily emulsion adjuvants.

Antigen quantity Selection of antigen quantity for immunization varies with the properties of the antigen and the adjuvant selected. In general, microgram to milligram quantities of protein in adjuvant are necessary to elicit high titer antibodies. Antigen dosage is generally species, rather than body weight, associated. The so called “window” of immunogenicity in each species is broad but too much or too little antigen can induce tolerance, suppression or immune deviation towards cellular immunity rather than a satisfactory humoral response. Optimal and usual protein antigen levels for immunizing specific species have been reported in the following ranges:

1. rabbit, 50–1000 µg;

2. mouse, 10–200 µg;

3. guinea pig, 50–500 µg; and

4. goat, 250–5000 µg.

Optimal “priming” doses are reported to be at the low end of each range.

The affinity of serum antibodies increases with time (months) after injection of antigen-adjuvant mixtures and as antigen in the system decreases. Widely used antigen dosages for “booster” or secondary immunizations are usually one half to equal the priming dosages. Antigens should be free of preparative byproducts and chemicals such as polyacrylamide gel, SDS, urea, endotoxin, particulate matter and extremes of pH.

Peptide Antibodies When a peptide is being used to generate the antibody, it is extremely important to design the antigens properly. There are several resources that can aid in the design as well as companies that offer this service. Expasy has aggregated a set of public tools under its ProtScale page that require some degree of user knowledge to navigate. For a more simple peptide scoring tool there is a Antigen Profiler tool available that will enable you to score individual peptide sequences based upon a relation epitope mapping database of previous immunogens used to generate antibodies. Finally, as a general rule peptides should follow some basic criteria.

When examining peptides for synthesis and immunization, it is recommended that certain residues and sequences be avoided due to potential synthesis problems. This includes some of the more common characteristics:

• Extremely long repeats of the same amino acid (e.g. RRRR)

• Serine (S), Threonine (T), Alanine (A), and Valine (V) doublets

• Ending or starting a sequence with a proline (P)

• Glutamine (Q) or Asparagine (N) at the n-terminus

• Peptides over weighted with hydrophobic residues (e.g. V,A,L,I, etc…) Reactivity Investigators should also consider the status of nativity of protein antigens when used as immunogens and reaction with antibodies produced. Antibodies to native proteins react best with native proteins and antibodies to denatured proteins react best with denatured proteins. If elicited antibodies are to be used on membrane blots (proteins subjected to denaturing conditions) then antibodies should be made against denatured proteins. On the other hand, if antibodies are to be used to react with a native protein or block a protein active site, then antibodies should be made against the native protein. Adjuvants can often alter the nativity of the protein. Generally, absorbed protein antigens in a preformed oil-in-water emulsion adjuvant, retain greater native protein structure than those in water-in-oil emulsions. Asepticity Antigens should always be prepared using techniques that ensure that they are free of microbial contamination. Most protein antigen preparations can be sterilized by passage through a 0.22u filter. Septic abscesses often occur at inoculation sites of animals when contaminated preparations are used. This can result in failure of immunization against the targeted antigen.

Adjuvants

There are many commercially available immunologic adjuvants. Selection of specific adjuvants or types varies depending upon whether they are to be used for research and antibody production or in vaccine development. Adjuvants for vaccine use only need to produce protective antibodies and good systemic memory while those for antiserum production need to rapidly induce high titer, high avidity antibodies. No single adjuvant is ideal for all purposes and all have advantages and disadvantages. Adjuvant use generally is accompanied by undesirable side effects of varying

severity and duration. Research on new adjuvants focuses on substances which have minimal toxicity while retaining maximum immunostimulation. Investigators should always be aware of potential pain and distress associated with adjuvant use in laboratory animals.

The most frequently used adjuvants for antibody production are Freund’s, Alum, the Ribi Adjuvant System and Titermax. Freund’s adjuvants There are two basic types of Freund's adjuvants: Freund’s Complete Adjuvant (FCA) and Freund’s Incomplete Adjuvant (FIA). FCA is a water-in-oil emulsion that localizes antigen for release periods up to 6 months. It is formulated with mineral oil, the surfactant mannide monoleate and heat killed Mycobacterium tuberculosis, Mycobacterium butyricum or their extracts (for aggregation of macrophages at the inoculation site). This potent adjuvant stimulates both cell mediated and humoral immunity with preferential induction of antibody against epitopes of denatured proteins. Although FCA has historically been the most widely used adjuvant, it is one of the more toxic agents due to non-metabolizable mineral oil and it induces granulomatous reactions. Its use is limited to laboratory animals and it should be used only with weak antigens. It should not be used more than once in a single animal since multiple FCA inoculations can cause severe systemic reactions and decreased immune responses. Freund’s Incomplete Adjuvant has the same formulation as FCA but does not contain mycobacterium or its components. FIA usually is limited to booster doses of antigen since it normally much less effective than FCA for primary antibody induction. Freund’s adjuvants are normally mixed with equal parts of antigen preparations to form stable emulsions. Ribi Adjuvant System Ribi adjuvants are oil-in-water emulsions where antigens are mixed with small volumes of a metabolizable oil (squalene) which are then emulsified with saline containing the surfactant Tween 80. This system also contains refined mycobacterial products (cord factor, cell wall skeleton) as immunostimulants and bacterial monophosphoryl lipid A. Three different species oriented formulations of the adjuvant system are available. These adjuvants interact with membranes of immune cells resulting in cytokine induction, which enhances antigen uptake, processing and presentation. This adjuvant system is much less toxic and less potent than FCA but generally induces satisfactory amounts of high avidity antibodies against protein antigens. Titermax Titermax represents a newer generation of adjuvants that are less toxic and contain no biologically derived materials. It is based upon mixtures of surfactant acting, linear, blocks or chains of nonionic copolymers polyoxypropylene (POP) and polyoxyethylene (POE). These copolymers are less toxic than many other surfactant materials and have potent adjuvant properties which favor chemotaxis, complement activation and antibody production. Titermax adjuvant forms a microparticulate water-in-oil emulsion with a copolymer and metabolizable squalene oil. The copolymer is coated with emulsion stabilizing silica particles which allows for incorporation of large amounts of a wide variety of antigenic materials. The adjuvant active copolymer forms hydrophilic surfaces, which activate complement, immune cells and increased expression of class II major histocompatibility molecules on macrophages. Titermax presents antigen in a highly concentrated form to the immune system, which often results in antibody titers comparable to or higher than FCA.

Specol: Specol is a water in oil adjuvant made of purified mineral oil. It has been reported to induce immune response comparable to Freund's adjuvant in rabbit and other research animal while producing fewer histological lesions

Adjuvants and their modes of action

The trend towards the use of peptides and subunit proteins in modern vaccine design has necessitated the use of immunological adjuvants to achieve effective immunity. Aluminium hydroxide, a component of the diphtheria, tetanus and hepatitis B vaccines, was first described as an adjuvant over 60 years ago and is the only adjuvant currently approved for use in humans. It is also a common component of many veterinary vaccines. While this adjuvant is effective at enhancing antibody titres to antigens, the effectiveness of aluminium hydroxide is limited due to its inability to promote cell mediated immunity. Freund's Complete Adjuvant (FCA) has been used experimentally and does stimulate cellular immunity, but is unsuitable for human and veterinary use as it promotes, amongst other toxic side effects, local inflammation and granuloma formation at the site of injection. Thus, in recent years there has been a great deal of interest in developing novel, cheap, effective and safe adjuvants which stimulate cellular, as well as humoral immunity to be used with medical and veterinary vaccines. In addition, the recent unravelling of numerous immunological pathways has facilitated the rational development of new adjuvants and allowed a better understanding of the modes of action of traditional adjuvants. Mode of action of immunological adjuvants: some physicochemical factors influencing the effectivity of polyacrylic adjuvants. The adjuvant effects of different polyacrylic products and monomers were tested. Influenza vaccine was used as a model antigen. Addition of monomers resulted in a decrease in the antibody response, though adjuvant activity of the monomers should be expected according to some theories on adjuvant action. The particle size of the polymer adjuvants proved to be a very important parameter for adjuvant activity. Particles of 0.1 to 0.2 micron yielded a good adjuvant effect, whereas conglomerates or particles bigger than 0.5 micron yielded only poor or no adjuvant effects. The adjuvant effect of 0.1- to 0.2-micron particles was much more reproducible than rat of Al(OH)3. Attention is drawn to the importance of using physiochemically reproducible materials, such as polymer particles, for experimental work.

ANIMAL HANDLING AND RESTRAINT Animal Handling Skills-Professionalism and Safety _ The public watches us to learn how to properly handle animals. _ Being professional means being SAFE and HUMANE. _ Good animal handling skills prevent staff from being injured. _ Good animal handling skills reduce stress for the animal. Examples of Safe Animal Handling: _ Be aware of the special stressors for animals in the clinic setting. _ The clinic is extremely chaotic for any animal-there are an incredible number of smells and other stimuli and animals are likely to be confused and distressed. _ Many of our patients have lived entirely outdoors and have not been handled or examined before. They may not have any experience on a leash and may panic in response. _ Even the most social animal may exhibit aggression toward other animals, particularly in a strange environment and may redirect to nearby people when over-stimulated. _ Never put your face directly into the face of a dog or cat. _ Do not move in behind or crowd around a dog. _ Concentrate on the animal you are handling without being distracted by other activities. _ NEVER sit on the floor while handling/examining a dog. If the animal becomes aggressive or aroused you will be unable to move away or protect yourself and risk serious facial bites. _ Always be prepared to protect yourself or move away quickly in the event an animal becomes aggressive unexpectedly.

Safe and effective animal handling requires a thorough understanding of the normal behavior and responses of each species. Below is some general information on animal behavior and handling techniques. There is no substitute, however, for careful observation and experience. Communication Any animal exhibiting potentially aggressive behavior should have a kennel sign (CAUTION) posted to alert others who may be handling the animal. Specific alerts or recommendations should be written on the sign and in the medical record to provide staff and other volunteers with as much information as possible when handling the animal. Restraint or Control The first rule to keep in mind when handling any kind of animal is that the least restraint is often the best restraint. This does not mean that you give up your control, just that you use as little restraint as necessary while maintaining control of the situation. Every animal and every situation is different so as to what method works best in which situation. Before attempting to restrain an animal you should take a moment to allow the animal to become comfortable with you: _ Crouch down so that you are on their level. Do not sit on the ground as you will be unable to move away or protect yourself if necessary. _ Avoid direct eye contact but maintain safe visual contact with the animal _ Talk in soothing tones. Avoid high-pitched, excited talk. _ Try patting your leg or the ground, motioning the animal towards you. TYPES OF RESTRAINT VERBAL RESTRAINT: Many dogs know some commands or can at least recognize authority, even if the command is unfamiliar.Commands such as SIT, STAY, COME, DOWN, NO or even HEEL may be useful tools to encourage a dog to cooperate. Also, soft quiet words can calm a frightened animal. Yelling or screaming should never be used as it can cause the animal to become more fearful or aggressive. PHYSICAL RESTRAINT: TOOLS AND EQUIPMENT Leash: The most common tool used to handle animals in the clinic is the leash. Placed around a dog's neck it normally controls even the largest dog. In the event a dog refuses to cooperate with a leash - carry him. Some dogs have never seen a leash and will freeze up to the sensation around a sensitive area like the neck. Leashes can be abused; never drag or strangle an animal with a leash; if the animal starts to struggle, pulling and jerking away from you, she is probably not leash trained. Pause and let the dog calm down and try again after reassuring her. Sometimes a quick tug on the leash will encourage a fearful dog to walk. If the dog refuses to walk, apply a muzzle (if necessary) and carry her. When handling cats, a leash should be used as a back-up in the event the cat should become frightened and resist restraint. Make a figure-eight harness by looping the free end of a slip lead back through the metal ring. The looser loop is placed around the chest behind the cat’s front legs and the other loop placed around the neck with the metal ring/handle on top between the shoulders. This will prevent the cat from escaping or injuring someone should she get loose from your restraint. The harness should be put on at intake and can be left on the cat throughout their stay. EVERY animal being transported or handled in the clinic must ALWAYS wear a slip-lead. This includes puppies, cats and sedated animals. It is too easy for a frightened animal to get loose and escape. Animals presented on leash/collar should be transferred to a slip lead and the leash returned to the client so that it is not lost during the animal’s stay.

Your hand: A very effective form of restraint, your hands are sensitive to the amount of pressure that is being exerted on the animal and can be quickly modified to the situation. Hands can be used to gently stroke a dog or to firmly grasp a struggling cat. Although hands can be the most versatile, they are also the most vulnerable to injury. Recognizing when they would not be effective is very important. Towels: A towel or blanket is a very useful tool for cats and small dogs. A towel can be used to decrease an animal’s arousal by covering the head and body and can help protect from sharp claws. Come-a-long or control pole: The control pole is used to safely handle extremely aggressive dogs.Used appropriately it is an effective tool. Inappropriate or unskilled use can cause serious injury to the animal. The control pole may further distress an upset animal and should only be used when the handler or other's safety is genuinely threatened. Volunteers are NOT to use the control pole unassisted. If an animal is aggressive enough to warrant the use of a control pole an experienced staff member should be consulted for assistance as the animal will also be evaluated for chemical restraint options. Nets: The net is the primary tool used to handle fractious cats or wildlife. It allows for the safe handling and transfer of even the most aggressive small mammal. Effective use of the net requires some training and practice. If you need to handle a feral or fractious cat ask for assistance from a staff member. Muzzles: Muzzles are used when a snappy or potentially aggressive dog must be handled. There are nylon muzzles and plastic basket available. A leash or strip of rolled gauze can be used as a temporary muzzle. Because dogs often try to remove a muzzle, it is important that the muzzle be placed securely. A weak or poorly made muzzle may lead to a false sense of security and the possibility of being bitten. Even with a securely placed muzzle, appropriate handling must be used to prevent injury from an animal who resists.Muzzles designed for cats extend up to cover the eyes, reducing visual stimulation. For some cats these can be very useful for calming the animal and helping to protect the handler from injury, Drugs: For animals who are too aggressive or stressed to handle safely for procedures, sedation and/or general anesthesia may be necessary to allow treatment. If you are unable to handle an animal, notify a staff member to determine whether sedation is appropriate. When receiving an animal for surgery who exhibits difficult or aggressive behavior consult the Anesthesia Lead prior to kenneling the animal as we may opt to administer a pre-anesthetic sedative immediately and expedite the surgery process to minimize the animal’s time in the clinic. Credo: Never Let Go. The place where correct use of restraint is the most critical is when two people are handling the animal.This could be to perform a physical exam, administer anesthetic or to give medications. The "holder" is the person whose job it is to restrain the animal in such a way that the procedure can be accomplished with the least amount of stress to both handlers and animal. The specific amount of restraint used to control the animal is the key to safety for the handlers and comfort for the animal. Too much restraint can cause the animal to fight back, too little restraint can result in the handler or others being injured or in the animal escaping.

Restraint and Handling of Animals General Principles

The use of proper restraint and handling techniques reduces stress to animals and also to the researcher. Handling stress represents an experimental variable and should be minimized whenever

possible. Animals can inflict serious injuries to humans and to themselves as a result of improper handling.

• Animals experience stress as a result of shipping. All large animals must be allowed to acclimate to the facility for three days. During this time they may not be experimentally manipulated. Acclimation periods of up to one week are recommended for all animals.

• If a study will involve significant handling of animals it is recommended that the animals be acclimated to the handling. Prior to experimental manipulation, handle the animal on a regular basis in a non-threatening situation, e.g. weighing, petting, giving food treats. Most animals, even rodents will respond positively to handling and will learn to recognize individuals.

• Handle animals gently. Do not make loud noises or sudden movements that may startle them.

• Handle animals firmly. The animal will struggle more if it sees a chance to escape.

• Use an assistant whenever possible.

• Use restraint devices to assist when appropriate.

• Chemical restraint should be considered for any prolonged or potentially painful procedure.

Handling Methods

The methods described below will assist with performing basic manipulations. Alternate techniques may be needed for special procedures. Most of these methods are also demonstrated in video tapes available to investigator. For other information on animal handling or for individual training, contact RAR at 624-9100. An excellent website containing laboratory biomethodology for rodents and rabbits is also available with descriptions and pictures of drug administration, blood collection and sex determination.

Needle Re-Use Policy

The use of a new sterile needle and syringe for each animal when giving parenteral injections (intraperitoneal, subcutaneous, intravenous, intramuscular, etc.) is the recommended best practice to prevent the horizontal transfer of contamination between animals. However, the IACUC recognizes that there are some instances where it may be justified to use the same needle and syringe for multiple animals, usually in rodents. In those instances the Principal Investigator must provide justification to the IACUC and must adhere to the following guidelines. Use of the same needle and syringe may be permitted with justification on animals housed in the same cage. The needle must be assessed for continued sharpness and the presence of barbing or burring of the tip between animals. If dullness or needle deterioration is found, a new needle must be used.

MICE Tail restraint, as described below is adequate for examining animals and transfering them to another cage.

These methods may be used to perform minor, non-painful procedures such as injections or ear tagging.

RATS may be handled by the tail, with precautions similar to those used for mice, with emphasis on only grasping the tail base. Holding the tail distal to the base can result in a de-gloving injury to the tail that will require surgical repair or euthanasia.

This method should be used to restrain a rat for injections and other minor procedures.

HAMSTERS Because hamsters do not have tails, they must be grasped firmly by the loose skin of its back, or handled in a manner similar to the rat.

GUINEA PIGS rarely bite, but are very easily frightened and will vocalize and squirm to avoid restraint. The hind limbs must be supported at all times to prevent the animal from injuring its back.

RABBITS are very susceptible to lumbar spinal luxation, resulting in paralysis. It is necessary to support the animal's hindquarter at all times. Although rabbits seldom bite, they can inflict painful scratches with their hind legs. One way of lifting a rabbit is by grasping the skin over the shoulder with one hand and gently lifting it with the other arm cradling the body, the head nestled in the crook of your arm. Rabbits must never by lifted by the ears.

CATS are often cooperative enough to be restrained on a table by the loose skin at the back of the neck and hips, or with one hand restraining the body and the other restraining the head. A fractious cat may have to be wrapped in a heavy towel for restraint with any needed limbs carefully withdrawn for treatment.

GATE Study MaterialImmunotechnology (Biotechnology

Engineering)

Publisher : Faculty Notes Author : Panel Of Experts

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