As described in Chapter 1, it was not long after Louis Pasteur’s initial discoveries that the sources of immune protection were found in the blood and were called antibodies. It was demonstrated that blood serum containing antibodies to bacterial toxins such as those from tetanus or diphtheria could be transferred from an immune animal to a susceptible individual and so confer protection (Fig. 12.1). Thus the recipient was “immunized” without mounting an immune response—passive immunization. Horses were the major source of these “antitoxins” because of their size and ease of management. Passive immunization was widely employed in the 1920s and 1930s against human pathogens such as Streptococcus pneumoniae, Neisseria meningitides, and Haemophilus influenzae, in addition to tetanus and diphtheria. With the advent of cheaper and easier to use antimicrobials and antibiotics such as penicillin and streptomycin, it fell into disuse. Passive immunization only persisted for use in toxin-mediated diseases such as tetanus and botulism, virus diseases such as rabies, and in snake envenomation (Table 12.1). It is now staging a comeback. Polyclonal antibodies generated in immunized animals and monoclonal antibodies generated in the laboratory are increasingly employed in the treatment of diverse animal and human diseases. TABLE 12.1 ■ The major antibody in mammalian serum is a protein called immunoglobulin (Ig)G. This is a Y-shaped protein of about 160 kDa. In passive immunization, whole or semipurified serum, or IgG obtained from an immune animal, is injected into or fed to another animal. If it is injected into an animal of the donor species then the injected antibodies will simply be removed through normal catabolic processes. If IgG is injected into an animal of a different species it will act as a foreign antigen and trigger an immune response. Such a response will result in its prompt elimination. It is therefore highly desirable to minimize the antigenicity of IgG. The simplest way of doing this is to treat the IgG with a protease such as papain or pepsin. These split the IgG molecule into two or three fragments. The first fragment to be cleaved off is the “tail of the Y” (Fig. 12.2). This fragment can be crystalized and so is called the Fc fragment. It does not contribute to toxin or virus neutralization so it can be discarded. The rest of the IgG molecule consists of the two joined arms of the Y is called Fab’2. This fragment retains the antibody activity. Further proteolytic digestion separates this into two antigen-binding fragments each called Fab. These too are functional. Elimination of the Fc region greatly reduces the antigenicity of the preparation although the smaller fragments do have a shorter half-life than intact IgG. As discussed later, modern molecular techniques also make it possible to alter the nonantigen-binding parts of immunoglobulins so that they too are identical to the recipient species and almost completely eliminating their antigenicity. A natural immune response to a complex antigen such as a bacterium or virus activates large numbers of B cells that in turn generate a diverse mixture of antibodies, each with a different antigen-binding specificity. Most pathogens have a complex structure and present the immune system with many different epitopes. As a result, multiple B cell clones are stimulated to respond. These clones produce polyclonal antibodies. Polyclonal antibodies with their mixture of specificities can bind collectively to many different antigens. (This is in contrast to monoclonal antibodies that are derived from a single clone of B cells and bind only a single targeted epitope.) Passive immunization requires that antibodies be produced in donor animals by active immunization and that these antibodies then be given to susceptible animals to confer immediate protection. Serum containing these antibodies may be produced against a wide diversity of pathogens. For instance, they can be produced in cattle against anthrax, in dogs against distemper, or in cats against panleukopenia. They are most effective when protecting animals against toxigenic organisms such as Clostridium tetani or Clostridium perfringens, using antisera raised in horses. Antisera made in this way are called immune globulins and are commonly produced in young horses by a series of immunizing injections. The clostridial toxins are proteins that can be denatured and made nontoxic by treatment with formaldehyde. Formaldehyde-treated toxins are called toxoids. Donor horses are initially injected with toxoids, but once antibodies are produced, subsequent boosters may contain purified toxin. The responses of the horses are monitored, and once their antibody levels are sufficiently high, they are bled. Bleeding is undertaken at intervals until the antibody level drops, when the animals are again boosted with antigen. Plasma is separated from the horse blood, and the globulin fraction that contains the IgG antibodies is concentrated, titrated, and dispensed. When an egg develops in the ovary of chickens, it contains a rich food source, the yolk. The yolk also contains a high concentration of chicken antibodies called IgY. IgY is the avian functional equivalent of mammalian IgG. Approximately 30% of the chicken’s IgY (but only 1% of its IgM or IgA) will transfer from the plasma to the yolk. Thus a single egg yolk may contain up to 250 mg of IgY. If the hen is first vaccinated, then their eggs will contain high levels of antibodies against that antigen. If these yolk antibodies are simply fed to a mammal they will confer local immunity. Passive immunization by feeding egg yolk immunoglobulins is a relatively simple and economical method of protection against some enteric diseases. For example, chicken egg yolk antibodies can protect calves against diarrhea caused by group A rotaviruses. Seven days of IgY treatment significantly suppressed virus shedding, duration of diarrhea, and disease severity when compared with untreated calves. Similar benefits have been recorded in piglets and poultry. Dried egg yolk powder from chickens has been administered to newborn puppies in milk replacer before closure of intestinal absorption (first eight hours after birth). Puppies supplemented in this way show significantly greater weight gain compared with controls. Weaned puppies receiving hyperimmune egg powder from chickens immunized against Escherichia coli and salmonella in the form of food supplementation had improved fecal quality and increased fecal IgA. Likewise egg powder containing antibodies to canine parvovirus 2 (CPV2) protected puppies against CPV2 challenge. Spray-dried blood plasma is used as a feed additive for pigs. It contains high concentrations (20%) of immunoglobulins. It has been shown to improve weight gain and resistance to some pathogens. Thus it also protects against E. coli colonization. The beneficial effects appear to reside in the immunoglobulin fraction. However, it is possible that viruses such as porcine epidemic diarrhea virus may survive the spray-drying process. Pooled abattoir blood plasma is another possible source of purified IgG. Fed to piglets for seven days postweaning, it reduces the severity of postweaning diarrhea. The major immunoglobulin in bovine milk is IgG. When casein is precipitated from milk during cheese manufacturing the liquid whey that remains contains small amounts of protein, 10% of which is IgG. However large volumes of whey are needed to obtain significant amounts of immunoglobulin for passive immunization. Antitetanus immunoglobulin (also called tetanus immune globulin or tetanus antitoxin) for veterinary use is produced in hyperimmunized healthy horses. Notwithstanding its equine origin, it can be used in cattle, sheep, pigs, dogs, and cats, as well as in horses. It is available in vials of 1500 and 15,000 units, and it contains thiomerosal and/or phenol to inhibit microbial growth. Deep, dirty wounds, especially when contaminated with soil or manure and tissues are devitalized sites where Clostridium tetani can grow and secrete its toxin. This toxin must be neutralized if clinical tetanus is to be avoided. Antitoxin should also be administered to nonimmune animals after castration, docking, and any surgical procedure conducted at sites where tetanus is known to be present. The half-life of equine IgG ranges from 27 to 39 days. Tetanus antitoxin given intramuscularly provides immediate immunity that lasts about 7 to 14 days in species other than horses. To standardize the potency of different immune globulins, comparison is made to an international biological standard. In the case of tetanus immune globulin, this is done by comparing the dose necessary to protect guinea pigs against a fixed amount of tetanus toxin with the dose of the standard preparation of immune globulin required to do the same. The international standard immune globulin for tetanus toxin is a quantity held at the International Laboratory for Biological Standards in Copenhagen. An international unit (IU) of tetanus immune globulin is the specific neutralizing activity contained in 0.03384 mg of the international standard. Tetanus toxoid may also be measured in limes flocculation (Lf) units. These are determined by an in vitro flocculation test. They measure the quantity and antigenicity of a toxoid but not its potency. One Lf unit is the amount of toxoid neutralized by 1.4 IU of tetanus immune globulin. Tetanus immune globulin is given to animals to confer immediate protection against tetanus. At least 1500 IU of immune globulin should be given subcutaneously or intramuscularly in the neck to horses and cattle; at least 500 IU to calves, sheep, goats, and swine; and at least 250 IU to dogs. The exact amount should vary with the amount of tissue damage, the degree of wound contamination, and the time elapsed since injury. Tetanus immune globulin is of little use once the toxin has bound to its target receptor and clinical disease appears. Notwithstanding this, some veterinarians seek to improve its prognosis by administering high doses of antitoxin, 10,000 to 50,000 units to horses and cattle, and 3000 to 15,000 units to goats and sheep. Animals with slow-healing puncture wounds may be given a second dose in seven days. Although immune globulins give immediate protection, some problems are associated with their use. For instance, when horse tetanus immune globulin is given to a cow or dog, as described earlier, the horse proteins will be perceived as foreign, elicit an immune response, and be rapidly eliminated. If repeated doses of horse immune globulin are given to an animal of another species, this may provoke IgE production and allergic reactions. Additionally, the presence of high levels of circulating horse antibodies may interfere with active immunization against the same antigen. This is a phenomenon similar to that seen in newborn animals passively protected by maternal antibodies. Mixtures of different monoclonal antibodies directed against multiple toxin epitopes are now being tested as possible replacements for polyclonal antiserum. These are more readily standardized than polyclonal antisera. When tetanus began to kill large numbers of soldiers during the First World War, the use of tetanus antitoxin increased dramatically. Physicians gradually increased the amount of antitoxin administered to severely wounded soldiers. However, soldiers who had received a very large dose of equine antitetanus serum developed a characteristic illness about 10 days later (Fig. 12.3). This was called serum sickness and consisted of a generalized vasculitis with erythema, edema, urticaria of the skin, neutropenia, lymph node enlargement, joint swelling, and proteinuria. The reaction was usually of short duration and subsided within a few days. A similar reaction can be produced experimentally in rabbits by administration of a large intravenous dose of antigen. The development of sickness coincides with the formation of large amounts of immune-complexes in the circulation. The experimental disease may be acute if it is caused by a single, large injection of an antigen, or chronic if caused by multiple small injections. In either case, animals develop glomerulonephritis and arteritis. For this reason, tetanus immune globulin of human origin is now preferred for the prevention of tetanus in people whenever possible.
Passive immunization
Function
Examples
Antibacterial
Escherichia coli (+K99)
Rhodococcus equi
Streptococcus equi
Salmonella typhimurium
Trueperella pyogenes
Antitoxins
Clostridium botulinum Type B
Clostridium perfringens types C and D
Clostridium tetani
Crotalidae (rattlesnake) antivenin
Antiviral
Bovine rotavirus-coronavirus
West Nile Virus
Immunoglobulins
Polyclonal antibodies
Immunized donor animals
Chicken egg yolk
Blood plasma
Milk whey
Antitoxins
Clostridium tetani
Serum sickness
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Passive immunization
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