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The coccidia are protozoan that enter the host as sporozoites inside an oocyst and are relleased in the digestive tract. Each sporozoite invades an intestinal cell and multiples into hundreds of merozoites that burst the host cell. Each merozoite invades a new cell and repeats the cycle of multiplication. The second generation of merozoites invades new cells but, this time, most of them develop into sexual forms that fuse to form a zygote. The zygote surrounds itself by a protective membrane becoming an oocyst, leaves the host, and develops into sporozoites in a few days.
The extensive cell destruction causes disease and sometimes death. Avian coccidiosis produces annual losses of 200 to 300 million dollars only in the United States. The traditional control consists of the administration of preventive drugs which is expensive, leaves chemical residues in the meat, pollutes the environment, and promotes development of parasite resistance to the drug. An alternative procedure of control was necessary.
1. Live vaccines
Beach et all in 1925 were the first to report that an infection with coccidia turned the birds resistant to a challenge. Johnson in 1927, 1928 demonstrated that the resistance lasted at least for 6 months and was effective only against the species used for the first infection. Goldsby and Eveleth in 1950 were able to produce effective resistance by infectiog chicks and treating them with a subcurative dose of coccidiostats in the feed. Edgar et al. (1951) developed a practical system to infect 3 to 5 day old chicks with several coccidia species in the water or feed, and limit the multiplication of parasites in the host with a prophylactic dose of coccidiostats in the feed. A variation of this system was used in a commercial vaccine (CocciVac) introduced in 1952 by Dorn and Mitchell Laboratories, Opelika, Alabama. lt is currently manufactured by Sterwin Laboratories, Millsboro, Delaware.
The vaccine contains a mixture of oocysts of 8 coccidia species (Eimeria tenella, E. necatrix, E. hagani, E. acervulina, E. maxima, E. brunetti, E. praecox, arad E. mivati) that is administered in the water to chicks 4 to 14 days of age. The devellopment of resistance depends on an initial moderate infection followed by other moderate infections that are generated by the contamination established by the first infection. To avoid heavy infections, the litter must be maintained at 25–30% humidity (damp but not wet to the touch, with the consistence of freshly cut grass) from the 5th day of vaccination. Higher humidity favors the survival of oocysts and lower humidity generate dust and promote infections by inhalation. Since birth and for 2 to 4 weeks after vaccination, the chicks are given vitamins K and A in the feed (4-8 g and 12-18 units per ton, respectively) to control hemorrhage and favor tissue regeneration. The manufacturer does not recommend the use of coccidiostats because they interfere with the development of resistance. Still bouts of coccidiosis could occur in 7 to 23% of the establishments that use this method but the vaccine is regarded as appropriate for commercial use and has been used extensively.
CocciVac was an important advancement for aviculture (and the only possible with the technology of the time) but still had drawbacks. Because it is a live vaccine, its standardization is difficult and its preservation precarious. Because it contains virulent strains, its application requires special precautions, its efficacy is variable since occasionally pathogenicity predominates over immunogenicity, it may introduce new species into a region, and the birds may fail to gain weight or lose weight for a couple of days after vaccination. Improvements in the administration of the vaccine or the use of attenuated or irradiated parasites (Shirley and Long, 1990) may ameliorate some of these inconveniences but a live vaccine has inherent weaknesses.
2. Possibilities of a molecular vaccine
Murray et al. (1985) reported that the oral or intramuscular administration of an extract of E. tenella sporozoites conferred resistance against a homologous challenge. Furthermore, an extract of E. acervulina protected against E. tenella or E. maxima challenges. This report heralded the possibility of a molecular vaccine and promoted a huge revival of research on coccidial Immunization. Much of this work is still going on, however, and the industrial secrecy connected with patent applications often prevent publication. Danforth (1989) has reviewed many reports of identification of antigens of sporozoites, merozoites, and sexual stages of Eimeria spp. using either serum of resistant birds or monoclonal antibodies. E. tenella is the favored species because it is the most pathogenic for birds. In turn, sporozoites are the preferred parasitic forms because they are comparatively easy to obtain and blocking of sporozoites should abort the infection. The use of serum of resistant birds demonstrates up to 45 antigens in extracts of the parasite but it is difficult so say which is relevant to protection. The use of monoclonal antibodies, on the contrary, permits the identification of a monoclonal antibody that affects the parasite and the verification of the corresponding antigen. In this technique, parasitic forms are injected into a mouse, and primed B lymphocytes are extracted from the mouse and fused to murine myeloma cells. The resulting (hybrid) cells multiply in vitro like that myeloma cell and produce antibody like the lymphocyte. The antibodies (called "monoclonal" because originated from a single B cell or clone) are collected and assayed for antiparasitic activity. Those that kill or inhibit the parasite can be used as reagents to purify the respective antigens by affinity chromatography. With this type of techniques, Files et al. (1987) made a monoclonal antibody that inhibited the penetration of E. tenella sporozoites in its host cell. The corresponding antigen was purified and showed to have a 25 kdalton molecular weight. Immunization of chicks with this antigen generated antibodies that inhibited sporozoite invasion and reduced the lesions of an homologous challenge. Ellis and Johnson (1992) have reviewed much of the work done with this antigen.
There exits the belief in immunoparasitology that only the antigens on the surface of the live parasite are accessible to antibodies or effector immune cells. Protective antigens, then, should be surface molecules. The identification of surface molecules is normally performed by incubation of parasites with radioactive salts that do not penetrate the cell. Any radioactive protein found subsequently in the parasite extract, must have been on the surface. Using this technique, the 25 kdalton antigen wa found to be located on the surface of the sporozoites. The amino acid composition of this antigen was studied, and genomic and DNA libraries viere prepared. The gene that codified for the antigen was isolated, incorporated into a plasmid, and use to infect Escherichia coli cells (Brothers et al., 1988). Unfortunately, the protein was expressed by the bacterial cells as an inclusion body instead of being secreted. Assays with other systems resulted in a protein that could be solubilzed. Inoculation of chicks with this recombinant protein with strong adjuvants produced antibodies that inhibited the invasion of host cells by E. tenella sporozoites. Other authors have produced other recombinant proteins from E. tenella (antigens GX3262 and GZ 3264 by Miller et al. [1988], and antigen 5401 by Danforth et al. [1989] that also induce partial resistance when used to vaccinate chicks. Jenkins et al. (1991) produced a recombinant antigen of E. acervulina and caused partial resistance to an homologous challenge by infectiog chicks with the bacteria that expressed the protein.
An industry as extensive as the avían industry needs a vaccine that can be administered easily to an enormous number of birds. A solution is to administer the antigen inside a replicating organism that is infections but apathogenic for the birds. Jerkins et al. (1991) used E. coli expressing the antigen. The complementary gene for the 25 kdalton antigen was also incorporated into the genome of vaccinia virus which, in turn, was used to vaccinate chicks. Although there is no direct proof that the virus expressed the antigen, birds vaccinated 3 times developed certain resistance to homologous infections. Embrex, Inc. has developed a machine (Inovoject) that allows the infection of antigens to up to 20,000 incubation eggs per hour. The machine is being assayed with the GX3264 antigen (Fredericksen et al., 1989).
Some specialists think that many years will pass before a practical molecular vaccine for avian coccidiosis becomes a reality (Grane et al., 1991). The current technical possibilities and the enthusiastic support of the industry, however, are reasons for optimism.
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