Introduction

Transmissible diseases of domestic animals must have been a constant concern of humans since the beginning of confined rearing, some 11.000 years ago. Only in the second half of the XIX century, basic knowledge of the biology of infectious agents and host responses to infeccion permitted to start applying rational procederes of control. Reasonably effective antiparasitic drugs became available only in the first decades of the XX century and antibiotics started to be used less than 50 years ago. Pressured by the need to identify diseases quickly and accu­rately and prevent their spread, microbiologists turned to immunology. By the turn of the century, they had already developed efficacious serologi­cal and immunization techniques. Parasitologists, on the contrary, had simple direct methods to demonstrate the presence of parasites and reasonable effective antiparasitic drugs (suramin, arsenic salts, carbon tetrachloride, oil of chenopodium, arecoline bromhydrate, tetrachloroethylene, phenothiazine, etc.) almost from the beginning. Furthermore, the first researchers found that eukaryotic organisms (parasites, sensu strictum) were biologically much more complicated than prokaryotic organisms (bac­teria and viruses) which made the study and inter­pretation of their immunology considerably more difficult. Many parasites, for instance, have diverse stages that produce common and unique antigens released at different concentrations in various host tissues. Extracts of parasites contain hundreds of potentially antigenic molecules with an overwhelming predominance of irrelevant over protective anti­gens. On many occasions, the protective antigens are only moderately immunogenic and the irrelevant antigens compete successfully for the attention of the immune system. Many parasites have the ability to elude or abrogate host immunity. To make things worse, most parasites cannot be culture in vitro. Without the pressing need of developing methods for diagnosis and immunization and lacking the thechnics to solve the existing riddles parasite im­munology lagged behind bacterial and viral immu­nology.

Although primitive vaccines against avian coc­cidiosis, bovine babesiosis and dictyocaulosis, and canine ancylostomiasis started being used in the 1950s and 1960s, the major single contribution to the development of parasite immunology came from the situation of malaria in the 1960s. The application of DDT to the control of mosquitos in 1942 and of chloroquine and primaquine to the treatment against Plasmodium in 1943 brought great optimism about the possibilities to fight malaria. In 1950, the World Health Organization predicted that malaria would be eradicated in 40 years. By the end of the 1960s, however, mosquitoes had develop resistance to most chlorinated and or­ganophosphorus insecticides and many Plasmodium strains were resistant to antimalaric drugs. When an alternative or complement to the tradition­al control of malaria was sought desperately, Nus­senzweig et al. in 1967 reported that a single injec­tion of irradiated parasites protected 86% of mice against a malaria challenged. The promise implied in this report (which has not been fulfilled yet) put parasite immunology in the fore front of applied research.

The recent advences of immunological concepts and techniques and the development of biotechnol­ogy have provided the necessary tools to undertake the study of parasite immunology with possibilitis of success. On the other hand, the progress of medicine and the greater expectations from or for patients have stimulated work in this area. As a result, these last years have seen an unprecedented development in the knowledge of immunity to para­sitic infections. In this review, we will survey the latest advances in the development of vaccines against some protozoal and arhropod diseases of veterinary importance. We will also comment on older methods when they are still in current use.

Avian Coccidiosis

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.

Babesiosis

The babesias (formerly called 'piroplasms') is a group of protozoan parasited of the red blood cells that are transmitted biologically by ticks. The most economically important species are those that affect bovines, particularly Babesia bigemina and B. bovis (synonym, B. argentina) because of their wide geo­graphic distribution. The disease causes fever, mal­aise, and anemia. Hemoglobinuria is common with B. bigemina and signs of nervous central system compromise with B. bovis. The animals that survive the infection remain as healthy carriers, probably for life. They develop a resistance to the infection and the disease that is proportional to the number of prior exposures. This peculiarity has been known to farmers for many years and stimulated Pound in Australia and Connaway and Francis in the Unites States, at the end of the XIX century, to attempt the production of artificial infections in bulls intro­duced to enzootic areas to facilítate the development of resistance. Modifications of this method are still used in Latin America and other regions under the name of premunization.

1. Premunization

The term premunition defines a resistance that per­sists only while the agent continues stimulating the immunity of the host. lt has been also called 'infec­tion-immunity'. Elimination of the infection is soon followed by lost of resistance in most hosts. Premunization is the artificial production of premunition. Bovines of enzootic areas generally acquire light infections in the first 6 months of life (when they are still relatively insuscetible) and develop some degree of resistance without showing overt signs of disease. Later infections strengthen the resistance. As a result, most of the cattle of enzootic areas carry some parasites in their blood but, in the absence of openly unfavorable cir­cumstances, they resist the disease caused by local strain. On the contrary, cattle introduced from areas free of Babesias or with different strains may ac­quire the disease and die. When the value of the animals justifies it, the introduced cattle are iso­lated, injected with blood from Babesia carriers, and examined twice a day for fever and presence of parasitemia. Fever usually appears 5 to 8 days after infection and parasitemia a couple of days later. Soon after the appearance of signs, the animals are treated with subcurative doses of diminazene aceturate ('Berenil', 0.5 to 0.25 mg/kg, in­travenously) or dipropionate of imidocarb ('Im­idocarb', 0.5 to 0.25 mg/kg, subcutaneous or in­tramuscular). Either treatment kills enough parasites to prevent serious disease but allows the survival of enough organisms to induce resistance to natural challenges (Kuttler, 1981). Some specialists treat as soon as fever appears to avoid severe reactions. Others way until 2% of the erythrocytes are infected to obtain a more effective resistance.

Refinements have been incorporated to this tech­nique. B. bigemina, B. bovis, and even Anaplasma marginale can be injected simultaneosly, and the dose of parasites can be standardized (10 to 100 million organisms of each species per animal). When anaplasma is inoculated, the treatment should be done with Imidocarb which is effective against the rickettsia, or wait until the second wave of fever and parasitemia (commonly during or after the third week of infection) that corresponds to the signs of anaplasmosis and treat with tetracyclines. In places were premunization if used frequently, it is common to maintain donor bovines, often splenectomized, with monospecific infections. effective blood can be preserved in liquid nitrogen.

Premunization has allowed the introduction of quality cattle into enzootic areas for over a century but it has a number drawbacks. It is expensive, occasionally causes disease or death, the induced resistance is variable, permits the transmission of other blood-borne pathogens, may sensitize against erythrocyte isoantigens and cause neonatal hemolytic anemia, and prevents eradication of the parasites. Although a more refined system of immunization is desirable, premunization still supplies de needs of large geographic regions (Markovics et al., 1991).

2. Vaccination with attenuated parasites

The frequent failure of premunization in Australia stimulated search for a more standardized immuni­zation procedure. An attenuated Australian vaccine against B. bovis started to be used in 1964 but the first reasonably comprehensiva report appeared only 13 years later. Dalgliesh et al. (1990) have written a more recent review. At least 109 parasites of 5 different strains of B. bovis are inoculated into a splenectomized calf and its blood is collected for 3 days starting on the 3rd or 4th day of infection. This blood is usad to infect another splenectomized calf and the procedure continuas until obtaining blood for the vaccine. Rapid passage for 10 calvas appreciable decreases the virulence of the protozoan for non splenectomized calvas, 30 to 40 passages appear to be a good compromise between loss of virulence and preservation of immunogenicity, more than 60 passages cause loss of infectivity for the tick but also a severa reduction of the immuno­genicity for calves. Passage of the attenuated para­sites by non-splenectomized bovines regenerates the original virulence. The spleen is known no play a role in protection to primary and secondary infec­tions, more important for B. bigemina than for B. bovis (Carson and Phillips, 1981). Ristic et al. (1984) found that B. bovis passed more than 8 times by splenectomized calves multiplied faster, were circular instead of piriform, and lack the membrana knobs that allow the parasite to attach to the endo­thelium.

The blood collected for vaccination is diluted in 25% bovine plasma in a glucosaline solution that mimics the concentration of bovina plasma, at 107 parasites per dose (2 ml). En Australia, this vaccine is preparad in government laboratories. Although its shelf life is only a few days, its ample use (about 700,000 doses per year) justifies its continous pro­duction. Nowadays, the vaccine is shipped frozen and the farmer must thaw it only immediately before use. It is preferable to administer it to animals in their first year of life. Vaccinated animals com­monly undergo slight fever and parasitemia 8 to 9 days after inoculation but do not show signs of disease. In case of severa reactions, imidocarb treat­ment is recommended. Vaccination can be repeated every 6 months but one or two vaccines commonly produce sustained resistance. Field observations have shown that when almost 18% of the nonvacci­nated animals acquired the disease, only about 1 % of the vaccinated got sick. Anaplasma marginale has been incorporated to the vaccine in certain parts of Australia.

The first attempts at producing a similar vaccine against B. bigemina showed that this species did not respond to rapid passage through splenectomized calves with reduction of virulence. On the contrary, some evidence was found that strains kept in the laboratory were more pathogenic than the field strains. This was not a problem in Australia where infection with B. bigemina is often asymptomatic but invalidated the vaccine in other areas. Only in 1981, Dalglish et al. (1981) could develop an attenu­ated strain of B. bigemina appropriate for use in a vaccine but the investigations with purified antigens had already begun.

Vaccination with strains attenuated by rapid pas­sage in splenectomized bovines was an important advance over premunization and produced better results than vaccination with parasites attenuated through irradiation. Yet the vaccine still causes oc­casional cases of abortion, hemolytic neonatal dis­ease, and severa babesiosis. Eventually, virulence of the vaccinal parasites may reappear and immuno­genicity may be lost (Bock et al., 1992). The precau­tions to present transmission of hematogenous infec­tions and to prolong shelf life are still important concerns. These drawbacks were an important in­centive for research toward molecular vaccines.

3. Possibilities of a molecular vaccine

Major obstacles to the vaccination against babesiosis with purified antigens were to find an appropriate source of antigen, and produce the antigen in useful quantities. The initial attempts to isolate antigens from infectad erythrocytes were disheartening be­cause of the difficulty of avoiding contamination with host's antigens and because the amount of antigen obtained was minimal. Some degree of im­munity was obtained, however, by inoculation (of B. bovis antigens present in the sera of infectad animals or cruda antigens obainted from infectad erythrocytes. Only the development of a system to rear Babesias in vitro (Erp et al., 1980; Vega et al., 1985) allowed to obtain antigens in the quantities necessary to advance research. Montenegro-James et al. (1985), for example, showed that a culture­-derived B. bovis antigen produced resistance to challenges with heterologous strains. The current cultura techniques produce Babesia parasites that do not appear to differ in virulence or immunogenicity with those from splenectomized cal­ves (Jorgensen et al., 1989). Once Babesia antigens were produced in relative abundance, the applica­tion of modem research techniques was un­avoidable. Wrigh et al. (1992) have reviewed the work done in the last years. Immunization with crude extracts or selected fractions of B. bovis in­duced as much resistance as natural infections. The antigens in the protective fractions were used to prepared monoclonal antibodies and these to iso­lated the corresponding antigens. Three antigens (GST–12D3, GST–11C5, and GST–21B4) produced a resistance that reduced the parasitemia of a chal­lenge by more than 95%. None of the protective antigens showed to be immunodominant but the immunodominant antigens al] immunosuppressors or nonprotective. Gill et al. (1987) cloned a large (220 kdaltons) immunodominant antigen that also proved not to be protective. As expected of parasities with a long coevolutionk with the host (Barriga, 1981), the antigens that generate protec­tion appear to be only moderately immunogenic. The protective antigens were produced ad recombi­nant proteins and assayed for their protective power. Individually, each antigen protected satis­factorily against the fever and parasitemia but weakly against the anemia of a challenge. Use of a mixture of the three antigens had a synergistic effect but they still generated less protection against anemia that a commercial attenuated vaccine. Brown et al. (1993) made a recombinant antigen from B. bovis merozoited that specifically stimu­lates T herper cells. It is hoped that the incorporation of this antigen to a vaccine would increase the immune response. The Australian government and private companies have invested substantial sums in the development of a practical system to vaccinate against babesiosis so it is likely that research will continue until a successful conclusion.

Vaccination of bovines with antigens from a su­pernate of B. bigemina cultures generated partial resistance against B. bigemina and B. bovis (Toro­Benitez et al., 1988). An antigen from merozoites of B. bigemina ('p58') was identified and purified with a monoclonal antibody that inhibited the pene­tration of parasites to the host cells (Mishra et al., 1991). Rabbits immunized with this antigen gener­ated antibodies that also inhibited the penetration of merozoites. Immunization of calves with this anti­gen and two other recombinant proteins of B. bigemina merozoites ('gp45' and 'gp55') resulted in a significant reduction of the parasitemia of a challenge (McElwain et al., 1991).

Using the concepts and techniques learned in the research for immunization against bovine babe­siosis, a system for the in vitro culture of B. canis was recently developed. Antigens obtained from this culture produced between 70 and 100% protec­tion when used to vaccinate dogs (Moreau et al., 1988). Rhone Merieuz Laboratories, of Lyon, France, have used these antigens in the production of a commercial vaccine against canine babesiois ('Pirodog').

Theileriosis

Theileria is a genus of protozoan closely related to Babesia. The main theilerias of bovines are T. parva, that causes the East Coast Fever in the East­ern half of Africa between the south of Sudan and the north of South Africa, and T. annulata, the causes the Mediterranean Cost Fever around the Mediterranean, middle East, India, Soviet Union, and other parts of Asia (Irvin, 1987). Like the Babesias, the theilerias are transmitted by ticks. The vector inoculates sporozoites that invade and multi­ply in the lymphocytes to form schizonts. The schi­zonts generate numerous merozoites that leave the lymphocyte to invade and multiply in the erythro­cytes. These forms in the red blood cells are still called 'piroplams' and are the origin of the infec­tion of new ticks. Lymphocyte parasitism causes blastogenesis, proliferation, and destruction of the cells. Erythrocyte parasitism is intense and destroys the cells in theileriosis annulata but not in theil­eriosis parva. Both theilerias cause fever, monspe­cific proliferation of lymphocytes, and debilitation. T. annulata also causes fever.

Cattle from enzootic areas commonly develop some degree or resistance against theilerioisis but tatúe from areas free of the parasite or regions with different strains of theilerias mar andergo severe disease and high mortality when introduced into infested areas. Animals that recover remain resistant for lile but, particularly in the case of T. annulata, mar continue as carriers of very low parasitemias for years. The traditional control of theileriosis con­sists of eliminating the vector by regular acaricide treatments of the animals at risk. The economic and ecologic cost of this technique and the certainty that the ticks will soon develope resistance to the acari­cides recommend the search for alternative methods of control.

1. Live vaccines

a. Limited infections

The first published assay of vaccination against T. parva was by Spreull in 1914. Because T. parva is difficult to transmit with infected blood, Spreull used homogenates of lymph nodes and spleen of infected cows. Of 283,000 bovines vaccinated with a dose of supposed low infectivity, 25% died of theileriosis, and 70% of the survivors developed diverse degrees of protection to the disease. The complexity of the inoculum preparation, high mor­taly, and variable results conspired against the rou­tine use of this method. Sergent et al en 1924 used the same method suscceflly against T. annulata in South Africa. Because T. annulata is easily trasnmitted with infected blood, they used inocula­tions of limited doses of blood from bovines in­fected with a strain of low virulence. The same method was later adopted in Israel but, to increase protection, they infected a strain of low virulence followed by a strain of high virulence one or two months later. In an extensive assay, mortality due to vaccination was only 1 to 3% but the field mortality dropped from 13% for nonvaccinated cattle to virtu­ally 0% for vaccinated animals. Results have been less satisfactory in other assay, apparently due to the difficulty of establishing adequate vaccination doses.

b. Sporozoite vaccines

Although it was known that inoculation of sporozoites induced resistance to a challenge, this technique coult not be used routinely until it was found that tetracyclines administered during the incubation pe­riod suppressed the manifestations of theileriosis and methods were developed to collect and pre­serve sporozoites from infected ticks. The tech­nique of infectiog animals with measured doses of sporozoites and treating them simulatenousky is called 'infection-treatment'. Introduced in 1965, it is the menthod in current use to vaccinate against T. parva (Radley, 1981). The method consists of pre­paring large amounts of an inoculum obtained by maceration of infectedf ticks, standardizing its in­fectivity by assays in susceptible calves, and storing aliquots of the inoculum in liquid nitrogen. The animals to vaccinate receive a potentially lethal dose of this inoculum and a simultaneous dose of oxytetracycline (5 mg/kg/day for four or more days) or of a long acting oxytetracycline (20 mg/kg, once or twice) to limit the proliferation of the parasite and permit the development of immunity. Differently from the new anti-theileria drugs (e.g., parvaquone, halofuginone), tetracyclines are not effective once the symptoms appeared. Because geographic strains of T. parva rarely cause cross protection, the inocula commonly are prepared with strains from different origins. This practice, however, facilitates the intro­duction of new strains.

About a decade later, a sporozoite vaccine against T. parva was Introduced. Pipano (1989) described the technique for preparation. The dose is the equivalent to 1 to 10 infected ticks per animal, depending on the concentration of sporozoites in the preparation. Because the vaccine causes violent dis­ease in cattle, appropriate treatment must be admin­istered at the same time; oxytetracycline, long last­ing oxytetracycline, or buparvaquone (2.5 mg/kg, intramuscular, once). Because there is a satisfactory schizont vaccine available against T. parva that does not require the cost and inconvenience of an associate treatment, the sporozoite vaccine has not become popular.

c. Schizont vaccine

The central problems for the production of limited infections is to obtain the appropriate inoculum and adjust the dose so it causes strong resistance but not severe disease. Both problems were solved to a large extent by the development of practical tech­niques to grow theilerias in lymphocyte cultures. The culture of T. annulata started in 1945 alhough it took several years to develop fully. The culture of T. parva started in 1971. Once it was possible to obtain important quantities of schizonts and prepare standardized doses, schizonts were assayed to pro­duce resistance to theileriosis.

It was soon found that bovines rejected a large part of the heterologous lymphocytes infected with T. parva. For this reason, 100 million infected lym­phocytes (equivalent to 100 ml of culture) per animal were necessary to establish an infection that would generate satisfactory resistance (Dolan et al., 1984). This inconvenience has prevented the development of a schizont vaccine against T. parva. In the case of T. annulata, doses of only 10,000 to one millon infected lymphocytes per animal caused minimum clinical effect but satisfactory immunity. Furthermore, repeated cultures of T. annulata at­tenuates the infectivity for calves but preserves im­munogenicity. Depending on the strain, 25 to 300 passages may be needed to obtain satisfactory at­tenuation. The protective value of the vaccine is not related to antibody level and must be verified by vaccination assay followed by infection. However, level of antibodies may be useful as a first approach to evaluate the success of the immunization. Vac­cination or infection with completely attenuated T. annulata cause no clinical signs or detectable parasitemia but generate abundant antibodies. In­oculation of dead schizonts, on the contrary, produces little antibody response. Pipano (1989) has written an excellent description of the techni­ques to produce a T. annulata schizont vaccine. In essence, the parasites are obtained from infected bovines or ticks, cultured through several passages in bovine lymphocytes, the vaccine is prepared and assayed, and then administered. The vaccine is distributed fresh (refrigerated), with 5 million schizonts per dose, or frozen, with 10 million schi­zonts per dose. The fresh vaccine is shipped in ice and should be maintained at 4°C and used within 5 days from production. The frozen vaccine is shipped in liquid nitrogen and should be thawed at 40°C within 30 minutes of its administration. The schizont vaccine protects strongly against ho­mologous strains and less strongly against heterolo­gous strains. It also protects better from (artificial) infections with schizonts than from (artificial o natural) infections with sporozoites. Field protec­tion is very good, however, and lasts from 1 to 3.5 years. Vaccinated animals do not transmit the para­site (because the vaccine rarely produce in­traerythrocytic forms) but does not prevent red blood cell infection with field parasites thus it does not eradicate the infection. Standardized schizont vaccines against T. annulata are produced currently in China, India, and Russia. The Russian vaccine (Thelecine, Medexport, Moscow) contains one mil­lion parasitized cells per dose and is shipped in liquid nitrogen. The Office Intemational des Epi­zooties has published standard for the preparation and assay of this vaccine.

d. Piroplasm vaccine

There has been no interest in developing a vaccine against the intraerythrocytic stages because they do not cause disease or protects against the highly pathogenic intralimphocytic stages. Also, preven­tion of red blood cell parasitism would stop the infection of ticks and the transmission of the infec­tion to susceptible cattle. Although this appears desirable, it implies loss of the herd resistance that natural infections confer to autochthonous cattle. The normal balance between the presence of a trans­missible pathogen and of hosts reasonable resistant to it in a region is called 'enzootic stability'. Com­monly, decreased transmission results in fewer resistant hosts which causes severe outbreaks when transmission returns to its normal level. This phenomenon is seen in babesiosis and theileriosis when a dry year reduces tick population and trans­mission but the next rainy year restore normal vec­tor density. Unless a region is prepared to go through the efforts of maintaning eradication, preservation of enzootic stability may be more con­venient.

2. Possibilities of a molecular vaccine

Studies of immunity to theileriosis indicate that al least part of the resistance to sporozoites is mediated by antibodies (Musoke et al., 1982) whereas resis­tance to schizonts is mediated by helper (CD4+) and cytotix (CD8+) lymphocytes (Morrison et al., 1987). Resistance against heterologous (vaccinal) schizonts seems to be an immune reaction to external antigens because it appears at the beginning of the second week of vaccination and does not have genetic restrictions. Resistance against autologous (infec­tion) schizonts, on the other hand, seems to be a reaction to internal antigens because it appears by the third week and is genetically restricted (see below).

Most antigen work with theileria has been done with sporozoites because their antigens era aesly identified by reactions with the sera of hyperin­fected animals. The observation that serum of ani­mals repeatedly infected with T. parva inhibited the penetration of sporozoites into the host cells stimu­lated the production of the corresponding mono­clonal antibodies. A monoclonal antibody that ex­hibited this same property was used to purify an antigen ('p67') which later was produced as a re­combinant protein. Nine calves were vaccinated with this protein and 6 developed as a recombinant against an homologous challenge (Musoke et al., 1992). Williamson et al. (1989) prepared a mono­clonal antibody that inhibited the penetration of T. annulata sporozoites, purified the corresponding an­tigen, and produced it as a recombinant protein. Vaccination of rabbits with this protein generated sera that also inhibited the penetration of sporozoi­tes. Other studies have shown antigens unique to merozoites or common to sporozoites, schizonts, and merozoites (Kachani et al., 1992).

A vaccine that acts only on sporozoites is not reliable because some parasities could scape an­tibody, particularly when titers are low. On the other hand, antigens unique to schizonts, which probably are the responsible for resistance to the disease, are difficult to identify. In general, antigens originated externally to the host body (like those of sporozoites and vaccinal schizonts) are processed by macro­phages that lini them with their own class II MHC (Major Histocompatibility Complex) moleculas to initiate an antibody response. Antigens originated inside that host cells (like those of infection schizonts) must be linked with class I MHC moleculas of the host cell to initiate a cytotoxic lymphocyte response. This peculiarity creates two problems for the design for vaccines against internal antigens. The first is the identification of the cor­responding antigens; because there is no antibodies which can be utilized as probes, the researcher must identify the CD8+ lymphocyte stimulating Complex (fragment of parasite protein + class 1 MHC molecule) located on the surface of the infected lymphocytes. In the current state of technology, this task is quite difficult. The second is that, once iden­tified and purified, these antigens must be ad­ministered in a from that they can be incorporated inside host cells that posses the class 1 MHC mole­cules. This is also a difficult task in the current state of the art. Helper cells (CD4+) assist in the produc­tion of antibodies and cytotoxic cells. Brown et al. (1990) identified three antigens (of 4.2, 12, and 43 kdaltons, respectively) that stimulate these cells. There are still several obstacles to overcome before a molecular vaccine against theileriosis becomes a reality but the studies are reasonable advanced and the current technology offers many opportunities.

African Trypanosomiases

The African trypanosomes are blood parasites of African vertebrates, including humans. At the be­ginning they were transmitted by tsetse flies (Glossina spp.) but some species had freed themselves from vector dependency and have migrated to other continents (Trypanosoma evansi, T. vivaz, T. eguiperdum). Human African trypanosomiases (sleeping sickness) are caused by T. rhodesiense and T. gambiense, currently considered as subspe­cies of T. brucei. More than 50 million people are at risk of infection and 20 to 30 thousands actually ac­quire it every year. African trypanosomiases of bo­vines (nagana) are caused mainly by T. congolense, T. vivax, and T. brucei. Nagana negates the full use of about 10.4 million km2 of African land that could support 125 million of heads of cattle of high pro­ductivity.

African trypanosomiases are typically chronic diseases. Even the 'acute' sleeping sickness caused by T. rhodesiense can last for months in the un­treated patient. The human infection is invariably lethal if not treated. The infection of bovines recently introduced into an area of enzootia is generally fatal but some animals are able to control the parasitemia and survive with a mod­erare parasitec load. This ability, known as 'try­panotolerance', is inherited and largely dependant on immunological mechanisms (Murray et al., 1982). Trypanotolerance is more frequent among animals naturally selected through centuries in the area of enzootia. Among the domestic cattle breeds of Africa, the N'Dama and Muturu are the most resistant.

Despite the immense potential gains of using the lands infested vvith trypanosomes for cattle raising, the control of African trypanosomiases is still based on vector destruction and preventive or curative treatment of the affected animals. The current situ­ation indicates that these methods are only moder­ately effective. Vector destruction is difficult be­cause there are 22 species of Glossina that live in different habitats and the authorities are reluctant to risk ecological damage by the use of massive insec­ticide applications. Cattle treatment is expensive and promotes the selection of parasites resistant to the drug. Although the design of an effective vac­cine is highly desirable, formidable obstacles exit.

1. Antigenic variation

The first attempts at vaccination against African trypanosomes were done by Koch and Ehrlich at the beginning of the century. Later researchers assayed immunization with dead, formolated, or irradiated parasites, or with crude or partially purified parasite extracts. In all successful cases, resistance was al­ways expressed against the homologous strain, never against heterologous stabilates (Shapiro, 1989). About the same time, it was observed that parasitemias were fluctuating and that the antigens characteristic of each wave changed along the infec­tion. Soon it was discovered that this was a property of each individual parasite rather than an expression of the heterogeneity of the inoculum. It is known today that this 'antigenic variation' is due to chan­ges in the composition of a glycoprotein of 55 to 65 kdaltons that covers the external surface of the parasites ('variant surface glycoprotein' or VSG). These proteins appear when the parasites become infective for the vertebrate host in the salivary gland of the fly and persist while the parasites remain in the blood of the vertebrate (Persons et al., 1983). VSGs are codified by possibly a thousand genes (Van Der Ploeg et al., 1982) that are expressed sequentially but without a predetermined order. Each VSG may have some nine epitopes (Hall and Esser, 1984) and their differences in amino acido composition confer the protein diverse antigenic specificities ('`variant antigen type' or VAT). Up to 12 VATs have been demonstrated in trypanosomes from the fly salivary gland and up to 100 in trypanosomes from the vertebrate blood stream. Many more are theoretically possible, though (Steinert and Pays, 1986). In an initial infection, protective antibodies are generated against the VSG existing at the moment of the infection (Sendaslion­ga and Black, 1982). The combined action of anti­bodies, complement, and phagocytic cells rapidly dstroys the parasites that exhibit that specificity (VAT) but these are soon replaced with new parasites that exhibit a different specificity (Doyle, 1977). Thus, successive waves of parasites with different antigenic specificities and development of the corresponding protective antibodies generate the prolonged and fluctuating parasitemia charac­teristic of African trypanosomiases. It was thought at the beginning that antigenic variation was in­duced by the action of antibodies on the parasites but this idea proved wrong when it was observed that the variation also occurred in vitro cultures, in the absence of antibodies (Doyle et al., 1980). On the other hand, some specificities ('predominant antigens') appear more frequently than it would be expected if the change were random. Despite its enormous importance, the stimulus that induces the passage to a new specificity is not clear yet.

The VSGs that have been isolated show cross reactivity due to homologies of the aminoacids and carbohydrates of the carboxylic end. In the living parasites, however, this cross reactivity is not ob­served because the carboxylic end is buried deeply into the parasite surface and inaccessible to the immune system. Animals particularly resistant to the disease, or that mount a very rapid immune response, or that are treated repeatedly, may develop antibodies to many VSGs and turn resistant to the infection. As a fact, there is evidente that trypano­tolerance operates through rapid and effective im­mune responses that prevent high parasitemia and accumulate antibodies against many VSGs (Murray et al., 1982).

The infection also induces formation of antibod­ies to nonvariable antigens but it has not been pos­sible to show that these antibodies are protective. The evidence appears to indicate that the VSG cov­ers the parasite totally and prevents access of the immune system to other subjacent antigens. The response to non–variable antigens would occur only when they are released by dead parasites but the corresponding immune effector mechanisms would not have access to these antigens in the living para­sites. It is also possible that VSGs suppress the responses to these antigens by a mechanism of anti­genic competition. Otherwise, nonvariable antigens could be a potential source of polyvalent vaccines since a 77 kdalton nonvariable antigen has been shown to be common to T. brucei, T. congolense, and T. vivax (Webster and Shapiro, 1990).

2. Nonspecifc Ivmphoproliferation

Although antigenic variation is possibly the major obstacle to the deisgn of a vaccine against African trypanosomes, there are other characteristics of the infection that are also regarded as impediments to artificial immunization. Fox example, the African trypanosomes have the ability to stimulate non­-specifically the host immune system. The parasites inoculated by the vector fly stir local inflammatory and immune reactions in the skin that form a 'chancre' after 5 to 10 days. The protozoa multiply locally and then spread through the blood stream producing an intense proliferation of lymphocytes and macrophages, particularly B cells, with an im­portant increase of circulating IgM (Morrison et al., 1982). Part of the immunoglobulin is directed to parasite antigens but another part is directed against host antigens, or is nonspecific. The nonspecific lymphoproliferation appears to be caused by a polyclonal factor produced by macrophages that ingest parasitic materials (Sacks et al., 1982). Lym­phoproliferation has an important role in the pathogenicity of trypanosomiases but is effect on the course of protective immune reactions is still unknown.

3. Immunodepression

Chronic trypanosomiasis causes a loss of lympho­cytes in the lymph organs and reduces the ability to produce antibodies or cell–mediated immunity to new antigens (Hudson and Terry, 1979). The production of IgM specific for the parasites con­tinues interrupted, however. Several theories have been proposed to explain this phenomenon but the observations point to a probable dysfunction of the macrophages or T lymphocytes caused by parasite materials (Bagasra et al., 1981; Sztein and Kierszenmaum, 1991).

4. Potential for a vaccine

Most specialists consider that antigenic variation, nonspecific lymphoproliferation, and immune depres­sion are formidable obstacles to the design of a vaccine and believe that vaccination against African trypanosomiases is only a remote possibility (Mur­ray and Urquhart, 1977; Lee and Maurice, 1983). Lymphoproliferation and immune depression, how­ever, have been studied mostly in laboratory ani­mals. Their manifestations are considerable milder in cattle (Stephen, 1986) and they certainly do not prevent the production of antibodies lethal to try­panosomes. Cattle has been protected against ho­mologous infections by infection followed by treat­ment, or by inoculation of irradiated parasites or VSGs (Wells et al., 1983). This demonstrates that artificial immunization is feasible and that the major (and, perhaps, the only) obstacle to an effective vaccine is antigenic variation.

On the other hand, it is possible that antigenic variation is not as unbeatable as it appears. A few animals recover spontaneously from trypanosorni ases despite antigenic variation. Perhaps the careful study of the immune reactions of these animals with modern technology may show tracks in the trypanosome armor. A common specificity to several VSGs, a break in the variable cover that would allow to reach nonvariable antigens, the find­ing of surface nonvariable antigens, the ability to manipulate the mechanism of variation, are pos­sibilities worth consideration. Webster and Shapiro (1990) proposed to look for receptors for parasitic endocytosis that would permit access to the parasit, or to search for moleculas responsible for the patho­geny which would permit to overcome the disease. For example, only 12 specificities are known to exist in the trypanosomes injected by the fly (Crowe et al., 1983). Cloning and combination of these 12 VSGs in a vaccine are well within the possibilities of modern technology. Presumably, the correspond­ing antibodies would avert the infection from the beginning. Pearson et al. (1988) obtained ara antigen from trypanosomes from the fly's gut ('procyclin') and raised the corresponding antibodies in rabbits. A similar technique could generate 'altruistic vac­cines', this is, vaccines that do not protect the indi­vidual but kill the parasites in the vector and prevent transmission.

There is no promising solutions for a vaccine to African trypanosomes yet but modern technology is pregnant with possibilities and the magnitude of the problem justifies any effort.

Boophilus Ticks

Ticks are hematophagous parasites of many verte­brates. They consitute important agents of disease of humans and their domestic animals, by them­selves and as vectors of other pathogens. The con­ventional control of ticks consists of applying acari­cides to the animals at risk. This method is expensive due to the price of the drug and of the installations for appropriate application. Many owners of small farms cannot afford the cost so their animals remain untreated and a constant source of infestation for the region. The main problem, how­ever, is the regular appearance of resistance to acari­cides. In Australia, for example, Boophilus ticks have developed resistance successively to arsenic, chlorinated hydrocarbons, organophosphorus, car­bamates, amidines, and synthetic pyretroids (Nolan and Schnitzerling, 1989). This situation has forced to look for alternatives or complements to the con­ventional tick control and has stimulated tremen­dously investigations toward a vaccine.

Because of their wide geographic distribution, their parasitism of cattle, and the diseases they can transmit, the most important ticks are those belong­ing to the genes Boophilus. B. annulatus was eradi­cated from the United States through a campaign that lasted from 1909 to 1942. lt is estimated that reintroduction of the tick would cause losses of over a billion dollars per year nowadays. Horn and Arteche (1985) estimated that B. microplus causes losses of 800 million dollar per year in Brazil. For these reasons, most of the work to devise ara anti­tick vaccine has been centered around B. microplus.

Farmers have known for many years that old are less affected by ticks than young animals. Many experiments with laboratory animals and a few with cattle in the 1960s and 1970s demonstrated that tick infestations effectively induced some degree of protective immunity. Brossard in 1976 showed that the injection of tick salivary glands in calves produced some resistance. Roberts and Kerr in 1976 demonstrated that the protection was transmisible with serum. Allen and Humphrey en 1979, im­munized bovines with intestinal and genital tracts of Dermacentor andersoni ticks and found that the vaccinated animals inhibited the fertility of the ticks that fed on them. Natural infestations produced only partial resistance and for a time it was believed that this depended on a cutaneous hypersensitivity that caused the drop of the ticks. Some investigators assumed that immunization with the antigens that induced resistance in infestations would also pro­duce only partial resistance and hypersensitivity (Wikel, 1988). Nowadays we know that most protective antigens of parasites are weakly im­munogenic and easily inhibited by stronger antigens inoculated simultaneously (Damian, 1989). The poor immune response to natural infestations, then, is probably due to the competition of strong an­tigens irrelevant to resistance that are inoculated at the same time as the weak protective antigens. Presumably, inoculation of purified natural protec­tive antigens with appropriate adjuvants should elicited satisfactory responses. On the other hand, it now appears that hypersensitivity does not play a role in anti–tick immune protection in bovines. Nonetheless, some investigators proposed im­munization with tick materials that were potentially antigenic but were not inoculated during infestation, such as the digestive and intestinal tracts. These materials have been called 'novel or occult antigens' (Wikel, 1988; Willadsen y Kemp, 1988). A series of experiments by Australian researchers demonstrated that the inoculation of tick extracts produced satisfactory resistance in about half the immunized calves, that feeding of ticks on these calves caused lesions in the tick intestine, and that he respective antigen was located in the plasmatic membrane of the arthropod's gut cells (Kemp et al., 1989). Vaccination with tick intestinal tissue in­duced a resistance manifested mainly as a severe reduction of tick fertility (Opdebeeck et al., 1988). The responsible antigen, with a molecular weight of 89 kdaltons and called 'Bm86', was finally purified through a complex procedure that required about I kg (circa 40,000 specimens) of ticks to obtain 0.1 mg of protein (Willadsen et al., 1988). Vaccination of calves with this antigen caused a resistance mani­fested as an increase in the mortality of the ticks that fed on these animals and a reduction of the fertility of the ticks that survived. The protective power of the vaccination was expressed as the reduction in reproduction of the ticks against controls, including mortality and reduced fertility. Bm86 was subse­quently produced as a recombinant protein in bacte­ria, fungi, and insect cells. While the native antigen induced a protection slightly over 90%, the recom­binant antigens caused protection between 53 and 91% (Tellan et al., 1992). These figures are prob­ably insufficient for field vaccination yet but a mix­ture of different protective antigens could improve them. Although putative protective antigens from salivary glands have been reported for other tick species (Barriga et al., 1991), they have not been studied for Boophilus. The salivary antigens appear to be more effective to prevent feeding of the ticks and almost equally effective to depress fertility (Sa­hibi et al., 1993). This is an important consideration because inhibition of feeding would prevent trans­mission of pathogens. An aspect of tick immunity that has started to be studied only recently is the ability of the arthropod to depress or elude host immunity (Barriga et al., 1993). We do not know yet what impact this could have on the efficacy of a vaccine.

Myiases

Myiases are infections or infestations of vertebrates by larvae of flies. Some myiases are accidental but other are obligatory. In this last case, the larva must spend some time in the host tissues to complete is life cycle. Among the obligatory myiases, the most common are those by flies of the genera Cochliomyia and Dermatobia in several species of hosts, Oestrus in ovines, Gasterophilus in equines, and Hypoderma, principally in bovines. The traditional control of myiases is through insecticide treatment of the animals at risk. This method is expensive, must be repeated at short intervals, leaves residues in milk and meat, pollutes the envi­ronment, and promotes development of resistance of the flies to the insecticide. These drawbacks have simulated the search for alternative or complemen­tary methods of control. An alternative that has been very successful in the case of Cochliomyia is the rearing, sterilization with irradiation, sterile males in nature eventually results in extermination of the population. This technique is effective against other fly species (Kunz et al., 1990) but is too expensive and requires the rearing of millions of specimens which is difficult or impossible with other species that frequently cause myiases. Immunization has been studied as an alternative for control at least in one of this cases.

The first publication about immunization against insects was probably by Schlein and Lewis in 1976, who immunized rabbits with fly extracts and ob­served that the flies fed on these animals showed abnormalities in their development. The existing evidence indicates that infestation by hematophagous insects causes some degree of protection against reinfestations but weaker than that induced by ticks. This may be so because the briefer feeding time of insects does not favor the inoculation of enough antigen to generate effective responses. Also, this brevity prevents the sustained action of antibodies against the antigens in the fly's salivary gland. Im­munization with occult antigens may not be as ef­fective with insects as it has been with ticks. While digestion in ticks occurs inside intestinal cells after phagocytosis, digestion in insects occurs in the in­testinal lumen. This means that the insect intestine has a low pH and proteolytic enzymes that could denature or digest the ingested antibodies before they have a chance to act.

In myiases, the association of the arthropod with the host tissues goes from a superficial contact (Oestrus), to expensive skin destruction (Cochliomyia), to systemic migrations (Hypoderma). The respective immune responses must vary in a similar manner. In many cases, however, the arthropod is completely surrounded by host tis­sues or fluids for several days which facilitates the action of the elements of immunity on the parasite. The most advances studies of immunization against myiases are those against Hypoderma spp.

Field observations had suggested that infection with Hypoderma caused some degree of protection against later infections. Evstafjev (1982) sys­tematized this information and found that the degree of protection depended on the number and parasitic load of prior infections. Resistance was found to be particularly effective against the first instar larvae (Liebisch and Frauen, 1989). Infected animals had immediate and delayed type skin reactions to larval protein (Pruett and Barret, 1984), circulating anti­bodies that persisted for 14 weeks after cure (Sinclair et al., 1984), and cell-mediated responses to parasite antigens and polycional mitogens (Baron and Weintraub, 1987). Baron and Weintraub (1986) immunized calves with crude extracts of H. lineatum first instar larvae or with a supernatant of an in vitro culture and infected them with the homologous parasite or H. bovis. Both treatments reduced the parasitic load by either species to half of what was in non-immunized controls. Study of the protein composition of the first instar larvae of H. lineatum showed that the principal proteins were 3 serine proteinases of about 24 kdaltons (Pruett et al., 1988). They were called 'hypodermins A, B, and C'. These enzymes help in the liquefaction of the host tissues during larval migration and also consume complement (Boulard and Bencharif, 1984). This last activity may well be a mechanism of protection of the larvae against host immunity. The main protective antigen appear to be hypoder­min A because vaccination of calves with it induced 90% resistance (Pruett et al., 1989) whereas vacci­nation with the 3 hypodermins combined caused only 95% protection (Baron and Colwell, 1991). There is cross resistance between H. lineatum and H. bovis and their hypodermins (particularly hy­podermin C) cross react also. This suggests that a polyvalent vaccine may be possible. The Livestock Insect Laboratory of the US Department of Agricul­ture in Kerrville, Texas, has recently produced hypodermin A as a recombinant protein and is cur­rently assaying it in a vaccine.

Conclusion

Some of the fundamental steps in the design of a parasitic vaccine are: 1) to identify the protective antigens, 2) to produce them in useful quantities, and 3) to administer them in the best suitable man­ner. Until recently, these were almost unsurmount­able problems that held back progress in immuniza­tion against parasites. In the last decade or so, he corresponding solutions appeared. Modem ap­proaches (Barriga el al., 1991) and techniques (e.g., monoclonal antibodies) have facilitated the identifi­cation of protective antigens, and cloning methodol­ogy is permitting to produce them in quantities appropriate for assay and even for commercial dis­tribution. We did not discuss here the advances in administration of delivery of antigens to the host but new adjuvants or the use of replicating organisms (viruses and bacteria) to carry the antigens into the host are changing vaccination (Spriggs and Koff, 1991). New concepts in immunology are perfecting our understanding of the regulation of the immune response and work with antigens is revealing the necessary epitope structure to trigger different kinds of immune cells. Skeptics are ready to point out that there is no recombinant commercial vaccine against parasites on the market yet but if recent history is of any value to predict the futuro, this situation will not remain like that for long.

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Recibido el 15 de septiembre de 1993.