Family Eimeriidae

Members of this family have a single host. Schizogony and gametogony take place within the host cells, and sporogony ordinarily occurs outside the host's body. The oocysts and schizonts lack an attachment organ. The oocysts contain 0, 1, 2, 3 or many sporocysts, each containing 1 or more sporozoites. The microgametes have 2 flagella. The genera are differentiated by the number of sporocysts in their oocysts and the number of sporozoites in each oocyst.

Morphology. The morphology of a typical oocyst, that of Eimeria, is shown in Fig. 23. The oocyst wall is composed of 1 or 2 layers and may be lined by a membrane. It may have a micropyle, which may be covered by a micropylar cap. Within the oocyst in this genus are 4 sporocysts, each containing 2 sporozoites. There may be a refractile polar granule in the oocyst. There may be an oocyst residuum or a sporocyst residuum in the oocyst and sporocyst, respectively; these are composed of material left over after the formation of the sporocysts and sporozoites. The sporocyst may have a knob, the Stieda body, at one end. The sporozoites are usually sausage- or comma-shaped, and may contain 1 or 2 clear globules.

Structures of sporulated Eimeria oocyst

Location. Most coccidia are intracellular parasites of the intestinal tract, but a few occur in other organs such as the liver and kidney. Each species is usually found in a specific location within the intestinal tract; some are found in the cecum, others in the duodenum, still others in the ileum, etc. They may invade different cells in these locations. Some species are found in the mucosal cells at the tips of the villi, others in the crypts and still others in the interior of the villi. Their location within the host cell also varies. Some species are found above the host cell nucleus, while others are found beneath it and a few occur inside it. Some species enlarge the host cell only slightly, while others cause it to become enormous. The host cell nucleus is also often greatly enlarged even tho it may not be invaded.

Life Cycle: The life cycles of the Eimeriidae are similar, and can be illustrated by that of Eimeria tenella, which is found in the ceca of the chicken (Fig. 24).

Life cycle of the chicken coccidium, Eimeria tenella

It was first worked out in a classic paper by Tyzzer (1929). The oocysts are passed in the feces; at this time they contain a single cell, the sporont. They must have oxygen in order to develop to the infective stage, a process known as sporulation or sporogony. The sporont, which is diploid, undergoes reduction division and throws off a refractile polar body. The haploid number of chromosomes is 2 (Walton, 1959). The sporont divides to form 4 sporoblasts, each of which then develops into a sporocyst. Two sporozoites develop within each sporocyst. Sporulation takes 2 days at ordinary temperatures. The oocysts are then infective and ready to continue the life cycle.

When eaten by a chicken, the oocyst wall breaks, releasing the sporozoites. The factors which cause excystation have not been determined. Itagaki and Tsubokura (1958) found that pancreatic juice did not cause excystation of E. tenella, and Landers (1960) was unable to induce excystation by treating the oocysts of E. nieschulzi from the rat with pepsin, trypsin, pancreatin, pancreatic lipase or bile. Ikeda (1960), however, reported that pancreatic juice did cause excystation of E. tenella, and that trypsin was the responsible enzyme.

According to Challey and Burns (1959) and Pattillo (1959), the sporozoites first enter the cells of the surface epithelium. Pattillo (1959) observed passageways, which he called penetration tubes, in the striated border and epithelium thru which the sporozoites passed. They deploy along the basement membrane and then pass thru it into the lamina propria. Here they are engulfed by macrophages and carried by them to the glands of Lieberkuhn. They then leave the macrophages and enter the epithelial cells of the glands, where they are found below the host cell nucleus, i.e., on the side away from the lumen. We do not know how common this method of penetration is among the coccidia; Van Doorninck and Becker (1957) first found it in E. necatrix of the chicken.

Once in a glandular epithelial cell, each sporozoite rounds up and becomes a first generation schizont. By a process of asexual multiple fission (schizogony), each schizont forms about 900 first generation merozoites, each about 2 to 4 u long. These get their name from the Greek word for mulberry, which they resemble before they separate. They break out into the lumen of the cecum about 2.5 to 3 days after infection. Each first generation merozoite enters a new host cell, and rounds up to form a second generation schizont, which lies above the host cell nucleus. By multiple fission it forms about 200 to 350 second generation merozoites about 16 u long. These are found 5 days after infection. Some of them enter new intestinal cells, round up to form third generation schizonts, which lie beneath the host cell nuclei and produce 4 to 30 third generation merozoites about 7 u long.

Most of the second generation merozoites, however, enter new host cells and begin the sexual phase of the life cycle, known as gametogony. Most of these merozoites turn into female gametes (macrogametes), which simply grow until they reach full size. Some of the merozoites turn into male gametocytes (microgametocytes). Both the macrogametes and microgametocytes lie below the host cell nuclei. Within each microgametocyte a large number of tiny biflagellate microgametes are formed. These break out and fertilize the macrogametes.

The resultant zygote lays down a wall around itself in the following way: The macrogametes contain one or two layers of eosinophilic plastic granules in their cytoplasm; these are composed of mucoprotein (Kheisin, 1958). They pass to the periphery, flatten out and coalesce to form the oocyst wall after fertilization. The formation of this wall marks the transition of a fertilized macrogamete into an oocyst. According to Monne and Honig (1954), the outer layer of the oocyst wall is a quinone-tanned protein and the inner layer is a lipid coat firmly associated with a protein lamella.

The oocysts then break out of their host cells, enter the intestinal lumen, and pass out in the feces. The prepatent period, from the time of infection to the appearance of the first oocysts in the feces, is 7 days. Oocysts continue to be discharged for a number of days thereafter, due to the fact that the sporozoites do not all enter the host cells immediately but may remain in the lumen for some time, and also because many of them are retained in a plug of material in the ceca for some days before they are eliminated.

In the absence of reinfection, coccidial infections are self-limiting. Asexual reproduction does not continue indefinitely as it does, for example, in Plasmodium. In E. tenella, 3 generations of merozoites are produced; in other species there may be 1, 2 or 4. After this, the life cycle enters its sexual phase; the oocysts are formed, eliminated from the body, and the infection is over. Reinfection may take place, but the host develops more or less immunity following primary infection.

The number of oocysts produced in an animal per oocyst fed depends in part on the number of merozoite generations and the number of merozoites per generation. A single oocyst of E. tenella containing 8 sporozoites is theoretically capable of producing 2,520,000 second generation merozoites (8 x 900 x 350), each of which can develop into a macrogamete or microgametocyte.

In E. bovis of cattle, there is only a single asexual generation, but a giant schizont containing about 120,000 merozoites is formed (Hammond et al. 1946). In the rat, E. nieschulzi is theoretically capable of producing 1,500,000 oocysts per oocyst fed, E. miyairii 38,016, and E. separata only 1536 (Roudabush, 1937). In E. nieschulzi there are 4 generations of merozoites, while in the latter two species there are only 3, and fewer merozoites are usually produced in each than in E. nieschulzi. In the rabbit, E. magna produces 800,000 oocysts per oocyst fed, E. media produces 150,000 and E. coecicola 100,000 (Kheisin, 1947, 1947a).

The actual numbers of oocysts produced per oocyst fed are usually considerably lower than the theoretical ones0 If the host is resistant or immune, it destroys many merozoites, and many others pass out in the feces before they have time to enter host cells. The infecting dose is also an important factor in determining the number of oocysts produced. The greater the infecting dose, the smaller the number of oocysts usually produced per oocyst fed. For example, Hall (1934) obtained a yield of 1,455,000 oocysts of E. nieschulzi per oocyst fed when the infecting dose was 6 oocysts, 1,029,666 when it was 150 oocysts, and 144,150 when it was 2000 oocysts. If the infecting dose is too small, however, smaller numbers of oocysts are produced. Hall (1934) found that when only a single oocyst was fed, the yield was 62,000.

Similarly, Brackett and Bliznick (1950, 1952) found that with E. acervulina of the chicken, 9000 oocysts were produced per oocyst fed when the infecting dose was 200 oocysts, 35,000 to 72,000 when it was 2000 oocysts, 35,000 when it was 10,000 oocysts, and 7,600 when it was 20,000 oocysts. With E. maxima of the chicken, they found that 11,500 oocysts were produced per oocyst fed when the infecting dose was 200 oocysts, 2,250 when it was 2000 oocysts, and 940 to 2900 when it was 10,000 oocysts. With E. necatrix of the chicken, they found that 50,000 oocysts were produced per oocyst fed when the infecting dose was 200 oocysts, and 2400 when it was 2000 oocysts. With E. tenella of the chicken, they found that the maximum number of oocysts produced per oocyst fed in numerous experiments was 400,000. However, in one series of 2-week-old chicks this figure ranged from 1200 for chicks fed 40,000 oocysts to 80,000 when the infecting dose was 50 oocysts.

All the factors responsible for these results are not known. More effective mobilization of the host's defenses is probably important, but lack of enough epithelial cells to parasitize, sloughing of patches of epithelium, increased intestinal motility with resultant diarrhea and elimination of merozoites before they can reach a cell, and entrapment of merozoites in tissue debris and cecal cores may also play a part.

Pathogenesis. While many species of coccidia are pathogenic, many others are not. Pathogenicity depends on a number of factors, some of which are probably still unknown. Among those which might be mentioned are the number of host cells destroyed per infecting oocyst (which depends upon the number of merozoite generations and the number of merozoites per generation) and the location of the parasite in the host tissues and within the host cells. The size of the infecting dose or doses, the degree of reinfection, and the degree of acquired or natural immunity of the host are also important.

Even with a pathogenic species, the final effect on the host depends on the interplay between many factors; it may range from rapid death in susceptible animals to an imperceptible reaction in immune ones.

If disease is present, the signs are those of a diarrheal enteritis. There may or may not be blood in the feces, depending on the parasite species and severity of infection. Affected animals gain weight poorly, become weak and emaciated, or may even die, depending again on the parasite species and the size of the infecting dose. Young animals are much more commonly affected than older ones. Those animals which recover develop an immunity to the particular species which infected them. However, this is not an absolute immunity, and recovered adult animals are often continuously reinfected so that they carry light infections which do not harm them but which make them a source of infection for the young. In addition, under conditions of stress their immunity may be broken down and they may suffer from the disease again.

Differentiation of Species. Both morphological and biological characters are used to separate the species of coccidia. Both the endogenous and exogenous stages of the life cycle may differ morphologically. However, since the endogenous stages of many species are unknown, the structure of the oocyst is most commonly used. The feeling is sometimes expressed that the oocysts have so few structures that not many species can be distinguished morphologically, but conservative calculation shows that at least 2,654,208 morphologically different oocysts are possible in the genus Eimeria alone (Levine, 1961).

A second group of criteria is the location of the endogenous stages in the host. This has been discussed above. Host specificity is a third criterion. This varies with the protozoan genus and to some extent with the species. In general, the host range of Isospora and Tyzzeria species is relatively broad. Several members of the same host order may be infected by the same species of these genera. For example, Isospora bigemina occurs in the dog, cat, ferret and mink, while Tyzzeria anseris has been found not only in the domestic goose and several other members of the genus Anser, but also in the Canada goose and Atlantic brant (both Branta) and whistling swan (Olor). On the other hand, the host range of Eimeria species is relatively narrow. A single species rarely infects more than one host genus unless the latter are closely related.

Cross-immunity studies are also used in differentiating the coccidia of a particular host species from each other. Infection of an animal with one species of coccidium produces immunity against that species but not against other species which occur in the same host.

Diagnosis: Coccidiosis can be diagnosed by finding the coccidia on microscopic examination. There are several pitfalls in diagnosis. Each species of domestic and laboratory animal has several species of coccidia, some of which are pathogenic and some of which are not. Since an expert is often needed to differentiate between some of the species, the mere presence of oocysts in the feces, even in the presence of disease signs, is not necessarily proof that the signs are due to coccidia and not to some other agent.

Following recovery from a coccidial infection, an animal is relatively immune to reinfection with the same species. This immunity is not so solid that the animal cannot be reinfected at all, but it does mean that the resultant infection will be low-grade (except possibly under conditions of stress) and will not harm the host. Such low-grade infections are extremely common, i.e., the animals have coccidiasis rather than coccidiosis. Hence, the presence in the feces of oocysts of even highly pathogenic species of coccidia does not necessarily mean that the animal has clinical coccidiosis.

On the other hand, coccidia may cause severe symptoms and even death early in their life cycle before any oocysts have been produced. This occurs commonly, for example, with E. tenella of the chicken and E. zurnii of the ox. Consequently, failure to find oocysts in the feces in a diarrheal disease does not necessarily mean that the disease is not coccidiosis.

The only sure way to diagnose coccidiosis, then, is by finding lesions containing coccidia at necropsy. Scrapings of the lesions should be mixed on a slide with a little physiological salt solution and examined microscopically. It is not enough to look for oocysts, but schizonts, merozoites, gametes and gametocytes inside the host cells must be sought for and recognized.

Some species of coccidia can be identified from their unsporulated oocysts, but study of the sporulated oocysts is often desirable. Oocysts can be sporulated by mixing the feces with several volumes of 2.5% potassium bichromate solution, placing the mixture in a thin layer in a Petri dish and allowing it to stand for 1 day to 2 weeks or more, depending on the species. The potassium bichromate prevents bacterial growth which might kill the protozoa, and the thin layer is necessary so that oxygen can reach the oocysts.

Treatment. The first compound found effective against coccidia was sulfur, which was introduced by Herrick and Holmes (1936). Later, Hardcastle and Foster (1944) introduced borax. Neither of these compounds was a satisfactory anticoccidial drug. Sulfur interferes with calcium metabolism, causing a condition known as sulfur rickets in chickens, while borax is only partially effective and in addition is toxic in therapeutic doses.

The first practical anticoccidial drugs were the sulfonamides, of which the first to be used was sulfanilamide, introduced by P. P. Levine (1939). Since that time many different drugs have been used, particularly against Eimeria tenella of the chicken. These include not only sulfonamides but also derivatives of phenylarsonic acid, diphenylmethane, diphenyldisulfide, diphenylsulfide, nitrofuran, triazine, carbanilide, imidazole and benzamide. Several thousand papers have probably been published on coccidiostatic drugs, and their use in poultry production is so common in the United States that it is difficult to obtain a commercial feed which does not contain one or another of them. They are used to a considerably lesser extent for other classes of livestock.

None of these drugs will cure a case of coccidiosis once signs of the disease have appeared. They are all prophylactic. They must be administered at the time of exposure or soon thereafter in order to be effective. They act against the schizonts and merozoites and occasionally against the sporozoites, preventing the life cycle from being completed. They are not effective against the gametes. Hence, since exposure in nature is continuous, these drugs must be fed continuously. This is usually done by mixing them with the feed or water.

Nowhere is a knowledge of the normal course of the disease more important than in interpreting the results of treatment of coccidiosis, and nowhere is the controlled experiment more important than in research in this field. This disease is self-limiting not only in the individual patient but also in a flock or herd. In a typical outbreak of coccidiosis, signs of disease appear in only a few animals at first, the number of affected animals builds up rapidly to a peak in about a week, and then the disease subsides spontaneously. In the early stages, most farmers do little, thinking that the condition is unimportant and will soon be over. Once more animals become affected and losses increase, it takes a little time to establish a diagnosis, so treatment is often not started until the outbreak has reached its peak. Under these circumstances, it matters little what treatment is used - the disease will subside. This is the reason why so many quack remidies used to get glowing testimonials from satisfied users.

A similar course of events is encountered by the small animal practitioner. The patient with coccidiosis is not brought to him until it is already sick. By this time it is too late for any anticoccidial drug to be of value, altho supportive treatment and control of secondary infections may be helpful. If the patient recovers, however, whatever drug happened to be used is often given undeserved credit. Such drugs are like Samian clay, which was Galen's favorite remedy. He said that it cured all diseases except those which were incurable, in which case the patient died.

Collins (1949) described the "four-pen test" which should be used in evaluating coccidiostats and other drugs. The birds in one pen are infected with coccidia and treated with the compound under test. Those in the second pen are infected and untreated, those in the third pen are uninfected and treated, and those in the fourth pen are uninfected and untreated. Comparison of the first 2 pens determines whether the compound has any effect on the coccidia; the third and fourth pens are used to determine whether the drug has any effect on the chickens themselves and to make sure that no extraneous infection has taken place.

After an animal has been receiving a coccidiostatic drug for some time during exposure to infection, it develops an immunity to the coccidia. This occurs because the sporozoites are not affected by the drug but invade the tissue cells and stimulate the host's defenses.

After coccidiostats had been mixed in poultry feeds for a number of years, it was inevitable that drug resistant strains of coccidia would appear. The first report of this was by Waletzky, Neal and Hable (1954), who found a field strain of Eimeria tenella resistant to sulfonamides. Cuckler and Malanga (1955) reported on 40 field strains of chicken coccidia which were resistant to one coccidiostat or another, and drug resistance is now a well-known complicating factor in the use of these agents. A race has developed between the coccidia and the pharmaceutical houses, and some day, horribile dictu, we may be reduced to sanitation to control coccidia.

Mixed Infections. All domestic animals have more than one species of coccidia. Some are highly pathogenic, others less so, and still others practically non-pathogenic. Pure infections with a single species are rare in nature, so the observed effect is the resultant of the combined actions of the particular mixture of coccidia and other parasites present, together with the modifying effects of the nutritional condition of the host and environmental factors such as weather and management practices.

In the remainder of this chapter, each species of coccidium in a particular host animal will be taken up first, and then a general discussion of coccidiosis in the host will follow.