Endogenous Development and Histopathological Differences Among the Chicken Eimeria: A Review Open Access

Coccidiosis remains one of the most significant diseases affecting poultry production globally. It is well recognized that parasites of the genus Eimeria are ubiquitous wherever chickens are raised, and the infections they produce influence growth, feed efficiency, mortality and the susceptibility to other diseases. As a result, the economic impact of coccidiosis is likely greater than any other disease affecting poultry production, with a recent global estimate of costs in the range of $14 billion annually (1).

The pervasive nature of coccidial infection requires the consistent use of prophylactics to limit its effects in chicken production. Anticoccidial medication and/or live coccidiosis vaccination are mainstays in the struggle to keep the disease in check and are the only reasonable means by which the poultry industry can maintain profitability in the face of these infections. Because these methods of control have shortcomings that limit their effectiveness in practical situations, the occurrence of coccidial infection, whether in a mild, subclinical form or as overt clinical disease, occurs frequently. In both situations, technical personnel are required to determine the cause of these occurrences and provide basic information on the species of Eimeria that are involved.

Although diagnosis of coccidial infection is seemingly a straightforward process using traditional methods of lesion scoring (2) and oocyst morphology (3), these methods are not well correlated with flock performance (4,5). Under field conditions, broilers are often infected with more than one Eimeria species that may infect similar sites in the intestinal tract, a factor that may influence the diagnosis of the species involved. Additionally, common species such as Eimeria praecox and Eimeria mitis do not produce gross intestinal lesions, making their evaluation difficult or impossible even by the most experienced personnel. Because coccidial infections are likely to occur in the presence of other intestinal pathogens, coccidial lesions may be more difficult to identify, affecting the accuracy of the diagnosis and the scores applied. Therefore, the traditional methods of identification, although relied upon and widely used by field specialists around the world, are affected by multiple variables that bring to question their accuracy and reliability.

To offset the shortcomings described above, molecular techniques, including PCR, are currently being applied in the diagnosis of Eimeria species. By using segments of oocyst-extracted DNA, these methods provide insight into differences among Eimeria that facilitate speciation and the detection of subclinical infections (6,7). Studies by Haug et al. (8) and Carvahlo et al. (9) demonstrated the improved sensitivity of PCR compared to standardized methods by improving the recognition of Eimeria species circulating in a flock. Additionally, the ability to detect Eimeria maxima and E. praecox, species that are often difficult to identify grossly, was greatly enhanced (9). Perhaps the greatest advantage of these procedures is their ability to establish the genetic uniqueness of specific Eimeria field samples that have expanded our understanding of the species infecting chickens (10). Thus, the advantages of molecular identification are clear, but application in the diagnostic setting may be limited by regional availability and relative cost compared to other methods.

Although the sensitivity of molecular diagnostic procedures has improved greatly in recent years, the use of standard histopathological methods remains an effective and reliable approach to diagnosis. In recent studies designed to compare the accuracy of three methods of diagnosis (lesion scoring/oocyst morphology, molecular methods, and histopathological analyses), Balestrin et al. (5) indicated that when each is evaluated independently, histopathological assessments proved to be most accurate for demonstrating the presence of a coccidial infection but were deficient in defining the species involved. However, when histopathology was combined with the other methods of diagnosis, accuracy of species identification improved, and was greatest when PCR and histopathology were combined. Based on this information, histopathological evaluation was considered a requisite for improved reliability of lesion scoring and morphological assessments, and provided important supportive information, such as intensity of infection and degree of intestinal damage, when PCR was employed (5).

Thus, histopathological evaluations of Eimeria are of growing significance, not only as a stand-alone diagnostic tool, but also as a means of support to other commonly used methods of diagnosis. Additionally, a review of the literature indicates that a concise summary of endogenous development of the Eimeria species of the chicken is lacking. With these details in mind, the objective of this review is to discuss the main histopathological features in the development of Eimeria in the digestive tracts of chickens to facilitate the accurate diagnosis of these infections. This paper also provides an accessible bibliography for those intending to pursue the study of this topic in greater detail.

Cogent publications dealing with the endogenous development and histopathological features of Eimeria infections in chickens were reviewed. As a part of this process, information on the pathogenic phases of the life cycles is presented. Accompanying these details are diagrams and micrographs that elucidate the size, location, and significance of these forms. Tabular comparisons of these data are also included.

Familiarization with the general life cycle of Eimeria in chickens is essential for understanding the nuances and pathological effects of each species. In addition, many terms have been used to describe the various life cycle stages of Eimeria. In this paper, certain terms having the same meaning will be used interchangeably. The most common examples are schizogony and merogony, schizonts and meronts, gametogony and gamontogony, microgametocytes and microgamonts, and macrogametocytes and macrogamonts. Subtle variation of these terms may also occur in some passages. Consulting basic reference material on these topics as well as the details of the Eimeria life cycle in chickens will assist those unfamiliar with these essentials. Our descriptions also rely upon familiarity with four major areas of the intestinal tract of chickens. As shown in Fig. 1, these are the duodenum, jejunum, ileum, and ceca. Occasionally, reference is made to specific areas of the lower digestive tract such as cecal necks, rectum, and the bursa of Fabricius. We also refer to specific areas of the enterocyte as sites where endogenous development is likely to occur. Since the host cell nucleus is usually the most prominent histological feature of the enterocyte, the position of a parasite in relation to the nucleus describes this location efficiently. Thus, terms such as “apical to” or “below the host cell nucleus” indicate specific locations where parasites can be identified.

The focus is this work is to illustrate and review the developmental and histopathological differences among the chicken Eimeria. The reader will note that we extensively use the landmark research of E. E. Tyzzer (11,12) as the basis for our descriptions of the histopathology of these important parasites. His seminal research on the Eimeria species of chickens remains cogent and has influenced researchers in the field for nearly a century.

Until about 20 yr ago, nine species of Eimeria had been recognized as parasites of the domestic fowl (Gallus gallus domesticus). However, with the contributions of Gasser et al. (13) and Vrba et al. (14), specialists in coccidial genetics reduced this number to seven (E. acervulina, E. praecox, E. mitis, E. maxima, E. necatrix, E. brunetti, and E. tenella), emphasizing that the species status of both Eimeria mivati and Eimeria hagani remained questionable. Since current research supports these findings, they continue to be classified as nomina dubia (15). Consequently, they will not be considered further here.

Recently, three operational taxonomic units (OTUs) of Eimeria were identified from field samples originating on commercial broiler farms in Australia and Africa (10,16). Subsequent research on these OTUs, followed by confirmation of their global distribution (15,17,18) indicate that these OTUs are to be considered new Eimeria species (15). The names applied to these new Eimeria are E. lata, E. nagambie, and E. zaria (15). Thus, at this juncture, 10 species of Eimeria are known to parasitize the chicken. Brief comments on the pathogenicity of these new coccidia and what is known of their endogenous development are presented below.

Recent surveys (19,20) indicate that E. acervulina is likely the most common of the chicken Eimeria, a fact that has changed little since estimates of its distribution were first reported (21). Although early studies did not illustrate or emphasize the pathogenic nature of E. acervulina (11), research in the last half century has proven the negative effects of this species on broiler performance and nutrient uptake. As the primary coccidial parasite of the duodenum, E. acervulina poses a significant threat to optimal broiler performance because of its effects on nutrient absorption and utilization. Studies have shown that the digestibility of amino acids, fats, minerals, vitamins, and xanthophylls is impaired during E. acervulina infections (22,23,24,25,26). Because intestinal hemorrhage and mortality are not clinical signs associated with E. acervulina, infections with this species are often overlooked, and subsequently recognized when feed conversion and economic return are calculated. It is clearly the most insidious of the chicken Eimeria.

Eimeria acervulina is a parasite of the upper intestinal tract (Fig. 1Ai), and its primary site of infection is the proximal section of the duodenum. In severe infections, parasites can be found in the midgut and on occasion in the lower digestive tract. Following oocyst exposure, sporozoites are found in the duodenal crypts, followed soon thereafter by four generations of schizogonous development (27). Generation I and II schizonts are located in cells of the crypts and in subsequent generations in enterocytes at the villar base (generation III) and then on the sides and tips of duodenal villi (generation IV). Schizonts III and IV are found apical to the host cell nucleus. All schizonts are small, with the generation I schizont being the largest at about 10 µm. According to Vetterling and Doran (27), none contain more than 32 merozoites.

Macrogamonts are approximately 14.5 × 19 µm and microgamonts about 7 × 8 µm and are always found apical to host cell nucleus, near the brush border. Developing oocysts are of similar dimensions to macrogamonts and occur apical to the nucleus. There are no subepithelial forms of the species.

Tyzzer’s description of the species (11) emphasizes that E. acervulina develops in “colonies,” that is, where clusters of parasitized cells, each containing one or more developing parasite, are found in duodenal segments (Fig. 1Ai, 1Aii). The epithelium in these cases is nearly completely covered by cells containing gamonts and oocysts, an occurrence associated with the nutritional impairments noted above.

Eimeria acervulina also influences the rate at which enterocytes are replaced along the sides of the villus. Studies by Fernando and McCraw (28) indicated that duodenal cell turnover was more rapid in infected birds than controls, a fact that produced immature cells lining the villi. Their studies also demonstrated that E. acervulina influenced the structure of the villus and crypts, with villar truncation and deepening of the crypts a common finding in infected birds. Severe infections produced denuding of the duodenal epithelium (28). Pathogenic and nutritional effects are directly related to the intensity of the infection (11,26).

Tyzzer’s original description of the parasite (11) clearly emphasizes its pathogenic effects in the midgut. His report noted that the species is less prolific but likely more detrimental to the host than similar infections with E. acervulina or E. mitis. Although Tyzzer’s report (11) described the gross appearance of orange mucoid exudates, swelling, inflammation, and ballooning of the midgut, subsequent studies by Long (29), Stephens et al. (30), and Idris et al. (31) showed that microscopic features of sloughed jejunal mucosa and villus atrophy were important outcomes of E. maxima infections. The studies of Idris et al. (31) indicated that villus height is routinely reduced by 50% compared to controls. Schneiders et al. (32) described the effects of E. maxima on the desmosomes of intestinal enterocytes and related these effects to cellar sloughing and increased porosity of the intestinal mucosa. Similar studies have shown that brush border nutrient transporters are also affected by E. maxima infections (33). Obviously, these effects bring about changes in nutrient uptake and performance, while increasing the potential for necrotic enteritis (34) and other bacterial complications (32). It is issues of this nature that make E. maxima a source of primary concern in modern broiler production.

Sporozoite invasion and development of schizont I take place in the jejunal crypts. However, the subsequent asexual stages that develop in the jejunal epithelium have been challenging to describe, and uncertainty exists today on the number and location of these asexual stages. Investigators have estimated that three (29,35), four (36), or as many as five (37) stages of merogony occur. From review of these publications, it seems clear that first- and second-generation schizonts develop in the crypts and at the tips of jejunal villi, respectively. However, schizonts III and IV vary greatly in size and shape and are therefore difficult to separate visually (37). Dubey and Jenkins (37) also described a fifth-generation schizont located in the lamina propria. Since this form occurred simultaneously with both micro- and macrogamonts, questions of its function were posed (37). A plausible explanation of this observation was offered by Schneiders et al. (32), who illustrated a biphasic increase in jejunal E. maxima DNA during infection that was followed by a corresponding pattern of oocyst passage occurring at day 7 and again at days 9 and 10 postinfection. Although the question of numbers of asexual generations in E. maxima awaits confirmation, it seems that resolution of this long-standing issue is likely.

Irrespective of the uncertainties described above, all schizogonous stages are small (on average 7.8 × 6.0 µm), containing no more than 16 merozoites per schizont. Although schizont I parasitizes the crypts, subsequent generations appear in the epithelial layer of the jejunum, usually apical to the host cell nucleus (37). Tyzzer (11) noted that these schizonts were smaller and more difficult to identify than those of E. acervulina. None of the authors cited above could attribute meaningful pathological changes to the development of these schizonts.

There is little doubt that the pathological effects of E. maxima are associated with the large sexual forms of the species. As asexual development progresses toward the production of micro- and macrogamonts, the location of parasite-laden enterocyte changes from the epithelial surface to the lamina propria. Accompanying this change is a notable increase in the size of both male and female gamonts, with mature gametocytes achieving a length of about 35 µm. Tyzzer (11) noted that the mature microgameteocyte of E. maxima usually exceeds the length of the E. maxima oocyst (30.5 µm on average), which is generally recognized as the largest of the chicken Eimeria. He suggested this measurement as a point of species differentiation.

Since microgameteocytes are highly basophilic and macrogametes eosinophilic upon staining with hematoxylin and eosin, they can be identified readily in the lamina propria of midintestinal villi (Fig. 1B). Thus, size and location of macrogamonts, microgamonts, and oocysts is the simplest means of species identification.

Eimeria tenella produces hemorrhages in the ceca that are readily identified by the passage of blood in the feces. Observations of this type are often followed by morbidity and mortality, usually about a day or two after fecal blood is first observed. Postmortem examination reveals ceca containing frank blood with petechia and hemorrhages in the cecal walls. It is often possible to see colonies of second-generation schizonts appearing as small white opacities on the serosal surface of the ceca. In the latter phases of infection, sloughing of the cecal mucosa occurs and is usually followed by the formation of cecal cores. Often the cecal walls become thickened, usually because of lymphocytic infiltration and the development of scar tissue. In chronic cases, oocysts entrapped within an inspissated core of luminal debris may provide the last evidence of a past infection. Under conditions where severe infection occurs, or when infections are complicated by secondary anaerobic bacterial infections, morbidity, mortality and lesion scores have been shown to increase (38). In addition, correlations between the severity of E. tenella infections and bacterial diversity of the cecum are now well established and demonstrate that taxa belonging to the order Enterobacteriaceae increase with the severity of the E. tenella infection (39).

It should be clear that E. tenella develops deep within the tissues of the ceca. In fact, soon after ingestion of oocysts, sporozoites have infected cells of the cecal crypts. First-generation schizonts develop in cells of the crypts soon thereafter and reach maturation about 2 to 3 days postinfection. These first-generation schizonts contain many first-generation merozoites (Tyzzer’s estimate was up to 900), which upon release invade other cells in the crypts. The invaded crypt cells, now containing developing second-generation schizonts, increase in size, become rounded, and no longer form a continuous layer. These cells then actively migrate to the surrounding connective tissue and in heavy infections occupy much of the lamina propria (11,40). The second-generation schizonts then increase in size (Fig. 1Di), often achieving dimensions of 50 × 40 µm or more, and may contain as many as 350 second-generation merozoites (11). Given their size and number, it should be clear that the hemorrhage and tissue destruction that typifies E. tenella infection are associated with the development and maturation of the second-generation schizont. Although a third asexual generation occurs, the number of schizonts produced is quite small. Tyzzer (11) did not attribute meaningful pathological changes to the development of this form, but it has been shown to be an essential segment of the life cycle (41). Extensive lymphocytic infiltration of the ceca accompanies the process of asexual development.

Micro- and macrogamonts occur in the cells of the cecal glands during the sixth day of infection (Fig. 1Dii); oocysts are released following their maturation, usually about 7 days postinfection. The numbers of merozoites produced during schizogony I and II should be an indication of the biotic potential of E. tenella. It is a highly prolific species.

Johnson’s original descriptions of E. necatrix (39) were brief, so much so that Tyzzer et al. (12) provided the key features of its life history as well as a reliable description of the pathological effects of E. necatrix. As clearly pointed out in the later reference, infection with E. necatrix greatly resembles infection with E. tenella, with parasite morphology, reactions of the host, and the hemorrhage produced all similar to those of E. tenella (12). Of course, a key difference is that the mid- small intestine is the primary site of pathological involvement. Like E. tenella, weight loss, morbidity, and mortality are common effects produced by E. necatrix, but it is the profuse intestinal hemorrhage that separates this species from other Eimeria of the chicken.

Eimeria necatrix develops deep within the tissues of the midgut, and soon after oocyst ingestion, sporozoites have entered cells of the crypts of Lieberkühn. The first two generations of schizonts develop in the crypts, and, as with E. tenella, the crypt cells containing second-generation schizonts then migrate to the adjacent lamina propria of the midgut (40). This schizont then develops into an especially large form that is readily visible even at low magnification (Fig. 1Ci). Johnson (42) reported that this schizont can have dimensions of 63 × 85 µm, clearly the largest schizont of the chicken Eimeria. Tyzzer et al. (12) noted that the colonies associated with E. necatrix infections consist of groups of these schizonts (Fig. 1Ci), often appearing as white opacities on the serosal surface of the midgut.

Eimeria necatrix is unique because further asexual multiplication and gametogenesis take place in the ceca (Fig. 1Cii). Thus, merozoites produced by second-generation meronts do not parasitize available cells in the midgut but seek out epithelial cells in the cecum for further replication. Generation III schizonts are small and contain few merozoites, causing little damage to the cecal epithelium. Unlike E. tenella, the number of oocysts produced by E. necatrix is comparatively small (12).

Hemorrhagic enteritis is a prominent sign of E. necatrix infections; it is often accompanied by ballooned intestines and a thickened mucosal surface. According to Stockdale and Fernando (43), hemorrhage occurs just prior to and during the development of the second-generation schizont. A rapid increase in mortality may also occur at this point (44). Lymphocytic infiltration of the midgut is also significant.

Since there are no gross lesions that are consistently produced by E. mitis (11,45), it often goes unrecognized when gross examinations and bird health assessments are carried out. However, E. mitis has been identified frequently in commercial operations when PCR is employed (19,46). Since the parasite has been shown to affect production performance (45,47), it seems likely that E. mitis exerts an effect on productivity that is not fully recognized by routine surveillance.

Studies by Novilla et al. (45) demonstrated distinct villar damage, cellular sloughing, and reduced villar length. These changes bring about a reduction in the absorptive surface of the mid-intestine and, as expected, the impaired performance responses noted by several investigators. There is no evidence that E. mitis affects mortality.

Novilla et al. (45) indicated that the ileum is the preferred site of infection. Consistent with Tyzzer’s original descriptions (11), Novilla et al. (45) showed that some development took place throughout the intestinal tract, with cells of the jejunum, ceca, rectum, and bursa of Fabricius harboring parasites. In the studies of Novilla et al. (45), a heavily parasitized group also showed some development in the duodenal loop. These authors described four asexual generations of E. mitis, with the first two generations occurring at the base of the villi or in the crypts. Subsequent asexual development occurred in enterocytes of the upper half of the villus. Changes in villar structure and length noted above have been associated with the development of these asexual forms (45).

Tyzzer (11) indicated that E. mitis often occurs in individual enterocytes scattered throughout the epithelium (Fig. 1Gi, 1Gii); there is no evidence of E. mitis forming colonies of parasitized cells. In Tyzzer’s diagrammatic representation of the species (11), schizonts and gamonts developed in close proximity to the host cell nucleus, with developmental stages appearing slightly above or slightly below the nucleus.

Although E. praecox is generally considered a mildly pathogenic species with few gross lesions produced, several authors have reported the upper intestinal tract becomes pale, and pigment loss is evident (48,49). Clear mucoid exudates appear during the early phases of development. The effects of E. praecox infections on growth and feed conversion in broilers have been shown to be variable, with some investigators reporting few, and others demonstrating marked changes in performance (49,50,51,52,53). Allen and Jenkins (49) suggested that differences among E. praecox strains likely accounted for this variation. Their studies (49) indicated that under challenge, bird performance and pigmentation were adversely affected, and plasma levels of NO₂^- and NO₃^-, markers for intestinal inflammation, appeared during the early stages of parasite development. Reperant et al. (53) indicated that infections with E. praecox worsened the effects of E. acervulina infection on performance.

Eimeria praecox is a parasite of the upper digestive tract and is commonly identified in duodenal sections. The crypts are not parasitized by E. praecox (48). There are four schizogonous generations (48,54), and as described by Tyzzer et al. (12) and Salish (48), schizonts and subsequent sexual stages occur below the host cell nucleus (Fig. 1E). Table 1 shows that the size of the schizonts is generally larger than those of E. acervulina. Since all developmental stages are confined to the epithelium (48), and colony formation does not occur (12), extensive tissue damage is not common (12,48,55).

Despite its original description in 1942, the first histological assessments of the species were reported some years later by Boles and Becker (56). Supportive studies by Pout (57), Ryley et al. (58), and Hein (59) added important details to the original studies of histopathology. The workers above showed that first-generation schizonts of E. brunetti develop throughout the intestinal tract and may even develop in the upper intestine and the duodenal loop. Although a few second-generation meronts may develop in the region around the yolk stalk diverticulum, E. brunetti is largely a parasite of the lower intestinal tract. Infections are normally distal to Meckel’s diverticulum, often extending into the necks of the ceca and the rectum. The crypts are not parasitized.

According to Boles and Becker (56), the first-generation schizonts are large (20–30 µm) and appear to be the only forms that are found in the upper intestine; subsequent asexual stages, gamonts, and oocysts develop distal to Meckel’s diverticulum. Irrespective of location, the large first-generation schizont is found below the host cell nucleus, close to the basement membrane. In the lower tract, second-generation schizonts are not as large as the first (maximal size is about 20 µm) and are commonly found in epithelial cells near the tips of the villi. In heavy infections they extend down the sides of each villus. Boles and Becker (56) reported that these parasites are not confined to specific regions of the enterocyte but are scattered throughout the epithelial cell. The investigations of Kawahara et al. (60) indicate that schizonts, gamonts, and oocysts develop in both epithelial cells and in the lamina propria of the lower intestine, cecal necks, and rectum (Fig. 1Fi, 1Fii).

Levine (61) and Ryley et al. (58) reported that mild infections of E. brunetti produce no gross lesions, but gross intestinal changes become evident as the severity of infection increases. Lesions vary from serosal petechia to redness on the surface of the mucosa to villar truncation leading to exposure of the lamina propria (57,58,59). Severe infections are known to have a profound effect on bird performance (58,59).

It is important to recognize that most pathological changes with E. brunetti occur with the development of schizonts, gamonts, and oocysts, some of which may be in the lamina propria. Normally they are located close to the villar tips. As previous studies have shown (56,57,58,59,60), cellular sloughing, villus atrophy, and denuding of the epithelial surface are common outcomes of E. brunetti infections. It is these changes to the intestine that account for most of the losses in production performance associated with this species.

As mentioned above, three new operational taxonomic units (OTUs) of Eimeria were recognized in chickens and are now considered new species (E. lata, E. nagambie, and E. zaria). Initially, these organisms were found in chickens reared on farms only in the Southern Hemisphere (62), but current reports indicate that they have been detected in many poultry-producing countries (15,18,19). Apparently, these species were first identified in commercial operations where live coccidiosis vaccines were frequently used (6,16). Producers noted consistent coccidiosis problems during vaccine usage, and further investigations led to the identification of these OTUs as the cause of the problems. Data now show that each species is immunologically and genetically distinct from the seven established Eimeria described above. Recent data also show that each has the potential to affect performance and productivity (15). Since current live coccidiosis vaccines do not contain these species, live vaccination offers no protection from these coccidia (15).

At this juncture, a paucity of data exists on the natural history of E. lata and E. nagambie. Data that are available have been summarized by Blake et al. (15), with emphasis placed on standardized parasitological differences from the seven established chicken Eimeria, their genetic and immunological uniqueness, and their effects on broiler performance. Details of their endogenous development and histopathology have not been addressed (D. Blake, pers. comm.). It is hoped that future work on these parasites will better characterize their life cycles and pathological effects in the chicken.

Preliminary work on E. zaria has been carried out (15) and shows that the parasite is found in the duodenum and upper ileum. Its oocysts are small, displaying similarities to those of E. mitis (15). However, the oocyst index for E. zaria is 1.17, compared to 1.0 for E. mitis. It appears that all developmental stages occur in the duodenal/ileal epithelium (Fig. 2); subepithelial stages have not been identified. In preliminary assessments of its pathogenicity, Blake et al. (15) reported that doses of 5000 and 100,000 oocysts reduced body weight gain by 16% and 31%, respectively. Gross pathology was considered mild with watery intestinal contents (S. Spiro, pers. comm.). Gross intestinal lesions do not occur, and initial results have shown no evidence of intestinal inflammation, villar blunting, or other adverse host responses (15).

Unlike coccidia parasitizing some other hosts, Eimeria infections in the chicken are confined to the digestive tract, with developmental forms occurring in the cells of the absorptive epithelial surface, the crypts of Lieberkühn, or the lamina propria. Additionally, Eimeria of the chicken display site specificity in the intestinal tract that serves as a guidepost for accurate diagnosis (3,11). As shown in Table 1, E. acervulina is primarily a parasite of the duodenum, E. maxima of the jejunum, E. tenella of the ceca, etc., but some overlap in location exists for each species. Nonetheless, location of the infection is an important criterion in determination of the infective species.

The concept of site specificity can be extended to intracellular sites of development as well. Numerous authors have shown that certain Eimeria species prefer specific locations within the enterocyte for the development of various endogenous stages (Fig. 1). For example, the gamonts and oocysts of E. acervulina are most often found apical to the host cell nucleus in duodenal epithelial cells. Eimeria praecox, the other common coccidial parasite of the duodenum, develops below the nucleus, close to the basement membrane. However, as Eimeria develop within the epithelial cell, schizonts and gamonts increase in size. The result is that the nucleus may be displaced within the cell, making its use as a “landmark” more difficult. Even so, predilection for sites within the parasitized enterocyte are well documented for E. acervulina, E. praecox, schizont I of E. brunetti, and, to a certain extent, E. mitis. These locations should be used to facilitate speciation where possible.

Eimeria acervulina, E. maxima, E. tenella, and E. necatrix are very likely to be encountered in routine histopathological assessments. Fortunately, these four species are histopathologically distinct so by combining an assessment of site of infection, the type and location of specific endogenous stages, and the presence or absence of hemorrhage (Fig. 1; Table 1), separation of these species can be ensured. As noted above, key to this separation are the different areas of the intestinal tract that are parasitized and the stages of parasite development within them. Since E. maxima and E. necatrix are both jejunal parasites, the key identifiers for E. maxima are sexual stages and oocysts in the lamina propria of the villi, whereas the large schizonts of E. necatrix are found at the base of the villi close to the muscularis. Additionally, since gametogenesis and oocyst production for E. necatrix occur in the ceca, oocysts identified in tissues of the midgut are those of another species.

Since both E. acervulina and E. praecox are parasites of the duodenum, separation of these species may be more challenging. However, differences in colony formation and developmental site within the duodenal enterocyte are usually sufficient to separate these species (E. acervulina forms colonies and is found apical to the host cell nucleus). In distal sections of the gut, E. brunetti parasitizes the lamina propria of lower intestine, cecal necks, and rectum. Eimeria mitis, the other common parasite of the lower intestine, is confined to the epithelial surface.

On occasion, both E. praecox and E. mitis may be found in the duodenum, and since each is confined to epithelial cell development (lack subepithelial forms), speciation may be especially difficult. According to Novilla et al. (45), E. mitis is normally a parasite of the lower gut and develops in the duodenum only during extremely heavy infections. Therefore, examination of the rest of the digestive tract for E. mitis will be informative. McDougald and Fitz-Coy (63) also suggest determination of oocyst index (oocyst length/oocyst width) for effective separation of these species. Because the oocysts of E. mitis are round, they have an index that is close to 1.0, a detail that is different from E. praecox, whose index is about 1.25. McDougald and Fitz-Coy (63) discussed other methods such as determination of prepatent period that may be needed for conclusive separation of these species. Obviously, these techniques go beyond routine histopathological evaluation.

Coccidial researchers have long discussed the factors associated with pathogenicity of the Eimeria of the chicken. As with many other pathogens, some strains of the same Eimeria species exhibit differences in the ability to infect tissue and produce disease. Especially for the highly pathogenic species, differences in pathogenicity are likely related to variation in the number, size, and/or location of one more of the endogenous stages of development. As has been shown repeatedly when field strains are compared to attenuated strains of Eimeria, lower pathogenicity is associated with reductions in the number and/or size of specific endogenous stages (64).

Because coccidial infections are self-limiting, the severity of infection is directly correlated to the numbers of oocysts ingested. Obviously, the effects of increased oocyst consumption are most evident when evaluating the effects of the highly pathogenic species, with greater production losses and mortality usually occurring at elevated exposure levels. Similar trends occur with the milder pathogens (E. acervulina, E. praecox, and E. mitis), but production losses are usually not as severe. In this same vein, it is common for studies involving Eimeria to employ treatments where graded levels of oocysts are administered in order to determine the level at which specific pathological effects occur. Similarly, oocyst passage (production) is also correlated to the numbers of sporulated oocysts consumed. Exceptions are usually noted in heavy infections where the number of intestinal cells available for coccidial replication is limited (a phenomenon known as the “crowding effect”).

There is little doubt that of the seven commonly encountered species, four have the ability to penetrate deep within the intestinal tissues and bring about alterations in tissue structure that impair growth and/or result in hemorrhage. As previously described, E. maxima, E. necatrix, E. tenella, and E. brunetti have stages that develop below the epithelial surface, and in the case of E. maxima, E. necatrix, and E. tenella, the forms that develop are of sufficient size to affect the structure of host tissue. The hemorrhage produced during E. necatrix and E. tenella infections is the result of both the size and number of their respective schizonts and the host’s response to the presence of these stages. Oxidative stress, free radical production, and NO-induced vasodilation occur as a consequence of the host’s recognition of these forms. Each is a known contributor to the tissue damage and hemorrhage that are the obvious signs of infection (65,66). Similar host responses occur during the development of the large sexual stages of E. maxima, although the effects produced may not be of the same magnitude as those recorded for E. tenella (66). Although E. brunetti is known to infect subepithelial tissue and cause villus atrophy and denuding of the lower intestine (57,60), we are not aware of research that depicts the host’s cellular responses to E. brunetti that induce the pathological effects noted above. Regardless, it seems likely that similarities would occur.

Notably, E. tenella and E. necatrix are known to affect the integrity of the basement membrane. Tyzzer (11,12) observed that the crypt cells parasitized by second-generation schizonts of E. necatrix and E. tenella changed in structure and appearance. These modifications were then followed by their separation from other non-infected crypt cells leading to their migration from the crypt to the adjacent lamina propria. In this process, the infected crypt cells pass through gaps in the basement membrane (40), facilitating or allowing their movement to the adjacent reticular tissue. Fernando et al. (40) speculated that the breakdown of the basement membrane was parasite induced primarily because of the short duration required for its degradation (2–4 hr). Despite this finding, they were unable to define a mechanism by which this process occurred, although they suggested that parasite produced enzymes could be involved. We are not aware of further research to support or refute this contention, but establishing its accuracy and/or separating it from a host-induced response would be of great interest.

Tyzzer (11) observed similar processes occurred in birds infected with E. maxima, but his descriptions are not nearly as detailed as they are with E. tenella and E. necatrix. He reported that enterocytes parasitized by E. maxima gametocytes become rounded, and the entire cell is then displaced into the lamina propria. No additional comments were made, and to our knowledge, no supplemental studies dealing with the integrity of the basement membrane have been carried out during E. maxima infections. It would be interesting to determine whether the E. maxima–induced changes to the jejunal desmosomes reported by Schneiders et al. (32) might allow gamont-parasitized enterocytes access to the lamina propria, producing responses similar to those recorded for the hemorrhagic species. Additional work on these effects is clearly warranted.

For the parasites of the epithelial surface, E. acervulina, E. praecox, and E. mitis, tissue involvement is much more limited, with loss of large sections of the absorptive surface (through villar truncation and/or denuding) occurring only in the most severe infections. As described previously, colony formation associated with E. acervulina infections often brings about changes in nutrient digestibility that affect growth, feed conversion, and carcass yield. Overall, however, infections with these species are milder than those where tissue involvement is common.

The key histopathological features of seven established Eimeria of chickens are described and compared in this review. Three new Eimeria species have been detected in many poultry-producing countries, and with the possible exception of E. zaria, there is insufficient information at this time to fully characterize their endogenous development.

The salient points of histopathological identification for the traditional Eimeria are the following:

E. acervulina. All life cycle stages occur in the epithelium of the duodenum. Development of meronts and gamonts occurs apical to the host cell nucleus close to the brush border. “Colonies” (clusters) of numerous parasitized enterocytes containing gamonts and oocysts occur in the duodenum.

E. maxima. Large gameteocytes and oocysts develop in the lamina propria of the villi of the midgut. Schizonts of E. maxima are very small and located near the brush border of enterocytes lining jejunal villi. Schizonts are difficult to identify because of their small size and variable shape.

E. tenella. Very large second-generation schizonts form colonies in the lamina propria of the ceca. The hemorrhage produced by these forms can be extensive and may be followed by the appearance of cecal cores. Oocyst production occurs in the cecal glands.

E. necatrix. Very large second-generation schizonts develop in the lamina propria of the mid gut. “Colonies” (clusters) of these schizonts develop close to the muscularis and create small white opacities that are visible on the serosa. Hemorrhage occurs during the development of the second-generation schizont. Oocysts develop in the cecal epithelium.

E. praecox. Primary site of development is the duodenum and is usually confined to the mid- and upper portions of the villus. Asexual development and gametogenesis take place below the level of the host cell nucleus. There are no subepithelial stages, and colonies do not occur. The crypts of Lieberkühn are not parasitized.

E. mitis. Developmental stages occur primarily in the ileum. Parasites develop at or below the level of the host cell nucleus (in the center of the enterocyte). All life cycle stages occur in enterocytes; colonies do not occur, and parasites are normally scattered throughout the ileal epithelium.

E. brunetti. Developing first-generation schizonts can be found throughout the intestine; these forms are large (about 30 µm) and located near the basement membrane of the enterocyte. Second-generation schizonts are smaller (20 µm) and are located in the lower intestinal tract, the rectum, and the necks of the ceca. Gamonts and oocysts develop in the lamina propria of these same regions. In the lower intestine, damage to the villar tips and villus atrophy are associated with the development of second-generation schizonts, gamonts, and oocysts. Cells of the crypts of Lieberkühn are not parasitized by E. brunetti.

The authors are grateful to Dr. Damer P. Blake of the Royal Veterinary College, London, UK for commenting on the manuscript. We also appreciate the assistance of Phibro Animal Health Corporation for supporting the Open Access format for this paper.

OTU =

operational taxonomic unit;

PCR =

polymerase chain reaction;

USD =

United States dollars