Pathology and pathogenesis of disseminated visceral coccidiosis in cranes

Abstract

Disseminated visceral coccidiosis (DVC) caused by Eimeria spp. was recognized as a disease entity in captive sandhill cranes (Grus canadensis) and whooping cranes (Grus americana) in the late 1970s. While most avian species of Eimeria inhabit the intestinal tract of its host, the crane eimerians, Eimeria reichenowi and Eimeria gruis, invade and multiply systemically and complete their development in both digestive and respiratory tracts. In DVC, cranes, especially chicks, may succumb to acute infections resulting in hepatitis, bronchopneumonia, myocarditis, splenitis, and enteritis. Cranes may also develop chronic, subclinical infections characterized by granulomatous nodules in various organs and tissues.

This paper reviews the pathology and pathogenicity of natural and experimental DVC in sandhill and whooping cranes. Naturally infected birds appeared clinically normal, but progressive weakness, emaciation, greenish diarrhea, and recumbency before death were observed in birds administered doses ≥10×10³ sporulated oocysts per os. At necropsy, naturally infected birds had nodules in the mucosa of the oral cavity and the esophagus, and in thoracic and abdominal viscera. Experimentally infected birds necropsied less than 7 days after infection (a.i.) had no gross lesions. Birds examined later had hepatosplenomegaly, liver mottling, lung congestion and consolidation with frothy fluid in airways, and turgid intestinal tracts with hyperemic mucosa. From 28 days a.i., grossly visible granulomatous nodules were seen in the esophagus, heart, liver, cloaca, and eyelids. By light microscopy, the basic host response was a granulomatous inflammation with non-suppurative vasculitis affecting many organs and tissues. With time, multifocal aggregates of mononuclear cells, many laden with asexual coccidial stages, increased in size and number. Widespread merogony resulted in morbidity and death, particularly in birds administered 20×10³ sporulated oocysts. Ultrastructural examination revealed developing asexual coccidian stages in the cytoplasm of large lymphocytes or monocytes within a parasitophorous vacuole, often indenting the nucleus. Oocysts and gametocytes were found in the intestines by 12 days a.i., and in the esophagus, trachea, bronchi, and lung by 14 days a.i., indicating that crane eimerians can complete their life cycle at these sites. Thus, DVC in cranes could be a useful animal model for the study of eimerian extra-intestinal stages and the evaluation of potential systemic anticoccidial drugs.

Introduction

The sandhill crane (Grus canadensis) and the endangered whooping crane (Grus americana) are the only cranes native to North America. The whooping crane is symbolic of efforts to conserve endangered species by the Patuxent Wildlife Research Center (PWRC) (Laurel, MD, USA). Sandhill cranes have been used as surrogate research subjects at PWRC to yield techniques used in the captive propagation program of the whooping crane. As a result of captive propagation and protection of cranes and their habitat, the whooping crane population increased from a low of 21 birds in 1941 to about 450 wild or captive birds in late 2003.

According to Levine (1973), (1982), coccidia are parasitic protozoan parasites of the family Eimeriidae, suborder Eimeriorina in the phylum Apicomplexa, especially species in the genera Eimeria and Isospora. Coccidiosis, an economically important disease characterized by morbidity, mortality and reduced production efficiency of affected poultry and other livestock, is caused primarily by Eimeria spp. (Long, 1973). Eimeria spp. of birds and mammals are known to have strict host, tissue, and cell specificity. In nature, eimerian infections occur by hosts ingesting sporulated oocysts that contain four sporocysts, each containing two sporozoites. Released in the intestinal lumen by digestive processes, sporozoites penetrate epithelial cells and become established at preferred sites. The sporozoites then divide during an asexual cycle referred to as schizogony (merogony) in which merozoites are produced in a set number of generations by sequentially infected cells that rupture and die. The final merogonic stage gives rise to micro-gametocytes and macro-gametocytes, which fuse to form the zygote (gametogony or sexual phase). An oocyst wall forms around the zygote as it develops and matures. The oocysts are released into the intestinal lumen and passed with the feces onto the ground where they sporulate under favorable conditions of moisture and temperature. Ingestion of a few sporulated oocysts results in a mild or subclinical disease (also called coccidiasis). Severe disease occurs following the ingestion of large numbers of oocysts since there is greater tissue damage by multiplying life cycle stages in the intestine. Consequently, anorexia, digestive disturbances, dehydration and blood loss, as well as increased susceptibility to other disease agents, occur in coccidiosis.

Eimeria reichenowi and Eimeria gruis are common parasites of wild whooping cranes and six subspecies of sandhill cranes (Courtney et al., 1975; Forrester et al., 1978). Parker & Duzynski (1986) described a new species, Eimeria bosquei, found in two of 212 fecal samples from sandhill cranes wintering in New Mexico, USA. Although in most species of mammals and birds Eimeria spp. are generally localized in the intestinal tract (Pellerdy, 1974), a systemic infection resulting in disseminated visceral coccidiosis (DVC) occurs in cranes (Novilla et al., 1981). This disease entity was first recognized at PWRC in 1977/1978 (Carpenter et al., 1979). In DVC, granulomatous nodules may form in many tissues and organs or may result in hepatitis, bronchopneumonia, myocarditis, splenitis, and enteritis (Carpenter et al., 1980, 1984).

The purpose of this paper is to review the pathology and pathogenesis of natural and experimental DVC in sandhill and whooping cranes.

Clinical and Parasitologic Findings

Except for the presence of small white raised nodules in the oral cavity noted during routine clinical inspection, there were no physical signs of DVC in naturally infected adult sandhill cranes (Carpenter et al., 1979, 1984; Novilla et al., 1981, 1989). The clinical presentation of the single naturally infected whooping crane was lethargy and severe diarrhea. Two whooping crane chicks were found dead without any premonitory signs and the other chick was submitted for necropsy in a moribund state (Carpenter et al., 1980). Greater sandhill crane (Grus canadensis tabida) chicks experimentally infected with 10×10³ to 65×10³ pooled sporulated oocysts of E. reichenowi and E. gruis or E. reichenowi alone manifested progressive weakness, emaciation, greenish diarrhea, dyspnea, and recumbency before death 8 to 10 days after infection (a.i.) (Carpenter et al., 1984; Novilla et al., 1981, 1989). A dose of 20×10³ oocysts produced illness and deaths in all the unmedicated controls and amprolium-medicated and clazuril-medicated Florida sandhill cranes by 10 days a.i. (Carpenter et al., 2003). In contrast, no clinical signs or lethality was recorded during a 14-day observation period of two Florida sandhill cranes (Grus canadensis pratensis) administered 25×10³ E. gruis oocysts per os (Augustine et al., 1998). One of two birds similarly dosed with E. reichenowi died without premonitory signs at 20 days a.i. and the other bird was observed to be listless the day before recumbency and death at 23 days a.i. (Augustine et al., 2001).

Sampling of feces from naturally infected greater and lesser (Grus canadensis canadensis) sandhills yielded E. reichenowi and E. gruis oocysts (Novilla et al., 1981) and a new species, E. bosquei (Parker & Duzynski, 1986). Whooping crane fecal samples yielded only E. reichenowi-type oocysts. In 7-day-old experimentally infected greater sandhill cranes, fecal passage of unsporulated oocysts was observed from the 12th day a.i. (Carpenter et al., 1984). Augustine et al. (1998), (2001) demonstrated sporozoites of E. reichenowi and E. gruis as early as 6 h a.i. in the distal jejunum by immunofluorescent techniques using monoclonal antibodies elicited against Eimeria spp. of chicken and turkey that cross-reacted with developing stages of the crane eimerians. Trophozoites or young schizonts were observed in Giemsa-stained tissue smears from 5 days a.i. and were found primarily in the intestines as well as the liver, spleen, and lungs (Novilla et al., 1989). Over the next 5 days, multifocal aggregates, as well as transmural and perivascular infiltrates, of mononuclear cells (MNC) contained developing meronts. Although widespread merogony occurred from 8 to 10 days a.i., the epithelial lining cells of intestinal villi were not infected until 9 days a.i. The prepatent period was 12 days a.i., and asexual and sexual stages with formation of oocysts were prominent by 14 days a.i. In chicks given 5×10³ and 20×10³ oocysts, the entire digestive tract (from the oral cavity to the cloaca) was infected.

Sporozoites or merozoites were infrequently observed in peripheral blood smears from experimentally infected birds and were not seen in naturally infected adult cranes. This is probably because of the low level of exposure and the transitory nature of the parasitemia. Positive blood smears, with large merozoites in the cytoplasm of monocytes and/or large lymphocytes, were observed from 9 days a.i., consistent with the widespread merogony at this stage of DVC (Novilla et al., 1989).

Gross Pathologic Findings

At necropsy of naturally infected adult sandhill cranes, parasite granulomas were the predominant gross lesion (Carpenter et al., 1979, 1984; Parker & Duzynski, 1986). Carpenter et al. (1979) first observed the granulomas in the oral cavity as grayish-white nodules, 0.5 to 3.0 mm in diameter. Similar nodules were disseminated throughout many tissues and organs, including: the surface and parenchyma of the liver, lung, spleen, kidneys, heart; adventitia of the blood vessels, including the carotid and femoral arteries; serosa and mucosa of the esophagus, proventriculus, gizzard, intestines, and cloaca; the mesentery and parietal peritoneum; submucosa of the trachea and main stem bronchi; epimysium and parenchyma of pectoral and cervical muscles; and subcutaneous tissues of the thoracic and cervical regions. The number of nodules varied; one bird had less than nine; three birds had 10 to 49; and two birds had more than 50. Greater numbers of nodules were found on the oral mucosa and liver in a later survey (Parker & Duzynski, 1986).

The adult whooping crane at PWRC that was examined was thin and dehydrated. At necropsy, urate deposits on the heart and kidneys, mottling of the liver and congested spleen and intestines were observed together with nematodes in the lumen of the intestines and gizzard. Two of three, 13-day-old to 18-day-old whooping crane chicks, were found dead and the other chick was submitted in extremis. Necropsy findings from all three chicks were mottling of the liver with 1 mm white spots on the surface and parenchyma, slight congestion and enlargement of the spleen, and hyperemia of the duodenal mucosa with nematodes and acanthocephalans in the intestinal tract. In addition, two chicks had lung consolidation, frothy fluid in the airways, and gapeworms in the trachea and bronchi, and one chick had 1 to 2 mm whitish spots on the ventricular myocardium (Carpenter et al., 1980; Novilla et al., 1981, 1989).

As for experimentally infected birds, none of the four sandhill crane chicks exposed to contaminated ground died during a 4-week observation period (Novilla et al., 1981; Carpenter et al., 1984), suggestive of low-level exposure to sporulated oocysts. Two birds orally administered 10×10³ oocysts had congested and edematous lungs, and one had gapeworms in the trachea at necropsy 8 days a.i. Other birds intubated with more pooled sporulated oocysts (from 20×10³ to 65×10³) died or were killed in extremis by 10 days a.i. (Carpenter et al., 1984, 2003; Novilla et al., 1989). Lesions typical of DVC including hyperemia of the intestines, mottled liver and spleen, congested lungs and grayish-white nodules in multiple organs were seen in many birds. Of the birds gavaged with E. reichenowi only, all that were alive past 7 days a.i. were thin; one died 10 days a.i., and three were moribund and euthanized at 9 days a.i. (one bird) and 10 days a.i. (two birds). Necropsy findings included slight hepatosplenomegaly with multiple grayish-white foci and pulmonary congestion and edema (Novilla et al., 1981, 1989).

Light Microscopic Findings

The basic host response to DVC in cranes was granulomatous inflammation with focal aggregates of lymphocytes and macrophages laden with developmental stages in many tissues and organs.

Naturally infected birds

In adult sandhill cranes, disseminated granulomas, focal chronic hepatitis, chronic focal phlebitis, focal myositis, and enteritis were the major lesions (Novilla et al., 1981, 1989; Carpenter et al., 1984). The granulomas were composed of inflammatory cells surrounded by a thin fibrous capsule. The cellular infiltrate included macrophages, lymphocytes, plasma cells, and a few heterophils. Numerous pale basophilic, round to oval bodies 5 to 10 μm in diameter that were uninucleated and multinucleated, were scattered throughout the granulomas. Most of the organisms considered to be developing meronts were in the cytoplasm of macrophages and appeared to have been viable; however, a few had vacuolation of cytoplasm suggestive of degeneration. A few granulomas did not contain meronts. In the liver of some birds, lymphoid aggregates protruded into the lumen of portal and hepatic veins and nearly occluded a few of the smaller hepatic veins. Other veins, including those in the lungs and kidneys and a few small veins near the thyroid, were similarly affected. Lesions in the small intestines were characterized by moderate to severe multifocal infiltration of the lamina propria by macrophages, lymphocytes, and few heterophils with gametogonic stages of coccidia in epithelial lining cells. Developing coccidial stages were also found within macrophages in granulomatous infiltrates and in lymphatics or capillaries in the lamina propria and tunica muscularis.

Histologic changes in major organs of the whooping cranes were similar in all cases (Carpenter et al., 1980; Novilla et al., 1981). There was inflammation and mild focal necrosis of the intestinal villi associated with coccidian oocysts and gametocytes in the epithelium. Asexual stages were in the lamina propria within the cytoplasm of macrophages. In one whooping crane chick, developing schizonts and merozoites were abundant near foci of necrosis in the mucosa, submucosa, and muscularis. Parasitized macrophages aggregated around vessels in the muscularis and serosa of the intestines. Lungs had multifocal areas of consolidation, and parabronchi and bronchioles contained a pleocellular exudate. In one chick, airways contained coccidian oocysts similar to those seen in the gut. Multifocal areas of granulomatous inflammation were seen in the liver, heart, spleen, kidneys, and bursa of Fabricius. In addition, there were extensive areas of necrosis in the liver and spleen. Many macrophages in the granulomatous foci contained intracytoplasmic organisms similar to those seen in the intestines and lungs. Except for nephritis and secondary visceral gout, the lesions in the adult whooping crane were identical to those in the chicks.

Experimentally infected birds

Dose and time factors probably influenced the abundance, distribution, and severity of lesions (Carpenter et al., 1984; Novilla et al., 1989). There were no histologic changes until 5 days a.i. At this time, small focal aggregates of MNC were observed in the parenchyma of major organs. Over the next 5 days, lymphoid aggregates increased in size and number with widespread merogony, morbidity, and death by 9 days a.i. In surviving birds necropsied at 14 days a.i., gamonts and oocysts were found not only in the small intestine, but also in the esophagus, trachea, and lungs. Vasculitis and focal cellular aggregates were consistently observed. Small granulomas, consisting mainly of MNC and associated with veins or lymphatics, were present in all visceral organs, particularly the liver, lung, heart, spleen, kidney, thymus, bursa of Fabricius, and skeletal muscle, and were well-developed by 20 days a.i. Severe necrotizing enteric inflammation extended to the tunica muscularis and serosa. Large numbers of gamonts, oocysts, and meronts, some of which were large, with large merozoites, were present among necrotic intestinal glands. Parasite granulomas were grossly visible at 28 days a.i. and clusters of degenerating gamonts and/or oocysts were found in the lamina propria.

Parasite granulomas were numerous and were the most important lesion observed by 49 days a.i. Granulomas were seen in the oral mucosa, esophagus, proventriculus, and other parts of the alimentary tract, heart, and skeletal muscle. Other areas affected included the palpebral conjunctivae, dermis, cloaca, and the internal nares and turbinates. In contrast to what was seen in cranes necropsied earlier, fewer asexual and sexual stages were found in the intestinal mucosa. However, the MNC infiltration of the mucosal and muscular layers of the intestine was extensive.

Electron Microscopic Findings

Ultrastructural studies showed that the predominant cell type parasitized in DVC was large MNC (Novilla et al., 1989). The MNC resembled large lymphocytes or monocytes with large electron lucent nuclei and peripherally clumped nuclear chromatin. The relatively abundant cytoplasm also contained mitochondria, rough and smooth endoplasmic reticulum, Golgi apparatus, centrioles, polyribosomes, and occasionally lysozomes. The cell nucleus, surrounded by a double membrane with irregular outlines, was often indented by the sporozoan parasite developing within a parasitophorous vacuole. The uninucleated and multinucleated organisms were generally merozoites and meronts (Carpenter et al., 1984; Parker & Duzynski, 1986; Novilla et al., 1989).

Discussion and Conclusions

The basic host response in DVC is granulomatous inflammation. This change is primarily because of the infiltration of phagocytic MNC (large lymphocytes or monocytes) laden with developing, primarily asexual, stages of coccidia in many organs and tissues. The consequent rupture of infected host cells initiates a necrotizing inflammation, principally in the intestines, liver, and spleen. The repeated and widespread merogony is considered directly responsible for the development of clinical disease and mortality of young cranes in captivity and probably in the wild (Novilla et al., 1981, 1989).

In birds surviving the acute and subacute stages of DVC, the granulomatous inflammation leads to the formation of grossly visible nodules as the coccidial infection becomes chronic. The parasite granulomas induced experimentally had similar morphology to those found in naturally infected cranes. In these cranes, focal granulomas and nodules in many tissues were the major lesions (Carpenter et al., 1984; Parker & Duzynski, 1986). In the earlier survey, juvenile birds had a higher prevalence and greater number of granulomas than did the adult cranes. These birds were also reported to have a low intestinal coccidial burden. Except for one whooping crane adult that died in captivity (probably from complications of dehydration, nephritis and visceral gout), no deaths in captive adult cranes have occurred from naturally occurring DVC. Thus, the parasite granulomas may indicate that cranes as they mature are able to tolerate the infection by eimerian coccidia. Since the parasite granulomas contained uninucleated and multinucleated parasites that appeared to have been viable, they may be the source of merozoites that re-infect various organs and tissues of the host, prolonging the eimerian infection (Novilla et al., 1981, 1989).

Severe, widespread infections usually occurred in experimentally infected sandhill crane chicks, moribundity and death coinciding with the peak of merogony (8 to 10 days a.i.). However, recent infectivity studies by Augustine et al. (1998), (2001) with E. reichenowi and E. gruis resulted in light infections, no overt or minimal clinical signs, and delayed deaths at 20 and 23 days a.i. from E. reichenowi infection only. The difference was attributed to the age and storage condition of the oocyst inocula. Although only a few eimerian species affecting birds and mammals are known to have extra-intestinal phases in their life cycles (Table 1), parenteral development may be a common occurrence for the genus Eimeria (Overstreet, 1981; Ball et al., 1989). A widespread, systemic infection by E. reichenowi and E. gruis occurs in captive sandhill and whooping cranes and wild sandhill cranes (Novilla et al., 1981). Infection by either or both species results in DVC, but it is not known whether E. bosquei plays a role in the disease. In crane coccidiosis, oocyst passage occurred by 12 days a.i. and gamonts and oocysts were found in the trachea, bronchi, lungs, esophagus, and intestines by 14 days a.i. (Carpenter et al., 1984; Novilla et al., 1989). Since there were no sexual stages in the intestines until after 9 days a.i., birds that die prior to the prepatent period could present a diagnostic problem. For differential diagnosis, careful consideration of other protozoan infections (i.e. Amoeba, Leucocytozoon, Sarcocystis, and Toxoplasma), granulomatous nodules, and neoplasia is important.

Table 1. Eimerian coccidia that develop in extraintestinal tissues of birds and mammals

Species	Host(s)	Tissue	Reference
Eimeria stiedai	Rabbit	Liver	Pellerdy (1969)
Eimeria tenella	Chicken embryos	Liver	Long (1971)
Eimeria deblieki, Eimeria spp., respectively	Pig, chamois	Liver	Desser (1978)
Eimeri hiepei	Mink	Bile ducts	Ball et al. (1989)
Eimeria adenoides	Turkey	Gall bladder	Critchley et al. (1986)
Eimeria arloingi/Eimeria christenseni/Eimeria crandalis	Goats	Mesenteric lymph node	Lima (1979)
Eimeria arloingi/Eimeria faurei/Eimeria ninakohlyakimovae	Sheep and goats	Mesenteric lymph node	Lotze et al. (1964);
Eimeria neitzi	Impala	Uterus	McCully et al. (1970)
Eimeria gaviae	Common loon	Kidney	Montgomery et al. (1978)
Eimeria truncata	Geese	Kidney	Gajadhar et al. (1983)
Eimeria boschadis; Eimeria christianseni; and Eimeria somaterie	Mallard duck; mute swan; and, common eider, respectively	Kidney	Ball et al. (1989)
Eimeria reichenowi; Eimeria gruis	Sandhill cranes; whooping crane	Widespread	Novilla et al. (1981)

That crane eimerians can complete their life cycle in both digestive and respiratory tracts is unique among avian Eimeria spp. and differs from that reported in isosporoid species, including Isospora serini and Toxoplasma gondii. The systemic infection with I. serini is characterized by asexual stages in intestinal and extra-intestinal tissues and gametocytes and oocysts in the intestines only (Box, 1977, 1981). While I. serini is monoxenous, affecting canaries only, T. gondii undergoes widespread merogony in a wide variety of hosts, including man, but it can only complete its life cycle in the digestive tract of felines (Dubey, 1977). Thus, DVC in cranes could be a useful animal model for the study eimerian extraintestinal stages and for the evaluation of potential systemic anticoccidial drugs.

Acknowledgements

The authors thank T. K. Jeffers, D. M. Hoover, V. P. Meador, and R. Y. Reams for their help and encouragement, and J. F. Horn, S. L. White, K. W. Wightman, and K. K. Newport for their excellent technical assistance.

Appendix

Translations of the abstract in french, german and Spanish are on the Avian pathology website