Protozoans

Last updated on September 13, 2010

Phylum Sarcomastigophora

Phylum Microsporida: Encephalitozoon, Enterocytozoon

Phylum Myxozoa (Myxobolus in fish)

Phylum Ciliophora: Balantidium, Ichthyophthirius, Pneumocystis

Phylum Apicomplexa

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Phylum Sarcomastigophora

The phylum Sarcomastigophora includes protozoans with flagella or pseudopodia. There are two subphyla: Mastigophora (flagella in trophozoites) and Sarcodina (which use pseudopodia for locomotion).{3930}

It is easy to confuse protozoans with some fungal diseases on histology. Stains used to help make the diagnosis include Gomori methenamine silver, PAS, Gridley fungus, and mucicarmine. Some examples are:

Histoplasma capsulatum and Candida albicans resemble Leishmania amastigotes, both appearing on H&E as tiny circular organisms with rings around the edges and eccentric nuclei. If stained with GMS, the fungi stain black.

Rhinosporidium seeberi of dogs causes nasal polyps. The large sporangium can be confused with Leucocytozoon megaloschizonts. Use of GMS will stain the wall of the mature fungal sporangium but not immature stages. Gridley fungus stain will make the mature sporangium and spores bright pink, as will mucicarmine stain.

Emmonsia crescens and Sporothrix schenckii produce very thick-walled adiaspores resembling cysts of Besnoitia in NHPs and other species. Again, GMS will stain the fungal walls black.

Coccidioides immitus and Prototheca may be confused with Sarcocystis. GMS and Gridley fungus stains will stain the fungal organisms.

Cryptococcus neoformans, Blastomyces dermatidis, and Paracoccidioides brasiliensis may be confused with coccidians, for example in the eye, skin and liver. Mucicarmine stains the fungi bright pink, as does Gridley fungus stain.{3930}

Hemoflagellates (family Trypanosomatidae, order Kinetoplasida)

Trypanosoma

There are two main species of Trypanosoma that are of veterinary importance. T. brucei is transmitted by the tsetse fly. The trypomastigotes remain in plasma and tissues where they cause anemia and degenerative, necrotic, inflammatory changes. T. cruzi is transmitted by reduviid bugs (kissing bugs) via their feces. The metacyclic forms deposited by the bug enter the bloodstream and go to cardiac muscle, where they transform into epimastigotes or amastigotes (unique to T. cruzi) which can be seen histologically. Myocarditis and megacolon are the disease entities caused by T. cruzi (Chagas disease). A cluster of amastigotes in cardiac muscle is called a pseudocyst; many will be found within myocardial cells. On H&E sections, amastigotes have basophilic kinetoplasts (bar-shaped structures) and somewhat resemble very large neutrophils. In peripheral blood smears stained with Giemsa, the trypomastigotes of T. cruzi are C-shaped, with large kinetoplasts and visible flagella and an undulating membrane. T. brucei is not crescent-shaped and the kinetoplast is smaller. {3930}

Experimentally, it is known that in mice infected with both mouse hepatitis virus (MHV) and T. cruzi, the trypanosomiasis is worsened, with more severe effects on lymphoid organs. The implication is that subclinical MHV infection may confound research results with trypanosomiasis.{3931}

There is some question of whether T. cruzi exists naturally outside of the Western Hemisphere, since there have been a few reports of trypanosomiasis in Old World macaques, a baboon and a gibbon. Certainly infection of New World primates is most common. Control of the insect vectors is the only means of elimination. Transmission is by sexual contact, blood exposure, or transplacental transmission{4149}. There are many other species of Trypanosoma that have been identified in NHPs, but only T. cruzi is known to be pathogenic.{3941} The best method of detection is PCR, which identified positive squirrel monkeys better than blood smears or ELISA (26% vs. 10% in wild-caught monkeys). {4149} This is an ABSL2 organism; workers should wear a face shield or use a biosafety cabinet when working with cultures or infected blood to prevent droplet exposure.{3950}

Leishmania

Leishmania infects humans, dogs, and rodents. Squirrel monkeys are a good model for Leishmania donovani, due to protracted infection{4075}. The sandfly is the vector. Disease is found in Central and South America, central Asia and central Africa. Amastigotes occur in the sandfly, and promastigotes in the host animal macrophages, which spread the disease when the sandfly takes on a blood meal. After many cycles of reproduction in the host, either cutaneous, visceral or mucocutaneous leishmaniasis occur. At first the host is asymptomatic; later there are hepatic and splenic enlargement, ascites and alopecia. This is an ABSL2 organism; workers should wear a face shield or use a biosafety cabinet when working with cultures or infected blood to prevent droplet exposure.{3950}

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Enteric Flagellates: Trichomonas, Giardia, Pentatrichomonas, Spironucleus

There are only two forms of flagellates, the motile trophozoite found usually in the duodenum and the cyst form which is the source of infection for the next host. Flagellates are often hard to see attached to the microvilli of small intestinal epithelial cells; it is difficult to see the nuclei and flagella.

Giardia

Giardia is the most common flagellate in animals (G. muris, G. agilis, G. duodenalis=lamblia=mobilensis). Zoonoses in humans are most often from contact with beavers, muskrats, voles, cats, dogs, sheep, goats, pigs and cattle. It must be handled using ABSL2 precautions{3950}. The trophozoite has two nuclei and four pairs of flagella. Large numbers of protozoans are commonly found in feces of NHPs, including Giardia and Trichomonas; however their significance as pathogens is unknown{2765}. Giardia muris causes a noninvasive infection in mice, rats and hamsters. Animals with heavy infections may appear unthrifty{3551}{2764}. G. duodenalis occurs sporadically in laboratory rabbits but is not considered pathogenic or zoonotic. However, G. duodenalis=G. mobilensis in NHPs has been incriminated in causing diarrhea in all types of NHPs, although the simple finding of organisms in feces does not prove cause.{3941} Rabbits have been used to cultivate Giardia spp. for experiments{3937}. NHPs can be treated with quinacrine, metronidazole (Flagyl) or furazolidone{3941}.

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Tritrichomonas

Tritrichomonas mobilensis is a newly-isolated flagellate of squirrel monkeys that may be a cause of diarrhea, based on having found it retrospectively in the lamina propria, submucosa and crypts of the cecum and colon. The organism caused cytopathic effects in tissue culture, and was shown by the standard "subcutaneous mouse inoculation assay" to be invasive and virulent. Both immediate microscopic exam and culture of feces are needed for diagnosis. The organisms are lanceolate, 7-10mm x 1½ - 3mm, and have 3 long anterior flagella and an undulating membrane. Incidence is near 100% in squirrel monkeys.{3941}

Tritrichomonas muris is not considered to be pathogenic in any species (mice, rats, Syrian hamsters and gerbils), but it is a useful biomarker of contamination in colonies because the infective dose is only 5 pseudocysts. The pseudocysts form from trophozoites in the fecal matter; they are rounder and don't have the typical morphology of trophozoites found in direct smears (spindle shape, 3 anterior flagella and 1 posterior flagella, axostyle that looks like a tail). Pseudocysts survive for up to 7 days outside the host in moist environments.{4195}

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Pentatrichomonas

Pentatrichomonas has a single nucleus rather than two. One of the flagella is attached along the body wall to form an undulating membrane. Spironucleus (Hexamita) is smaller and lacks an undulating membrane. It is a common protozoan of mice, rats and hamsters, like G. muris; however it inhabits the crypts of Lieberkuhn. Young, stressed, and immunoincompetent mice are unthrifty.{3551}

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Spironucleus

Spironucleus muris is common in mice and rats. Cysts that resemble Easter eggs are passed in the feces and can also be seen in the large intestine. Disease is usually subclinical, and often occurs in conjunction with other pathogens, especially MHV. Trophozoites are found in the crypts and may distend them. The mucosa becomes hyperplastic and trophozoites may invade the lamina propria.{3763}{2764} Trophozoites move fast in a zig-zag pattern and are seen histologically in the duodenal crypts of Lieberkuhn.{4756} They are smaller than either Giardia or Tritrichomonas, measuring 3-4 x 10-15um, and have 6 anterior and 2 posterior flagellae. The prepatent period is 5 days. At least one lab has developed a PCR test for Spironucleus.{4772}

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Trichomonas

Trichomonas caviae infects guinea pigs and can be detected in fecal smears using Protargol stain. It resembles Giardia except it is uninucleate.{3930} Trichomonas spp. were listed as the most common parasite of hamsters in the old blue book{3566}.

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Ichthyobodo

Ichthyobodo necatrix is a fish parasite that inhabits the skin and gills. The common name, "blue slime disease", refers to the increased mucus produced in response to infestation. Treatment is copper sulfate or formalin.{3779}

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Histomonas meleagridis

The life cycle of Histomonas in poultry is complex. Turkeys pass Heterakis roundworm eggs that contain Histomonas trophozoites inside, and these are ingested by earthworms, which are in turn picked up by other birds. The trophozoites are released in the bird’s cecum from the roundworm eggs, become amoeboid and pleomorphic in form and invade the liver and intestinal wall. Grossly the liver has characteristic depressed lesions (they look like sliced black olives) and is hemorrhagic. The cecum is enlarged and hemorrhagic. The trophozoites are seen in clusters of 3-5 roughly circular organisms in the tissues, and stain more brilliantly with PAS than H&E.{3930}

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Amoebae 

Entamoeba, Balamuthia

Entamoeba is an important parasitic amoeba with a direct life cycle. Cysts are ingested and develop 8 trophozoites, which invade the lumen of the intestine. E. invadens affects reptiles, and E. histolytica infects man (amebic dysentery) and other animals including NHPs (both New World and Old World) and dogs. It is an ABSL2 organism, and there have been lab-acquired infections.{3950} As with trichomonads, the organisms appear to be commensal and cause disease only when they invade the mucosa. They can cause petechial hemorrhages and large, classic flask-shaped intestinal ulcers. Histologically they measure from 10-50µm in diameter, have clear chromatin plaques and a circular dark eosinophilic endosome that is eccentrically located.{3930, 2765, 4150} The organisms stain poorly with H&E, but bright red with PAS.  Only pathogenic Entamoeba histolytica ingest RBCs, which can be used to differentiate them on histology. Clinical signs in NHPs include lethargy, weakness, dehydration, weight loss, anorexia, vomiting and diarrhea. Diagnosis is by direct fecal exam while the sample is still warm. The treatment (which usually isn't necessary anyway) is metronidazole (Flagyl){3941}, chloramphenicol, tetracycline, chloroquin or diiodohydroxyquin (CL Davis 2001). E. muris is considered a nonpathogen in mice, rats and hamsters{3551}.

A new amoebic organism has been named Balamuthia mandrillaris since it was first isolated in 1990 from a baboon that died of suppurative meningoencephalitis at the San Diego Zoo. A few other cases have since been described in NHPs, and there have been 60 reported cases in humans. All humans had amoebic meningoencephalitis. In tissue sections of brain the trophozoites cannot be distinguished from those of Acanthamoeba without further studies.{3941}

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Phylum Microsporida

Microsporidia are obligate intracellular protozoans that affect a large number of species. In general, they are small, oval, and stain with Gram stain, Giemsa, modified trichrome and chemoluminescent agents. Morphologically, they have polar filaments or polar tubules. The spores survive in the environment and are probably the transmissible form. Mature spores have thick chitin coats. Spore morphology is used to differentiate species, as is host cell/parasite morphology.

There are two general disease patterns: acute clinical disease frequently causing death in neonatal animals (especially dogs, foxes and squirrel monkeys), and a chronic persistent infection that is clinically silent. Protection is mediated by T cells.

In humans, important microsporidian diseases are caused by Enterocytozoon bienusi, Encephalitozoon intestinalis, Encephalitozoon hellum, and Encephalitozoon cuniculi. Other species include Nosema, Pleistophora, Trachipleistophora hominis, Thelohania apodemi, and Microsporidium. {4169}

Encephalitozoon cuniculi

This is the most recognized genus of the several microsporans that infect animals including fish. It is in the family Pleistophoridae, which also includes Enterocytozoon bienusi and Microsporidian sp.{3941}. HIV patients sometimes are infected by species of Encephalitozoon that are indistinguishable from E. cuniculi. Transmission is by carnivorism or is spread through the urine. After ingestion of the spores, the sporont within is taken up by peritoneal macrophages, unspecified brain cells or renal tubular epithelial cells where it undergoes asexual reproduction. Migration from the gut to other viscera does not occur, as it does with other microsporidia. The resulting sporoblasts secrete a thick resistant spore wall. A parasitophorous vacuole is present after schizogony occurs in peritoneal macrophages. Spores range from 2-15mm. They have dark-staining annular masses when stained with H&E, Brown & Hopps Gram stain (dark red ovals with blue polar bodies), or acid-fast stain such as Ziehl Nielsen (dark blue rings around immature spores and bright pink staining mature spores).{3930}{3941}

There are several reports of encephalitozoonosis in guinea pigs, with lesions confined to the brain and kidneys. The disease is not significant unless the histology interferes with research objectives. Lesions must be differentiated from those of toxoplasmosis, which is zoonotic.{3559}

Encephalitozoonosis is widespread in rabbits, and interferes with research. It is usually a latent disease; occasionally rabbits exhibit CNS signs such as convulsions, tremors, torticollis, paresis and coma. Grossly, the kidneys have small dents or whitish foci. The organism causes chronic inflammation, fibrosis and tubular dilatation in the kidney. The most characteristic lesion is granulomatous encephalitis in the brain, with organisms not often identifiable within the lesions. Perivascular cuffing of vessels with mononuclear cells often occurs. Mature spores measure 1.5 x 2.5mm and have a thick cell wall. Unlike Toxoplasma gondii which is Gram negative, E. cuniculi in histologic sections is Gram positive. The table below lists some other stains used to differentiate the two. The diagnosis is made postmortem on histology, can be picked up serologically (IFA, CF, ELISA), or by using the India ink immunoreaction (nonspecific binding of carbon to rabbit heavy chains of IgG on the spores) or other stains of urine and infected body fluids.{3561, 2764, 3937}

Nude or scid mice can be implanted with segments of neonatal rabbit intestine and then inoculated with E. cuniculi grown in vitro, as a means of studying pathogenesis of parasitic infection in immunocompromised humans. The microspora invade many organs including the brain, liver, respiratory tract, spleen, salivary glands, and gastrointestinal tract.{3933}

E. cuniculi infects many other mammals including mice, rats, guinea pigs, dogs, squirrel monkeys and immunocompromised humans. Animals ingest spores or cannibalize each other to become infected. In mice, the organisms proliferate asexually in peritoneal macrophages, as E. cuniculi is an obligate intracellular parasite. Spores disseminate hematogenously to the brain and other organs. In the few squirrel monkeys that have been infected, there was little evidence of clinical disease; one animal displayed seizures for a month.{3941} Fulminant infection causes lymphocytic meningoencephalitis and focal granulomatous hepatitis. Unlike rabbits, mice do not develop interstitial nephritis. The infection is diagnosed by cytology of ascitic fluid, histologic evaluation of Goodpasture’s stained tissues, or by skin testing and serology. {3551}

Encephalitozoon intestinalis is a human parasite that has also been grown in the rabbit intestinal xenograft (to nude mice) model. In AIDS patients, it infects the small bowel and biliary tract. It causes widespread disease, including nephritis. Although the organism can be grown in vitro, it does not infect nude or scid mice.{4169}

Enterocytozoon bienusi

Enterocytozoon bienusi is a newly-described cause of chronic diarrhea in rhesus macaques. The organisms are ubiquitous in nature, but cause opportunistic infections in SIV-positive macaques. Hepatitis lesions consist of hyperplasia of biliary epithelium, fibrosis and infiltrates of lymphocytes and plasma cells. The organisms are very difficult to find on sections.{3770} E. bienusi was also identified in human AIDS patients in 1985. It has been experimentally transferred to immunosuppressed gnotobiotic pigs.{4169} 

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Phylum Myxozoa (Myxobolus)

Parasites in this phylum affect poikilotherms including fish and annelids. The spores have 1-2 sporoplasms within them and 1-6 polar capsules. Myxobolus has two sporoplasms with teardrop-shaped polar capsules, and the entire spore is also teardrop-shaped or oval. The spore stains intensely blue with Giemsa, and is acid-fast, but stains weakly with H&E. Myxosporidia cause several serious diseases of fish. The intermediate hosts are tubifex worms. Heavy infestations are seen in the cartilage, muscle, gut wall, gills, brain, skin and excretory system. {3930}

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Phylum Ciliophora

Although many ciliates are normally found in animals, only a few are associated with pathology. Ciliates may travel through the blood vessels after death to be found in liver histologic sections, and should not be confused with pathologic organisms. Balantidium coli is found in NHPs (New World, Old World and great apes), rodents and pigs. The trophozoites have a large kidney or heart-shaped nucleus and a contractile vacuole. They are usually found in the large intestine where there is no associated mucosal change. Only occasionally will B. coli invade the mucosa and cause mild to severe enteritis.{3779} The cysts are large (40-60 mm) and heavily ciliated. In great apes, severe ulcerative enterocolitis has been attributed to infection with B. coli. The organism is also zoonotic, causing diarrhea in people. Treatment is with metronidazole (Flagyl), tetracycline, or diiodohydroxyquin (Yodoxin).{3941, 4762}

Ichthyophthirius spp. are fish pathogens which partially penetrate the skin, develop a cyst wall, then drop off and release new trophozoites that infect other fish. Ichthyophthirius are 75-100µm in diameter, uniformly ciliated, and have a distinctly C-shaped nucleus.{3930} The life cycle is completed in 7-14 days. Treatment is formalin or copper sulfate to kill the free swimming theronts.{3779}

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Pneumocystis carinii

Pneumocystis is a taxonomically unclassified organism; some place it with yeasts and others with the protozoa. The life cycle is not yet understood, however the entire life cycle takes place in the lung of many mammalian species and is direct. Transmission is likely by aerosols without the need for contact, as well as vertically{3937}. This ubiquitous opportunistic organism can be activated by immunosuppression (i.e. by administration of steroids to rats or mice) and fills the lungs with trophozoites and cystic structures. The lungs are firm and enlarged, and do not collapse. Histologically the lung is solid with massive mononuclear infiltration. On H&E a characteristic foamy fine granular eosinophilic exudate fills the alveolar space, but organisms can’t be seen. The material stains intensely with PAS. Parasites are best demonstrated with Gomori methenamine silver (GMS) stain. Some are spherical and 2-6mm in diameter, while others are collapsed. Look for cysts with 8 intracystic bodies when stained with Giemsa. Infection is diagnosed by histology of sections stained with H&E and GMS, PAS, or by serology.{3551}{2764} P. carinii is occasionally observed in association with respiratory disease in macaques{2765}. It can cause pulmonary lesions and clinical disease in weanling rabbits, although most recovered. LDH and triglycerides were elevated. PCR proved superior to staining methods for detection of pneumocystosis. Tests to detect circulating antigens have not been useful, nor have antibody detection tests since most patients are immunocompromised{3937}.

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Phylum Apicomplexa

Coccidia (Eimeria, Isospora, Cryptosporidium, Sarcocystis and Toxoplasma) are the most economically important protozoans in animals in the US. The species were historically classified by the number of sporozoites contained within sporocysts.{3930}

Eimeria

There are over 1000 species of Eimeria, all of which are quite host-specific and have a direct life cycle. Unsporulated oocysts are shed in the feces and they sporulate in the environment to become infectious. Following ingestion, sporozoites excyst in the intestine and invade epithelial cells. They round up to form trophozoites which undergo asexual reproduction (merogony or schizogony). Merozoites form sexual stages which unite to form oocysts.{3930}

Histologically, the sporozoite is a banana-shaped structure with a single nucleus, refractile bodies and PAS-positive granules. It is motile and infects intestinal epithelial cells. The trophozoite is the intracellular, rounded, uninucleate stage. It divides to produce a meront or schizont containing 2-100,000 nuclei. Each nucleus becomes a merozoite, which is a motile infectious stage structurally similar to a sporozoite. Male gametes (microgametes) are multinucleate, and each nucleus becomes a sperm-shaped flagellate structure. Female gametes (macrogametes) are uninucleate with peripheral eosinophilic granules.{3930} Oocysts measure 30-40 by 16-25mm{3937}.

Coccidiosis is most common in young animals, with necrosis and hemorrhage localized to the intestinal tract.

Coccidiosis and encephalitozoonosis are the most significant protozoal diseases of the rabbit. In rabbits, Eimeria stiedae infects the epithelial cells of the bile ducts, causing hepatic coccidiosis with anorexia, weight loss, diarrhea and hepatomegaly. The prepatent period is 15-18 days. Liver lesions are the result of dilated bile ducts filled with coccidia and yellow exudate. Histologically, the bile duct epithelium undergoes marked hypertrophy and cystic enlargement. If the bile duct ruptures there will be inflammation and fibrosis. Adjacent liver cells are compressed by the lesions. Globulins, bilirubin, ALT and AST are increased.

Several other species of Eimeria (E. perforans, followed by E. irresidua, E. magna, and E. media) cause intestinal coccidiosis in rabbits, limited to the small intestine. Signs, when present, include diarrhea, weight loss, and perhaps intussusception; rabbits may die of dehydration and secondary bacterial overgrowth. Grossly there are multiple white areas in the wall of the small or large intestine. Although all species of rabbits can be infected, there is likely no cross-transmission among species. Rabbits are infected by ingesting sporulated oocysts, but autoinfection through coprophagy does not occur. Diagnosis is by fecal flotation. Coccidiosis is best treated with sulfonamides, which act as coccidiostats until the rabbit’s immune system can eradicate the infection and produce life-long immunity. They may be administered in the drinking water (0.02%) or in the feed (0.03%). Monensin may also be effective. Problems with vaccine development include inadequate shelf-life and difficulties in delivery; a genetic vaccine may be successful. Raising rabbits in scrupulously clean or barrier conditions is the most practical means of control. Washing cages with hot water and detergent, and treatment of the environment with 10% ammonia are effective against oocysts.{3561}{3937}

In guinea pigs, E. caviae causes diarrhea and death with thickening of the colon and petechial hemorrhage. Disease is more often seen in guinea pigs under stress, after shipping, or those with vitamin C deficiency. Sulfamethazine in the drinking water acts as a coccidiostat.{3559}

In Europe, E. muris is a common pathogen of mice, causing diarrhea and catarrhal enteritis when the infestation is heavy{3551}. Several other species have been reported in laboratory and wild mice (E. falciformis, E. musculi, E. schueffneri, E. krijgsmanni, E. keilini, and E. hindlei), where they cause enteritis, typhlitis, and colitis with bloody diarrhea.{3763}

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Isospora

Isosporans are similar to eimerians but their sporocysts are different in configuration (there are 2 sporocysts within each sporulated oocyst, and each contains 4 sporozoites). Some species can have stages in extraintestinal tissues of the host, and have been referred to as Cystoisospora spp. Isospora are found in carnivores, passeriform birds, amphibians, reptiles, rodents, humans, NHPs and swine. Diagnosis is often by examination of intestinal mucosal smears, as disease may develop before oocysts are shed in the feces. With Cystoisospora, the accidental host shows no signs; monozoic cysts develop in extra-intestinal sites (i.e. lymph nodes).{3930} It is handled using ABSL2 precautions, as lab-acquired infections have occurred.{3950}

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Cryptosporidium

Cryptosporidia are highly resistant to drying and chemical disinfection (they will survive in full-strength Clorox bleach). There is a high potential for zoonotic transmission, and there are no drugs to treat the disease. Therefore, care in protective clothing and personal sanitation are critical in preventing human and animal disease.{3941, 4780} It is an ABSL2 organism, and there have been lab-acquired infections; use of calves to produce cryptosporidia almost invariably results in human infection.{3950} 

Mammals, birds, reptiles, fish and amphibians can all be infected with Cryptosporidium. C. parvum infects only mammals, C. baileyi and C. meleagridis infect only birds, and C. serpentis infects only reptiles. Morphologically, the cryptosporidia are very similar to other coccidians but are smaller, about 1-5mm, and are located at the microvillar surface. Diagnosis is made by histopathology and by fecal floats stained with fluorescent antibody, auramine or acid-fast stains. Methylene blue, PAS (they are negative), and iodine are also used (CL Davis 2001). The trophozoites are seen just under the surface of the epithelial membranes, never within cells or beneath the epithelium. The villi can be blunted and fused, variable in height, and there may be necrosis of individual epithelial cells{3941}. A fecal flotation specimen in sugar water and viewed under phase contrast makes the unstained oocysts refractile.{3930}

Generally, cryptosporidiosis will resolve spontaneously in a healthy patient, but it can cause serious small intestinal diarrhea in the immunocompromised{3770}. In transgenic mice it has been shown that intestinal gamma-delta T cells are necessary for protection against infection{2736}.

Mice: C. muris and C. parvum in mice are opportunistic, with C. muris found in the stomach and C. parvum in the small intestine.{3763}

Rabbits: C. parvum is found in rabbits but is considered an incidental finding. Its only significance is as a potential for zoonotic spread to humans.{3937}

Guinea pigs: A new species, Cryptosporidium wrairi (guess where that came from?) has been shown to be pathogenic for guinea pigs, but the disease is subclinical. It causes a greasy rough haircoat, lethargy, diarrhea, weight loss, and emaciation. Acute edema of the small intestine is caused by organisms located in the brush border of enterocytes.{3775}

NHPs: Serious disease occurs only in young, debilitated or immunosuppressed (by SIV or SRV) animals. Transmission is fecal-oral. In normal animals there may be diarrhea, anorexia, and weight loss, but these resolve spontaneously. It is a commonly-encountered opportunistic infection in macaques with SRV or SIV, in whom it may cause generalized infection with inflammation in conjunctiva, trachea, bronchioles, bile ducts, gallbladder, and the pancreatic duct. In ductal tissue there is often marked hyperplasia and fibrosis.{4150} The disease in infant macaques is clinically, histologically and microbiologically indistinguishable from the disease seen in young children. A reproducible model has been used in pigtailed macaques (M. nemestrina) to study infectious dosage.{3941}

The first case of cryptosporidiosis in the stomach of an NHP was published in 2002{2758}. C. muris and C. parvum were both discovered by Tyzzer. C. muris infects the stomach of mice and rats and is considered insignificant, while C. parvum causes gastroenteritis in animals and man and grows in the small intestine. These were found serendipitously in the stomachs of 15 cynos from Covance that had been part of a clinical safety assessment study using immunosuppressive drugs. All the stomachs also contained an organism resembling Helicobacter heilmannii, but no correlation was clear. Not enough DNA was collected to distinguish which species of Cryptosporidium was present. The structure generally resembled that of C. muris.

In snakes, a consistent clinical picture is hypertrophic gastritis with regurgitation of meals. The stomach may be so thickened that the body wall bulges.{3570} 

In man, the disease has occurred in immunodeficient patients as an opportunistic infection. Large epidemics have occurred from contaminated drinking water. Disease also occurs in people handling infected calves. It is refractory to antibiotic treatment.{3934, 4780} 

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Family Sarcocystidae

This family of coccidia all has oocysts with 2 sporocysts in the intestine of a definitive host, and undergoes asexual stages in an intermediate host, in which there is usually pathology. Genera included in this family are Toxoplasma, Besnoitia, Hammondia, Sarcocystis, Frenkelia, and Atoxoplasma.

Toxoplasma gondii

T. gondii is a Gram negative coccidian parasite named for Ctenodactylus gondi, a North African rodent discovered at the same time as a host. The definitive host is the cat; the mouse and all warm-blooded animals can be intermediate hosts. Toxoplasma is transmitted by oocysts in cat feces (10µm long), ingestion of meat containing cysts, or transplacental transmission of tachyzoites. Animal workers have been infected by contacting peritoneal fluid{3950}. On ingestion by the intermediate host, the oocysts excyst in the intestine, releasing sporozoites which multiply in the intestine and lymph nodes as tachyzoites. These 4-6 um crescent-shaped organisms must be differentiated from coccidia and Plasmodium. They undergo many divisions while spreading throughout the tissues, eventually forming a cyst. Cysts are most commonly found in the brain, liver, muscles and retina. They are spherical or elongate, 10-100um long, thin-walled structures containing a few to hundreds of slender PAS-positive bradyzoites. If another non-feline host ingests the cysts, they revert to tachyzoites and repeat the cycle.

In the cat, bradyzoites undergo a cycle of both sexual and asexual reproduction in the epithelial cells of the intestine. An extraintestinal cycle similar to the one in intermediate hosts also occurs in the cat. Eventually unsporulated oocysts are shed in the feces, becoming sporulated in the environment.{3930} Toxoplasma is an ABSL2 organism.{3950}

Focal necrosis is the most characteristic histologic lesion in toxoplasmosis. Pneumonitis is the most common lesion in fatal toxoplasmosis of dogs and cats, whereas in sheep and goats it is placental necrosis and abortion; the fetus dies of encephalitis. In the placenta, it can be hard to recognize the cysts in the cotyledons among the degenerating host cells.{3930} In New World monkeys, which are more susceptible than other NHPs, necropsy findings include dehydration, cardiomegaly, myocardial necrosis, hemorrhagic lymphadenopathy, pulmonary edema and many other nonspecific signs. In fact, toxoplasmosis should be ruled out in any NHP with nonspecific acute illness.{3941}

Necrosis and granulomatous inflammation occur in the mouse intestine, mesenteric lymph nodes, eyes, heart, adrenals, spleen, brain, lung, liver, placenta and muscles. These lesions are caused by both tachyzoites contained within pseudocysts and bradyzoites contained within true parasitic cysts which are resistant to proteolytic enzymes. Cysts contain numerous crescent-shaped bradyzoites. Animals commonly recover from toxoplasmosis and develop humoral and cellular immunity.{3551}

Diagnosis is based on serum immunochemical tests such as CF, HA, Sabin-Feldman dye test (methylene blue dye), IFA, and ELISA. Histology of lesions is helpful, including immunoperoxidase staining. Morphology of the organisms by light and electron microscopy is also useful. Wright-Giemsa stains of peritoneal exudate, mesentery or omentum can be examined for characteristic organisms.{3551}{3937}

In rats, T. gondii causes subclinical disease. Intracellular organisms are found in several tissues, including lung and spleen; cysts are found in the CNS.{2764}

Serologic surveys indicate that toxoplasmosis is more common in the guinea pig than in the rat, yet histologic evidence is rarely seen. Lesions include myocarditis, focal encephalitis, mild pneumonia, and focal histiocytosis in lymph nodes and spleen.{3559}

Prevalence of antibodies has been claimed to be as high as 50% in clinically normal rabbits using the Sabin-Feldman dye test. Toxoplasmosis can be acute, chronic or latent in rabbits. In acute disease, rabbits show abrupt onset of anorexia, fever, and ocular and nasal discharge with increased respiratory rate. Over 2-8 days they become lethargic, have convulsions, become paralyzed in the hindquarters, and die. There is generalized necrosis of organs noted in the reticuloendothelial elements and the vascular connective tissue. Trophozoites may be found intra- or extracellularly. In the chronic form, they become anorectic and emaciated, anemic, sometimes paralyzed, and may die suddenly or recover. Organs are edematous and have scattered necrotic foci. There is pronounced reticuloendothelial hyperplasia in the lymph nodes, spleen, liver and CNS. The latent form is characterized by cysts in the CNS, which must be differentiated from those of Encephalitozoon.{3937}

Treatment, if elected, includes sulfadiazine, pyrimethamine, sulfones, tetracyclines, spiramycin and 1,2-dihydrotriazones. Control involves removing cat feces within 24 hours before any oocysts can sporulate, freezing and/or cooking meat fed to animals, strict sanitation, and keeping a reasonable distance between feline cages and those of other mammals.{3937}{3941}

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Sarcocystis

This genus of coccidian requires an intermediate host (herbivore) for the asexual cycle and encystation, with the definitive host (carnivore) carrying only the sexual cycle. In the herbivore, excysted sporozoites are released in the intestine and migrate to the arteries, where they develop into meronts. Merozoites released from those meronts migrate to capillaries, where they undergo another generation of meronts. The next generation of meronts enters the cytoplasm of mononuclear leukocytes for another round of endodyogeny (two progeny forming within a parent cell). Those merozoites are released and enter the heart, skeletal muscle and neural tissue to produce noninfective sarcocysts. The intermediate host may develop pathologic lesions in blood vessels, myocardium, GI tract, urinary bladder, skeletal muscle, lung, liver and kidney. At necropsy, lesions may include hemorrhage in all organs, serous atrophy of fat, yellowish fluid in body cavities, watery blood, generalized petechial hemorrhage, and alternate pale and dark striping or mottling of skeletal muscle. Sarcocysts may be grossly visible as white nodules or streaks (Miescher’s tubes{3941}). Merozoites may be seen within leukocytes in peripheral blood. Sarcocysts have PAS-negative walls with PAS-positive granules in the zoites. Mature sporozoites are acid-fast. Histologically the cysts vary in size and may be oval or round. Diagnostically, Sarcocystis schizonts are differentiated from other coccidia by the lack of a parasitophorous vacuole and the presence of a multilobed nucleus.{3930}

S. muris has a similar life cycle to T. gondii, but is considered rare or nonexistent in laboratory mice and rats.{3551}{2764}

Oryctolagus cuniculus has only rarely been diagnosed with Sarcocystis, but the disease is common in Sylvilagus. The definitive host for S. cuniculi is the cat. Infected cottontails are asymptomatic; on necropsy there are grossly visible white streaks in the muscles with no inflammatory response unless the cyst is ruptured. Smears stained with Giemsa demonstrate the motile, banana-shaped organisms (Rainey’s corpuscles{3941}). The cysts have radial spines. Human infection has only sporadically been reported.{3937} Sarcocystis spp. is an ABSL2 organism, and there have been lab-acquired infections.{3950} 

In Old and New World NHPs, sarcocystosis is considered a common incidental finding at necropsy. Lesions are often seen in the tongue or esophageal muscle.

S. falcatula is the cause of equine protozoal encephalitis, a newly-described disease of American horses. The opossum (Didelphis virginiana) is the definitive host, and the usual intermediate hosts are passerine birds (budgies, sparrows). The horse is considered an aberrant host. The disease is characterized by formation of cysts in neural tissues of the CNS.{3930}

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Neospora caninum

This protozoan was recognized in 1988, having been misdiagnosed as T. gondii prior to that time. The definitive host is unknown. Transplacental transmission is the only known mode, and may occur through several generations. It causes abortion in dogs, cows, sheep, goats and horses. Tachyzoites cause focal necrosis followed by inflammation most often in the brain.{3930}

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Frenkelia

These coccidians have a rodent intermediate host and a raptor definitive host. Lesions in the rodent are related to merogony in the liver, with hepatic necrosis. Cysts growing in the CNS may cause pressure necrosis of surrounding tissue.{3930}

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Atoxoplasma

This bug infects passerine birds 2-9 months old, causing GI and neurological signs. There is a case report of acute respiratory failure in a canary with no gross lesions. Macrophages contained dark bodies in the liver that stained lavender with PAS, but not at all with Gram or Giemsa stains. Macrophages were also infected in the lung and jejunal villi, where they formed cystic cells in apical enterocytes. There is no treatment. Atoxoplasma serini is more pathogenic in canaries than Isospora canaria, with which it is easily confused. Treatment is to eliminate sick birds based on fecal exam.{4041}

Besnoitia

Intermediate hosts include bovids (Africa, Asia, Europe), equids (Africa, Mexico), reindeer and caribou (N. America), rodents and opossums (U.S.), and lizards and opossums (C. America). Cats are the definitive hosts for some species of Besnoitia. Cysts in the intermediate hosts develop in connective tissues, especially the skin, conjunctiva, mesentery, and scrotum. They may grow to be very large, several millimeters in diameter. Chronic lesions result from displacement of adjacent organs as well as granulomas from rupture of the cysts.

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Klossiella muris

This organism is a renal coccidian of mice, equids (K. equi) and guinea pigs (K. cobayae). Sporocysts are ingested and release sporozoites to the bloodstream. They enter the endothelial lining of the vessels in the kidney tubules and undergo schizogony. Mature schizonts rupture into Bowman’s capsule and release merozoites, which enter the convoluted tubules for the sexual phase of the life cycle. Sporocysts are eventually passed in the urine. K. muris is usually nonpathogenic and asymptomatic. Heavily parasitized kidneys may have tiny gray foci on the cortical surface, which are areas of necrosis with perivascular inflammation. Detection is by histology. The sporonts have a central eosinophilic mass with oval sporoblasts radiating from it, giving it a wheel-like appearance.{3551}{3930}{3763}

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Hepatozoon

This coccidian is in the family Haemogregarinidae, with stages occurring in the digestive tract of the invertebrate host (ticks, mites, lice, tsetse flies, mosquitoes) and in the liver and spleen of vertebrates (rodents, squirrels, canids, felids, raccoons, mink, carnivores, birds, reptiles). Ingestion of the invertebrate which has become infected by blood-sucking is the means of infection for vertebrates. Schizonts form in the liver cells, with several generations occurring there. The final generation produces merozoites that enter mononuclear leukocytes to become microgamonts and macrogametes. Lesions vary widely, from mild cellular infiltration to necrosis of liver and spleen. In rats, ingestion of the spiny rat mite Laelaps echidninus infects the host; hepatosplenomegaly, anemia, emaciation and death can occur{2764}. As with Klossiella, zoites bud from a central residual mass producing a radial appearance.{3930}

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Family Plasmodiidae

This family includes coccidians that have motile zoites and naked sporozoites. The family includes Haemoproteus, Leucocytozoon (both found in birds), Hepatocystis and Plasmodium.

Plasmodium

Plasmodium spp. infect most higher vertebrates. Transmission is through the bite of the female Anopheles mosquito as well as other arthropod hosts. In the mosquito, the sexual phase takes place, with zygotes transforming into motile ookinetes. These enter the gut wall and produce oocysts which undergo sporogony to produce thousands of sporozoites that are infective for mammals. The sporozoites enter hepatocytes to produce schizonts; these undergo asexual reproduction to produce tens of thousands of tissue-stage merozoites. Merozoites invade erythrocytes and transform into trophozoites, the feeding or ring stage. Eventually a schizont with 8-32 merozoites develops; the cell ruptures and the merozoites infect other RBCs. Some merozoites in RBCs transform into microgamonts and macrogametes, which are transmitted to the mosquito.

Almost all NHPs are naturally infected with a species of Plasmodium, except for the rhesus, tamarin (Callithrix), marmoset (Saguinus), and owl monkey (Aotus). In the natural host there is usually no disease. In the aberrant host, however, severe disease, debilitation and death occur. Clinical signs include cyclic fever (quotidian=24 hours, tertian=48 hours, quartan=72 hours), depression, anemia, anorexia and weight loss. Fever coincides with rupture of RBCs and release of toxic products.{3941} The lesions of malaria are anemia, brown-tinged skin and mucous membranes due to malarial pigment, splenomegaly, and darkening of the viscera also due to malarial pigment (hemazoin).{3930} Hepatosplenomegaly, hyperplasia of lymphoid elements, and myeloid hyperplasia of bone marrow are seen.{3941} Owl monkeys (Aotus) and squirrel monkeys (Saimiri sciureus) are used in malaria research, for example to examine differences in hematologic parameters during infection with either P. falciparum (owl monkeys), which produces very large numbers of merozoites, or P. vivax (squirrel monkeys), which parasitizes only young red cells and therefore was thought to have different effects. In both cases, erythrocytes, platelets, and granulocytes decreased and hemoglobin and hematocrit decreased, but lymphocytes and monocytes increased. Perhaps in addition to parasitemia, the animals had some autoimmune component and/or anemia from renal disease{3630}.

Squirrel monkeys are naturally susceptible to their own species of Plasmodium which are similar to those in humans. New World monkeys are infected by P. brasilianum (similar to P. malariae) and P. simium (similar to P. vivax).  There is monoclonal antibody cross-protection between P. brasilianum and P. malariae.  Similarly, malarial parasites from Old World monkeys have been studied in squirrel monkeys. P. inui is a quartan parasite from macaques that infects Bolivian squirrel monkeys. P. fragile is found in Macaca radiata that is similar to P. falciparum and has been transmitted to splenectomized squirrel monkeys. It has also been used in squirrel monkeys for vaccine trials. P. knowlesi infects both NHPs and man. Exoerythrocytic (EE) bodies appear in large numbers in the liver of infected squirrel monkeys and are useful for serological studies and vaccine assessment.{4075} 

Human malarial parasites include P. ovale, malariae, vivax and falciparum. P. ovale is similar to P. vivax and has been transmitted to squirrel monkeys with production of useful EE bodies in the liver. P. malariae, the cause of quartan malaria, is harder to transmit to squirrel monkeys, with slow but steady rise in parasitemia but no ability to infect mosquitoes. Splenectomized Bolivian squirrel monkeys have been used to maintain P. malariae to produce antigens. P. vivax causes tertian malaria. The Salvador I strain of P. vivax has great potential for vaccine production, as has the Thai 561 strain, and both are used to infect Bolivian squirrel monkeys. P. falciparum causes malignant tertian malaria. It is the newest malaria and is least efficient as a parasite since it tends to kill its host, causing the most severe form of malaria. The owl monkey was a primary model for studies of P. falciparum, but their scarcity caused several strains to be adapted to squirrel monkeys for investigation. Three basic types of vaccine are currently being developed: blood stage, sporozoite, and transmission-blocking. To date there has been variable success in developing any successful vaccines.{4075} Plasmodium is handled as an ABSL2 agent. Workers should wear a face shield or use a biosafety cabinet when handling blood or infected cultures, to prevent droplet exposure{3950}.

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Hepatocystis

These organisms parasitize arboreal tropical Old World mammals, squirrels, fruit bats, and lower NHPs; deer mice are also susceptible but man is not. It is endemic in Old World NHPs including baboons (a case report appeared in 2001 in CTLAS){4498}. The invertebrate host is the Culicoides midge, which deposits sporozoites into the circulation. These enter the hepatocytes and develop into meronts, which may reach 4-6mm in diameter. Liver lesions have a thick hyaline rim surrounding a colloid-filled center. The lesions may be seen radiographically as small radio-opaque foci{4498}. Merozoites develop at the periphery of this large meront, which are released when the cyst ruptures and then invade erythrocytes to resemble the ring stage of Plasmodium. Sexual stages also develop in the RBCs and enter the mosquito for fertilization and sporogony. It can be difficult to differentiate Hepatocystis from Plasmodium; it is accomplished by staining techniques and histologic structure. When stained with Giemsa, microgamonts of Hepatocystis have an unusual nucleus with a large, oval pink nucleoplasm occupying >1/3 of the parasite.{3930} Because schizogony occurs in the liver rather than the RBCs, Hepatocystis does not cause the cyclic fever seen in malaria. There are apparently no adverse health effects in NHPs. Grossly one may see some random grayish white foci approximately 1-2 mm across that are the mature merocysts. Rupture causes inflammation and subsequent fibrosis.{3941}{3770}

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Babesia

This parasite infects domestic animals including cattle, sheep, goats, horses, swine, dogs and cats, as well as wild animals and man. Ticks are the vector. The trophozoites enter the circulation and multiply in erythrocytes only. Within RBCs the parasites may be round, ovoid, elongate, or amoeboid. They appear also in pairs or tetrads as merozoites. Sexual stages have not been identified, forming the basis for the family Babesiidae. The primary lesion is erythrocyte destruction and anemia. The anemia is at first normocytic, later becoming macrocytic with reticulocytosis. Lesions may be seen in liver and kidney with hemosiderin deposition. Differential diagnosis on blood smears includes Ehrlichia, Anaplasma, and Theileria.{3930}

Two organisms in the family Babesiidae have been found in NHPs: Babesia pitheci in Old and New World monkeys in Africa only, and Entopolypoides macaci in Old World monkeys and great apes. Neither caused significant disease, but in splenectomized animals, marked increase in parasitemia and intensity of anemia and icterus occur. Even if the vector is controlled, blood-borne transmission may occur. There is no public health significance with either parasite, as infections in man are unknown.{3941}

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©1999, Janet Becker Rodgers, DVM, MS

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Comments? Send an email to rodgers@uky.edu