An Overview of Cryptosporidiosis

John Bryan, DVM and Kenneth S. Latimer, DVM, PhD

Class of 2007 (Bryan) and Department of Pathology (Latimer), College of Veterinary Medicine, The University of Georgia Athens, GA 30602-7388

Introduction

Cryptosporidium muris was initially described in 1907 within gastric epithelial cells of laboratory mice and served as the forerunner to future discoveries of additional Cryptosporidium spp.¹  A second Cryptosporidium sp. was observed within cecal epithelial cells of a chicken in 1929.² This organism was initially identified as another case of Cryptosporidium muris; however, it was re-examined again in 1961 and the name of the organism was changed to Cryptosporidium tyzzeri. The name of this organism subsequently was altered to Cryptosporidium meleagridis out of deference to a 1955 study.² This final incarnation remains to this day.

Fortunately, such ambiguities of nomenclature were superfluous to future discoveries of Cryptosporidium sp. in other animal species. Unfortunately, it was discovered that avian cryptosporidial organisms could efficiently infect several additional species of birds including Budgerigars, Ducks, Finches, Geese, Jungle Fowl, Parrots, Peacocks, Pheasants, Quail, and Turkeys.³ Mammalian groups susceptible to cryptosporidial infection include cattle, sheep, goats, deer, pigs, dogs, cats, rabbits, rodents, and primates among others.⁴ Moreover, Cryptosporidium spp. also have the ability to infect reptiles and fishes.^5,6

The ability of Cryptosporidium spp to infect simians includes human beings (Homo sapiens). Thus, the potential for zoonotic transmission cryptosporidial infections from infected animals to humans is an ever-present concern.⁷ Individuals particularly at risk for zoonotic transmission of cryptosporidiosis are those whose lives or profession often place them in constant proximity to potentially infected animals. Horizontal, non-zoonotic transmission may come from other high-risk groups of humans.^8,9  A key concern, especially in humans, is the development of cryptosporidial infections in immunocompromised individuals.⁴ Whereas immunocompetent individuals infected with Cryptosporidium sp. may experience a self-limiting disease course of approximately ten days, individuals whose immune systems have been compromised may endure a chronic, protracted course of disease lasting years.⁴ Consequently, the zoonotic and opportunistic character of Cryptosporidium infections relates directly to its threat as a public health issue.¹⁰

The taxonomy of Cryptosporidium sp. is briefly presented below.

Phylum - Apicomplexa

Class - Sporozoasida

Subclass - Coccidiasina

Order - Eucoccidiorida

Suborder - Eimeriorina

Family - Cryptosporidiidae

Genus - Cryptosporidium

Species - based on hosts

Species - Based upon current taxonomy, there are four well recognized Cryptosporidium sp. However, other new species also have been described. Taxonomy will probably be revised and improved as newer methods of genetic molecular analysis are applied. The four major species of this parasite are designated C. parvum, C. muris, C. baileyi, and C. meleagridis.

C. parvum is primarily an intestinal parasite that infects both humans and young cattle. Oocysts range from 4.0 μm to 5.0 μm in diameter.

C. muris primarily infects the gastric mucosa of mice (Mus musculus). Oocysts are approximately 8.0 μm in diameter.

C. baileyi primarily infects the respiratory tracts of avian species, usually chickens (Fig. 1).

C. meleagridis primarily infects avian species as well; however, its oocysts are smaller than those of C. baileyi. Moreover, C. meleagridis primarily infects the gastrointestinal tract.

Since the descriptions of the above Cryptosporidium spp. in the medical literature, several other species have been discovered. Each of these new species occupies a somewhat unique niche based upon preferred host, but cross-infectivity may occur, especially between mammalian species.¹⁰

Figure 1. Cytologic touch imprint of trachea. Mature oocysts are large, granular, and eosinophilic (black arrows). Immature oocysts are smaller and basophilic (white arrows). Chicken, Cryptosporidium baileyi, tracheal imprint, Wright-Leishman stain.

Life Cycle

Cryptosporidium spp. are classified taxonomically within the same group as other coccidian parasites such as Eimeria, Toxoplasma, and Isospora spp. A key difference between Cryptosporidium spp. and other coccidia is that Cryptosporidium organisms progress through a life cycle characterized by the host’s expulsion of double-walled, sporulated, immediately infective oocysts. Moreover, thick walled cryptosporidial oocysts are strongly resistant to most adverse environmental conditions such as extremes of temperature and/or humidity. Infective oocysts may persist in the environment for six months or more.¹¹ The life cycle of Cryptosporidium sp. progresses through five phases as follows (Fig. 2):

Figure 2. Life cycle of Cryptosporidium sp. Courtesy of Dr. Mark Goodwin and Dan Biesel, The University of Georgia, College of Veterinary Medicine, c. 1987.

1. Excystation (Infective Sporozoite Release) - Thick-walled oocysts are ingested by the host and reach the intestine where sporozoites excyst and penetrate the microvillus borders of ileal enterocytes. This stage of development occurs in a parasitophorous vacuole, characterized as an intracellular but extracytoplasmic space. Other Eimeriorina do not exhibit such behavior and confine themselves to intracellular, intra-cytoplasmic domains.²

2. Merogeny (Asexual Multiplication) - Once encysted within enterocytes, Cryptosporidium sp. sporozoites produce type I meronts, each of which contains  six to eight merozoites. This asexual development allows type I meronts to self replicate and help to perpetuate the infection. Type I meronts may also continue development to type II meronts. Type II meronts contain only four merozoites. With the help of a feeder organelle, these merozoites mature to continue sexual development of the organism.²

3. Gametogeny (Gamete Formation) – Microgametocytes (containing ~16 gametocytes), and the singular macrogametes originate from the type II meronts.^2,12 These stages represent the sexual reproduction of Cryptosporidium sp.

4. Fertilization- A microgametocyte fertilizes the macrogamete to form a zygote. At this point in the life cycle, Cryptosporidium spp. also diverge from other coccidia as explained below.

5. Oocyst Wall Formation and Sporogony (Sporozoite Formation) - Approximately 80% of the zygotes formed during sexual reproduction continue to develop as thick-walled oocysts. These oocysts sporulate within and are released from the host cell. Once free of the host cell, these oocysts pass from the body in the feces where they are infective and can immediately transmit the infection to another host. The remaining ~20% of zygotes will form a unit membrane in place of an oocyst wall that surrounds sporozoites. These zygotes will not be shed with the feces; they excyst within the intestinal lumen releasing free sporozoites. These sporozoites continue the parasitic infection by penetrating another host enterocyte (autoinfection).²

Pathogenesis

Most signs of disease associated with cryptosporidiosis stem from the organism’s ability to invade, encyst, and prosper within the microvillous portion of the host enterocyte. However, C. baileyiin birds and some Cryptosporidium spp. species in humans may present with signs involving the respiratory system.

General Gastrointestinal Changes – Ingestion of sporulated oocysts or autoinfection by infective, free sporozoites results in a relatively consistent clinical presentation across species. Cryptosporidium spp. invade the microvillous surface of enterocytes primarily in the distal small intestine and large intestine.¹³ Following the development of the parasitophorous vacuole just under the enterocyte membrane, Cryptosporidium infections result in atrophy and fusion of affected intestinal microvilli (Fig. 3). Villous atrophy may be associated with severe malabsorptive diarrhea characterized by copious volumes of fluid feces that may contain blood, mucus, and/or bile.^7,13 The infective stage of disease corresponds with the onset of diarrhea, as oocysts are shed within the watery stools. The infectious stage of disease may persist for several days following the cessation of diarrhea.¹³

Figure 3. Scanning electron micrograph of luminal surface of the intestine showing displacement of microvilli by Cryptosporidium sp. Cockatoo, intestine, C. meleagridis, scanning electron micrograph.

Cryptosporidium spp. may be classified as primary or secondary invaders within a given host. As a primary invader, it may infect and cause disease in immunocompetent hosts. As a secondary invader, Cryptosporidium spp. may infect immunocompromised hosts. Continued growth of cryptosporidial organisms results from a deficient host immune system that would otherwise eventually terminate the infection. Immunocompromised hosts also may develop concurrent secondary bacterial and/or fungal infections. Poor sanitary conditions, especially overcrowding, significantly favor the spread of cryptosporidial infections. In these cases, whether in calving operations or child daycare, diarrhea often presents as the sentinel sign or symptom of disease.¹⁴

Diagnosis of Cryptosporidiosis

Various diagnostic procedures have been developed to diagnose cryptosporidiosis. These techniques include fecal flotation and fecal smear examination following acid-fast (Ziehl-Neelson) or immunocytochemical staining.¹² Phase-contrast microscopy may allow more rapid identification of Cryptosporidium spp. oocysts because these structures are non-refractile by light microscopy. At least 1.0 x 105 to 1.0 x 107 oocysts / ml of feces may be recovered from patients with diarrhea.¹²

Other techniques to identify Cryptosporidium oocysts in feces include formalin-ethyl acetate (FEA) sedimentation and direct staining of  oocysts with Kinyoun’s modified carbolfuchsin (Fig. 4) or crystal violet.¹⁴ Fluorescent antibody staining and enzyme-linked immunoabsorbent assay (ELISA) antigen testing of fecal samples also may identify Cryptosporidium spp. oocysts. However, these tests primarily have been largely restricted to use in larger ruminant species such as cattle, goats, and sheep. Polymerase chain reaction (PCR) testing also has been employed to diagnose cryptosporidiosis in human patients.¹⁴

Figure 4. Mature cryptosporidial oocysts appear deep fuchsia. Kinyoun’s modified carbolfuchsin technique, oocysts, C. meleagridis.

Lastly, intestinal (most species) or gastric (reptiles) biopsies may be of benefit in the diagnosis of cryptosporidiosis (Fig. 5). In gastric biopsies, oocysts may be lost from the gastric surface during routine tissue processing; however, organisms usually can still be found in the proximal gastric glands.

Figure 5. Small cryptosporidial oocysts line the luminal surface of the intestine. Mild heterophilic inflammation also is present. Chicken, intestine, cryptosporidiosis, hematoxylin and eosin stain.

Treatment of Cryptosporidiosis

Any proposed treatment of cryptosporidiosis depends upon whether the patient is immunocompetent or immunodeficient. Immunocompetent patients require only supportive care because their infections are usually self-limiting. Immunosuppressed patients often require treatment to suppress or eliminate cryptosporidiosis. Supportive treatment may include fluid and electrolyte replacement, depending upon the degree of fluids and electrolyte loss from diarrhea. Eflornithine, oral bovine dialyzable extract, or hyperimmune bovine colostrum may be used to treat cryptosporidial infection. Paromomycin, an aminoglycoside antibiotic, has also shown some efficacy in the treatment of cryptosporidiosis infections in humans, calves, rats, dogs, cats, and mice.¹⁴ In addition, azithromycin (a macrolide antibiotic) has demonstrated some efficacy in the treatment of cryptosporidiosis in immunosuppressed rats and one human being.¹⁴

Zoonotic Concerns

Individuals working in close proximity to or in physical contact with animals at risk of developing Cryptosporidium infections also are at an increased risk of contracting the infection. Examples of higher-risk groups include veterinarians, veterinary students, veterinary technicians, farm workers, animal handlers, and those working with potentially infected animal materials and products. Self-limiting infection usually occurs in immunocompetent individuals that contact cryptosporidiosis. In contrast, people with immunodeficiency are at a particularly high risk of contracting cryptosporidiosis and may suffer a prolonged course of disease. Education and vigilance in disease surveillance are the keys to preventing cryptosporidiosis.

References

1. Goodwin MA. Cryptosporidiosis in Chickens and Turkeys – A Review. Georgia Poultry Laboratory, Oakwood, Georgia 30566, USA.

2. Goodwin MA. Cryptosporidiosis in Birds – A Review. Avian Patho18:365 – 384, 1989.

3. Goodwin MA, Brown J. Intestinal Cryptosporidiosis in Chickens. Avian Dis 33:770 - 777, 1989.

4. Duncan FJ, Amorosino CS. Cryptosporidiosis and the Healthy Host. New Engl J Med 312:1319 – 1320, 1985.

5. Sanford SE. Enteric Cryptosporidial Infection in Pigs: 184 Cases (1981 – 1985). J Am Vet Med Assoc 190:695 – 698, 1987.

6. Anderson BC, Donndelinger T, Wilkins RM, Smith J. Cryptosporidiosis in a Veterinary Student. J Am Vet Med Assoc 180:408 - 409, 1982.

7. Ley DH. Avian Cryptosporidiosis – An Emerging Disease. Avian Medicine Section, Department of Food Animal and Equine Medicine, School of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606.

8. CDC Fact Sheet for the General Public. Cryptosporidium Infection (Cryptosporidiosis). Division of Parasitic Diseases, Department of Health and Human Services, Centers for Disease Control and Prevention, 2006.

9. Moon HW, Woodmansee DB. Cryptosporidiosis: Zoonosis Update. J Am Vet Med Assoc 189:643 – 646, 1986.

10. Xiao L, Fayer R, Ryan U, Upton SJ. Cryptosporidium Taxonomy: Recent Advances and Implications for Public Health. Clin Microbiol Rev 17: 72 – 97, 2004.

11. Current WL. Cryptosporidiosis. J Am Vet Med Assoc 187: 1334 – 1337, 1985.

12. The Merck Veterinary Manual, 9th ed. Merck & Company, Inc., New Jersey, 2005, pp. 168 - 170.

13. Smith BP. Large Animal Internal Medicine, 3rd ed. Mosby Publishing, St. Louis, 2002, pp. 350 – 357.

14. Greene C (ed). Infectious Diseases of the Dog and Cat, 2nd ed. W.B. Saunders Co., Philadelphia, 1998. pp. 518 - 520.

Acknowledgement

The image of the watercolor "Mice on Library Books" by Henry Grant Plumb (1847-1930) courtesy of www.georgeglazer.com, George Glazer Gallery, New York City.

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