Lab 10 cryptosporidiosis


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  • Crytosporidium parvum is an intracellular but extracytoplasmic, below the cell,s outer membrane, protozoan parasite that causes self-limiting diarrhea in immunocompetent individuals. Currently more than 20 species of Cryptosporidium have been identified, infecting a wide range of hosts. In humans, the majority of cryptosporidiosis cases in most countries are caused by C. hominis or C. parvum Both the parasite and the disease are commonly known as "Crypto.
  • Until recently, giardiasis was the most frequently reported waterborne disease (112,113). Cryptosporidiosis has now overtaken (go past) giardiasis, with a point estimate of 420,000 annual waterborne cases in the United States (2). Cryptosporidium and Giardia are major causes of diarrhoeal disease in humans, worldwide and are major causes of protozoan waterborne diseases.
  • As few as 10 to 100 oocysts can initiate an infection. In patients with a normal immune system, the disease manifests itself with watery diarrhea, cramps, nausea and anorexia, lasting ten to fifteen days. In immunocompromised people, such as those receiving immunosuppressant drugs or those infected with HIV-1,( especially those with CD4 counts <200/µl ) symptoms are more severe. The disease is prolonged, and diarrhea can persist for months, even years.
  • They are “intracellular but extracytoplasmic. Worldwide in temperate and tropical zone More prevalent in rural areas of U.S. More animal contact The main reservoir for Cryptosporidium is domestic animals. [5] Cryptosporidium parvum can reside in 150 different species of mammals such as cattle, sheep, goats, deer, mice and pigs. Non-biting cyclorrhaphan flies are another reservoir. Cryptosporidium parvum is ubiquitous, infecting most mammals, and is highly infectious. Thus everyone is at some risk of acquiring cryptosporidiosis. Prevalence rates of cryptosporidiosis in diarrheal illness range from a few per cent in cooler, more developed countries (0.1-2% overall), to 0.5-10% in warmer, less developed countries. Recent data suggest that 3-4% of AIDS patients in the USA and Europe will become infected with cryptosporidiosis during their symptomatic period. In the developing countries the equivalent numbers are much higher up to 50% in hospital patients with AIDS.
  • One major outbreak in Milwaukee in 1993 affected over 400,000 persons ( 111 deaths: mostly the immunocompromised, but also elderly and children ). Transmission of C. parvum from household pets is extremely rare, but there is a definite correlation between calves and humans--approximately 50% of calves shed oocysts and the pathogen is present on upwards of 90% of all dairy farms (Juranek, 1995). Animal-person transmission there is a definite correlation between calves and humans. pitfall occurs at a high frequency in day-care centers, where infants or younger children are clustered within classrooms, share toilets and common play areas. Milwaukee Illness Watery diarrhea (93%) Median stools/day=12 (range 1 to 90) Abdominal cramps (84%) Fever (57%) Vomiting (48%)
  • Infected hosts produce huge quantities of infective stages (oocysts), which are long-living and highly resistant to standard water disinfecting. Methods. Resistant to chlorine, drying, progressive freezing, salt water. The hardy oocyst has been found to be resistant to a number of environmental stresses, including chlorination during drinking water treatment . Therefore water filtering (.1-1.0 microns in diameter) and boiling are the only known means of eliminating the pathogen from the water supply. Oocysts infective for 2-6 months in environment
  • Oocyst-contaminated water, soil, and vegetables cause infections. The prepatent period is about 4-22 days
  • Sporulated oocysts, containing 4 sporozoites, are excreted by the infected host through feces and possibly other routes such as respiratory secretions .  Transmission of Cryptosporidium parvum and C. hominis occurs mainly through contact with contaminated water (e.g., drinking or recreational water).  Occasionally food sources, such as chicken salad, may serve as vehicles for transmission.  Many outbreaks in the United States have occurred in waterparks, community swimming pools, and day care centers.  Zoonotic and anthroponotic transmission of C. parvum and anthroponotic transmission of C. hominis occur through exposure to infected animals or exposure to water contaminated by feces of infected animals .  Following ingestion (and possibly inhalation) by a suitable host , excystation occurs.  The sporozoites are released and parasitize epithelial cells (, ) of the gastrointestinal tract or other tissues such as the respiratory tract.  In these cells, the parasites undergo asexual multiplication (schizogony or merogony) (, , ) and then sexual multiplication (gametogony) producing microgamonts (male) and macrogamonts (female) .  Upon fertilization of the macrogamonts by the microgametes (), oocysts (, ) develop that sporulate in the infected host.  Two different types of oocysts are produced, the thick-walled, which is commonly excreted from the host , and the thin-walled oocyst , which is primarily involved in autoinfection.  Oocysts are infective upon excretion, thus permitting direct and immediate fecal-oral transmission. Note that oocysts of Cyclospora cayetanensis , another important coccidian parasite, are unsporulated at the time of excretion and do not become infective until sporulation is completed.  Refer to the life cycle of Cyclospora cayentanensis for further details.
  • Pathology / clinical symptoms: Infections are self-limiting in immune competent individuals. In this case symptoms are standard gastrointestinal with cholerae-like watery or mucus diarhea, and varying degrees of cramping as in giardiasis. Immune compromised patients, especially those with AIDS, develop severe diarhea leading to electrolyte imbalance, dehydration and sometimes death. Severe cases result in destruction of intestinal microvilli, In immuno-compromised patients, the extent of infection can extend from esophagus to rectum, and include hepatic and respiratory systems.
  • Acid-fast stain of infected feces Direct immunofluorescence antibody stain using monoclonal antibody to oocyst wall
  • Acid-fast stain of infected feces Direct immunofluorescence antibody stain using monoclonal antibody to oocyst wall
  • A: Agarose gel (2%) analysis of a PCR diagnostic test for detection of Cryptosporidium parvum DNA. PCR was performed using standard ABI protocol. Sample: For PCR testing, samples from suspected birds are collected with fresh cloacal and fecal samples. Swabs must be sterile and transported in a correct medium. Multiple specimens collected over period of time are required for best results. Environmental samples can also be submitted to help evaluate the environment. Handling: Prior to shipping samples should be stored at 4 C. (refrigerator). Samples must be shipped in a padded envelope or box. Samples may be sent by regular mail, but overnight is recommended.
  • Course of Disease:   In healthy individuals,  its a self-limiting disease where symptoms persist for 2-4 days then go away.  The young and immuno-compromised run a risk of death due to severe dehydration (symptoms can last for weeks to even a lifetime).  Also, the pulmonary form can prove to be fatal.              Treatment:  There is no treatment at this time, other than bed rest and plenty of fluids.
  • Lab 10 cryptosporidiosis

    1. 1. University of Sulaimani School of Science Department of Biology Practical Parasitology 2 nd stage Lab 10: Cryptosporidiosis Enteric Apicomplexa: Cryptosporidium parvum
    2. 2. <ul><li>Objectives: Students should be able to: </li></ul><ul><li>Identify oocyst stage in a stool smear. </li></ul><ul><li>Describe trophozoite, sporozoite, and oocyst stages of C. parvum. </li></ul><ul><li>List methodes of transmission and diagnosis. </li></ul>Cryptosporidium parvum
    3. 3. <ul><li>Cryptosporidium parvum is another apicomplexan parasite </li></ul><ul><li>It is an enteric, anthroponotic and zoonotic parasite </li></ul><ul><li>One of the three most common diarrhea-causing pathogens in the world. </li></ul><ul><li>One of the most common causes of waterborne illness in the world. </li></ul><ul><li>Causes a self-limiting diarrhea in immunocompetent individuals. </li></ul>Cryptosporidium parvum
    4. 4. <ul><li>G.D: Cosmopolitan </li></ul><ul><li>Definitive Host: Human </li></ul><ul><li>Reservoir Hosts: kittens, puppies, goats, calves, mice, …. Etc. </li></ul><ul><li>Disease: Cryptosporidiosis . </li></ul><ul><li>Habitat: Epithelial cells of the gastrointestinal tract, mainly jejunum </li></ul><ul><li>Transmission: Fecal-oral route. </li></ul>Cryptosporidium parvum
    5. 5. Transmission <ul><li>Food and water </li></ul><ul><ul><li>contamination of drinking water </li></ul></ul><ul><ul><li>Swimming pools </li></ul></ul><ul><ul><li>untreated groundwater or well water </li></ul></ul><ul><ul><li>an infected person or an asymptomatic carrier contaminates a food supply </li></ul></ul><ul><li>Animal-person transmission </li></ul><ul><li>Person-person (anthroponosis) transmission </li></ul><ul><ul><li>occurs at a high frequency in day-care centers. </li></ul></ul>
    6. 6. <ul><li>Trophozoites are round or oval intracellular, but exocytoplasmic forms </li></ul><ul><li>have a single prominant nucleus </li></ul><ul><li>Measure 2.0 to 2.5 µm in diameter. </li></ul><ul><li>They are transitional stages from sporozoites and merozoites to meronts </li></ul>Trophozoites TEM
    7. 7. Sporozoites Cryptosporidium parvum <ul><li>Sporozoites are generally crescent or bow shaped, </li></ul><ul><li>with the anterior end slightly pointed and the posterior end rounded. </li></ul><ul><li>Measure 4.9 by 1.2 µm </li></ul><ul><li>Each sporozoite contains a prominent nucleus in the posterior third of the body. </li></ul>TEM
    8. 8. <ul><li>Oocysts are double walled, rounded or ovoid and measure 5 to 6 µm in diameter.  </li></ul><ul><li>Contain bow-shaped 4 sporozoites, are sometimes visible inside the oocysts, indicating that sporulation has occurred. </li></ul>sporozoite release from the C oocyst
    9. 9. Life cycle: Cryptosporidium parvum
    10. 10. rounded SEM of type 1 meront SEM of type 2 meront
    11. 11. Mouse small intestine infected with Cryptosporidium parvum . Small bodies seem to be on the surface of epithelial cells are various developmental stages of C. parvum enveloped by host cell membrane. H & E stain.
    12. 12. TEM of Immature oocyst of C. parvum . TEM of Merozoites of C. parvum. SEM of oocysts of C. parvum .
    13. 13. TEM: Meront stage of Cryptosporidium parvum under asexual division (endopolygeny). TEM: Mature type I meront including eight merozoites.
    14. 14. Symptoms <ul><li>In immunocompetent patients, include : </li></ul><ul><ul><li>Frequent, watery diarrhea (1-2 week duration), yet self-limiting </li></ul></ul><ul><ul><li>Nausea, Vomiting , Abdominal cramps, and Low-grade fever </li></ul></ul><ul><li>For immunocompromised persons, the illness is more severe: </li></ul><ul><ul><li>Debilitating, cholera-like diarrhea (up to 20 liters/day), electrolyte imbalance, dehydration and sometimes death. </li></ul></ul><ul><ul><li>Severe abdominal cramps, Malaise, Low-grade fever, Anorexia and Weight loss. </li></ul></ul>
    15. 15. Diagnosis <ul><li>Microscopy with an acid fast stained stool smear, which will stain the oocysts bright red. </li></ul><ul><ul><li>Another form of microscopy is fluorescent microscopy using monoclonal antibody to oocyst wall </li></ul></ul>acid-fast oocyst stain Direct immunofluorescence antibody stain
    16. 16. Diagnosis <ul><li>Enzyme immunoassay (ELISA), for the detection of cryptosporidial antigens in stool samples, . has greatest sensitivity and specificity </li></ul><ul><li>Molecular methods using PCR </li></ul>
    17. 17. Diagnosis Lane S: Molecular base pair standard (100-bp ladder). Black arrows show the size of standard bands. Lane 1: C. parvum positive fecal specimen. The red arrow shows the diagnostic band for Cryptosporidium parvum zoonotic genotype (size: 435 bp). Cryptosporidium parvum
    18. 18. References <ul><li>Chiodini PL, Moody AD, & Manser DW. (Atlas of Medical Helminthology and Protozoology. 4 th ed. Churchill Livingstone, Edinburg. </li></ul><ul><li>Smith HV, Cacciò SM, Cook N, Nichols RA, Tait A. (2007). Cryptosporidium and Giardia as foodborne zoonoses. Vet Parasitol. 149(1-2):29-40. </li></ul><ul><li> </li></ul><ul><li> </li></ul><ul><li> </li></ul><ul><li> </li></ul><ul><li> </li></ul>
    19. 19. References <ul><li> </li></ul><ul><li> </li></ul><ul><li> </li></ul><ul><li> </li></ul>
    20. 20. Next Lab <ul><li>Blood-dwelling Apicomplexa: </li></ul><ul><li>Plasmodium spp. </li></ul>