Fascioliasis Leigh Tyndall BIOL 402

What is Fascioliasis? WHO has classified it as a neglected tropical disease Zoonosis –started off as an animal disease and spread to humans Largely effects the liver, specifically the bile ducts, but can occasionally end up in other random places…this is called ectopic fascioliasis Still considered a rare disease in humans

Fascioliasis is caused by trematode worms F. gigantica F. hepatica Hepatica – 20-30 mm long and 13 mm wide Gigantica – 25-75 mm long and 12 mm wide Adults are hermaphroditic and can produce thousands of eggs in a day

F. hepatica – Europe, America (specifically the south and west), Australia – more temperate areas F. gigantica – Asia and Africa – more tropical areas (NOTE: this map seems to have gotten it backwards…all my other sources say it’s the other way around) 2.4 million people infected 180 million people are at risk A lot of cases are probably not reported, especially in Africa (note question marks) This is mostly in the developing countries, and not developed ones, as a neglected disease, it has largely been eliminated from the developed world and so is largely forgotten in the developing world

The Fasciola Life Cycle Fluke eggs are contained in the feces of the infected organism. Once it gets into the water by fecal contamination or washed out by heavy rains, the embryo (miracidium) begins to develop (and not before, though it can last for a while in the feces) The eggs hatch to release larvae (called miracidium) The larvae have to find a host immediately because they cannot survive longer than 24 hours on their own, so they infect snails (usually the Lymnaea) Inside the snail, they transform into a sporocyst and migrate to the digestive gland, also known as the liver when they hatch, they produce larvae (rediae) that are much more active than the sporocyte and so damage the liver considerably more. Once fully developed into the third stage within the snail liver (cercariae), these organisms then escape inot the water These cercariae encyst 1-2 minutes to 2 hours onto aquatic plants and develop into metacercariae. Humans and animals are infected by eating these plants uncooked; the immature flukes are released from the metacercariae cysts within an hour in the small intestine. The flukes penetrate the intestinal wall and travel throught the abdominal cavity to the liver It usually takes the flukes 4-6 days to reach the liver; after they do, the burrow into the liver where they grow in size 7 weeks into the infection, the flukes reach the bile ducts in the liver where they grow and mature into adults 8 weeks into the infection, eggs are found in the bile and then feces of the infected organism NOTE: the more flukes that infect, the longer it takes them to mature

There are several ways to get infected… raw aquatic plants- ingesting encysted forms of the fluke in water plants (such as raw watercress, water chestnuts, or bamboo shoots). Drinking water with the unattached larvae in it or eating from dishes to which the larvae have connected Fecal-oral transmission Potential human-to-human transmission? (-WHO) Reservoirs aquatic plants Snails (intermediate hosts) domestic farm animals (pigs, cows, sheep, donkeys, horses, camels, goats etc)

There are several ways to get infected… Fecal-oral transmission raw aquatic plants- ingesting encysted forms of the fluke in water plants (such as raw watercress, water chestnuts, or bamboo shoots). Drinking water with the unattached larvae in it or eating from dishes to which the larvae have connected Potential human-to-human transmission? (-WHO) Reservoirs aquatic plants Snails (intermediate hosts) domestic farm animals (pigs, cows, sheep, donkeys, horses, camels, goats etc)

There are several ways to get infected… Fecal-oral transmission raw aquatic plants- ingesting encysted forms of the fluke in water plants (such as raw watercress, water chestnuts, or bamboo shoots). Drinking water with the unattached larvae in it or eating from dishes to which the larvae have connected Potential human-to-human transmission? (-WHO) Reservoirs aquatic plants Snails (intermediate hosts) domestic farm animals (pigs, cows, sheep, donkeys, horses, camels, goats etc)

There are several ways to get infected… Fecal-oral transmission raw aquatic plants- ingesting encysted forms of the fluke in water plants (such as raw watercress, water chestnuts, or bamboo shoots). Drinking water with the unattached larvae in it or eating from dishes to which the larvae have connected Potential human-to-human transmission? (-WHO) Reservoirs aquatic plants Snails (intermediate hosts) domestic farm animals (pigs, cows, sheep, donkeys, horses, camels, goats etc)

There are several ways to get infected… raw aquatic plants- ingesting metacercariae forms of the fluke in water plants (such as raw watercress, water chestnuts, or bamboo shoots). – Drinking water with the unattached larvae in it or eating from dishes to which the larvae have connected And how do the worms get into the water in the first place? Potential human-to-human transmission? (-WHO) Reservoirs Aquatic plants Snails (intermediate hosts) domestic farm animals (pigs, cows, sheep, donkeys, horses, camels, goats etc) Next slide is gross

Fasciola (along with most helminths) easily evade the physical barriers to infection Most physical immune barriers (mucus membranes, skin, gastric acid, body temperature) aren’t effective against Fasciola - they are ingested usually - gastric acid helps the worms excyst from the metacercariae

Fasciola have evolved several ways to avoid rest of the immune system as well. First they go to the bile ducts in the liver. Once there, they is safe from any immune response and the liver bile protects any eggs or antigens that might present themselves as they travel away from the liver Before then: the fluke has a one surface membrane covered by many glycoproteins. The composition of this glycocalyx changes as the fluke develop, most likely because of the changes in environment it goes through and also to present the antibodies with a constantly changing target. In juveniles, this happens every 3 hours or so. Another theory is that the flukes secrete immunosuppressive proteins that regulate or modify the host immune response but these proteins have not yet been identified. It could also be that the flukes secrete proteinases that cleave the antibodies sent to catch them

In the first phase of fascioliasis, the worms migrate across the liver Called the acute phase hemorrhaging and inflammation, fever, abdominal pain, gastrointestinal problems, occasionally coughing and chest pain Lots of scarring where the migration has occured Here you can see a lesion that’s migrating across the liver There is also a latent phase, which is asymptomatic and can last for months or years

The chronic phase affects liver function Chronic phase: can develop months to years after the initial infection reach bile ducts – can get thickening and calcification of said ducts, inflammation leads to thickening of ducts and gallbladder, fibrosis – which is the creation of excess connective tissue in an attempt to heal eventual blockage due to parasites or parasite fragments which results in biliary colic pain – similar to having a stone in your gall bladder can also cause jaundice and anaemia This is why they call it liver rot It is assumed that humans are not the ideal host, because a lot of the flukes die in the liver parenchyma without reaching the bile ducts. Very low mortality rate in humans- if they do die (a few cases have been reported) it’s usually because the bile duct is blocked However, they can die of complications, such as bleeding from the haemmorhaging

Fascioliasis is usually diagnosed by finding eggs in the feces Best test: Detecting eggs in stools is the best but this is difficult and unreliable in humans - humans are not the ideal host and so flukes may exist but if they don’t grow to maturity, then they won’t release eggs ultrasound, CT scans and MRI – these are very expensive though Immunoserological tests(like some of the papers we’ve read) used to find it in your blood

Two doses of triclabendazole will treat both stages of fascioliasis Triclabendazole at 10mg/kg body weight once “drug of choice. The drug works by binding to the Beta tubulin and therefore preventing the polymerization of the molecule tubulin into the microtubules, which support the cell. This makes movement of secretory vesicles impossible and therefore the fluke can’t renew that glycoprotein coat and keep the integrity of the membrane. Also, the cells can’t move For humans, the drug is called Egaten OR bithionol at 30-50 mg/kg every other day for 10-15 doses (taken orally); might take two treatments 80-100% chance of being cured! WHO and Novartis Pharma AG (pharmacy company) have an agreement where countries in need can apply for the drug and will receive it for free

There is already resistance to triclabendazole, though… Resistance in Australia and Ireland A paper came out in 2006 which showed some thought had gone into this issue, as triclabendazole is currently the only drug available for human treatment. Think there might be different isotypes of Beta tubulin, In comparing proteins taken from susceptible and resistant flukes shows that resistant flukes have similar beta tubulin protein sequences as beta tubulin in nematodes resistant to their own version of bendazole.

Societal Effects In places with unsafe water, poor sanitation, limited access to basic healthcare like most of these neglected tropical diseases, once you get it you’re out of commission for a while Detrimental to animal husbandry High risk of reinfection because of contaminated pastures and it’s really hard to get rid of an infestation This is a problem because, as previously mentioned, the methods of diagnosis are either unreliable in humans

Several molecules have been looked at as possible vaccines Fatty Acid Binding Proteins GST Capthepsin L Cerceriae – free-swimming larval stage of a Trematode Interesting: when lymphoid cells were taken from vaccinated sheep and put into un-exposed sheep, they conferred resistance to infectioion Irradiated attenuated cercariae and metacercariae Effective in cattle and rats, not so much in sheep Found antigens from sera from sheep injected with irradiated metacercariae: 1. Most promising: Fatty Acid Binding Proteins: found by George Hillyer in 1970s – protected from many flukes (including related S. mansoni) involved in binding and transportation of hydrophobic lipids (bile acids!) 2. GST: glutathione S-transferases – cellular detoxification, found at high levels in all helminth parasites, so presumably it’s important for their survival and therefore a good target 3. Capthepsin L: protease, promotes tissue penetration, aids in acquiring nutrients, immune evasion NOTE: None of these has been tried in humans

Measures should be taken to prevent infection by the flukes Control of aquatic plant industry Wash aquatic plants in vinegar Health Education Clean water

Image References Slides 2 and 3 Wikipedia: http://en.wikipedia.org/wiki/Fasciolosis (copyright: Flukeman-http://commons.wikimedia.org/wiki/User:Flukeman) WHO: http://www.who.int/neglected_diseases/diseases/fascioliasis/en/index.html Water Secrets Blog: http://watersecretsblog.com/archives/children%20dirty%20water.jpg Parasites World: http://parasites-world.com/fasciola-hepatica-in-human-eye/ Slide 4: http://iranhelminthparasites.com/species/fasciola.htm Slide 5: Food and Agriculture Organization of the UN: www.fao.org/docrep/ 004/T0584E/T0584E05.gif Slide 6: CDC website: http://www.dpd.cdc.gov/dpdx/HTML/Fascioliasis.htm Slide 7: Water Aid.org: http://www.wateraid.org/images/cm_images/uk/what_we_do/the_need/problems_for_women/woman_drinking_dirty_water_UG_45151.jpg\ Slide 8: Nassau Lake: http://nassaulake.org/Water%20Chestnut.jpg Slide 9: Hardwood Bamboo Flooring: http://www.hardwood-bamboo-flooring.com/wp-content/upload/Bamboo-300x273.jpg Slide 10:Essentially England: http://www.essentially-england.com/images/watercress_2.jpg Slide 11: FAO website http://www.fao.org/docrep/006/Y4743E/y4743e07.jpg Slide 12: http://en.wikipedia.org/wiki/Fasciolosis (copyright: Flukeman-http://commons.wikimedia.org/wiki/User:Flukeman) Slide 13: http://www.fao.org/docrep/003/t0756e/T0756E06.htm Slide 14: http://radiographics.rsna.org/content/28/5/1307/F5.large.jpg Slide 15: http://en.wikipedia.org/wiki/Fasciolosis (copyright: Flukeman-http://commons.wikimedia.org/wiki/User:Flukeman Slide 16: http://www.nhm.ac.uk/resources-rx/images/liver-fluke-egg_52390_1.jpg Slide 17: Triclabenzadole: wiipedia http://en.wikipedia.org/wiki/File:Triclabendazole.png Vietnamese Institute of Malariology, Parasitology, and entomology quynhon: http://www.impe-qn.org.vn/impe- qn/vn/portal/InfoDetail.jsp?area=58&cat=1174&ID=2809 Slide 18: Tubulin: Brennan GP, Fairweather I, Trudgett A, Hoey E. 2007. Understanding triclabendazole resistance. Experimental and Molecular Pathology 82:104-9. Slide 19: Patients coming for treatment: http://www.impe-qn.org.vn/impe-qn/en/portal/InfoDetail.jsp?area=58&cat=1067&ID=511 Cows: http://www-naweb.iaea.org/nafa/aph/stories/2010-fasciola-project.html Cow with flukes http://www.nhm.ac.uk/nature-online/species-of-the-day/fasciola-hepatica/index.html Slide 20: Vaccine: http://alignmap.com/wp-content/Graphics/vaccine.gif Slide 21: Market: examiner.com: http://www.examiner.com/x-7707-Infectious-Disease-Examiner~y2009m9d14-Liver-flukes-showing-its-presence-in- the-Vietnamese-highlands

Sources Brennan GP, Fairweather I, Trudgett A, Hoey E., et al. 2007. Understanding triclabendazole resistance. Experimental and Molecular Pathology 82:104-9. Dalton JP, editor. 1999. Fasciolosis. New York: CABI Publishing. 544 p. Hanna REB. 1980. Fasciola hepatica: Glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity. Experimental Parasitology 50(1):103-14. UK’s Natural History Museum: http://www.nhm.ac.uk/nature-online/species-of-the- day/fasciola-hepatica/index.html Turkington C, Ashby B. 1998. Encyclopedia of infectious disease. New York: Facts on File, Inc. 370 p. WHO website: http://www.who.int/neglected_diseases/diseases/fascioliasis/en/index.html