Nematodes

1. Nematodes • Are surrounded by a secreted multilayered cuticula, have only longitudinal muscles which are organized in quadrants and a ‘pressurized’ pseudocoel • A simple linear intestine made up of columnar cells with a muscular pharynx that pumps in food against the internal pressure • A linear reproductive tract along which germ line cells go through meiosis and mature into gametes (amoeboid sperm, eggs surrounded by an impermeable shell).

2. Nematodes • Believe in order and fate. They develop to a defined number of cells and follow a highly deterministic developmental program. • Four larval stages and adult, molt at each developmental step. The L3 stage can developmentally arrest and ‘hang in’ waiting for better times or the next host. • Different life cycle stages can differ dramatically in their metabolism (aerobic larvae, anaerobic adults)

3. Nematodes or round worms 2 GI Nematodes: Trichuris, Ascaris & Hookworms Worm expulsion and the Th2 immune response The hygiene hypothesis of allergy Trichinella & the first modern public health campaign

4. The importance of gastro-intestinal (GI) nematode infections • Roughly halve of all people are currently infected with GI-nematodes • Infection is generally associated with low mortality, but there can be considerable morbidity

5. The importance of GI nematode infectioins Children are especially at risk to suffer from GI nematode related morbidity.

6. The WHO helminth control program for school aged children • Children are continuously exposed to infection • Children grow rapidly and are especially susceptible to infection and the associate morbidity • Chronic helminth infection seems to have a negative impact on cognitive development • 10 cents annually per child covers drug cost • The program is heavily based on drug treatment

7. Trichuris trichuris (human whip worm) • Adults live in cecum where the female worm produces 20,000 eggs per day which are shed with the feces • Embryonation occurs within 3 weeks in soil • Infection is through ingestion of eggs, larvae hatch and penetrate the mucosa and tunnel it • Larvae grow and molt 4 times until mature

8. Trichuris • Adults remain embedded with their thin front end in the mucosa • Infections with less than 10 worms are asymptomatic • Moderate to heavy infections cause abdominal pain, diarrhea, weight loss • Heavily infected children may have profuse bloody diarrhea, cramps, tenesmus and urgency which may result in rectal prolapse • Adults can live for years so worm burden can build up Trichuris vulpes (dog whip worm in the intestine of a heavily infected dog)

9. Trichuris • Diagnosis by demonstration of characteristic eggs (“champagne corks”) • In moderate infections eggs might be hard to find and concentration techniques might be required • Treatment with mebendazole • Sanitary disposal of human feces prevents disease

10. Ascaris lumbricoides -- the human round worm • Large intestinal nematodes (Ascaris lumbricoides infects humans, A. suum infects pigs, the species are almost indistinguishable, but have clear host specificity) • Males are 15-31 cm and have incurved posterior end, females are 20-49 cm • The anterior end of both sexes shows three lips • One billion people infected

11. Ascaris • Fully embryonated eggs are swallowed and L2 hatches in the stomach and penetrate stomach or duodenal mucosa • Larvae enter blood stream and leave through alveoli into lung • Larvae molt several times in the lungs L3/L4 move up and get swallowed • 2-3 months after infection the adult worms start laying eggs (200,000 daily) • Eggs are shed with the feces and embryonate within 2-3 weeks

12. Ascaris • Infection depends on fecal contamination of food, water or soil • Eggs are sensitive to sun light but otherwise extraordinarily resistant (ascarosides - special glycolipids secreted by the embryo) • Fertilized eggs are shorter and rounder than unfertilized

13. Ascaris • Occasional pulmonary symptoms • Intestinal phase mostly asymptomatic, but worms can lead to a developmental slow down in children • Dangerous complications are mostly observed in children under 10 • Volvulus, a mass of knotted worms obstructing the intestine • Penetration of the bile duct and liver by adult worms • Penetration of the intestinal wall, followed by peritonitis • Wandering and obstruction can be an unwanted side effect linked to certain medications

14. Acylostoma duodenale & Necator americanus -- human hookworms • Small nematodes (1-1.5 cm) • Head is slightly bend (hook) and the ‘mouth’ carries characteristic teeth (Ancylostoma) or plates (Necator, note that these are not real teeth but cuticular formations of the ‘buccal capsule’) • The posterior end of the male worm is elaborated into a copulatory bursa

15. Hookworms • Ancylostoma is found in Europe around the Mediterranean, on the West coast of South America and in parts of China and India • Necator is found over much of the western hemisphere, Africa and South East Asia • More than a billion people infected

16. Hookworms • Adult worms live in the small intestine and female lay 5-10000 eggs a day over 5 years • Eggs are passed with the feces • Larvae develop outside the body and molt twice • The filariform or L3 larvae move to the surface in search for a host • If they come into contact with the host they penetrate the skin, enter blood vessels and leave the circulatory system into the alveoli • The lavae move up the trachea into the esophagus, are swallowed and finally reach the intestine, where they molt twice more before they reach maturity Infectious L3

17. Hookworms • Skin penetration and associated secondary bacterial infection can result in “ground itch” • Pulmonary phase is usually asymptomatic • Intestinal phase: worms attach to the mucosa and feed on blood. Worms continuously move to new places exacerbating bleeding

18. Hookworms • The main concern with hook worm disease is blood loss • 0.03 ml (N.a.) to 0.26 ml (A.d) per worm, up to 200 ml per day in heavy infections • Chronic heavy infection results in anemia and iron deficiency • Together with malnutrition infection can severely stunt growth and development in children • Anemia leads to weakness and fatigue in adults • Antihelminthic treatment with mebendazole

19. Creeping eruption or cutaneous larva migrans • Humans occasionally get infected by L3 larvae of dog and cat hookworms (e.g. Ancylostoma caninum or brazilienzis again by skin penetration) • The larvae can not establish a productive infection in humans, but wander about in the subcoutaneous tissue, causing significant inflammation and painful swelling • Responds well to treatment

20. Hookworms • Acylostoma was commonly found in coal mines throughout Europe • Heavy infection rates due to ‘perfect’ conditions for transmission in mines could result in severe anemia and pathology • At the turn of the 19th century, severe hookworm disease was an officially recognized occupational hazard in German coal mines and miners with the disease were eligible for workman’s compensation

21. Hookworms • Hookworms were wide spread in the Southern USA (read Jimmy Carter’s boyhood biography for detail on hookworm and other infectious diseases in rural Georgia) • Hookworm control programs were a big part of the public health campaign in the South during and right after World War II • 1972 12% of school children from rural costal Georgia tested positive for hookworm infection Nurse brings hookworm medicine to a rural Alabama family in 1939 (National Library of Congress photo LC-USF34- 051377-D)

22. The immune response and GI nematodes • Nematode infections are usually chronic and have high rates of reinfection • This suggests that the worms have developed effective ways to a) avoid getting killed by the host and b) they are not inducing overly strong inflammatory responses, which could kill the host • However there are data from human epidemiological studies which indicate that a certain degree of protective immunity might develop (at least in some diseases) • The many variables in the study populations make it hard to develop sound models.

23. Mouse models to study GI nematode immunity • The use of a mouse model allows control over many factors variable in human studies • The mouse immune system is very well studied and many reagents are available • A huge collection of so called knock-out mice with defined defects in specific aspects of their immune system can be used/

24. Lymphocytes are required for worm expulsion • Mice are infected with Trichuris muris eggs • After 11 days many worms can be detected, but after 33 days all adult worms are cleared • SCID mice lack T and B cells and can not expel worms • Reconstitution of SCID mice with naïve lymphocytes from a normal mouse restores ability to control infections • This approach of ‘taking it apart and then putting it back together’ has been very powerful to dissect the immune system and its responses to various pathogens

25. A quick review of Dr. Tarleton’s immunology intro Worm expulsion requires T-cells, more specifically a certain flavor of T helper cells

26. T-helper cells can develop into two flavors (TH1 or 2) • The immune system and its cells make important choices • The cells of the immune system talk to each other through messenger proteins called cytokines • T-cells both make and respond to many cytokines • Stimulation with different cytokines leads to the development of two types of T-helper cells specialized for orchestrating two very different immune responses • Th1 and Th2 strongly down-regulate each other • This polarization has important consequences for the downstream response and can spell life or death

27. TH1 cells promote cytotoxicity and destruction of intracellular pathogens • TH1 cells orchestrate a response directed to inhibit intracellular pathogens like viruses, certain bacteria (e.g. TB), or certain protozoan parasites (Leishmania - see later lecture) • They secret Interferon gamma which activates intracellular killing mechanisms • They activate macrophages and cytotoxic T-cells

28. TH2 cells promote neutralizing antibodies and mast cell activity • TH2 cells suppress the activation of macrophages and activate eosinophiles and mastcells • TH2 cells promote a strong antibody response based on neutralizing IgGs and IgEs • A TH2 response is most effective to combat extracellular pathogens

29. The key to expulsion is mounting the “right” response • Certain inbred mouse strains mount a protective response to Trichuris muris resulting in expulsion (resistant), others develop chronic infections with worm persisting in the gut (susceptible) • Susceptible mice develop a strong immune response, but it’s a Th1 response • If however these susceptible mice are treated to artificially shift to a Th2 response they clear the infections • The immune system is not hard wired but makes important choices which can control the outcome of an infection

30. Physiological changes induced by anti-worm immunity • Recent work show strong effects of the anti-worm immune response on the gut physiology • Instead of directly killing the worms these physiologial changes appear to render the intestine inhospitable for the parasite • Increase in number of goblet cells (these are the cells in the intestinal epithelium which produce mucus - strong red stain in the histology image) • Increase in mucin secretion • Increase in intestinal mobility • Increase in water influx into the intestinal lumen (some of these seem to be directly induced by cytokines and immune cells like mast cells)

31. Worm expulsion • It is associated with a type 2 (TH2) response • The effector mechanisms include immunological and physiological processes • Many worms have found ways to suppress or modulate the immune system • This allows them to dodge an effective immune response and/or prevent the potential demise of their mammalian host due to massive inflammation

32. Immune modulation by worms can be detrimental or beneficial • Numerous recent epidemiological studies show that certain vaccines are less effective in children that are infected with worms than those that have been cured using drugs (these are mostly vaccines that require a robust TH1 response) • This is backed up by many studies in mice that use vaccination or co-infection • On the other hand worm infection can dampen autoimmune diseases and allergies (diseases due to an ‘over-active’ immune system)

33. The Hygiene Hypothesis • There has been a considerable increase in the diagnosis of autoimmune diseases and allergies over the second half of the 20th century • Prevalence of allergies in urban areas appears higher than in rural environments • Environmental factors like pollution, nutrition etc. can be important for specific allergies but have shown little consistent overall association with allergies and autoimmunity • Childhood infections though show strong negative correlation with both autoimmune disease and allergies

34. Inverse correlation of type I diabetes and chronic infectious diseases Red delineates areas which harbour six or more of the low mortality neglected diseases (filariasis, leprosy, onchocerciasis, schistosomiasis, soil-transmitted helminths, and trachoma). Yellow delineates areas where there are relatively high incidences of T1D (> 8 per 100 000/year). Non coloured areas delineate where T1D < 8 per 100 000/year and where the ‘neglected diseases’ are not endemic.From: Zaccone et al. Parasite Immunol. 2006 28:515–523.

35. Inverse correlation of allergies with infections • Among kids in various studies in different areas of the world around 30% have antibodies against dust mite allergen (suggesting they all are exposed) • But whereas asthma is found in 12% of kids from Europe and Australia, only 3% have asthma in Gambia and Nigeria

36. The Hygiene Hypothesis • High pathogen burden stimulates the immune system to develop a robust regulatory network that keeps inflammation in check • Worms set up long lasting chronic infections, they induce strong TH2 responses and promote regulation of this process • Understanding this process in detail could point to new allergy interventions

37. The Hygiene Hypothesis: Treatment by worm infection? • Several clinical trials using Trichuris suis have been conducted to treat human Crohn’s disease or ulcerative colitis (autoimmune inflammation of the intestine) • T. suis does not productively infect humans yet modulates the immune response • Most studies show clinical improvement for a significant fraction of the patients

38. The Hygiene Hypothesis: Treatment by worm infection? • One example (double blind study with 54 patients suffering from acute ulcerative colitis): The patients received either a placebo or 2500 T. suis ova every 2 weeks for 12 weeks. • 43.3% of the patients given T. suis improved compared with those given placebo (16.7%). • The study also included a 12 week crossover limb where patients originally on placebo where switched to T. suis and those on T. suis were switched to placebo. In the crossover limb, 56.3% of the patients given T. suis improved compared with 13.3% of patients given placebo (see Elliot et al. (2007) International Journal for Parasitology 37: 457-464 for detail) • Don’t try this at home!

39. Trichinella spiralis • Trichinosis is caused by infection with Trichinella spiralis • (additional morphologically indistinguishable species have now been described for sylvatic cycles) • Infection of all hosts occurs through larvae encysted in muscle tissue (carnivorism, salvage and or cannibalism is needed to maintain transmission)

40. Trichinella • The larva is freed from its nurse cell, and enters the mucosa of the small intestine • Larvae grow, molt 4 time and young adults copulate within 32 hours of infection • The females give birth to live L1 larvae while tunneling the epithelium • Females die after 4-16 weeks

41. Trichinella • The larvae enter blood vessels and are carried throughout the body • When they pass skeletal muscle (they prefer highly active muscles) they leave the vessels and penetrate the muscle and the muscle cells

42. Trichinella • The larvae enters a muscle cell and lives as an intracellular parasite developing in the cytoplasm of the host cell • The larvae manipulates the host cell to its needs (probably by secretion of suitable effector proteins, the molecular mechanism is not well understood) • The end product is a nurse cell • A fine net of blood vessels forms around the nurse cell (angiogenesis)

43. Trichinella • The host cell looses its myofilaments and several additional subcellular changes occur • Both host cell and worm are enclosed by a collagen capsule (collagen mRNA has been detected in nurse cell, but some authors suggest the capsule is secreted by surrounding fibroblasts

44. Trichinella • Strong dose dependence of symptoms (>1200 larvae can be lethal) • Activity of the females can cause inflammatory reaction in the mucosa, leading to diarrhea and abdominal pain • Parasitized muscle exhibits intense inflammation, cyst start to calcify in humans after 6 months

45. Trichinella • Muscle invasion stage begins in the second week after infection • Fever and perorbital edema are followed by myalgia (muscle pain) and weakness • Characteristic splinter hemorrhages can be found under finger nails • Fever and chills can persist for weeks • Headache is common and dizziness may develop • Muscle swelling, aching and tenderness occurs often • Deaths are rare and due to myocarditis (inflammation of the heart muscle), encephalitis and pneumonia (larvae in the diaphragm)

46. Trichinella control was one of the first effective modern public health measures • Trichinella was discovered in 1835 and linked to human disease 1860 • Several large outbreaks in Germany resulted in the formation of an expert commission in Berlin which included Rudolf Virchow one of the co-discoverers of the Trichinella life cycle • Virchow suggested mandatory inspection of all meat at the slaughterhouses a practice that was introduced in Prussia in 1877 • This has greatly reduced trichinellosis as human health problem

47. Trichinella • Domestic infection through pigs has become rare in the US and Europe • Meat control, rat control, prompt removal of dead animals, limit access of wildlife to pigs • Almost all case now are sylvatic transmission from game

48. Aniskiasis • Humans are accidental host of several nematodes which reach maturity in marine mammals • Infection through consumption of uncooked fish • Larvae often penetrate gastric and intestinal mucosa causing pronounced inflammation which can mimic Crohn disease • Rapid onset of symptoms often within hours • Diagnosis by endoscopy, surgical or endoscopic removal of larvae in cases with severe symptoms