Paper No. : 08 Biology of Parasitism Module : 25 Life Cycle, Transmission and Pathogenicity of Ancylostoma duodenale.

Transcription

1 Paper No. : 08 Module : 25 Development Team Principal Investigator: Co-Principal Investigator: Paper Coordinator: Content Writer: Content Reviewer: Prof. Neeta Sehgal Head, Department of Zoology, University of Delhi Prof. D.K. Singh Department of Zoology, University of Delhi Dr. Pawan Malhotra ICGEB, New Delhi Dr. Sarita Kumar Acharya Narendra Dev College, University of Delhi Prof. Rajgopal Raman Department of Zoology, University of Delhi 1

2 Description of Module Subject Name Paper Name ; Zool 008 Module Name/Title Module Id Keywords Gastro-Intestinal Nematodes M25 Ancylostoma, Filariform, Dermatitis, Ground itch, Anaemia, Ancylostomiasis Contents 1. Learning Outcomes 2. Introduction 3. Discovery of Ancylostoma 4. Geographical Distribution of Ancylostoma 5. Characteristic Features of Ancylostoma 5.1 Habit and Habitat of Ancylostoma 5.2 Morphological Features of Adult Ancylostoma 6. Life Cycle and Development of Ancylostoma 6.1 Copulation 6.2 Fertilisation 6.3 Passage of the Eggs from the Infected Host 6.4 Hatching of the Larvae 6.5 Development of Larva in Soil 6.6 Entrance into a New Host 6.7 Larval Migration after Entrance 6.8 Maturation into Adults 7. Mode of Infection of Hookworm 8. Factors Affecting the Transmission of Hookworm 8.1 Critical Stages in Life Cycle 8.2 Geographic Features 8.3 Lack of Sanitation 8.4 Unawareness 9. Pathogenicity 9.1 Pathogenic effects caused by Larvae 9.2 Pathogenic effects caused by Adults 10. Diagnosis of Ancylostomiasis 2

3 10.1 Microscopic Examination 10.2 Study of Duodenal Contents 10.3 Clinical Diagnosis 11. Immune Response in Hookworm Infection 11.1 Immune Response to Invasive Larvae 11.2 Immune Response to Adults 12. Prophylaxis and Treatment of Hookworm Infection 12.1 Prophylaxis 12.2 Treatment 12.3 Vaccines against Hookworms 13. Summary 1. Learning Outcomes After studying this Unit, you will be able to realize the medical importance of Ancylostoma identify the parasite and distinguish between male and female from its morphological features recognize the monogenetic nature of parasite and comprehend its life cycle understand the pathogenic effects and the diseases caused by worm be aware of the host immune response against the parasite gain knowledge of genomics and proteomics of Ancylostoma appreciate the various methods of prevention and control of parasite 2. Introduction Ancylostoma is a thread-like sanguivorous parasitic nematode of human beings. It lives in the small intestine of man and is commonly known as Old World Hook Worm. It is well known as the causative worm of ancylostomiasis or Hook worm disease which is characterized by severe anaemia and gastrointestinal problems. 3. Discovery of Ancylostoma Records of hookworm exist as early as the third-century B.C. when the authors of the Hippocratic Corpus described a disease characterized by intestinal disorders, pale complexion and liking to eat dirt. Ancylostoma was discovered by an Italian Physicist, Angelo Dubini (December 8, 1813 March 28, 1902). He noticed the worm in 1938, when he was doing an autopsy of a woman. In succeeding years, he observed the parasite during other autopsies too and named the parasite Ancylostoma. He also described the hookworm s teeth in great detail and published his findings in Annali universali di medicina in

4 Gradually, the relation between the worms and disease was established in 1854 by Wilhelm Griesinger. Thereafter, in 1879 that the Italian doctor, Edoardo Perroncito established the factual connection while investigating the diseases in miners. The entry of worms in the human body was discovered by Arthur Looss in 1898, when he accidentally infected himself. (a) (b) Fig. 1 (a) Wilhelm Griesinger, (b) Edoardo Perroncito Source: Geographical Distribution of Ancylostoma Ancylostoma is largely distributed in the tropical and sub-tropical regions of the world, especially in areas where humidity and temperature are favourable for their development, i.e. extending from parallel 36 N to parallel 36 S. Occurrence of the hookworm has been reported from India, Bangladesh, Europe, Sri Lanka, Egypt, Pacific Islands, North Africa, Southern states of US and Central and North China. In India it is more prevalent in Punjab, Haryana and Himachal Pradesh. 4

5 Fig. 2 Prevalence of hookworm infection worldwide Illustration: Margaret Shear, Public Library of Science (Diemert et al., 2008) 5. Characteristic Features of Ancylostoma Ancylostoma is a pseudocoelomate (coelomic cavity is not surrounded by mesoderm) cylindrical, thread-like nematode. The body is covered with a thick cuticle with syncytial epidermis. The systematic position of Ancylostoma is as under: Phylum : Nematoda Class : Phasmidia Genus : Ancylostoma Species : 5.1 Habit and Habitat of Ancylostoma The adult hookworms reside in the small intestine of human beings particularly in the jejunum. Though called, it is found less often in the duodenum and rarely in the ileum. It has been found on rare occasions in pigs. The adult worms anchor the wall of the small intestine by their anterior ends. 5.2 Morphological Features of Adult Ancylostoma Adult Ancylostoma are elongated, cylindrical and slender worms. They are greyish-white in colour but when freshly passed out from the body, the worms appear reddish brown due to ingested blood. The anterior end of the worms is bent slightly dorsally, because of which they are called hook worms. A few characteristic features of the worms are as follows: 5

6 Buccal Capsule: The worms possess well developed buccal capsule which is lined with a very hard chitinous substance. It is provided with six teeth; four teeth are hook-like present on ventral surface while two teeth are knob-like present on the dorsal surface. Fig. 3 Buccal capsule of Ancylostoma Source: Cervical Papillae: Minute finger-like cervical papillae is present, a little away from the anterior extremity, on each side of the body. Cephalic Glands: Worms contain two kinds of cephalic glands which are connected to the oesophagus. These are a small oesophageal gland and two pear-shaped cervical glands. The secretion of the oesophageal gland prevents clotting of the ingested blood. Sexual Dimorphism: Male and female Ancylostoma are separate and exhibit distinct sexual dimorphism. The female is larger than the male. It has a straight and pointed caudal end while male has a wider caudal end because of the presence of bursa copulatrix (copulatory bursa). Copulatory bursa is an invagination of the body-wall around the genital aperture and is supported by 13 rays. It has two protrusible spicules which help in sperm transfer during the copulation. Also, as in other nematodes, the digestive system and reproductive system of female adults open separately, via anus and gonopore; whereas, male Ancylostoma has single opening called cloaca for the both the systems. 6

7 Fig. 4: Outline structure of adult Ancylostoma (a) Female; (b) Male Source: The summary of the differences in the characteristic features of male and female Ancylostoma is presented in Table 1. Table 1: Difference between Male and Female Ancylostoma Characteristic Features Male Ancylostoma Female Ancylostoma Size Smaller than the female adult Longer than the male adult Length About 8 mm in length About 12.5 mm in length Posterior End Expanded in umbrella like manner, contains copulatory bursa Tapered end without any copulatory bursa Opening at posterior end Cloaca (Common opening of digestive and reproductive system) Anus (Opening of digestive system). Reproductive system opens separately Reproductive opening Opens with the cloaca At the junction of posterior and (Gonopore) middle third of the body Copulatory spicules Two pairs at the anal end Absent 7

8 6. Life Cycle and Development of Ancylostoma dodenale Ancylostoma is a monogenetic parasite. It completes its life cycle in single host, human being. 6.1 Copulation Copulation occurs in the intestine of the human beings. During copulation, the worms appear like a Y- shaped figure because of the position of the genital openings. The copulatory bursa of male is applied on the vulva of female. Male releases sperms in the female body which stores them in the seminal receptacles. 6.2 Fertilisation Fertilisation of the gametes takes places in the seminal receptacle of females. Fertilised eggs have the following distinctive features: Elliptical and colourless 65 µm in length and 40 µm in width. Surrounded by a hyaline shell membrane Contains a segmented ovum and a clear space between ovum and shell membrane The eggs are pushed into the uteri, pass into vagina and are laid through gonopore. During the movement, the ovum undergoes segmentation. (a) (b) Fig. 5: Eggs of Ancylostoma ; (a) Unsegmented stage (b) 4-celled stage Source: egg.jpg 6.3 Passage of the eggs from the infected host 8

9 A single female worm lays about 10 to 20 thousand eggs per day. These eggs are passed out of the human body along with the faeces. These eggs are not infective to another individual. 6.4 Hatching of the larvae The larvae hatch from the eggs in hours. Eggs need moist soil, warmth (27 C) and sufficient oxygen as optimum conditions for hatching. In case of adverse condition, eggs do not develop further. 6.5 Development of larva in soil First Stage larva: The newly hatched larva is called rhabditiform larva or first juvenile stage. It has been named so because of similarity to another nematode, Rhabditis. It is about 250 µm long, and possesses a rounded anterior end and a sharp caudal end. The larva is highly active and feeds ravenously on the bacteria in faeces and organic matter present in the soil. Second Stage larva: The larva grows rapidly and moults twice in one week. First moult takes place on the 3rd day leading to the formation of second juvenile stage. Fig. 6: Rhabditiform larva of Ancylostoma Source: Third Stage larva: The second moult occurs on the 5th day and the larva attains a length of 500 to 600 µm. The oesophageal bulb disappears and the shedded cuticle is retained as the protective sheath. It is now called a filariform larva or third juvenile stage. These larvae live in the upper ½ inch of the soil. The larva though active but stops feeding and does not grow further. 9

10 Fig. 7: Filariform larva of Ancylostoma Source: Development from egg to filariform larva takes about 8-10 days. The third juvenile stage or filariform larva is infective to man. 6.6 Entrance into a new host The life span of filariform larvae is quite short. Most of these larvae die within 2 to 3 weeks, the maximum life span being 6 weeks. The larvae are highly sensitive to adverse environment conditions of desiccation and extremes of temperature. These can also be easily destroyed by exposure to direct sunlight. Consequently, they need another host for further development. The filariform larvae gain entrance into another human being by penetrating the skin. The larva casts off its membranous sheath and bores directly through the skin or indirectly through the hair follicles. 6.7 Larval migration after entrance After several hours of infection, the larvae migrate through various human organs. (A) Migration through Lungs: Initially, the larvae enter lymphatics or small venules within 24 hours. Those which are unable to reach the vascular spaces usually die or are phagocytosed. They are then passively carried to the right ventricle of the heart. On about 3 day they enter the lungs from the heart through pulmonary artery. They then migrate in the alveoli by boring the wall of pulmonary capillaries where they are arrested owing to their large size. (B) Entry into alimentary canal: 10

11 The fourth stage larva now moves upwards through the alveoli of the lungs and reaches trachea. They are then propelled into the pharynx and are swallowed When they reach the oesophagus, the third moulting occurs with the formation of a temporary buccal capsule bearing 4 small teeth. The larvae now migrate into the stomach and enter the duodenum between the 7th and the 10th day. By this time, the larva grows rapidly and attains a size of 2 mm x 0.13 mm. 6.8 Maturation into adults The growing larva settles in the jejunum and undergoes the 4th and final moult on the 15th day. During the moult, the temporary buccal capsule is replaced by definitive buccal capsule with teeth. It gets attached to the wall of small intestine and starts feeding on the host blood. The worm matures sexually in 3-4 weeks and start laying eggs during 6th week. The video depicting the morphology and movement of Ancylostoma larvae can be seen on 11

12 Fig. 8: Detailed life cycle of Ancylostoma Source: The duration between the infection and the first appearance of the eggs in the faeces is about 6 weeks. 7. Mode of Infection of Hook Worm An individual can get hookworm infection in various ways. Infection generally takes place when the man walks bare-footed on the soil contaminated with faeces. It is believed that the larvae are attracted by the warmth of the human skin and they enter through contact. The most common sites of entry are: (a) Delicate and thin skin between the toes (b) Dorsum of the feet (c) Inner side of the soles Larvae can also enter the body through soft hands and palms, especially in gardeners and miners. Infection may also take place during handling of faeces-soiled clothing s left damp for 4 or 5 days. The possibility of intra-uterine infections has also been reported. Infection can also take place by drinking water contaminated with soil containing filariform larvae. 8. Factors Affecting the Transmission of Hook Worm There are almost one billion infections with hookworm in the world today. Vast majority of these occur in sub-saharan Africa, Southeast, South and East Asia, and parts of South America. However, the transmission of hook worm depends on various factors. 8.1 Critical stages in life cycle The life cycle of hook worm has three critical factors that limit the epidemiology of human transmission. The eggs pass out into the environment in the feces of the human host. The larvae live in the soil during the first two larval stages of the life cycle. The larvae must make contact with the skin of a new human host. 12

13 Fig. 9: Hookworm disease by country (per 100,000 inhabitants) Source: Geographic Features no data less than more than 60 Hookworm infection is considered as one of the primary neglected tropical diseases. Its occurrence in humans can be dependent on certain geographic features; for example - soil and climate determine the distribution of hookworm species. 13

14 Soil Texture: The larvae of A. require sandy and loamy soils during first two moults resulting in the formation of infective L3 stage. The hookworm eggs laid in hard clayey soils do not hatch and remain non-infective. Soil Moisture: Hookworm eggs require humid soil and warmth for hatching. The hatching does not occur in dry and cool environment. Precipitation: The amount of rains also limits the worms to the tropical and subtropical regions of the world. These are more prevalent in the regions that receive significant amounts of precipitation on an annual basis. 8.3 Lack of sanitation Poor people often do not have resources for infrastructural development. Lack of adequate sanitary facilities and improper infrastructure spreads the contaminated faeces in the human environment. 8.4 Unawareness Farmers often use human faeces as a fertilizer to enhance the crop growth as it is readily available and provides a cheap but rich source of critical nutrients to the soil. In areas endemic with hookworms, farmers can easily get infections while using human faeces as fertilizer. 9. Pathogenicity Hookworm causes hookworm disease in human beings, called ancylostomiasis. The disease is characterized by severe anaemia. The pathogenic effects can be caused by larval stages as well as adult forms. 9.1 Pathogenic effects caused by larvae The larval forms of A. causes pathogenicity while entering into the body and migrating through the lungs. The effects caused are as follows: a) Ground Itch Penetration of hook worm larvae through the human causes intense itching and inflammation of the skin. This condition is followed by formation of skin lesions in the form of papules and blisters. It is called ground itch, miner's itch, foot itch, toe itch, dew itch or water itch. b) Creeping eruptions Creeping eruption is caused when filariform larvae of hookworms wander through the skin for several weeks. The infection is also called cutaneous larva migrans or sandworm disease. The migration of larvae causes severe itching, blisters and rashes which can grow up to 1 to 2 centimeters per day. The infection usually appears on the feet, legs, buttocks, or back that have been exposed to the contaminated ground. These rashes generally appear after 1 to 5 days of exposure. 14

15 Fig. 10: Photograph of right foot taken on date of presentation showing typical serpiginous lesion of cutaneous larva migrans. Source: Archer M. Late presentation of cutaneous larva migrans: a case report. Cases J Aug 12; 2:7553 Fig. 11: Dermatological examination showed an erythematous serpiginous intraepidermal tunnel on the left leg, caused by accidental percutaneous penetration of the larva. Source: Cutaneous larva migrans: a case report. Vano-Galvan S, Gil-Mosquera M, Truchuelo M, Jaén P - Cases J (2009) Jan 31;2(1):112. doi: / c) Lesions in the lungs When hook worm larvae migrate through the lungs, the larvae break through the pulmonary capillaries and enter the alveolar spaces. It results in noticeable eosinophilia (increased count of eosinophils). 15

16 Fig. 12: Eosinophilia Source: Further, while passing through the lungs, there may be spells of coughing with sore throat and bloody sputum. Fig. 13: Larvae of hookworms in lung tissues Source: Pathogenic effects caused by adults As discussed earlier, the adult worms live in the intestine of human beings and attach to the mucosa layer through buccal teeth. 16

17 Fig. 14: Highly magnified histological section showing hookworm (Ancylostoma sp) attached to the intestine Source: The attachment of the worms to the intestinal epithelium results in lesions and severe anaemia. The anaemia may occur because of the following factors: (i) Sucking of blood by hook worms for their nutrition (ii) Loss of blood from the sites of attachment causing chronic haemorrhages Each adult worm can draw 0.03 to 0.2 ml of blood. Imagine the amount of blood loss during heavy infections for prolonged durations!! Clinical Gastroenterology and Hepatology , DOI: ( /j.cgh ) Copyright 2006 American Gastroenterological Association Terms and Conditions Clinical Gastroenterology and Hepatology , DOI: ( /j.cgh ) Copyright 2006 American Gastroenterological Association Terms and Conditions 17

18 Clinical Gastroenterology and Hepatology , DOI: ( /j.cgh ) Copyright 2006 American Gastroenterological Association Terms and Conditions Fig. 15: Ulcers caused by hookworm adults; (A) nonreactive; (B) ulcers covered with a black clot; (C) dilated reddish portion at the anchorage point Source: Alarcon Fernandez et al. (2006) Loss of blood and severe anaemia results in the following effects: Epigastric tenderness and duodenal ulcers Abnormal appetite Geophagy development of taste towards earth, mud, lime etc. Hyperacidity due to hyper-secretion of gastric juices Constipation; sometimes fatty diarrhea Pale skin, and mucous membranes of the eyes, lips and tongue Puffy face with swelling of lower eyelids, feet and ankle oedema Protuberant abdomen and dry lusterless hair A person with such severe symptoms cannot survive long unless the worms are removed from the body. 10. Diagnosis of Ancylostomiasis Diagnosis of hook worm infection in the human beings can be done by a variety of methods Microscopic examination The microscopic examination of stool may demonstrate the presence of (a) adult worms passed out impulsively or after medication (b) hook worm eggs passed out through faeces. Each worm generally produces 30 eggs per gram of feces per day. The worm load is thus quantified by the counting of eggs. However, identification of species is carried out by examining the adult worms passed after medication Study of duodenal contents 18

19 Duodenal contents obtained by intubation may reveal the presence of eggs or adult A Clinical diagnosis The patients can generally be diagnosed through following clinical symptoms. a) Extremely high levels of eosinophilia b) Severe anaemia c) Occult blood and Charcot-Leyden crystals in the stool 11. Immune Response in Hookworm Infections Regardless of repeated and frequent exposure to hook worms, protective immunity often does not develop in humans. As a result, infections tend to be prolonged and occur in all age groups Immune response to invasive larvae Immune responses against hookworm larvae can be elicited in the hosts because of the following reasons: Larval invasion through the skin: On penetrating the skin, L3, i.e. infective stage of hookworm sheds its outer sheath. The larva also secretes certain enzymes that facilitate their entry and migration through tissues. Antibodies have been detected to L3 exsheathing fluid suggesting that the cast sheath and associated antigens unfocused the immune response from the potentially susceptible larval surface (Kumar and Pritchard, 1992). Death of the larva: Significant numbers of larvae die and disintegrate in the human body. They release an extensive range of immune-reactive molecules. Migration of larvae through the blood: Entry of larvae into the blood stimulates immune response though their stay in the bloodstream is short-lived. Entry of larvae into the organs: Entry of larvae into the heart, lungs and intestine secretes more antigens leading to immune response. Moulting in gut: The larvae moult in the gut for the third time and release more antigenic molecules that might have immune-modulatory properties. Cellular Response Eosinophils predominate in the inflammatory response to L3 stage of hookworm in tissues (Behnke, 1991). The inflammatory response is clearly indicated by peripheral blood eosinophilia observed in experimental human infections with A. (Nawalinksi and Schad, 1974). It was reported that the arrival and development of worms in the gut probably accelerated antigenic output because of the feeding larvae and adults Immune response to adults Adult feed on blood and mucosa of intestinal wall which also stimulates immune response. Response to worms has a characteristic T-helper type 2 profile resulting in 19

20 activated mast cells in the gut mucosa, increased levels of circulating IgE, and eosinophilia in the peripheral blood and local tissues. The endurance of adult worms is probably determined by genetics of worm instead of host immunity. However, many of the proteins released by the parasites seem to have immunomodulatory activity, presumably for self-protection. The larvae and adults of hookworms share many antigens. Thus, it becomes difficult to distinguish between anti-larval and anti-adult responses. 12. Prophylaxis and Treatment of Hookworm Infection 12.1 Prophylaxis The protection from hook worm infection may be carried out by adopting following measures: Personal protection preventing larval skin invasion by wearing boots and gloves Prevention of soil contamination by adequate sewage disposal Disinfection of faeces or contaminated soil Treatment of infected individuals 12.2 Treatment The hookworm disease can be easily treated by antihelminthic drugs. Commonly used drugs are: Mebendazole - A safe drug, 100 mg of which is given twice daily for three consecutive days. It clears 80% of worms in a single course and almost completely clears worms if two courses are administered at an interval of 2 weeks. Bephenium hydroxyl naphthoate - Single dose of 5g is prescribed empty stomach. Two or three courses may be required for complete cure. Pyrantel pamoate - A broad spectrum drug given in a single dose of 11 mg/kg body weight. Other drugs prescribed are: Thiabendazole Tetramisole Benzimidazoles Albendazole 20

21 However, treatment also requires dietary supplements to improve the general level of health of persons. Adequate diet and iron supplementation is very important which is an important constituent of many enzyme systems involved in energy metabolism, DNA synthesis and drug detoxification Vaccines against hook worms The development of vaccine against hook worms is a difficult task as natural immune response to hookworm infection is inadequate and does not confer protective immunity. Therefore, focus has shifted to recognize antigens essential for hookworm development and survival in the human host and developing these as subunit vaccines. Prior work has been done on L3 vaccines against canine hook worms. However, as canine vaccines were unable to induce complete protection against hookworm infection, it is unlikely that a vaccine based solely on larval antigen would suffice for a human vaccine. Consequently, a few L3 may reach the gut and develop into adult hookworms. Thus, the approach has been to develop vaccines that would target both the larval and adult stages of the hookworm life cycle (Table 2). Specific antigens produced by L3 are being identified. In addition, macromolecules required by adults to use human blood as a food source and that are essential to worm survival are being explored. Table 2: Candidate Hook worm vaccines Source: Plotkin et al. (2008) Hookworm vaccines. Clin Infect Dis.; 46(2): doi: /

22 Fig. 16: Treatment protocol of Hookworm infection Source: Table 3: Summary of Characteristic Features in the Life Cycle of Ancylostoma Feature Characteristic Mode of infection Infective stage Portal of entry Through skin invasion Filariform larva Skin 22

23 Migration of larva Site of localisation Pathogenic stage Pathogenesis Diagnostic stage Through lungs Small intestine Filariform larvae and Adult Ancylostoma Skin lesions, Dermatitis, Creeping eruption, Anaemia, Eosinophilia Ova and Adults 13. Summary Ancylostoma is a thread-like sanguivorous parasitic nematode of human beings which lives in the small intestine of man. Ancylostoma is largely distributed in the tropical and sub-tropical regions of the world. The anterior end of the worms is bent slightly dorsally, because of which they are called hook worms. The worms possess well developed buccal capsule. Male and female Ancylostoma are separate and exhibit distinct sexual dimorphism. Ancylostoma is a monogenetic parasite. It completes its life cycle in single host, human being. Copulation between male and female adults takes place in the intestine of man. The eggs are passed out of the human body along with the faeces. Development from egg to filariform larva takes place in the soil. The third juvenile stage or filariform larva is infective to man. The filariform larvae gain entrance into another human being by penetrating the skin. The larvae migrate through various human organs, lymphatics, heart and lungs; after which they enter the digestive tract through pharynx and mature into adults. The transmission of hook worm depends on various factors; such as stages of life cycle, sanitation, geographic features, etc. Hookworm causes ancylostomiasis in human beings characterized by severe anaemia. Larval penetration through the human skin causes Ground Itch, Creeping eruption, and lesions in the lungs. The attachment of the worms to the intestinal epithelium results in lesions and severe anaemia. Diagnosis of hook worm infection in the human beings can be carried out by stool test, clinical diagnosis and examination of duodenal contents. Immune responses against hookworm larvae and adults can be elicited in the hosts because of the several reasons. 23

24 The larvae and adults of hookworms share many antigens. Thus, it becomes difficult to distinguish between anti-larval and anti-adult responses, Protection from hook worm infection may be carried out by sanitation and personal protection. The hookworm disease can be easily treated by antihelminthic drugs, especially Mebendazole. The development of vaccine against hook worms is a difficult task as natural immune response to hookworm infection is inadequate and does not confer protective immunity. 24