Parasitology: Parasitic Protozoa PDF

Page 1 of 56 Parasitology Chapter 6 PARASITIC PROTOZOA Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 202...

Page 1 of 56 Parasitology Chapter 6 PARASITIC PROTOZOA Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 2 of 56 Chapter 6 PARASITIC PROTOZOA Introduction The name ‘proto-zoa’ literally means ‘first animals’ and in the earlier classification systems, protozoa are grouped as the basal members of the animal kingdom. However, they were recognized as a discrete assemblage on the basis of their unicellularlity and were assigned to the taxon Protozoa. Protozoa are single celled organisms that exist as structurally and functionally independent individual cells. They come in many different shapes and sizes ranging from an Amoeba which can change its shape to Paramecium with its fixed shape and complex structure. Protozoans have developed complex subcellular features (membranes & organelles) which enable them to survive the rigours of their environments. They live in a wide variety of moist habitats including fresh water, marine environments and the soil; some live in the body of other organisms Most protozoans are free living while some are parasitic that causes serious diseases and medical complication to humans and other animals. This module introduced students in Clinical Parasitology of parasitic Protists. Students will learn more about the different classification and characteristics of protozoans. Pathogenicity, epidemiology and treatment some protozoal infection are also discussed in this chapter.. Specific Objectives At the end of this chapter, the students should be able to: 1. Identify and describe each classes of Protozoa 2. Distinguished the different types of protozoa belong to their respective phyla 3. Learn about the causes, transmission and treatment of different protozoal infection 4. Gain knowledge on how to protect themselves and prevent the spread of different parasitic infections. Duration Chapter 6: Parasitic Protozoans = 3 hours (1 hour discussion; 2 hours assessment) Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 3 of 56 Lesson Proper PARASITIC PROTIST Major Characteristics of a Protozoa  A single cell performs all the functions: reproduction, digestion, respiration, excretion  Protozoa exhibit wide range of size (1 - 150 μm), shape and structure; yet all possess essential common features  They do not have cell wall; some however, possess a flexible layer, a pellicle, or a rigid shell of inorganic materials outside the cell membrane  They have the ability to move from one place to another during their entire life cycle or part of it using their locomotor organelles or by a gliding mechanism.  They are heterotrophic, (free-living forms ingest particulates, such as bacteria, yeast and algae; the parasitic forms derive nutrients from the body fluids of their hosts).  They reproduce by asexual means, although in some groups sexual modes also occur.  Most of the protozoa are completely nonpathogenic but few may cause major diseases such as malaria, leishmaniasis and sleeping sickness. STRUCTURE OF A PROTOZOA A typical protozoan cell is bounded by a trilaminar unit membrane, supported by a sheet of contractile fibrils enabling the cell to move and change in shape. Its cytoplasm is made of two portions:  Ectoplasm: Outer homogeneous part that serves as the organ for locomotion and for engulfment of food by producing pseudopodia is called as the ectoplasm. It also helps in respiration, discharging waste material, and in providing a protective covering of cell.  Endoplasm The inner granular portion of cytoplasm that contains nucleus is called endoplasm. The endoplasm shows number of structures: the Golgi bodies, endoplasmic reticulum, food vacuoles and contractile vacuoles. Contractile vacuoles serve to regulate the osmotic pressure. The Nucleus The nucleus is usually single but may be double or multiple; some species having as many as 100 nuclei in a single cell. The nucleus contains one or more nucleoli or a central karyosome. Their chromatin may be distributed along periphery (peripheral chromatin) or as condensed mass around the karyosome. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 4 of 56 REPRODUCTION Reproduction usually occurs asexually in protozoans; however, sexual reproduction occurs in ciliates and sporozoans. Asexual Reproduction in Protozoa  Binary fission: It is a method of asexual reproduction, by which a single parasite divides either longitudinally or transversally into two or more equal number of parasites. Mitotic division of nucleus is fo llowed by division of the cytoplasm. In amebae, division occurs along any plane, but in flagellates, division is along longitudinal axis and in ciliates, in the transverse plane.  Multiple fission or schizogony: Plasmodium exhibits schizogony, in which nucleus undergoes several successive divisions within the schizont to produce large number of merozoiles.  Endodyogeny: Some protozoa like Toxoplasma, multiply by internal budding, resulting in the formation of two daughter cells. Figure 6.1. Asexual Reproduction of Protozoans Sexual Reproduction  Conjugation: In ciliates, the sexual process is conjugation, in which two organisms join together and reciprocally exchange nuclear material (e.g. Balanlidium coli).  Gametogony or syngamy: In Sporozoa, male and female gametocytes are produced, which after ferti lization form the zygote, which gives rise to numerous sporozoites by sporogony (e.g. Plasmodium). Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 5 of 56 LIFE CYCLE OF A PARASITIC PROTOZOA The life cycle of protozoa can be:  Single host: Protozoa like intestinal flagellates and ciliates require only one host, within which they multiply asexually in trophic stage and transfer from one host to  another by the cystic form.  Second host: In some protozoa like Plasmodium, asexual method of reproduction occurs in one host (man) and sexual method of reproduction in another host (mosquito). CLASSIFICATION OF PROTOZOANS Medical important parasitic protozoan has been classified into kingdom Protista, subkingdom Protozoa then further divided into the following phyla (see Table 6.1. Classification of Protozoan, Levine et al., 1980): 1. Sarcomastigophora 2. Apicomplexa 3. Microspora 4. Ciliophora Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 6 of 56 Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 7 of 56 PHYLUM SARCOMASTIGOPHORA  includes many unicellular or colonial, autotrophic, or heterotrophic organisms. Organisms under this phylum uses flagellae, pseudopodia, or both for locomotion Phylum Sarcomastigophora has been subdivided into two subphyla based on their modes of locomotion: 1. Sarcodina (sarcos meaningflesh or body): It includes those parasites, which have no permanent locomotory organs, but move about with the aid of temporary prolongations of the body called pseudopodia (e.g. amebae). 2. Mastigophora (mastix meaning whip or flagellum): It includes those protozoa which possess whip-like flagella (e.g. Trypanosoma and Trichomonas). Subphylum Sarcodina: Class Lobosea (Amoeba) Amoeba is a single cell protozoon that constantly changes its shape. The word “amoeba” is derived from the Greek word “amoibe” meaning “change”. They constantly change their shape due to presence of an organ of locomotion called as “pseudopodia”. Classification of Amoeba based on Habitat 1. Intestinal amoebae: They live in the large intestine of humans and animals. Entamoeba histolytica is the only pathogenic species. Others are nonpathogenic such as— E. dispar, E. moshkovskii, E. coli, E. polecki, E. hartmanni, E. gingivalis, Endolimax nana, and Iodamoeba butschlii 2. Free-living amoebae: They are small free living and opportunistic pathogens. Examples are Acanthamoeba species, Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea Parasitic Amoeba Entamoeba histolytica E. histolytica was first described by Fedor Losch (1875) from Russia. It one of the leading parasitic cause of mortality The species name was first coined by Fritz Schaudinn in 1903. Morphology E. histolytica has three stages—(1) trophozoite, (2) precystic stage and (3) cyst stage (immature and mature). Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 8 of 56 Figure 6.2 A to C. Entamoeba histolytica (schematic diagram) (A) trophozoite; (B) precyst; (C) cysts Trophozoite  It is the invasive form as well as the feeding and replicating form of the parasite found in the feces of patients with active disease.  It measures 12–60 μm (average 15–20 μm) in diameter. The parasite, as it occurs free in the lumen as a commensal is generally smaller in size, about 15-20 μm and has been called the minuta form  It is large and actively motile in freshly-passed dysenteric stool, while smaller in convalescents and carriers.  Cytoplasm of trophozoite is divided into a clear ectoplasm and a granular endoplasm. Granular endoplasm looks as ground glass appearance and contains red blood cells (RBCs), white blood cells (WBCs) and food vacuoles containing tissue debris and bacteria. RBCs are found only in the stage of invasion  Pseudopodia: long finger like projections called as pseudopodia (organ of locomotion); which exhibits active, unidirectional rapid progressive and purposeful movement  Nucleus is single, spherical, 4–6 μm size, contains central dot like compact karyosome surrounded by a clear halo. Nuclear membrane is thin and delicate and is lined by a layer of fine chromatin granules.  The space between the karyosome and the nuclear membrane is traversed by spoke like radial arrangement of achromatic fibrils (cart wheel appearance).  Amoebic trophozoites are anaerobic parasites They lack mitochondria, endoplasmic reticulum and Golgi apparatus.  The trophozoites divide by binary fission in every 8 hours.  Trophozoites survive up to 5 hours at 37°C and are killed by drying, heat and chemical sterilization. Therefore, the infection is not transmitted by trophozoites. Even if live trophozoites from freshly passed stools are ingested, they are rapidly destroyed in stomach and cannot initiate infection. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 9 of 56 Precyst (intermediate stage between trophozoite and cyst.)  It is smaller to trophozoite but larger to cyst (10–20 μm)  It is oval with a blunt pseudopodia, Food vacuoles and RBCs disappear. Nuclear structures are same as that of trophozoite  Before encystment, the trophozoite extrudes its food vacuoles and becomes round or oval, about 10-20 μmin size. This is the precystic stage of the parasite.  It contains a large glycogen vacuole and two chromatid bars.  It then secretes a highly retractile cyst wall around it and becomes cyst. Cystic Stage  It is the infective form as well as the diagnostic form of the parasite found in the feces of carriers as well as patients with active disease.  It measures 10–20 μm (average 12–15 μm) in diameter. Nuclear structures are same as in trophozoites.  First, the cyst is uninucleated; later the nucleus divides to form binucleated and finally becomes quadrinucleated cyst  Cytoplasm of uninucleated cyst contains 1–4 numbers refractile bars with rounded ends called as chromatoid bodies (aggregation of ribosome) and a large glycogen mass (stains brown with iodine)  Both chromatoid body and glycogen mass gradually disappear, and they are not found in mature quadrinucleated cyst  Cysts are present only in the gut lumen; they never invade the intestinal wall. “Minuta” form of Entamoeba histolytica: They are the commensal phase of E. histolytica, living in the lumen of gut. They are usually smaller in size (trophozoite 12–14 μm and cyst < 10 μm) and often mistaken as E. hartmanni. Life Cycle  Host: E. histolytica completes its life cycle in single host, i.e. man.  Infective form: Mature quadrinucleated cyst is the infective form. It can resist chlorination, gastric acidity and desiccation and can survive in a moist environment for several weeks. Mode of Transmission  Feco-oral route (most common): By ingestion of contaminated food or water with mature quadrinucleated cysts  Sexual contact: Rare, either by anogenital or orogenital contact. (especially in developed countries among homo sexual males)  Vector: Very rarely, flies and cock roaches may mechanically transmit the cysts from feces, and contaminate food and water. Note: Trophozoites and immature cysts can be passed in stool of amoebic patients, but they can’t serve as infective form as they are disintegrated in the environment or by gastric juice when ingested. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 10 of 56 Figure 6.3. Life Cycle of Entamoeba histolytica Development in man (small intestine) Man acquires infection by swallowing food and water contaminated with cysts. As the cyst wall is resistant to action of gastric juice, the cysts pass through the stomach undamaged and enter the small intestine. When the cyst reaches cecum or lower part of the ileum, due to the alkaline medium, the cyst wall is damaged by trypsin, leading to excystation  Excystation: In small intestine, the cyst wall gets lysed by trypsin and a single tetranucleated trophozoite (metacyst) is liberated which eventually undergoes a series of nuclear and cytoplasmic divisions to produce eight small metacystic trophozoites  Metacystic trophozoites are carried by the peristalsis to ileocecal region of large intestine and multiply by binary fission, and then colonize on the mucosal surfaces and crypts of the large intestine  After colonization, trophozoites show different courses depending on various factors like host susceptibility, age, sex, nutritional status, host immunity, intestinal motility, transit time and intestinal flora  Asymptomatic cyst passers: In majority of individuals, trophozoites don’t cause any lesion, transform into cysts and are excreted in feces  Amoebic dysentery: Trophozoites of E. histolytica secrete proteolytic enzymes that cause destruction and necrosis of tissue, and produces flask shaped ulcers on the intestinal mucosa. At this stage, large numbers of trophozoites are liberated along with blood and mucus in stool producing amoebic dysentery. Trophozoites usually degenerate within minutes Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 11 of 56  Amoebic liver abscess: In few cases, erosion and necrosis of small intestine are so extensive that the trophozoites gain entrance into the radicals of portal veins and are carried away to the liver where they multiply causing amoebic liver abscess. Development in Man (Large intestine)  Encystation: After some days, when the intestinal lesion starts healing and patient improves, the trophozoites transform into precysts then into quadrinucleated cysts which are liberated in feces  Encystation occurs only in the large gut. Cysts are never formed once the trophozoites are excreted in stool  Factors that induce cyst formation include food deprivation, overcrowding, desiccation, accumulation of waste products, and cold temperatures  Mature quadrinucleated cysts released in feces can survive in the environment and become the infective form. Immature cysts and trophozoites are sometimes excreted, but get disintegrated in the environment. Figure 6.4. Excystation and Encystation of E. histolytica in the host body Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 12 of 56 Clinical Manifestation of Amoebiasis  Asymptomatic amoebiasis  About 90% of infected persons are asymptomatic carriers and excrete cysts in their feces. Now it is confirmed that many of these carriers harbor E. dispar. The remaining 10% of people (who are truly infected by pathogenic E. histolytica) produces a spectrum of diseases varying from intestinal amoebiasis to amoebic liver abscess.  Intestinal amoebiasis  Incubation period varies from one to four weeks. Intestinal amoebiasis is characterized by four clinical forms: 1. Amoebic dysentery: Symptoms include bloody diarrhea with mucus and pus cells, colicky abdominal pain, fever, prostration, and weight loss. Amoebic dysentery should be differentiated from bacillary dysentery 2. Amoebic appendicitis: Presented with acute right lower abdominal pain 3. Amoeboma: It present as palpable abdominal mass 4. Fulminant colitis: Presents as intense colicky pain, rectal tenesmus, more than 20 motions/day, fever, nausea, anorexia and hypotension. Figure 6.5. Complication of intestinal amoebiasis (cross section of intestinal wall)  Amoebic liver abscess - Presents with tender hepatomegaly, fever with weight loss, sweating and weakness, rarely jaundice, and cough. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 13 of 56 Diagnosis of Intestinal Amoebiasis  Stool microscopy by wet mount, permanent stains, etc.—detects cysts and trophozoites  Stool culture  Polyxenic and axenic culture  Stool antigen detection (copro-antigen)— Counter Current Immune Electrophoresis (CIEP), Enzyme Linked Immunosorbent Assay (ELISA), Immunochromatographic TestICT  Serology  Amoebic antigen— Enzyme Linked Immunosorbent Assay (ELISA)  Amoeboic antibody—Indirect hemagglutination Test (IHA), Enzyme Linked Immunosorbent Assay (ELISA) and Indirect fluorescent antibody Test (IFA)  Isoenzyme (zymodene) analysis  Molecular diagnosis - Nested multiplex Polymerase Chain Reaction (PCR) and real time PCR Diagnosis of Amoebic Liver Abscess Microscopy—detects trophozoites Stool culture Antigen detection—ELISA Antibody detection—IHA, IFA, ELISA, CIEP, CFT, SAT, CIA, etc Histopathology—PAS stain Molecular diagnosis—PCR Ultrasonography Treatment  Metronidazole or tinidazole is the drug of choice for intestinal amoebiasis and amoebic liver abscess  Other measures include fluid and electrolyte replacement and symptomatic treatment Prevention Preventive measures are as follows:  Avoidance of the ingestion of food and water contaminated with human feces  Treatment of asymptomatic persons who pass E. histolytica cysts in the stool may help to reduce opportunities for disease transmission. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 14 of 56 Subphylum Mastigophora: Class Zoomastigophora (Flagellates) Flagellates are protozoans that uses flagella for locomotion. Flagella are slender, long and thread-like extension of cytoplasm. Its intracellular portion is called as axostyle or axoneme. Flagella arise from kinetoplast (made up of copies of mitochondrial DNA) which in turn consists of: - Blepharoplast or basal body or kinetosome from which flagellum arises - Parabasal body, through which it passes as axostyle Classification based on habitat 1. Lumen-dwelling flagellates: Flagellates found in the alimentary tract and urogenital tract. 2. Hemoflagellates: Flagellates found in blood and tissues Table 6.2. List of Lumen-dwelling flagellates Lumen-dwelling flagellates Habitat Giardia lamblia Duodenum and jejunum Enteromonas hominis Large intestine Retortamonas intestinalis Large intestine Chilomastix mesnili Cecum Dientamoeba fragilis Cecum and colon Trichomonas tenax Mouth (teeth and gum) Pentatrichomonas hominis Ileocecal region Trichomonas vaginalis Vagina and urethra Table 6.3. List of Hemoflagellates Hemoflagellates Habitat Leishmania Blood and tissue Trypanosoma Blood and tissue Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 15 of 56 Lumen-dwelling Flagella: Giardia lambia Giardia lamblia was first observed by A.V. Leeuwenhoek in 1681 while examining his own stool. The parasite was named after Dr F. Lambl of Prague and Prof. A. Giard of Paris in 1859 Giardia can be differentiated to various species based on the origin of the host. G. lamblia (infects humans and other mammals), G.muris (infects mice), G.agilis (infects amphibians), G.psittaci (infects birds) and G. microti (infect voles). It is the most common protozoan pathogen and is worldwide distributed. The infection is endemic in places with very poor sanitation, especially in tropics and subtropics. Visitors to such places frequently develop traveler's diarrhea caused by giardiasis through contaminated water. Morphology Figure 6.6 A to C. Giardia lambia (schematic diagram) (A) Tropozoite front view, (B) Trophozoite Lateral view, (C) Cysts 1. Trophozoite The trophozoite has a falling leaf-like motility, usually measures 10–20 μm in length and 5–15 μm in width.  Shape: - In front view, it is pear shaped (or tear drop or tennis racket shaped) with rounded anterior end and pointed posterior end - Laterally, it appears as a curved portion of a spoon (sickle shaped) - It is convex dorsally while the ventral surface has a concavity bearing a bilobed adhesive disc. Hence, it appears as sickle shaped in lateral view  Trophozoite is bilaterally symmetrical; on each side from the midline it bears: - One pair of nuclei - Pair of median bodies - Four pairs of basal bodies or blepharoplasty (from which the axoneme arises) - Four pairs of flagella—two lateral, one ventral and one caudal pair of flagella - Pair of parabasal bodies (connected to basal bodies through which the axoneme passes) - Pair of axoneme or axostyle (the intracellular portion of the flagella). 2. Cysts (infective form) Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 16 of 56 - The cyst is small and oval, measuring 12 um x 8 um is surrounded by a hyaline cyst wall. - Its internal structure includes two pairs of nuclei grouped at one end. A young cyst contains one pair of nuclei. - The axostyle lies diagonally, forming a dividing line within cyst wall. - Remnants of the flagella and the sucking disc may Life Cycle of Giardia lambia  Host: Giaridia completes its life cycle in one host.  Infective form: Mature cyst.  Mode of transmission: Man acquires infection by ingestion of food and water contaminated with mature cysts or rarely by sexual route (mainly in homosexuals). Figure 6.7. Life of Giardia lambia (Source: https://commons.wikimedia.org/wiki/File:Giardia_life_cycle_en.svg)  Excystation: Two trophozoites are released from each cyst in the duodenum within 30 minutes of entry  Multiplication: Trophozoites multiply by longitudinal binary fission in the duodenum.  Adhesion: Trophozoites adhere to the duodenal mucosa by the bilobed adhesive ventral disc Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 17 of 56 - This is achieved by the microtubules of median bodies, contractile proteins and lectins present on the surface of adhesive disc that bind to the intestinal receptors (sugar molecules) - The trophozoites live in the duodenum and upper part of jejunum, feeding by pinocytosis - In active stage of the disease, sometimes the trophozoites are excreted in diarrhea stool  Encystation: Gradually when the trophozoites pass down to large intestine, encystation begins - Promoting factors for encystation are the conjugated bile salts, alkaline pH and cholesterol starvation - Encystation specific vesicles (ESV) appear in the cytoplasm that helps in processing and transportation of the cyst wall protein antigens to the exterior of the plasma membrane to synthesize the cyst wall - Encystation begins with retraction of the flagella followed by condensation of the cytoplasm and finally formation of the cyst wall - On maturation, nuclei divide to become four. The mature cysts excreted in feces can survive better in the environment and are infective to man. Pathogenicity  Infective dose: As few as 10–25 cysts can initiate the infection  Risk factors: Children are commonly affected. Other high-risk groups are elderly debilitated persons and patients with cystic fibrosis, poor hygiene, and immunodeficiency syndromes such as common variable hypoglobulinemia. However, association with acquired immunodeficiency syndrome (AIDS) patient is not been confirmed yet  Several pathogenic mechanisms have been postulated that include: - Trophozoites adhere to the duodenal mucosa and cause disruption of the intestinal epithelial brush border that leads to increase permeability and malabsorption - Very rarely, elaboration of enterotoxin such as cystein rich surface protein 136 (CRP-136)  Malabsorption: There could be various types which include: - Malabsorption of fat (steatorrhea)— leads to foul smelling profuse frothy diarrhea - Disaccharidase deficiencies (lactate, xylose)—leading to lactose intolerance - Malabsorption of vitamin B12 and folic acid - Protein loosing enteropathy Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 18 of 56  Antigenic variation: - Giardia undergoes frequent antigenic variation due to a cysteine rich protein on its surface called variant surface protein (VSP) - This helps the parasite in evasion of the host immune system and resistant to intestinal proteases which in turn leads to persistence of infection resulting into chronic and recurrent illness. Clinical features of Giardiasis Clinical course of giardiasis can be divided into three stages: 1. Asymptomatic carriers: Most infected persons are asymptomatic, harboring the cysts and spreading the infection 2. Acute giardiasis:  Incubation period varies from 1 week to 3 weeks (average 12–20 days). Symptoms may develop suddenly or gradually  Common symptoms include diarrhea, abdominal pain, bloating, belching, flatus and vomiting  Diarrhea is often foul smelling with fat and mucus but no blood  The acute stage lasts for 1 week but usually resolves spontaneously. Very rarely, in some children may last for months 3. Chronic giardiasis:  It may present with or without a previous acute symptomatic episode  Symptoms are intermittent and recurring  Common symptoms include recurrent episodes of foul smelling diarrhea, foul flatus, sulfurous belching with rotten egg taste, and profound weight loss leading to growth retardation  Uncommon symptoms such as—fever, presence of blood and/or mucus in the stools, and other signs and symptoms of colitis  Extra intestinal manifestations have been described, such as urticarial, anterior uveitis, salt and pepper retinal changes and arthritis. Diagnosis  Stool examination—detects cysts and trophozoites  Entero-test Antigen detection in stool (copro-antigen)—  ELISA, ICT  Antibody detection in serum—ELISA, IFA  Culture  Molecular method—PCR  Radiological findings—barium meal, X-ray Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 19 of 56 Treatment  Metronidazole (250 mg thrice daily for 5 days) is usually affective in more than 90% of cases of giardiasis  Tinidazole (2 g once orally) is more effective than metronidazole  Nitazoxanide (500 mg twice daily for 3 days) is an alternative agent for treatment of giardiasis  Furazolidone is given to children and paromomycin can be given in pregnancy  In patients with AIDS and hypogammaglobulinemia, giardiasis is often refractory to treatment. Prolonged therapy with metronidazole (750 mg thrice daily for 21 days) has been successful. Prevention Giardiasis can be prevented by:  Improved food and personal hygiene  Boiling or filtering of potentially contaminated water  Treatment of asymptomatic carriers  No vaccine is currently available. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 20 of 56 Lumen-dwelling Flagella: Trichomonas Trichomonas is the only flagellates that lacks the cyst stage. They exist as only trophozoites. There are three species of Trichomonas infect humans. 1. Trichomonas vaginalis is the only pathogen. It resides in the genital tract 2. Pentatrichomonas hominis: Non-pathogen, resides in large intestine 3. Trichomonas tenax: Nonpathogen, resides in mouth (teeth and gum). Trichomonas vaginalis  It is the most common parasitic cause of sexually transmitted diseases (STDs).  Females are commonly affected than males  It was first observed by Donne in 1836 from the purulent genital discharge of a female  Though it is a eukaryote, its metabolism is similar to primitive anaerobic bacteria.  Carbohydrate is utilized fermentative pathway. It is unable to synthesize fatty acid, sterols, purines and pyrimidines and hence depends on exogenous sources. Habitat In females, it lives in vagina and cervix and may also be found in Bartholin 's glands, urethra and urinary bladder. In males, it occurs mainly in the anterior urethra, but may also be found in the prostate and preputial sac. Morphology Trophozoites It is pear (pyriform) shaped, measures 7–23 μm and 5–15 μm wide (Fig. 4.6), resides in vagina and urethra of women and urethra, seminal vesicle and prostate of men.  It shows characteristic jerky or twitchy motility in saline mount preparation  It bears five flagella—four anterior Figure 6.8. Trophozoite of Trichomonas vaginalis flagella and one lateral flagellum called as recurrent flagellum as it curves back on the surface of the parasite and traverses as undulating membrane and stops halfway down the side of the trophozoite. It doesn’t come out free posteriorly  The undulating membrane is supported on to the surface of the parasite by a rod like structure called as costa Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 21 of 56  The axostyle runs down the middle of the trophozoite and ends in the pointed end of the posterior pole  It has a single nucleus containing central karyosome with evenly distributed nuclear chromatin and the cytoplasm contains a number of siderophore granules along the axostyle  The respiratory organelle is called as hydrogenosome. Life Cycle Life cycle of T. vaginalis is completed in a single host either male or female Mode of Transmission  The trophozoite cannot survive outside and so infection has to be transmitted directly from person-to-person. Sexual transmission is the usual mode of infection  Trichomoniasis often coexists with other sexually transmitted diseases like candidiasis, gonorrhea, syphilis, or human immunodeficiency virus (HIV).  Babies may get infected during birth.  Vaginal pH of more than 4.5 facilitates infection  Fomites such as towels have been implicated in transmission.  Trophozoites divide by longitudinal binary fission giving rise to a number of daughter trophozoites in the urogenital tract which can infect other individuals  Incubation period is roughly 10 days Pathogenicity  Trichomoniasis is the most common parasitic cause of STDs. It is worldwide in distribution and accounts for 10% of cases of vulvovaginitis  T. vaginalis particularly infects squamous epithelium and not columnar epithelium. It secretes cysteine proteases, adhesins, lactic acid and acetic acid, which disrupt the glycogen levels and lower the pH of the vaginal fluid. - It is an obligate parasite and cannot live without close association with the vaginal, urethral, or prostatic tissues. - Parasite causes petechial hemorrhage and mucosa! capillary dilation (strawberry mucosa), metaplastic changes and desquamation of the vaginal epithelium. - Intracellular edema and so called chicken-like epithelium, is the characteristic feature of trichomoniasis. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 22 of 56 Clinical Features Infection is often asymptomatic, particularly in males, although some may develop urethritis, epididymitis and prostatitis. Acute infection (vulvovaginitis):  Females are commonly affected and are presented as vulvovaginitis, characterized by profuse foul smelling purulent vaginal discharge. Discharge may be frothy (10% of cases) and yellowish green color mixed with a number of polymorphonuclear leukocytes  Strawberry appearance of vaginal mucosa (Colpitis macularis) is observed in 2% of patients. It is characterized by small punctate hemorrhagic spots on vaginal and cervical mucosa  Other features include dysuria and lower abdominal pain  In males, the common features are nongonococcal urethritis and rarely epididymitis, prostatitis and penile ulcerations Chronic infection: In chronic stage, the disease is mild with pruritus and pain during coitus. Vaginal discharge is scanty, mixed with mucus Complications: Rarely it is associated with complications like pyosalpinx, endometritis, infertility, t and cervical erosions. Low birth weight, neonatal pneumonia and conjunctivitis have been reported in infants born to infected mothers Diagnosis  Direct microscopy - Wet saline mounting - Permanent stain - Acridine orange fluorescent stain - Direct fluorescent antibody test  Culture— gold standard method  Antigen detection in vaginal secretion— ELISA, ICT, etc  Antibody detection—ELISA  Molecular method—PCR  Other supportive test  Raised vaginal pH  Positive whiff test Treatment  Metronidazole or tinidazole  Drug of choice, 2g, single dose is usually effective  Both the sexual partners must be treated simultaneously to prevent reinfection,especially asymptomatic males Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 23 of 56  Resistance to metronidazole:  Resistance is rare but has been reported: - 2.5–10% to metronidazole - Less than 1% to tinidazole  The mechanism of development of resistance to metronidazole is controlled by hydrogenosome  Metronidazole requires hydrogen as an electron acceptor which is provided by hydrogenosome present in T. vaginalis  In metronidazole-resistant T. vaginalis, the expression levels of the hydrogenosomal enzymes like ferredoxin are reduced dramatically, which probably eliminates the ability of the parasite to activate metronidazole  Resistance is relative and can be overcome with higher doses of oral metronidazole Prevention Trichomoniasis can be prevented by:  Treatment of both the partners  Safe sex practices like use of condoms  Avoidance of sex with infected person  Vaccine: There is no effective vaccine licensed so far. However, trials are going on targeting potential immunogenic antigens like 100 kDa protein. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 24 of 56 Hemoflagellates Hemoflagellates are the flagellated protozoa that are found in peripheral blood circulation. They complete their life cycle in two hosts, i.e. vertebrate host and insect vector; therefore, called as digenetic or heteroxenous parasites. Morphology Hemoflagellates have an oval to elongated body, nucleus, and a single flagellum arising from:  Kinetoplast: It consists of blepharoplasty and parabasal body connected by a delicate fibril (cytoskeleton). It lies tangentially or at right angle to the nucleus. It represents multiple copies of mitochondrial DNA  Axoneme (or axostyle): It extends from blepharoplast to the cell wall. It represents the intracellular portion (root) of flagellum Based upon arrangement of flagellum, they exist in four morphological stages: 1. Amastigote form: Round to oval, lacks flagellum, found in reticuloendothelial cells of man infected with Leishmania and Trypanohsoma cruzi 2. Promastigote form: Lanceolate shaped; kinetoplast is anterior to nucleus (antenuclear kinetoplast). Flagellum arises from the anterior end. It is found in the mid gut of insect vector. This is the infective stage of Leishmania to man 3. Epimastigote form: Elongated, kinetoplast is placed close to the nucleus (juxtanuclear kinetoplast). Flagellum arises from the lateral side and traverses the body as a short undulating membrane and comes out from the anterior end. This form is seen for Trypanosoma in insect vector 4. Trypomastigote form: Elongated and spindle shaped with central nucleus. Kinetoplast lies near the posterior end. Flagellum arises posteriorly and runs as long undulating membrane. It is the infective stage of Trypanosoma found in insect vector and peripheral blood of humans. Figure 6.9. Morphological Stages of trypanosome (Source: https://www.clinmicronow.org/doi/10.1128/9781683670438.MCM.ch140_1) Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 25 of 56 Hemoflagellates: Trypanosoma Trypanosomes are hemoflagellates that reside in peripheral blood and tissues of their host. Classification of Trypanosoma 1. Human Trypanosomes  Trypanosoma cruzi: It is the causative agent of South American trypanosomiasis (also called as Chagas’ disease) in man and transmitted by insect vector reduviid bug  Trypanosoma brucei: It causes African trypanosomiasis, transmitted by tsetse fly. It has three important subspecies out of which only two of them infect humans - Trypanosma brucei rhodesiense: It is the causative agent of East African sleeping sickness - Trypanosma brucei gambiense: It is the causative agent of West African sleeping sickness  Trypanosoma rangeli: It is a nonpathogenic species that rarely infects humans in South America. 2. Animal Trypanosomes  Trypanosoma brucei brucei: It causes “nagana”, a disease affecting cattle in Africa - T. congolense and T.vivax cause disease similar to that of T. brucei brucei  Trypanosma evansi: It causes “Surra” in horses and other animals. It is transmitted by flies (tabanidae and stomoxys). Many animal cases are reported in India  Trypanosma lewisi: It causes a harmless infection affecting rodents  Trypanosma equiperdum: It causes “Stallion’s disease” in horses. It is transmitted by sexual route (not by insect vector). Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 26 of 56 Trypanosoma cruzi  It is the causative agent of South American trypanosomiasis or Chagas’ disease.  Discovered by Brazilian scientist Carlos Chagas, isolated from reduviid bug (triatomine bugs) and blood of infected monkeys. Later on he found it causing human infection also. Hence the condition is named as Chagas’ disease. He named the parasite as T. cruzi after his guide Oswaldo Cruz. Habitat In humans, T. cruzi exists in two forms: (1) amastigote and (2) trypomastigote form.  Amastigotes are intracellular parasite found in reticuloendothelial cells of spleen, liver, lymph node, bone marrow, and myocardium. They are also found in cells of epidermis and striated muscles  Trypomastigotes are extracellular and found in peripheral blood. Epidemiology Chagas’ disease is mainly restricted to South and Central American countries like Brazil, Argentina, Venezuela, etc. Currently, it is estimated that 8 million people are chronically infected with T. cruzi and 14,000 deaths occur due to the illness every year It is a zoonotic disease, having many animal reservoirs like dogs, cats, opossums and rodents. Morphology  In vertebrate host, it exists mainly in two forms—(1) trypomastigote form and (2) amastigote form  In insect vector (reduviid bug), it exists as all four forms, i.e. (1) trypomastigote, (2) amastigote, (3) promastigote and (4) epimastigote form. Figure 6.10. Reduviid bug, isect vector of T.cruzi (Source: https://www.medicalnewstoday.com/articles/327250) Life Cycle Host: T. cruzi passes its life cycle in two hosts—(1) humans and (2) vector reduviid bugs or kissing bugs or triatomine bugs (Triatoma infestans, Rhodnius prolixus and Panstrongylus megistus). Infective form: Metacyclic trypomastigote form is the infective forms, found in feces of reduviid bugs. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 27 of 56 Mode of transmission:  Reduviid bugs are nocturnal in habitat and humans get infection when abraded skin, mucous membranes, or conjunctivae become contaminated with reduviid bug’s feces containing infective form of the parasite.  T. cruzi can also be transmitted by the blood transfusion, organ transplantation. From mother to fetus or very rarely by ingestion of contaminated food or drink, and most importantly by laboratory accidents. Figure 6.11. Life Cycle of Trypanosoma cruzi (Source: https://www.cdc.gov/parasites/chagas/biology.html) Development in Man  The parasite invades the reticuloendothelial cells and other tissues like muscle (cardiac, skeletal and GIT muscles) and nervous tissue and transforms into amastigote form.  In these tissues, the amastigotes multiply by binary fission forming a cyst like mass of growth known as pseudocyst  Many amastigotes within the pseudocyst are transformed into motile C shaped non- multiplying trypomastigote forms. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 28 of 56  On rupture of the pseudocyst, the trypomastogotes are liberated to blood. They are of two types: 1. Slender highly motile forms: Have an elongated nucleus, sub terminal kinetoplast and a short free flagellum. They are the invasive forms, eventually migrate to many organs, penetrate the cells and continue the life cycle 2. Broader less motile forms: Have an oval nucleus, terminal kinetoplast and a long free flagellum. They persist in the blood to be taken up by the insect vector during a blood meal. Development in Reduviid bug  Broader less motile trypomastigote forms are transmitted to reduviid bugs during the blood meal.  They transform into amastigote forms in the foregut.  In the midgut, the amastigote forms multiply by binary fission and divide to form epimastigote forms  They finally transform into metacyclic trypomastigote forms in the hindgut and are excreted in the bug’s feces  The insect cycle takes about 10–15 days (extrinsic incubation period). There is no transovarian transmission seen in the bugs and once infected, they retain the infection throughout the life by the molting cycles Pathogenicity and Clinical features Acute Chagas Disease It is characterized by:  Chagoma: An erythematous subcutaneous nodule is formed at the site of deposition of bug’s feces. It is painful, commonly occurs on face and may take 2–3months to resolve  Romana’s sign: When the parasites enter through conjunctiva, they occur in unilateral painless edema of the eye lid and conjunctivitis  Generalized lymphadenopathy and hepatosplenomegaly may also appear  Severe myocarditis and neurologic signs like meningoencephalitis occur occasionally, especially in children  Usually within 4–8 weeks, patient either recovers spontaneously or develops chronic T. cruzi infection. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 29 of 56 Chronic Chagas Disease Chronic Chagas’ disease manifests years or even decades after the initial infection. It occurs due to multiplication of the parasites in the muscles (skeletal, cardiac and GIT) and nervous tissue.  Cardiac form: Occurs in 30% of the patients. Patient develops dilated cardiomyopathy, rhythm disturbances like right bundle branch block, and thromboembolism  Gastrointestinal form: Involvement of muscles of GIT leads to megaesophagus (manifested as dysphagia, chest pain, and regurgitation) and megacolon (manifested as abdominal pain and chronic constipation)  Pulmonary form: Repeated episodes of aspiration pneumonitis are common (especially during sleep) in patients with severe esophageal dysfunction  Mixed forms are observed in 10% of the patients. Congenital Trypanosomiasis Rarely, T. cruzi can be transmitted transplacentally both in acute and chronic stage of the disease. It is manifested as low birth weight, still birth, rarely myocarditis and neurological alterations. Diagnosis (T. cruzi)  Peripheral blood microscopy by wet mount, thick or thin smear—detects trypomastigotes  Culture—NNN medium or Yager’s liver infusion tryptose medium  Antibody detection in serum—ELISA, IFA, CFT, RIPA  Antigen detection from serum, urine—by CLIA  Molecular methods—PCR  Animal inoculation—Mice  Xenodiagnosis—nymph of reduviid bugs Treatment Therapy for Chagas’ disease is still unsatisfactory. Only two drugs—(1) nifurtimox and (2) benznidazole have been available In acute disease: - Benznidazole is considered as the drug of choice in Latin America. The recommended oral dosage is 5 mg/kg per day for adults and 5–10 mg/kg per day for children for 60 days - Nifurtimox is given 8–10 mg/kg for adults and 15–20 mg/kg for children in four divided doses for 90–120 days In chronic disease: These drugs lack efficacy and may cause many side effects. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 30 of 56 Trypanosoma brucei T. brucei was first demonstrated by Sir Bruce in 1895 from horses suffering from “nagana”. Forde in 1902 had demonstrated the parasite in man whereas Kleine had demonstrated the parasite in the vector tsetse fly. The name “Trypanosoma” was coined by Dutton in 1902. T. brucei complex consists of three subspecies: 1. T. brucei gambiense: Agent of West African sleeping sickness 2. T. brucei rhodesiense: Agent of East African sleeping sickness 3. T. brucei brucei: Causes “nagana”, a disease affecting cattle in Africa. It doesn’t infect humans. Life Cycle Host: T. brucei passes its life cycle in two hosts. 1. The vertebrate host is man and other animals 2. Invertebrate host is the tsetse fly (genus Glossina). Both male and female flies bite man and serve as vectors. Glossina palpalis group serves as the vector for T. brucei gambiense whereas Glossina morsitans group is the vector for T. brucei rhodesiense. Figure 6. 12. Tsetse fly, invertebrate vector of T. brucei (Source: https://www.britannica.com/animal/tsetse-fly) Infective form: The metacyclic trypomastigote forms are found in salivary gland of tsetse fly Mode of transmission: By the bite of tsetse fly, trypomastigote forms are transmitted to the punctured wound from the saliva of the tsetse fly Development in Man  At the site of inoculation, they transform into long slender trypomastigote forms which multiply by binary fission  They transform into an intermediate stage and then into nondividing short stumpy form without free flagellum  Subsequently the parasites invade the blood stream resulting in parasitemia and migrate to various organs including CNS  The short stumpy forms are the infective form to the tsetse fly, hence the transformation of long slender trypomastigotes into short stumpy forms is critical for the transmission of the parasite. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 31 of 56 Development in Tsetse fly  The short stumpy trypomastigote forms are taken up by the tsetse fly along with the blood meal  They become long slender forms in the midgut and hindgut of the insect where they multiply and finally reach the salivary gland  They attach to the epithelial cells of salivary ducts and transform into broad epimastigote forms  Finally, the epimastigotes develop into metacyclic trypomastigote forms which are the infective forms to man  It takes around 3 weeks from the time of blood meal till the fly becomes infective (extrinsic incubation period), then the fly remains infected throughout the life. Figure 6.13. Life Cycle of T.brucei (Source: https://www.mcdinternational.org/trainings/malaria/english/DPDx5/HTML/TrypanosomiasisAfrican) Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 32 of 56 Pathogenesis and Clinical Features In general, T. brucei gambiense develops a chronic course with slow progression whereas T. brucei rhodesiense runs an acute course with rapid progression and early death Figure 6.14. Comparison between T.b. gambiense and T.b. rhodesiense Trypanosomal Chancre A self-limited inflammatory lesion may appear a week after the bite of an infected tsetse fly. Stage I Disease  It is characterized by a systemic febrile illness that occurs due to dissemination of the parasite through the lymphatics and bloodstream.  Lymphadenopathy is prominent in West African trypanosomiasis. The posterior cervical nodes are commonly involved and become soft, rubbery and nontender called as winterbottom’s sign  Pruritus, maculopapular rashes and transient edema are common  Delayed sensation to pain is noted (Kerandel’s sign)  Hepatosplenomegaly may be seen in few cases  Hematologic manifestations include moderate leukocytosis, thrombocytopenia, anemia and production high levels of polyclonal IgM. Stage II Disease It involves invasion of the CNS. The presence of trypanosomes in perivascular areas of CNS is accompanied by intense infi ltration of mononuclear cells.  Patient develops characteristic progressive daytime somnolence (hence called as “sleeping sickness”), with restlessness and insomnia at night Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 33 of 56  Other features include listless gaze, loss of spontaneity, and abnormal speech with few extrapyramidal signs like choreiform movements, tremors and fasciculations  CSF fi ndings include increased pressure, elevated total protein and pleocytosis, with frequently demonstration of trypanosomes. Antigenic Variation Trypomastigotes undergo periodic antigenic variation leading to frequent change of antigenic nature of variable surface glycoprotein (VSG) antigens present on their surface. This serves as the key mechanism of evading host immune response.  Genome of Tryponasoma brucei contains thousands of genes that undergo gene switching (like mutations, deletions, additions or recombinations) leading to formation of new variant antigenic types (VATs).  Every 5–10 days, a new wave of genes evolves, that code for a new batch of VATs. Host immunity is strain specific hence, is not able to eliminate the new waves of parasitemia. Diagnosis (T. brucei)  Direct microscopy—detects trypomastigotes - Serial blood sample examination - CSF examination - Lymphonode aspirate  Antibodies from serum and CSF—card agglutination test, ELISA, IFA  Antigen from serum and CSF—ELISA  Molecular method—PCR  Culture—inoculated into KIVI  Animal inoculation in mice Treatment The drugs used for treatment of African sleeping sickness are suramin and pentamidine. Alternate drugs are eflornithine, and the organic arsenical melarsoprol. Treatment is based on type of disease (West or East African) and presence or absence of CNS invasion Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 34 of 56 Hemoflagellates: Leishmania Leishmaniasis is caused by the obligatory intracellular protozoa of the genus Leishmania. Primarily it affects the reticuloendothelial system of the host. Leishmania species produce widely varying group of clinical syndromes ranging from self-healing cutaneous ulcers to fatal visceral disease. Leishmaniasis is mainly a zoonotic disease affecting dogs, foxes, jackals and rodents. Animal reservoir plays a major role for transmission; except in Indian subcontinent where it is anthropophilic affecting only humans. The parasite is transmitted by bite of the female sandfly vector. Classification Leishmania Leishmania has two subgenera L. Leishmania and L. Viannia. The main difference between the two subgenera is that promastigotes of the subgenus Viannia develop in the midgut and hindgut of sandfly where as that of subgenus Leishmania develop in the anterior portion of the alimentary tract of sandfly  Old world leishmaniasis: Affects Asia, Africa and Europe and transmitted by sandfly (Genus Phlebotomus)  New World Leishmaniasis: Affects Central and South America and transmitted by sandfly (Genus Lutzomyia) Figure 6.15. Sand fly, invertebratevector of Leishmania (Source: https://www.ecdc.europa.eu/en/disease- vectors/facts/phlebotomine-sand-flies) Clinical syndromes of leishmaniasis include:  Visceral leishmaniasis (VL)  Post–kala-azar dermal leishmaniasis (PKDL)  Cutaneous leishmaniasis (CL)  Diffuse cutaneous leishmaniasis (DCL)  Leishmaniasis recidivans (LR)  Mucocutaneous leishmaniasis (MCL) Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 35 of 56 Old World Leishmaniasis Leishmania donovani Leishmania donovani causes visceral leishmaniasis (VL) or kala azar (a hindi term meaning “black fever”) It was named after two scientists who discovered the parasite in the same year 1903: 1. Sir William Boog Leishman in London observed the amastigotes form of the parasite in the liver of a British soldier died at Dumdum, Kolkata. (Hence also known as Dum- Dum fever) 2. Sir Donovan who found the amastigotes in the splenic smear from a patient from Chennai Charles Nicolle, a 1928 Nobel laureate, at the Pasteur Institute of Tunis, characterized the new world VL and cultivated the etiologic agent. Morphology Figure 6.16. Morphological Structure of Leshmania (Source: https://www.pinterest.ph/pin/552676185505992462/) Amastigote form  It is an obligate intracellular form and the infective stage to vector, sandfly.  Found in reticuloendothelial cells like macrophages, neutrophils, endothelial  cells of liver, spleen, bone marrow, etc. of the vertebrate hosts like humans, dogs and rodents  Round to oval, 3-5 μm in size  Nucleus: It measures less than 1 μm, oval to round, located in center or side of the cell  Kinetoplast: Consists of copies of mitochondrial DNA. It is made up blepharoplasty and parabasal body connected by a delicate fibril (cytoskeleton). It lies at right angle to the nucleus Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 36 of 56  Axoneme: It extends from blepharoplasty to the cell wall. It represents the intracellular portion (root) of flagellum  There is no external flagellum and it is nonmotile  Vacuole: It is a clear space, lies adjacent to axoneme Promastigote form This is an extracellular form, infective stage to humans.  It is mainly found in sandfly and in culture  It is motile and contains single anterior flagellum  Pear shaped, 8–15 μm length  Nucleus is situated centrally and kinetoplast is placed near the anterior end transversely  Axoneme: Represents the intracellular portion of flagellum Life Cycle Host: Leishmania completes its life cycle in two hosts: 1. Vertebrate host (man, dog, rodents, etc.) 2. Insect vector (female sandfly): Phlebotomus argentipes Infective form: Promastigote forms present in the midgut (majority) or foregut (small proportion) of female sandfly Mode of transmission: By bite of an infected sandfly mainly during the late evening or the night time. Minimum 10–1,000 promastigotes per infective bite are required to initiate the infection In vertebrate hosts, including humans:  Promastigotes are regurgitated from the midgut rarely or directly discharged from foregut (proboscis) of the female sandfly into the skin of the vertebrate host  Promastigotes are phagocytosed by the skin macrophages and transform into amastigote forms within 12–24 hours  The amastigote forms inside the macrophages multiply further causing cell rupture and release into the circulation  Amastigotes are carried out in the circulation to various organs like liver, spleen and bone marrow and invade the reticuloendothelial cells like macrophages, endothelial cells, etc. In sandfly:  During the blood meal taken up by the sandfly, the amastigotes are ingested and transformed into promastigote forms in the insect midgut  Promastigotes multiply by longitudinal fission and pass through various stages such as: Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 37 of 56 - Amastigote → procyclic promastigote → nectomonad promastigote → haptomonad promastigote → leptomonad promastigote → metacyclic promastigote - The metacyclic promastigotes multiply in the midgut of vector by binary fission and a small proportion migrates to the foregut (proboscis). They infect a new host during another blood meal  The duration of the life cycle in sandfly varies from 4 to 18 days depending on the species Figure 6.17. Life Cycle of Leishmanis donovani (Source: https://www.cdc.gov/parasites/leishmaniasis/biology.html) Pathogenicity and Clinical Features Visceral leishmaniasis - Incubation period ranges from 2–6 months. - The hallmark of VL is a triad of fever, hepatosplenomegaly and pancytopenia Diagnosis  Microscopy (detects LD bodies)  Culture (detects promastigotes)  Antidbody detection in serum Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 38 of 56  Nonspecific tests to detect hypergammaglobulinemia  Molecular method—PCR  Leishmanin test (montenegro test) Leishmania Tropica Complex It includes three species:  L. tropica is reported from Western India (mainly Rajasthan), Middle East and Mediterranean coast. It mainly affects urban area hence known as agent of urban anthroponotic CL  L. aethiopica infects people from Ethiopia, Uganda and Kenya  L. major is reported from Middle East, India, China, Africa, and central and western Asia. It mainly affects rural area hence known as agent of rural zoonotic CL. Life Cycle The life cycle of the L. tropica complex is same as L. donovani except:  The species of vector sandfly are different: - L. tropica—vector is P. sergenti - L. aethiopica—vector is P. longipes - L. major—vector is P. papatasi  Reservoir of infection: - L. tropica—is man (anthrop onotic) - L. aethiopica—is Hyraxes (Zoonotic) - L. major—is rodents (zoonotic)  In humans, the amastigote forms reside in reticuloendothelial cells of skin (they do not migrate to viscera). Clinical Features Cutaneous Leishmaniasis It is caused by L. tropica complex. This condition is also known as “Oriental sore”, Delhi Boil, Aleppo Boil and Baghdad Button, etc  Oriental sore usually occurs on face and hands. It begins as papule, becomes nodular and finally it ulcerates. The margins of the ulcers are raised, painless and indurated.  Lesions may be single or multiple and vary in size from 0.5 cm to more than 3 cm Mostly, it heals spontaneously leaving behind a scar  There may be satellite lesions, especially in L. major and L.tropica infections Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 39 of 56 Leishmaniasis recidivans It is a granulomatous response occurs years after healing of primary sore due to L. tropical. Characterized by new lesions formed on the face, usually scaly, erythematous papules and nodules develop in the center or periphery of a previously healed sore. CMI is intact and skin test is positive. Very few parasites can be demonstrated in the smears from the lesions Figure 6.18. A and B. Real images showing clinical features of (A) cutaneous leishmaniasis; (B) leishmaniasis recidivans Diffuse cutaneous leishmaniasis It is a rare form of leishmaniasis, caused by L. amazonensis and L. mexicana in South and Central America (New World) and by L. aethiopica in Ethiopia and Kenya (old World). Characterized by the lack of a CMI response to the parasite Low CMI leads to widespread cutaneous disease—symmetric or asymmetric distribution of various lesions like papules, nodules, plaques, and areas of diffuse infiltration, non ulcerative lesions with heavy load of parasites Diagnosis  Microscopy—detects amastigotes  Culture—NNN medium  Montenegro test—positive except in diffuse CL Treatment  Supportive therapy  Specifi c antileishmanial drugs - Pentavalent antimonial, v Resistance to antimonials Amphotericin B, Paromomycin, Miltefosine Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 40 of 56 New World Leishmaniasis It is mainly caused by:  Leishmania Viannia (L.V.) braziliensis complex  Leishmania Leishmania (L.L.) Mexicana complex  L.L. chagasi (new world variant of L.L. infantum) The morphology and life cycle of new world Leishmania species are identical to that of L. donovani except:  Geographical distribution-restricted to central and south America  Vector: Lutzomyia species  Reservoir of infection: Dogs, foxes (zoonotic)  The amastigote forms in humans reside in reticuloendothelial cells of skin and mucus membrane (don’t invade viscera). Clinical Features L. mexicana complex infected people develop CL similar to those seen with old world cutaneous disease. L. mexicana causes a specific form of CL called as chiclero ulcer (or bay sore) characterized by persistent ulcerations in pinna seen in Central America among workers living in forests harvesting chicle plants to collect chewing gum latex. 30% of people are infected during the fi rst year of exposure L. mexicana and L. amazonensis produce Diffuse cutaneous leishmaniasis (DCL) similar to that is described earlier for L. aethiopica. Leishmania Viannia braziliensis - cause mucocutaneous leishmaniasis MCL and also cutaneous leishmaniasis (CL) similar to oriental sore but they are more severe Espundia (mucocutaneous leishmaniasis) L. braziliensis infects mucous membrane of the nose, oral cavity, pharynx or larynx months to years after the CL. It is seen in 1–3% of patients infected with L. braziliensis, more in males of age 10–30 years  The initial symptoms are often nasal stuffiness, erythema and mucopurulent discharge.  It may eventually involve the upper lip, buccal, pharyngeal, or laryngeal mucosa  Ulcerative lesions are formed with erosion of the soft tissue and the cartilages leading to loss of lips, soft part of nose and soft palate  Gradually, the nasal septum may be destroyed, resulting in nasal collapse with hypertrophy of upper lip and nose leading to development of “tapir nose” Forest yaws and uta: The cutaneous lesions of L. V. guyanensis and L. V. peruviana are known as forest yaws (pain bois) and uta respectively. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 41 of 56 Leishmania Leishmania chagasi L.L. chagasi is the new world variant of L. L. infantum.  Causes mediterranean VL and CL  Occurs in Central and South American region  It is zoonotic (canine reservoir)  Vector: Lutzomyia species  Age: Children are aff ected commonly Diagnosis  Microscopy—detects amastigotes  Culture—NNN medium  Montenegro test—Positive (except in DCL and active VL)  Antibody detection—Poorly sensitive in DCL and MCL Treatment  In contrast to Old World CL, systemic therapy is recommended for New World CL as the lesions are more chronic, multiple and shows tendency for mucosal involvement  Pentavalent antimonial is the drug of choice, administered as a dose of 20 mg/kg for 30 days  In case of relapse, liposomal amphotericin B (2–3 mg/kg for 20 days) or miltefosine (2.5 mg/kg for 28 days) are given. Prevention Vaccine Trials  Currently no vaccine is available for the prevention of leishmaniasis. However, several trials are going on.  Both killed and live-attenuated vaccine trials are on going targeting antigens derived from killed promastigotes  Trials for recombinant and synthetic vaccines are also on going using gp-63 antigen. Control Measures  Vector control measures to eradicate sandfly:  Personal prophylaxis by using insect repellents or bed nets  Control of canine or rodent reservoir  Phlebotomus doesn’t fly high above the ground level and it is nocturnal in habitat.  So, sleeping at top floors also can prevent transmission  Early treatment of all cases (mainly anthroponotic VL and PKDL cases). Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 42 of 56 Chapter 6 PARASITIC PROTOZOA Phylum Sporozoa Phylum Sporozoa (Apicomplexa) is distinguished morphologically by the presence of a specialized complex of apical organelles (micronemes, rhoptries, polar ring, conoids and dense granules) which help in invasion into the host cell. Sporozoa contains one Class Coccidea which in turn has three Orders Eimeriida, Haemosporida, Piroplasmida. Order Haemosporida and Order Pirop lasmida include the blood parasites belonging to genus Plasmodium (the causative agent of malaria) and Babesia (rare parasites infecting humans) respectively Malaria - Malaria is a mosquito-borne disease caused by a parasite. People with malaria often experience fever, chills, and flu-like illness The Causative Agent of Malaria More than 125 species of Plasmodium exist infecting wide range of birds, reptiles and mammals. However, human infection is mainly caused by five species such as: 1. P. vivax causes benign tertian malaria. (periodicity of fever is once in 48 hours, i.e. recurs every third day) 2. P. falciparum causes malignant tertian malaria. (severe malaria, periodicity of fever is once in 48 hours, recurs every third day) 3. P. malariae causes benign quartan malaria. (periodicity of fever is once in 72 hours, i.e. recurs every fourth day) 4. P. ovale causes ovale tertian malaria. (periodicity of fever is once in 48 hours, i.e. recurs every third day) 5. P. knowlesi causes quotidian malaria. (fever periodicity is once in 24 hours, i.e. recurs every day). It is a parasite of monkey but can also affect humans and many cases affecting man were recently reported from Asia. Figure 1.19. Plasmodium spp. under a Microscope (Source: https://www.medmastery.com/guide/malaria-clinical-guide/how-identify-type-malaria-blood-smear) Other Plasmodium species are mainly of animal importance like P. cynomolgi, P. simium, etc. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 43 of 56 Life Cycle Host: Plasmodium completes its life cycle in two hosts: 1. Female Anopheles (Anopheline) mosquito is the definitive host where the sexual cycle (sporogony) takes place 2. Man acts as intermediate host where the asexual cycle (schizogony) takes place Infective form: The sporozoites are the infective form of the parasite. They are present in the salivary gland of female Anopheles mosquito. When Plasmodium species is transmitted by blood transfusion or through placenta, merozoites act as infective form. Mode of transmission: Man gets infection by the bite of female Anopheles mosquito. Sporozoites from the salivary gland of the mosquito are directly introduced into the blood circulation. Rarely, it can also be transmitted by:  Blood transfusion  Transplacental transmission. In humans, the asexual cycle takes place through the following stages:  Pre-erythrocytic schizogony  Erythrocytic schizogony  Gametogony. Figure 6.20. Life Cycle of Plasmodium spp. (Source: https://www.cdc.gov/malaria/about/biology/ Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 44 of 56 Pre-erythrocytic Schizogony This stage occurs in liver and it is so named because it occurs before the invasion of RBC.  It is also called Exoerythrocytic stage or intrahepatic or tissue stage  The motile sporozoites leave the circulation within 30 minutes and enter liver  Attachment: The circumsporozoite protein present on the surface of sporozoites binds noncovalently to the receptors on the basolateral surface of hepatocytes facilitating the entry of sporozoites  After entering into hepatocytes, the spindle shaped sporozoites become rounded and lose their apical complex and transform into trophozoites  Trophozoite is the feeding stage of the parasite which later on undergoes several nuclear divisions (schizogony) and transforms into pre-erythrocytic schizont  Pre-erythrocytic schizont contains several merozoites; which are released outside on rupture and attack RBCs to perform erythrocytic schizogony  As only few hepatocytes are infected by Plasmodium, so hepatic damage doesn’t occur in malaria  Duration of preerythrocytic schizogony varies from 5 days to 15 days depending on  the species  Some sporozoites of P. vivax and P. ovale don’t develop further and may remain in liver as hypnozoites and cause relapse of malaria after many years  Relapse (reactivation of the infection via hypnozoites) should be differentiated from another phenomena seen in P. falciparum and P. malariae called as recrudescence (recurrence of infection with all malaria that lacks hypnozoites. This occurs when the infection (unless a new infection) has persisted in the blood at undetectable levels and then becomes detectable again) Erythrocytic schizogony  The hepatic merozoites after released from preerythrocytic schizont, attack RBCs.  Merozoites bind to the glycophorin receptors on RBC surface, enter by endocytosis and are contained within a parasitophorous vacuole inside the RBCs. The process of entry into RBC takes about 30 seconds  Trophozoite: Soon the pear shaped hepatic merozoites round up, lose their internal organelle and transform into trophozoites  Ring form: Early trophozoite form is known as ring form. It is annular or signet ring  appearance containing a central vacuole and peripheral thin rim of cytoplasm and a nucleus  Ring form occupies one third of RBC except in P. falciparum, where it occupies one sixth of RBC  Prepatent period: Ring forms are the first asexual form that can be demonstrated in the peripheral blood. The time interval between the entry of the parasite into man and demonstration of the parasite in the peripheral blood is called as prepatent period. It varies between the species; - P. vivax—8 days - P. falciparum—5 days - P. malariae—13 days - P. ovale—9 days Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 45 of 56  Malarial pigment  Plasmodium feeds on hemoglobin. The undigested product of hemoglobin metabolism like hematin, excess protein and iron porphyrin combine to form malarial pigment (hemozoin pigment)  The appearance of malarial pigment varies, mostly it is brown black in color and numerous (except in P. vivax it is yellowish brown in color and in P. falciparum, it is few in number)  Late trophozoite: Ring form enlarges and becomes more irregular due to amoeboid movement and transforms into late trophozoite or amoeboid form  Erythrocytic schizont: Late trophozoites become compact, vacuoles disappear, pigments scatter throughout cytoplasm and nucleus becomes larger and lies at the periphery. This form is known as erythrocytic schizont  Schizogony: Erythrocytic schizont undergoes multiple nuclear divisions (erythrocytic schizogony or merogony) and produces 6–30 daughter merozoites arranged in the form of rosette  Number of merozoites per mature schizont varies: - P. vivax—12–24 number (average 16) - P. falciparum—18–24 number (average 20) - P. malariae—6–12 number (average 8) - P. ovale—8–12 number (average 8)  RBCs then rupture to release the daughter merozoites, malarial pigments and toxins into the circulation which result in malarial paroxysm of fever at the end of each erythrocytic cycle  Each merozoite is potentially capable of invading a new RBC and repeating the cycle. Intraerythrocytic life cycle takes roughly 48 hours for P. falciparum, P. vivax and P. ovale, 72 hours for P. malariae and 24 hours for P. knowlesi  Incubation period: The time interval between entry of the parasite to the body and appearance of the first clinical feature is known as incubation period. It varies between the species: - P. vivax—14 days (ranges 8–17 days) - P. falciparum—12 days (ranges 9–14 days) - P. malariae—28 days (ranges 18–40 days) - P. ovale—17 days (ranges 16–18 days) Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 46 of 56 Figure 6.21. Life Cycle of Malarial Parasite (Asexual Stage) Gametogony  After a series of erythrocytic cycles, some merozoites after entering into RBCs, instead of developing into trophozoites, they transform into sexual forms called as gametocytes.  The gametocytic development takes place in the blood vessels of internal organs such as spleen and bone marrow and only the mature gametocytes appear in the peripheral blood  The gametocytes of all the species are round in shape except in P. falciparum in which they are crescent or banana shaped  They are of two types—(1) male gametocyte (or microgametocyte) and (2) female gametocyte (or macrogametocyte)  Microgametocytes in all the species are smaller in size, lesser in number, their cytoplasm stains pale blue, and nucleus is larger, stains red and diffuse  In contrast, macrogametocytes are larger, numerous, their cytoplasm stains deep blue, nucleus is small, red and compact  The time of appearance of gametocytes in the circulation from the first appearance of asexual forms (i.e. ring forms) in the peripheral blood varies between the species - P. vivax—4–5 days - P. falciparum—10–12 days Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 47 of 56 - P. malariae—11–14 days - P. ovale—5–6 days  Neither gametocytes don’t cause any clinical illness nor they divide  Individuals harboring gametocytes are considered as carriers or reservoirs of infection and play an important role in the transmission of the disease  A patient can be a carrier of several Plasmodium species at the same time  However, gametocytes are effective in transmission of the infection if they are: mature, viable, and present in sufficient density (usually 12 per cubic mm of blood) Mosquito Cycle  A female Anopheles mosquito during the blood meal, takes both the asexual forms and the sexual forms.  The asexual forms get digested whereas the sexual forms, i.e. the gametocytes undergo further development (hence considered as infective form of the parasite to mosquito)  Exflagellation: - Nucleus of the male gametocytes divides into eight flagellated actively motile bodies (15–20 μm length) called as microgametes. - Microgametes protrude out as thread like filaments, lash out for some time and then, break free - This process is called as exflagellation. At 28°C, it is completed in 15 minutes for P. vivax and 15–30 minutes for P. falciparum. Female gametocytes don’t divide and don’t undergo exflagellation but each undergoes maturation to form one macrogamete or female gamete  Zygote: The male microgamete fertilizes with the female macrogamete by fusion of their pronuclei and the zygote is formed. Fertilization occurs in about 30 minutes to 2 hours after the blood meal  Ookinete: Within 24 hours, the nonmotile rounded zygote transforms into vermicular motile elongated form with an apical complex (the ookinete stage). Till this stage, the development takes place in the midgut of the mosquito  Oocyst: The ookinete penetrates into the stomach wall of the mosquito and lies just beneath the basement membrane. It becomes rounded and covered by a thin elastic membrane to form oocyst. This is the stage was discovered by Sir Ronald Ross. Several thousands of mature oocysts can be found; each measuring 500 μm  Sporozoites: Oocysts undergo sporogony (meiosis) to produce thousands of spindle shaped sporozoites measuring 10–15 μm length with apical complex anteriorly  On rupture of the mature oocyst, the sporozoites are released and migrate to salivary gland  Mosquito is said to be infective to man only when the sporozoites are present in salivary gland. Once infected, it remains infective throughout the life  Extrinsic incubation period: Time required to complete the life cycle in mosquito is called as extrinsic incubation period and it varies from 1 week to 4 weeks. At 25°C, it is: - P. vivax—8–10 days Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 48 of 56 - P. falciparum—9–10days - P. malariae—25–28 days - P. ovale—14–16 days  Mixed infection: Different species of Plasmodium can infect the same mosquito which in turn can transmit mixed infections to man Figure 6.22. Life Cycle of Malarial Parasite (Sexual Stage) Pathogenesis Benign malaria Benign malaria is milder in nature, can be caused by all four species. It is characterized by a triad of febrile paroxysm, anemia and splenomegaly. a. Febrile paroxysm - Fever comes intermittently depending on the species. It occurs every fourth day (72 hour cycle for P. malariae) and every third day (48 hour cycle for other three species) - Paroxysm corresponds to the release of the successive broods of merozoites into the bloodstream, at the end of RBC cycle - Each paroxysm of fever is comprised of three stages: 1. Cold stage (patient feels lassitude, headache, nausea, intense cold, chill and rigor 2. Hot stage (Patient develops high grade fever of 39–41°C and dry burning skin. Headache persists but nausea diminish. 3. Sweating stage (Fever comes down with profuse sweating. Skin becomes cold and moist. Patient feels relieved and often asleep. This stage lasts for 2–4 hours) Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 49 of 56 Anemia After a few paroxysms of fever, patient develops a normocytic normochromic anemia. Various factors can attribute to the development of anemia such as:  Parasite induced RBC destruction—Lysis of RBC due to release of merozoites  Splenic removal of both infected RBC and uninfected RBC coated with immune complexes  Bone marrow suppression leading to decrease RBC production  Increased fragility of RBCs  Autoimmune lysis of coated RBCs Splenomegaly After a few weeks of febrile paroxysms, spleen gets enlarged and becomes palpable. Splenomegaly is due to massive proliferation of macrophages that engulf parasitized and nonparasitized coated RBCs. Falciparum malaria (malignant Tertian malaria) Pathogenesis of falciparum malaria Plasmodium falciparum possesses a number of virulence factors and its pathogenesis is different from other species. Hence the disease is more acute and severe in nature with more complications than the benign malaria. Sequestration of the parasites: P. falciparum has the ability to sequester (holding back) the parasites in the blood vessels of deep visceral organs like brain, kidney, etc. This leads to blockade of vessels, congestion and hypoxia of internal organs. Sequestration is mediated by:  Cytoadherence: It refers to binding of infected erythrocytes to endothelial cells. It is mediated by a specialized antigen called as P. falciparum erythrocyte membrane protein-1 (PfEMP-1)  Rosetting: It refers binding of infected erythrocytes to uninfected erythrocytes. PfEMP1 also plays an important role in rosetting, as it can adhere to complement receptor 1 (CR1) and blood group A antigen present on the uninfected erythrocytes  Deformability: Parasitized RBCs become more spherical and rigid, and are less filterable than uninfected cells - Since the parasites are sequestrated back in deep vessels, they can avoid frequent spleen passage, hence can escape splenic clearance - PfEMP undergoes frequent antigenic variation, thus helps the parasite in evading thehost immune response. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 50 of 56 Complication of P. falciparum  Cerebral malaria - Occurs due to plugging of brain capillaries by the rosettes of sequestered parasitized RBCs leading to vascular occlusion and cerebral anoxia  Renal failure - It occurs due to erythrocyte sequestration in renal microvasculature leading to acute tubular necrosis. It is common among adults than children  Pulmonary edema and adult respiratory distress syndrome - Severe falciparum malaria in adults may lead to non-cardiogenic pulmonary edema often aggravated by over hydration. Usually it doesn’t respond to antimalarial therapy mortality rate more than 80%  Tropical splenomegaly syndrome - abnormal immunologic response to repeated malaria infections Figure 6.23. A to C. Thin blood smear showing different forms of Plasmodium falciparum (A) multiple ring form and accole form; (B) double dot (head phone shaped) ring form; (C) gametocyte Figure 6.24. A to C. Thin blood smear showing different forms of Plasmodium vivax (A) ring form; (B) gametocyte; (C) schizont Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 51 of 56 Figure 6.25. Left to right. Thin blood smear showing ring form of Plasmodium malariae (left); Thin blood smear showing ring form of Plasmodium ovale (right) Laboratory Diagnosis of Malaria Microscopic tests: - Peripheral blood smear—Gold standard - Thick smear—more sensitive - Thin smear—speciation can be done - Fluorescence microscopy (Kawamoto’s technique) - Quantitative buff y coat examination Nonmicroscopic tests: - Antigen detection tests (RDTs) or ICTs— detects parasitic LDH, HRP-II, aldolase - Antibody detection—ELISA - Culture—RPMI 640 medium - Molecular diagnosis—PCR using PBRK1 primer Treatment Figure 6.26. Antimalarial drugs and their activity Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 52 of 56 Vaccination for Malaria Several vaccine trails are done in Africa, Asia and the United States. Despite of intense research, till date, there is no vaccine licensed for human use. The approaches are made targeting the various stages of malaria cycle. The main problems in malaria vaccine include:  The vaccine candidates are poor inducer of cell mediated immune response  Antigenic variation in malarial antigens such as PfEMP  Different immune mechanisms occur in different stages of malaria life cycle. Self-Learning Module in Genetics (BIO 4) Estigoy and Sazon.., 2022  Page 53 of 56 Balantidium coli Balantidium coli, is the largest protozoan and the only ciliated parasite of humans. Though it was observed by A.V Leeuwenhoek earlier while examining a dysentery stool but the proper description was given later by Malmsten (Sweden) in man in 1856.  Taxonomy: It belongs to the Phylum Ciliophora, Class Litostomatea, Order Vestibuliferida and Family Balantidiidae.  Habitat: It resides in the large intestine of man, pig (main reservoir) and other animals. Morphology It exists in two forms—(1) trophozoite (found in dysenteric stool) (2) cyst (found in carriers and chronic cases). Both the forms are binucleated having a large macronucleus and a small micronucleus.  Trophozoite  It is found in the active stage of the disease and considered as the invasive form.  It is oval shaped, 30–300 μm in length and 30–100 μm in breadth  The whole body is covered with a row of tiny delicate cilia (organ of locomotion)  Cilia present near to the mouth part appear to be longer and called as “adoral cilia”  Anterior end is narrow and the posterior end is broad  Anterior end bears a groove (peristome) that leads to a mouth (cytostome) followed by a short funnel shaped gullet (cytopharynx) extending up to one- third of the body  There is no anus  Posterior end is broad, round and bears an excretory opening called a cytopyge  The cytoplasm is divided into outer clear ectoplasm and inner granular endoplasm  The endoplasm contains: - Two nuclei: large kidney shaped macronucleus in the center and a small micronucleus lies in the concavity of the macronucleus - Two contractile vacuoles: lie side by side or one above the other. They maintain the proper osmotic pressure inside the cell - Numerous food vacuoles: It contains food particles like debris from host gut, bacteria, starch grains, fat droplets and occasional red blood cells (RBCs), etc. Digestion of the food particles takes place here.  Cyst  It is round, measu