1518784669M10PlasmodiumMorphologyandLifeCycleQuad1.pdf

Paper : 08 Biology of Parasitism Module : 10 Plasmodium: Morphology and Life Cycle Development Team Principal Investigator: Prof. Neeta Sehgal Department of Zoology, Uni...

Paper : 08 Biology of Parasitism Module : 10 Plasmodium: Morphology and Life Cycle Development Team Principal Investigator: Prof. Neeta Sehgal Department of Zoology, University of Delhi Co-Principal Investigator: Prof. D.K. Singh Department of Zoology, University of Delhi Paper Coordinator: Dr. Pawan Malhotra ICGEB, New Delhi Content Writer: Dr. Gauri Mishra1 and Dr. Lokesh Chandra Mishra2 1. Swami Shraddhanand College, University of Delhi 2. Hansraj College, University of Delhi Content Reviewer: Prof. Virender Kumar Bhasin Department of Zoology, University of Delhi 1 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  Description of Module Subject Name ZOOLOGY Paper Name Biology of Parasitism Module Name/Title Plasmodium: Morphology and Life Cycle Module Id M10; Morphology and Life Cycle Keywords Plasmodium, Infection, Species, Parasite, Erythrocytic Contents 1. Learning Outcomes 2. Introduction 3. Spectrum of Plasmodium Infection 3.1.Species of Malaria Parasite Afflict Human Beings 4. Morphology of Plasmodium 5. Life Cycle of the Malaria Parasite 5.1.Exo-erythrocytic Stages of Human Malaria Parasites 5.2.Erythrocytic Stages of Human Malaria Parasites 5.3.Sexual Cycle in the Mosquito 6. Summary 2 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  1. Learning Outcomes The module has been designed to make you understand:  General aspects of Plasmodium  Spectrum of Plasmodium infection  Species of Malarial parasite afflicting human beings  Morphology of different stages  Life Cycle of Plasmodium 2. Introduction Plasmodium, an intracellular endoparasitic protozoan which passes on to human beings by female Anopheles mosquito, is responsible for causing Malaria. It is commonly known as the malaria parasite. Malaria is one of the most dreaded diseases of tropical countries and remains as an epidemic in more than 100 countries (Figure 1). Deadly fevers-probably malaria, have been recorded since the beginning of the written word and references can also be found in Vedic writings in India. “Malaria” is an Italian word mala aria means “bad air”. It was so termed as it was thought that malaria was caused by breathing in bad air or gas over marsh or swamps. In 1880, French Army physician, Charles-Louis-Alphonse Laveran discovered Plasmodium, the causative organism for malaria and subsequently in the year of 1897, Sir Ronald Ross discovered malaria cysts in the stomach wall of Anopheles mosquito. For the same he was honored with Nobel Prize in 1902. In 1898, malaria transmission through the mosquito was established by an Italian scientist Giovanni Batista Grassi. These discoveries transformed the face of malaria research. 3 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  Figure 1: Global distribution of Malaria The systematic position of malarial parasite described by Mhelhorn and Walldorf (1988) is as follows: Kingdom Protista Sub Kingdom Protozoa Phylum Apicomplexa Class Sporozoa Sub Class Coccidia Order Haemosporida Sub Order Aconoidina Family Haemosporidae Genus Plasmodium 3. Spectrum of Plasmodium Infection There are more than 120 species of Plasmodium which are known to infect various groups of vertebrates. Few of them are as follows: Human parasites: There are four species of Plasmodium which are known to infect humans; Plasmodium falciparum, P. vivax, P. ovale and P. malariae. Out of four species mentioned, P. falciparum is the major cause of morbidity and mortality. Recently, P. knowlesi has also been reported to cause infection in human beings which is known to infect monkeys. 4 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  Rodent parasites: P. berghei, P. yoelii, P. vinckei and P. chabaudi are the four species known to infect rodents which can be differentiated on the basis of their erythrocytic cycle synchronicity and preference to infect differently aged RBCs. Simian parasites: There are twenty species of Plasmodium which are believed to infect monkeys or apes with a range of similar biological characteristic to that of human malarial parasites. e.g. P. cynomolgi, P. fragile, P. fieldi, P. hylobati, P. simiovale etc. Avian parasites: P. gallinaceum, P. lophurae, P. elongatum, P. relictum and P. fallax are the most common avian malarial parasites. These species differ from the mammalian parasites in having different site of pre-erythrocytic schizogony. The site of pre-erythrocytic schizogony in avian parasites is mesoderm while in mammals it is liver parenchyma. Reptilian parasites: P. wenyoni is the only species which infects snakes. Malarial parasites are very common in lizards while absent in crocodiles. 3.1. Species of Malarial Parasite Afflict Human Beings Various species of Plasmodium infect humans to produce different symptoms and periodicity which are as follows: Plasmodium vivax P. vivax causes benign tertiary malaria. Primarily, symptoms include headache, nausea, anorexia and vomiting. Other symptoms include perspiration, shivers and very high temperature. Plasmodium ovale P. ovale causes ovale malaria. The symptoms are comparable to benign tertiary malaria. If left untreated, it can last for about a year. Plasmodium malariae P. malariae causes quaternary malaria. The bouts of temperature are of 72 hour periodicity. The symptoms are much similar to benign tertiary malaria. Untreated cases can last about 20 years. 5 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  Plasmodium falciparum P. falciparum causes malignant tertiary malaria which is the lethal form of malaria. Symptoms are similar to flu with daily shivers, temperature, intense nausea, vomiting and diarrhoea. Crises reappear every 36 to 48 hours. In this, brain is more frequently affected. The unique property of causing agent is sequestration with endothelial wall of capillaries causing brain haemorrhage. This eventually leads to either coma or death of the infected individuals. Renal lesions are found in infected individuals. Because of vomiting and diarrhoea liver is also affected which causes rapid dehydration. 4. Morphology of Plasmodium The blood-stages of human Plasmodium species display different morphology and modification in the host erythrocyte. These morphological variations can be used to differentiate the four species (Table 1). P. falciparum blood stages are characterized by the existence of slightly smaller and numerous ring stages than the other species. Erythrocytes having multiple infections are seen frequently in P. falciparum as in the other species. Distinct crescent-shaped gametocytes appear late in the infection. P. vivax with engorged infected erythrocytes and granules 'Schuffner's dots', above the erythrocyte cytoplasm, manifests at caveola-vesicle complexes forming on the erythrocyte membrane. The trophozoite of P. vivax is ameboid in shape. The schizonts possess more than 20 merozoites. P. ovale also exhibits Schuffner's dots with an enlarged erythrocyte. It is difficult to discern the infection from P. vivax. P. ovale in general, is rather compact parasite than P. vivax. This insistence is most apparent in the growing trophozite stage. Merozoites are less per schizont. Elongated host erythrocytes are found in case of P. ovale. P. malariae exhibit compact stages and do not modify the host erythrocyte except few elongated trophozoites which stretch erythrocyte transversely to form a band like structure. Schizonts possess 8-10 merozoites, often arranged in a rosette pattern, with center having a tuft of pigment. A comparative account of various stages (Table 1) and the diagrammatic figures (Table 2) clearly represent the disparity in the morphological appearance of the four Plasmodium species. 6 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  Table 1: Characteristics of Plasmodium parasites Species P. falciparum P. vivax P. malariae P. ovale Host Cell Size Not enlarged Enlarged Not enlarged Enlarged Shape Round Round or oval Round Round or oval, Normal & Color Pale Normal Normal may turn dark Large red spots Small red dots few tiny dots abundant small red dots Stippling Maurer‟s dots Schüffner's dots Ziemann‟s dots James‟s dots Usually black or Golden brown Black or brown Pigment As of P. malariae dark brown granules coarse granules Parasite Large slightly Small, compact Regular shape staining dark, staining Strong affinity to Regular shape. amoeboid General features parasite. many form a band Size in between P. vivax and parasite. infections of across the P. malariae numerous single RBC infected RBC trophozoites Trophozoites, Common Stages Only rings and Schizonts, As in P. vivax As in P. vivax found in smear gametocytes Gametocytes fragile, small, 1.5 Single, Large 2.5 µm Double Similar to P. Ring stage µm, Prominent Similar to P. vivax, but compact chromatin and vivax but thicker thick chromatin multiple rings. Compact, Large, prominent Characteristic inconspicuous irregular vacuole band form, Compact rough pigment, large Trophozoite small vacuole, Chromatin as vacuole irregular clumps of chromatin rarely seen in dots or threads inconspicuous smear Large, filling the Nearly fills RBC, Small, compact RBC, segmented, segmented, Almost fills RBC, segmented, Schizont rarely seen in yellow brown pigment is dark pigment dark yellow brown blood smear pigment brown Bigger than Smaller than Fills enlarged RBC, kidney RBC, very few in RBC, round or shaped with blunt peripheral blood oval, compact Micro- round ends, film, round Same as in P. vivax. Very few in pale blue gametocyte reddish blue compact. Pale peripheral blood flim. cytoplasm, cytoplasm, smear blue cytoplasm. profuse brown with many fine Pigment and granules granules. chromatin as in 7 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  P. vivax Slender, nucleus Large, loose and Same as P.vivax, small, compact, Macro- fuzzy mass of low numbers Same as P.vivax, low numbers pigment granules gametocyte chromatin and appear after 12– appear after 12–14 days. closely smaller mass 14 days. aggregated Table 2: Diagrammatic illustration of the morphology of the different stages of the Plasmodium sps. life cycle in thin blood films Source: http://www.phsource.us/PH/PARA/Diagnosing_Medical_Parasites.pdf Structure of Plasmodium Merozoite Merozoite is an ovoid structure measuring approximately 1.5 micron in length and 1 micron in width. The apical end is like a condensed cone-shaped projection demarcated by the polar rings. At the anterior end of the Merozoite three types of membrane-bound organelles, Rhoptries (two prominent pear-shaped), micronemes (ovoid bodies) and dense granules (spheroid vesicles) are present (Figure 2). The function of 8 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  these organelles is related to the binding and entry of the Merozoite into the host cells. Merozoites are mainly short-lived and hence need to enter a new host erythrocyte just after the release. A trilaminar pellicle surrounds the merozoite, which is made of a plasma membrane and two closely associated inner membranes. Underneath this inner membrane complex is a row of subpellicular Microtubules, radiating posteriorly from the polar ring of the apical end. Both, the inner membrane complex and subpellicular Microtubules function as a cytoskeleton providing rigidity to the Merozoite. Mitochondrion are generally acristae or with very few cristae. The „apicoplast‟ (plastid) is believed to be the evolutionary homologue of the plant chloroplast. A single vesicular nucleus with a centrally located nucleolus is also present in Merozoite. Figure 2: Merozoite of Plasmodium The Infective Stage: Sporozoite The most adaptable of the invasive stages of the Plasmodium life cycle are the Sporozoites. It exhibits diverse behaviors, including gliding locomotion, invasion, migration and egress from target cells during 9 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  their passage from the mosquito vector to the vertebrate host. These functions are performed by rhoptries and micronemes, which are the only secretory organelles around the apical cap (Figure 3). Finally these sporozoites attack hepatocytes and change into exo-erythrocytic stages, continuing the cycle to the erythrocytic part in RBC. Figure 3: Typical structure of sporozoite of Plasmodium Source: http://www.kullabs.com 5. Life Cycle of the Malaria Parasite The life cycle of malaria parasite is very complex. It‟s a digenetic parasite, i.e. it requires two hosts to complete its cycle (Figure 4). The life cycle of all species of human malaria parasites is fundamentally the same. It consists of an exogenous sexual phase known as sporogony, which is multiplication stage in certain Anopheles mosquitoes and an endogenous asexual phase known as schizogony which is multiplication stage in the vertebrate host. The latter phase includes the development cycle in the red cells (erythrocytic schizogony) and the phase taking place in the parenchyma cells of the liver (pre-erythrocytic schizogony). The intermediate (secondary) host for the parasite is the vertebrate, while the mosquito is considered to be the definitive (primary) host as the sexual reproduction takes place in it. 10 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  Figure 4: Life cycle of malaria parasite (P. falciparum) Source: http://www.cdc.gov/parasites 5.1. Exo-Erythrocytic Stages of Human Malaria Parasites Sporozoites are the infective stage. Malaria infection in human starts with their injection into the blood stream during a blood meal by an infectious mosquito. The circulatory period of sporozoites is short for about 60 minutes, after which they enter the liver of the host actively. The sporozoites die shortly after invasion in the Kuppfer cells of liver, as these are resistant to sporozoites. Most of the sporozoites enter the hepatocytes to begin the asexual exo-erythrocytic schizogonic cycle. The liver trophozoite, a mononucleated round body, divides asexually to form a mature multinucleated schizont which finally releases a large number of merozoites. The number of merozoites formed at the end of the cycle is dependent on the species of the parasite. The merozoites are released into the sinusoids of the liver by the rupture of liver schizonts. 11 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  Released merozoites invade red blood cells. Two species of human malaria which parasite show relapses are P. vivax and P. ovale in which some of the liver trophozoites instantly start the exo-erythrocytic schizogony while others remain in a dormant stage and are termed as hypnozoites. 5.2. Erythrocytic Stages of Human Malaria Parasites The intricate and unstable spectrum of symptoms characterizing the disease in humans is due to Erythrocytic stages of malaria parasite. The presence of parasites in the blood of a patient allows the analysis of the infection and the differentiation of the various species. The time required to complete the erythrocytic cycle is a constant characteristic of the parasite species (Table 3). Merozoites initiate the blood phase of the life-cycle on rupture and discharge of liver schizonts into the circulation. The merozoites possess a single nucleus and adjacent cytoplasm with a diameter of 1 μm. It instantly invades an erythrocyte to develop in trophozoite stage. The young trophozoite feeds on erythrocyte, produces a vacuole which assumes the characteristic ring form. This stage is referred to as signet ring stage. Approximately in 18 hours expansion of cytoplasm and disappearance of vacuole takes place slowly, and there is an appearance of a characteristic parasitic pigment within the cytoplasm. Pigment, known as haemozoin (Yellowish brown malarial pigment, haemozoin), is formed due to the parasite ingestion of haemoglobin and decomposition of the same into protein and haematin. Protein is used as food whereas unused haematin form is toxic. Mature trophozoite possesses a single nucleus, a large cytoplasm without vacuole and inconsistent quantity of pigment. P. falciparum, P. vivax and P. ovale takes approximately 30 hours to start nuclear division after invasion while P. malariae takes approximately 40 hours. Nuclear division leads to the production of the schizont stage. Nuclear division continues until a definite number of merozoites are produced (Table 3). Table 3: Characteristic features of four species of human Plasmodia Species P. vivax P. ovale P. malariae P. falciparum Pre-erythrocytic cycle (days) 8 9 13 5-6 Pre-patent period (days) 11-13 10-14 15-16 9-10 Incubation period (days) 13 17 28 12 Number of merozoites per 10,000 15,000 2,000 40,000 tissue schizont Hypnozoites present present absent absent Erythrocytic cycle (hours) 48 50 72 48 12 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  A typical malaria paroxysm is determined by erythrocytic rupture to release the merozoites into the blood stream. Merozoites released into the circulation enter new erythrocytes to repeat the schizogony till the process is subdued by the specific immune response or by chemotherapy. Some of the merozoites differentiate into sexual forms (the gametocytes) in the course of erythrocytic schizogony which remain inside the RBCs (Figure 4.). Gametocytes become visible roughly from the third generation in case of P. vivax, P. ovale, and P. malariae while in case of P. falciparum it requires approximately 10 generations for appearance of gametocytes which perhaps reflects the slow maturation and the sequestration of the undeveloped stages in this species. Gametocytes of P. vivax, P. ovale and P. malariae are morphologically comparable to the late trophozoite while P. falciparum gametocytes present a crescent shape. In the peripheral blood two types of gametocytes, the female macro-gametocytes and the male micro-gametocytes are present. They can be distinguished on the basis of nuclear material which is dispersed in male parasite (preparing to ex-flagellation) while condensed in female parasite. 5.3. Sexual Cycle in the Mosquito Protein, required for egg formation of female Anopheles mosquitoes, comes through the blood meal. Throughout the life of the female mosquito, oviposition is continued which requires repetitive contacts with the vertebrate host for blood meal. These subsequent feedings allow malaria parasite‟s multiplication, maturation and transmission to other individuals. The sexual cycle starts with the ingestion of mature female and male gametocytes by a suitable species of Anopheles during a blood meal. In the midgut of insect, the female gametocytes get rid of the cover of erythrocytes to remain free in the extra- cellular space as macrogamete. The male gametocyte nucleus divides to form eight sperm like flagellated micro-gametes through ex-flagellation which arrive at midgut and actively move to fertilize a macrogamete. After fertilization a zygote is formed, which in approximately 15 hours, develops gradually into a motile ookinete. The peritrophic membrane and the epithelium of the midgut are ruptured by the ookinete to inhabit below the basal lamina of the outer gut wall. In 24-72 hours after the blood meal the ookinete develops into a non motile oocyst. Narrow and curved sporozoites are produced from oocyst on maturation which is actively motile and about 15 μm in length. The sporozoites travel and reach the salivary glands by making small perforations in the wall of cyst. Sporozoites penetrate the basal membrane of salivary gland and settle into the salivary duct. During the mosquito blood feeding, the salivary fluid content (possessing anti-clotting properties) with sporozoites is vigorously injected into the 13 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle  human host to initiate another asexual cycle. Mosquito, a poikilothermic host, affect the speed of the cycle as it strongly depends on the temperature and other climatic factors. 6. Summary Plasmodium is an intracellular endoparasitic protozoan and causes Malaria. Malaria is one of the most dreaded diseases of tropical countries. There are more than 120 species of Plasmodium which are known to infect various groups of vertebrates. Among those, Plasmodium falciparum, P. vivax, P. ovale and P. malariae can infect human beings. The malignant tertiary malaria caused by P. falciparum is the lethal one. The blood-stages of human Plasmodium species display diverse morphology and modification in the host erythrocyte. A fifth species, Plasmodium knowlesi, has recently been identified as a clinically significant pathogen in humans. This is an emerging infection in South East Asia. Merozoite is an ovoid cell with an apical condensed cone-shaped protrusion demarcated by the polar rings. Three types of membrane-bound organelles are present at the anterior end of the merozoite, which are Rhoptries (two prominent pear-shaped), micronemes (ovoid bodies) and dense granules (spheroid vesicles). These organelles functions in the binding and entry of the Merozoite into the host cell. The most versatile and infective stage of the Plasmodium life cycle is the Sporozoite. The life cycle of malaria parasite is very complex. It‟s a digenetic parasite, i.e. it requires two hosts to complete its cycle. It consists an exogenous sexual (sporogony) phase in Anopheles mosquitoes and an endogenous asexual (schizogony) phase in the vertebrate host. The vertebrate act as the intermediate or secondary host for the parasite, while the mosquito is considered to be the definitive or primary one as the sexual reproduction takes place in mosquito. Two species of parasite P. vivax and P. ovale show relapses where dormant stages (hypnozoites), are present. Erythrocytic stages include singlet ring stage, mature trophozoite (feeding stage) and schizonts. The female macro-gametocytes and the male micro-gametocytes can be distinguished on the basis of nuclear material which is dispersed in male parasite (preparing to ex-flagellation in mosquito) while condensed in female parasite. Mosquito, a poikilothermic host, affects the speed of the cycle since it strongly depends on the temperature and other climatic factors. Sporozoites, infective stage, find way to get accumulated in the salivary gland of Anopheles mosquito for next cycle in human. 14 Biology of Parasitism ZOOLOGY Plasmodium: Morphology and Life Cycle