ENTAMOEBA HISTOLYTICA

E. histolytica produces intestinal and extra- intestinal diseases. It is a leading cause of morbidity and mortality in developing countries. Amoebiasis is endemic in India. It can affect all age groups and both the sexes. Organism Characteristics E. histolytica exists as trophozoites, precysts and cysts. The trophozoites range in size from 18 to 40 μm (mean 25 μm) and do not have a definite shape. The trophozoites exhibit active unidirectional movement achieved

with the help of broad hyaline, finger-like pseudopodia. Their cytoplasm has an outer, clear ectoplasm and inner, granular endoplasm. The trophozoites of F. histolytica engulf red blood cells, bacteria, yeast and other debris. The presence of red blood cells in the cytoplasm is diagnostic of E. histolytica as it is the only intestinal amoeba to exhibit this characteristic.The trophozoites contain a single, spherical, 3 to 5 .tm nucleus. The nucleus has a delicate nuclear membrane. On the internal surface of the nuclear membrane there are minute granules known as chromatin dots. In the center of the nucleus is a single dense karyosome or nucleolus. The nuclear pattern of E. histolytica differentiates the

parasite from other species of amoeba. The nuclear details are evident on staining the trophozoites. The trophozoites of E. histolytica live in the mucosal folds of the large intestine and divide by binary fission. They may invade the intestinal wall. There are distinct invasive and non-invasive strains of E. histolytica. These strains vary according to their isoenzyme patterns.Other characters of virulence of E. histolytica include • Enhanced erythrophagocytosis • Reduced surface charge • Agglutination by concanavalin-A • Resistance to complement mediated lysis • Greater cytopathic effect on cell monolayers • Greater pathogenicity when inoculated into rat caecum or hamster liver • An ability to grow in semisolid media and axenic culture

Trophozoites when passed in faeces die on exposure to air. Gastric acid also destroys them. Therefore, trophozoites can not transmit infection. Thus, for transmission of infection and continuation of the species, E. histolytica has to change from a trophozoite to a cyst to enter a new host. In this process of encystation, the trophozoites become non-motile, rounded and devoid of inclusion. This is the precystic stage. The precysts are smaller than trophozoites and usually larger than cysts. They secrete a highly refractive cyst wall around themselves and become the cysts. The cysts of E. histolytica range in size from 8 to 22 μm (average 15 μm) and are spherical. Depending upon the maturity of the cyst, they may contain one, two or

four nuclei. The immature cysts have a single nucleus, diffuse glycogen mass and sausage- shaped chromatoid bodies. The

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glycogen mass serves as a food reserve. The chromatoid bodies contain ribonucleic acid. The cysts mature with two mitotic divisions of the nucleus to form sequentially binucleate and then quadrinucleate cysts. In this process the nuclear size is successively reduced from 5 μm to 2 μm. Simultaneously, the glycogen mass and the chromatoid bodies gradually reduce in size and ultimately disappear

immature cysts if passed in the faeces can mature outside the human body. The mature cysts can survive in moist environment for 10 days. They withstand the gastric acid and therefore act as the infective forms.

Life Cycle

E. histolytica has a simple life cycle. The mature cysts are the infective forms of E. histolytica. They infect humans through contaminated food or water. Contamination of food and water with the cysts occurs due to faeces, flies or unwashed hands of food handlers. Cross-connection between sewage and waterlines can lead to waterborne epidemics of amoebiasis. The cysts pass upto the ileo-caecal region; the surrounding alkalinity and the damage caused by trypsin to the cyst wall during the transit leads to excystation in this region. A quadrinucleate metacyst

emerges from a cyst. The metacyst rapidly divides to produce eight small amoebae (amoebulae or metacystic trophozoites). These amoebulae develop and mature to form trophozoites. The trophozoites establish themselves at areas of stasis in the large intestine. The static areas in the glandular crypts of the colon (particularly caecum) provide the optimum habitat.

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The trophozoites may live in the lumen without invading the intestinal mucosa in about 90% of infected persons. In such persons the trophozoites feed on colonic contents and mucus. The trophozoites encyst themselves when conditions like overpopulation of amoebae, pH change, extreme change in the available food and oxygen supply occur. The cysts pass in the faeces. Such cyst passers act as carriers and are responsible for maintenance and spread of infection in the community. This condition is eventually self-limiting; but, its course can exceed 10 years. The mean duration is 2 to 3 years. In the remaining 10% of the infected persons, amoebae invade the host tissue. Invasion is common during the first 4 months; but, it can be as trophozoites are frequently present in fresh stools’ of these persons. Encystation does not occur during the acute phase of dysentery. Encystation occurs later due to the factors mentioned above and the increased resistance developed by the host. Such persons recovering from dysentery and passing cysts in faeces are called convalescent carriers. The cysts passed by the asymptomatic and convalescent carriers continue the life cycle of E. histolytica..

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Domain: EukaryotaSuperphylum: AlveolataPhylum: ApicomplexaClass: AconoidasidaOrder: HaemosporidaFamily: PlasmodiidaeGenus: Plasmodium

Plasmodium is a genus of parasitic protozoa. Infection with this genus is known as malaria. The genus Plasmodium was created in 1885 by Marchiafava and Celli. Currently over 200 species in this genus are recognized and new species continue to be described.[1] [2]

Of the 200+ known species of Plasmodium, at least 10 species infect humans. Other species infect animals, including birds, reptiles and rodents. The parasite always has two hosts in its life cycle: a mosquito vector and a vertebrate host.

The genus is currently (2006) in need of reorganization as it has been shown that parasites belonging to the genera Haemocystis and Hepatocystis appear to be closely related to Plasmodium. It is likely that other species such as Haemoproteus meleagridis will be included in this genus once it is revised.

Plasmodium, the parasite responsible for human malaria, is among the most researched genera of parasites in the world. Despite extensive studies on possible control methods, infection in humans continues to grow in tropic and sub-tropic areas. Currently there are an estimated 500,000,000 infected persons, with 1-2 million dying annually. There are four types of Plasmodium which cause human malaria: Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, and Plasmodium malariae. All of these are transmitted to human hosts solely by way of Anophele mosquito vectors. Plasmodium is one of the oldest known parasites; its long history suggests a long, adaptive relationship with the human host. Today cases of the disease are increasing in non-malarious countries as more people travel to Africa, India, Brazil, and some Asian nations, where the mosquito vectors are most prevalent. Symptoms of the disease may go unnoticed or misdiagnosed; clinical signs include fever, chills, weakness, headache, vomiting, diarrhea, anemia, pulmonary and renal dysfunction, neurologic changes. Untreated malaria may result in death.

Cell Structure

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While the four major species of Plasmodium differ in some ways from each other, they all share the same complex life cycle involving the insect (mosquito) vector and the human host. When an infected Anophele mosquito bites a human, sporozoites are injected with the saliva. The sporozoites are 10 -15 µm in length and about 1 µm in diameter. They have a thin outer membrane, a double inner membrane below which lies the subpelicular microtubules. They have 3 polar rings and the rhoptries are long, extending half the length of the body. The micronemes, convoluted elongate bodies, run forward to the anterior of the sporozoite entering a common duct with the rhoptries. Mitochondria are located at the posterior end. After entering the circulatory system, the sporozoites make quick work of invading liver cells using the apical organelles (characteristic of all apicomplexans; for more details, see Apicomplexa).

Inside the host's liver cell the Plasmodium cell undergoes asexual replication. The products of this replication, called merozoites, are released into the circulatory system. The merozoites invade erythrocites and become enlarged ring-shaped trophozoites. In this stage the cells ingest the host cytoplasm and proteolyze hemoglobin into amino acids. Several rounds of nuclear divison yield a schizont. From these schizonts merozoites bud, which are released after rupturing the erythrocites. More erythrocites are invaded, and the cycle is reinitiated.

Sometimes instead of schizogony (as the schizont cycle is known) the parasites will reproduce sexually into micro- or macrogametocytes. Through gametogenesis these micro- or macrogametocytes morph into micro- or macrogametes. This may only occur after the gametocytes have been ingested by a mosquito. After said ingestion, the microgametocyte undergoes three nuclear divisions; the resulting eight nuclei become associated with thrashing flagella (this process is called exflagellation). The highly motile microgametes fuse with macrogametes and produce a zygote, which then develops into an ookinete. Once reaching the space between the epithelial cells and the basal lamina of the host, the ookinete develops into an oocyst. Asexual replication results in the production of a large number of sporozoites, which are released into the body cavity of the mosquito vector upon the maturation of the oocyst. The sporozoites are able to recognize the salivary gland of the vector, and are injected into the vertebrate host during the mosquito's blood meal, thus beginning the process over again. The four species of Plasmodium that affect humans are different morphologically, slightly in terms of their life cycles, in terms of their host erythrocite preferences, and varying clinical symptoms.

Phylum:ApicomplexaClass:SporozoaOrder:HaemosporidaFamily:HaemosporidaeGenus:PlasmodiumSpecies:vivax,ovale,malariae,and falciparum

Organism CharactericsMalarial parasite belongs to the genus plasmodium. There are 4 species in this genus that infect

humans.These are P.vivax,P.falciparum,P.ovale and P.malariae.P.falciparum causes the most dreadful disease. Almost all deaths from malaria are due to P.falciparum.

There are various morphological forms of malarial parasites. Out these, some occur in humans and the others in mosquitoes.Morphological forms seen in humans

1. Forms in Red blood cellsTrophozoites: Young trophozoites are called ring forms. They have a vacuole in the center At the later stage they are called amoeboid forms.Schizonts.(Greek-Schzein=dividing): Asexual multiplication in plasmodium sp. Is called schizogony. The form of malarial parasites seen during asexual multiplication inside RBCS IS CALLED SCHIZONT. During early stages of schizogony, nucleus divides repeatedly while the cytoplasm does not divide. This leads to formation of immature Schizonts in the RBCs. Later, the cytoplasm condenses around each daughter nucleus. At this stage the Schizonts is called

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mature Schizonts. Ultimately, each newly formed nucleus along with the surrounding cytoplasm is covered with a cell membrane. These newly formed morphological forms are the merozoites. Merozoites. These forms range from 0.5 to 2.5min diameter. Their number in the RBCs varies from 8 to 32 depending on the species of Plasmodium. Merozoites infect other RBCs to repeat schizogony.Gametocytes: After a variable number of schizogony cycles, gemetogony is initiated. In gemetogony, instead of merozoites, a gametocyte is formed inside an RBC. There are two types of gametocytes- female gametocyte and male gametocyte. They are the exit forms of malarial parasite. As these forms do not develop further, syngamy- sexual process – can not take place in human beings further development of gametocytes and syngamy takes place in mosquitoes.

2. Forms in liverSporozoites: These forms are slender, banana-shaped and measure 9 to 12m in length. They

are the infective forms for humans. They reach human beings through bite of infected mosquitoes.

Merozoites: These are end-products of schizogony. The merozoites come out of the liver cells and infect RBCs to initiate erythrocyte schizogony. It is very difficult to demonstrate these forms of malarial parasites in the liver as very few hepatic cells are affected.

Morphological forms seen in mosquitoesFemale gametes(Macrogametes): The female gametocytes mature in mid-gut of mosquito to develop into female gametes. One female gametocytes develop in to one female gamete.Male gametes(Microgametes):one microgamete produces 6 to 8 male gametes by a process of exflagellation. Exflagellation takes place in mid gut of mosquitoes.Öokinete: A male gamete fertilizes a female gamete to form a diploid zygote (syngamy).The zygote quickly elongates to become motile Ookinete which is 10 to 12m in length.Öocyst: Öokinete penetrate the gut wall and comes to lie on the haemocoel side of the gut, where it develops into an Öocyst. The Öocyst is covered by an electron – dense capsule . Size of the Öocyst increases with maturity. It may reach upto 60m.Sporozoites: Meiosis in the Öocyst results in development of haploid forms called Sporozoites. Sporozoites break out of the Öocyst into the haemocoel. They ultimately reach the salivary gland of the mosquitoes. These slender forms are infective to humans.

The morphology of various forms of malarial parasite varies according to the species.Life Cycle:

Plasmodium sp have a complex life cycle. They require two hosts to complete their life cycle. Humans act as the intermediate host and mosquitoes (female Anopheles) act as the definitive hosts.

The infective form of Plasmodium sp is the Sporozoites. These forms are present in saliva of the infected mosquitoes. When such mosquitoes bite human beings, Sporozoites pass along with the saliva through their proboscis. Mosquitoes inject saliva in the tissues of humans and other animals before they suck blood or tissue fluid as their meal. It is essential for the mosquitoes to inject saliva in tissues of its host as it facilitates the sucking action. These Sporozoites enter the blood stream and soon lodge in the liver. In the hepatocytes they multiply by schizogony (Pre erythrocytic schizogony).

Pre –erythrocytic schizogony results in production of merozoites. After their release from the hepatocytes, merozoites infect RBCs. Here the merozoites initiate erythrocytic schizogony.

After an indeterminate number of such cycles, merozoites initiate gemetogony instead of schizogony in the infected RBCs. In gemetogony,one merozoites develops into one gametocyte; either a female (macrogametocyte) or a male (microgametocyte).

Gametocytes do not develop further in human beings ; they are the exit forms. Gametocytes reach the mid-gut of definitive host, the female Anopheles mosquito, along with its blood meal. One macrogametocyte develops into one macrogamete while one microgamete produces 6 to 8 microgametes by the process of ex-flagellation.

One microgamete fertilizes a macrogamete to produce a zygote. The Zygote elongates and becomes an Öokinete. The Öokinete penetrates the gut wall and is located beneath basement membrane on the haemocoel-side of the gut wall. Here it converts into an Öocyst. Öocystgradually enlarges and ultimately ruptures to liberate Sporozoites in the haemocoel. Sporozoites migrate throught the body of mosquito. On contacting the salivary gland, Sporozoites enter its channel. From here they are injected into a new host at the next feed. The life cycle of the malarial parasite is thus completed.

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Malaria - The disease was described as early as 5th century ,B.C. There are four recognised species of Plasmodium, that are related to malaria: P. vivax ,p.ovale, P. malariae and P. falciparum, all transmitted by female Anopheles mosquito. This insect consumes human blood to acquire a component for the production of her eggs. The parasite has a very complex life cycle. It takes place partly in mosquito and partly in the human blood.

The parasite enters the blood stream in the sporozite form and immediately invades the liver, where it is very difficult to treat. After several hours, many of the merozites emerge and penetrate the red blood cells.  Thousands of RBCs rupture simultaneously and release tens of thousands of new parasites. At this Point, the victim suffers a malaria attack. There is severe cold, temperature rises rapidly to 104°F - 1O6°p and there is severe headache with mild delirium. After next two or three hours, massive perspiration ends the hot stage.

The patient as a sound sleep till next attack. During this period, the parasite enters a new set of RBC, and repeats the transformation cycle. Plasmodium vivax and P. ovale spend about 48 hr in RBCs and there is a 48 hr interval between attacks. This malaria is called tertian malaria(three days). For P. malariae, the cycle is of about 72 hr - therefore, quartan malaria (four days). The cycle of P. falciparum is not defined and attack may occur at irregular intervals. This type of malaria is called estivo autumnal malaria.

Death from malaria may be due to several factors, related to. loss of red blood cells. The anemia is substantial. The RBC fragments accumulate in the small vessels of brain, kidneys, heart muscle, liver and other vital organs, causing their blockage. Heart attacks, cerebral hemorrhages and venal shutdowns are not uncommon. Quinine is used to treat malaria since its discovery in 1640. Synthetic drug chloroquine is also used for the clinical phase of malaria, and primaquine for dormant phase. Recently mefloquine is also used. Most recently Walter Reed Army Institute for Research in Washington, U.S.A. has developed halofantrine, a new anti-malaria drug that is effective to all forms of multidrug-resistant malaria. Halfan, as the compound known, is shown to cure more than 95% for both, P. vivax and P. falciparum.

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Life cycle

Mosquitoes of the genera Culex, Anopheles, Culiceta, Mansonia and Aedes may act as vectors. The currently known vectors for human malaria (> 10 species) all belong to the genus Anopheles. Bird malaria is commonly carried by species belonging to the genus Culex. Only female mosquitoes bite. Aside from blood both sexes live on nectar, but one or more blood meals are needed by the female for egg laying as the protein content of nectar is very low. The life cycle of Plasmodium was discovered by Ross who worked with species from the genus Culex.

The life cycle of Plasmodium is very complex. Sporozoites from the saliva of a biting female mosquito are transmitted to either the blood or the lymphatic system[3] of the recipient. The sporozoites then migrate to the liver and invade hepatocytes. This latent or dormant stage of the Plasmodium sporozoite in the liver is called the hypnozoite.

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The development from the hepatic stages to the erythrocytic stages has until very recently been obscure. In 2006[4] it was shown that the parasite buds off the hepatocytes in merosomes containing hundreds or thousands of merozoites. These merosomes have been subsequently shown to lodge in the pulmonary capilaries and to slowly disintergrate there over 48-72 hours releasing merozoites. Erythrocyte invasion is enhanced when blood flow is slow and the cells are tightly packed: both of these conditions are found in the alveolar capilaries.

Within the erythrocytes the merozoite grow first to a ring-shaped form and then to a larger trophozoite form. In the schizont stage, the parasite divides several times to produce new merozoites, which leave the red blood cells and travel within the bloodstream to invade new red blood cells. The parasite feeds by ingesting haemoglobin and other materials from red blood cells and serum. The feeding process damages the erythrocytes. Details of this process have not been studied in species other than Plasmodium falciparum so generalisations may be premature at this time.

At the molecular level a set of enzymes known as plasmepsins which are aspartic acid proteases are used to degrade hemoglobin.

Most merozoites continue this replicative cycle, but some merozoites differentiate into male or female sexual forms (gametocytes) (also in the blood), which are taken up by the female mosquito.

In the mosquito's midgut, the gametocytes develop into gametes and fertilize each other, forming motile zygotes called ookinetes. The ookinetes penetrate and escape the midgut, then embed themselves onto the exterior of the gut membrane. Here they divide many times to produce large numbers of tiny elongated sporozoites. These sporozoites migrate to the salivary glands of the mosquito where they are injected into the blood and subcutaneous tissue of the next host the mosquito bites. The majority appear to be injected into the subcutaneous tissue from which they migrate into the capillaries. A proportion are ingested by macrophages and still others are taken up by the lymphatic system where they are presumably destroyed. The sporozoites which successfully enter the blood stream move to the liver where they begin the cycle again.

The pattern of alternation of sexual and asexual reproduction which may seem confusing at first is a very common pattern in parasitic species. The evolutionary advantages of this type of life cycle were recognised by Mendel.

Under favourable conditions asexual reproduction is superior to sexual as the parent is well adapted to its environment and its descendents share these genes. Transferring to a new host or in times of stress, sexual reproduction is generally superior as this produces a shuffling of genes which on average at a population level will produce individuals better adapted to the new environment.

Reactivation of the hypnozoites has been reported for up to 30 years after the initial infection in humans. The factors precipating this reactivation are not known. In the species Plasmodium malariae, Plasmodium ovale and Plasmodium vivax hypnozoites have been shown to occur. Reactivation does not occur in infections with Plasmodium falciparum. It is not known if hypnozoite reactivaction may occur with any of the remaining species that infect humans but this is presumed to be the case.

A single report of recurrence of P. falciparum in a patient with sickle cell anaemia exists [6] but this needs additional confirmation as liver forms are not known to occur in P. falciparum infections.

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