2 PARA 1 - Protozoa - Flagellates

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Parasitology 2.1

PROTOZOA - FLAGELLATESDr. FontanillaJanuary 6, 2014

UTLINEProtozoa

Nucleus and Cytoplasm Locomotor Organelles

Pseudopodia Cilia Flagella

Encystment

Feeding and Metabolism ClassificationFlagellates

Genus Trypanosoma Trypanosoma brucei Trypanosoma cruzi

Genus Leishmania Visceral Cutaneous Mucocutaneous

PROTOZOAWas once a phylum nameActually refers to a number of phyla

Explains diversity and complexity of membersCurrently used colloquially as a common noun

NUCLEUS AND CYTOPLASMProtozoa consist of a single cell

Many species contain more than one nucleus during all or portionsof their life cycles.

Nucleus and cytoplasm Like all cells, the bodies of protozoa are covered by plasma

membrane which may contain glycocalyx that may haveimmunologic importance

Pellicular microtubules or fibrils may course beneath the plasmamembrane. Examples:

o Kinetoplastid flagellates - microtubules underlie aflexible membrane

o Trypanosome and trichomonas - adjoining membraneshave a fibrous connection between them such asbetween the body and undulating membrane

Mitochondria Examples:

o Amoeba - branched tubular cristaeo Flagellates - a single, large bodyo Ciliates - arranged as elongated sausage-shaped

structures Golgi apparatus (dictyosome)

Examples:o Flagellates - large and/or multiple parabasal bodies in

association with kinetosomes, the “basal bodies” or“parabasal bodies”

Microbodies Peroxisomes contain oxidases and catalases. Examples:

o Trichomonas spp - hydrogenosomeso Kinetoplastida – glycosomes which contain glycolytic

enzymes used in carbohydrate and fat synthesizingpathway called glyoxylate cycle

Cytoplasmic matrix Low density colloid Can exist as fluid (sol state) or relatively solid (gel state) Central zone of cytoplasm

o ENDOplasmo SOL stateo Contains nucleus, mitochondrion, Golgi bodies

Peripheral zone of cytoplasmo ECTOplasmo GEL stateo More transparent than solo Maintains cell shapeo Base of flagella or cilia are embedded in the ectoplasm Protozoa, like fungi, are described as eukaryotes.

Eukaryotes - the genetic material, DNA, is carried on weldefined chromosomes contained within a membrane-boundnucleus

Nuclei - oval, discoid, or round that appear vesicular witirregular distribution of chromatino Examples:

CiliatesMicronucleus – reproductive; undergo meprior to sexual reproduction (conjugation)*some protozoan members reproduce basexual reproduction such as binary fission budding

Macronucleus – dense, elongated, chainlike; considered as somatic; function cell metabolism and growth; does not undmeiosis

Nucleoplasm – finely granular with aggregation of denchromatin

Endosomes – nucleoli; do not disappear during mitosis Nuclear envelope – consists of two membranes

LOCOMOTOR ORGANELLES Protozoa move by 3 basic types:

Pseudopods (amoeba) Flagella (flagellates)* Cilia (ciliates)*

*called undulopodia Some amoebas possess both flagella and pseudopods – transform

from flagellated to amoeboid cell occurs in response to environmentalconditions and is a recognized life cycle event.

Figure 1. Euglenoid Flagellum; Paramecium Cilia, Amoeba Pseudopodia

PSEUDOPODIA Temporary extensions of the cell membrane and are found in amoebas

as well as in a variety of cell types Movement by means of pseudopodia is a complex form of

protoplasmic streaming involving protrusion of the cell, adhesion tosubstrate, and subsequent contraction.

Pseudopodia may also be used for amoeboid feeding


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PARASITOLOGY 2.1

Figure 2. Mechanism of pseudopod feeding

An amoeba feeds on small organisms such as bacteria.↓

As an amoeba approaches food, pseudopodia form and eventuallysurround the food.

↓ The food becomes enclosed in a food vacuole.

↓ Digestive enzymes break down the food, and the nutrients diffuse into

the cytoplasm.

CILIA

Figure 3. Schematic representation of a ciliate

Structurally similar to flagellaWith a kinetosome and an axoneme composed of two central and nineperipheral microtubulesAppear to beat regularly, with a back-and-forth stroke in a two-dimensional plane

FLAGELLA

Figure 4. Cilia and Flagella Structure

Undulipodia Slender, whip-like structures Composed of a central axoneme and an outer sheath that is a

continuation of the cell membrane The axoneme consists of nine peripheral and one central pair of

microtubules. The axoneme arises from a kinetosome (basal body) which is similar to

centrioles of other eukaryotic cells. The flagellum may also be bent back along and loosely attached to the

lateral cell surface, forming a fin-like undulating membrane (anadaptation in a viscous environment).

A dark staining body, the kinetoplast found near the kinetosome; discmade of DNA circles (kDNA)

May be directed anteriorly, posteriorly or laterally regardless ofdirection of movement

Heterkonts are flagellates with two or more flagella with differeinstructures

Figure 5. Flagellum, kinetosome, and associated organelles are called the“ mastigont ” or “ mastigont system ”.

ENCYSTMENT Many protozoa can secrete a resistant covering and enter a resting

stage – cyst.

Conditions favoring encystment involve some adverse environmentalevents such as food deficiency, desiccation, increased tonicity,decreased oxygen concentration or pH, or temperature change.

During encystment, a cyst wall is secreted and starch or glycogen isincorporated in the cyst as energy source

During excystation, there is return to a favorable environment usuallyassociated with absorption of water from environment, cell swelling,activation of lytic enzymes and of normal physiologic pathways

In coccidians , the cystic form is an oocyst which is formed after gamunion and in which multiple fission occurs (sporogony) with cytokinesisto produce sporozoites.

Figure 6. Different flagellate morphologies



PARASITOLOGY 2.1

Figure 7. Different amoebae morphologies

FEEDING AND METABOLISMProtozoa lacking chloroplasts are all heterotrophic . They get energyfrom complex carbohydrates and nitrogen from amino acids.Mouth parts: Amoeba – temporary pseudopod

Ciliates – permanent cytostome Excretion of indigestible material Ciliates – cytopyge

CLASSIFICATIONProtozoa have been divided traditionally on the basis of their means oflocomotion, although this character is no longer believed to representgenuine relationships.

Flagellates (e.g. Giardia lamblia ) Amoeboids (e.g. Entamoeba histolytica ) Sporozoans (e.g. Plasmodium )

Apicomplexa Microsporidia

Ciliates (e.g. Balantidium coli )

FLAGELLATESPresence of several, long, thread-like extensions of ectoplasm calledflagella during their trophozoite stageFlagella arises from axoneme which is associated with a kinetoplastconstituting the neuromotor apparatusFree-living or parasiticFlagellates of importance to man:

Flagellates of the blood and tissues (to be discussed here) Hemoflagellates Requires a blood-sucking insect to complete life cycle Four basic morphologic forms differing in position of

kinetoplast, presence/absence of undulating membrane Flagellates of the digestive tract and genitals ( Trans 2.2)

ylum EuglenozoaClass Kinetoplasta

Order TrypanosomatidaGenera: Trypanosoma

LeishmaniaLeptomonas (no medical importance)Crithidia (in insects)

TRYPANOSOMAAll trypanosomes are heteroxenous .

During one stage of their lives, they live in the blood and/or fixedtissues of all vertebrate classes. During other stages, they live inthe intestines of bloodsucking invertebrates.

They are called hemoflagellates - laboratory culture media usuamust contain blood.

In the past (in manual), stages were named after the genera they mostresembled. (e.g. Leptomonad – for a stage resembling species of genLeptomonas)

The nomenclature used today refers to kinetoplast and nuclposition .

STAGES

Various species pass through amastigote , promastigote , epimastiand/or trypomastigote stages

Figure 8. Stages of Trypanosoma

Amastigote - basal body anterior of nucleus, with a short, essentiallynon-functional flagellum

Promastigote - basal body anterior of nucleus, with a long, detachedflagellum

Epimastigote - basal body anterior of nucleus, with a long flagellumattached along the cell body

Trypomastigote - basal body posterior of nucleus, with a long flagellumattached along the cell body

These names are derived from the Greek mastig , meaningreferring to the trypanosome's whip-like flagellum .

Figure 9. Trypanosoma VS Leishmania morphologies



PARASITOLOGY 2.1

The mammalian stages of T. brucei exist primarily in the bloodstream.In contrast, most of the mammalian developmental stages of T. cruzi and Leishmania spp. reside within the cytoplasm of a wide range ofhost cells or the phagolysosome of host macrophages respectively.Proliferative and non-proliferative (boxed) stages and the locations ofthe flagellum (blue) and kinetoplast (red) relative to the nucleus (grey)are indicated. T. cruzi and Leishmania amastigotes may undergoperiods of proliferative and non-proliferative growth.

CLASSIFICATIONTrypanosomes are divided into two broad groups based on thecharacteristics of their development in the insect hosts.

Section Salivaria - the species develops in the anterior portion ofthe digestive tract of the insect host

Section Stercoraria - the species develops in the vector’s hindgut

SECTION SALIVARIA – TRYPANOSOMA BRUCEIThe genus includes about 30 species and subspecies.Most of these are not associated with transmission of sleeping sickness,although many transmit animal trypanosomiasis to game and domesticlivestock.Members of the genus Glossina are divided conveniently into threegroups which are often given subgeneric status. These divisions are:

The fusca group (subgenus Austenina ) The palpalis group (subgenus Nemorhina ) The morsitans group (subgenus Glossina s.s.)

VECTORS - tsetse flies (Dipteran: Glossinidae; Genus: Glossina ) Tsetse flies are now restricted to continental Africa. About half of

Africa is infested, some 10.4 million square kilometres in all. Glossina species are large (6-15mm), narrow-bodied flies,

brownish or greyish in color, with a stout proboscis that projectswell forward in front of the head.

Figure 10. Tsetse Fly

3 subspecies that are morphologically indistinguishable: Trypanosoma brucei brucei Trypanosoma brucei gambiense Trypanosoma brucei rhodesiense

TRYPANOSOMA BRUCEI BRUCEIA bloodstream parasite of native antelopes and other African ruminantProduces a disease called nagana Humans are not susceptible .

TRYPANOSOMA BRUCEI GAMBIENSEThe etiologic agent of African sleeping sickness CHRONIC form of sleeping sicknessFound in west central and central AfricaVectors

G. palpalis and G. tachinoides - riverine flies Breed in shady, moist areas along rivers

Reservoirs Found mostly in domestic pigs , cattle , and dogs There is evidence that antelopes in certain areas may also ca

the parasite. Man-fly-man transmission is hence, more common in West and Centr

Africa. Asymptomatic persons can carry the parasites in their blood for long

periods and could be continuously infective for the vectors.

TRYPANOSOMA BRUCEI RHODESIENSE ACUTE form of sleeping sickness Found in east central and central Africa Vectors

G. morsitans, G. pallidipes, G. swynnertoni - inhabit the savannah and pupate in dry friable earth

Reservoirs Wild game mammals (bushbuck, hartebeest, lion, hyena) as well

as cattle The more virulent of the two, is thus, maintained in the most

resistant reservoirs, resulting in continuous selection of aggressivestrains.

LIFE CYCLE ofTrypanosoma brucei gambiense and Trypanosoma brucerhodesiense

During a blood meal on the mammalian host, an infected tsetse fly(genus Glossina ) injects metacyclic trypomastigotes into skin tissue.

The parasites enter the lymphatic system and pass into thebloodstream inside the host. They transform into bloodstreamtrypomastigotes which are carried to other sites throughout the body,reach other blood fluids (e.g. lymph, spinal fluid), and continue thereplication by binary fission.

The entire life cycle of African Trypanosomes is represented byextracellular stages. The tsetse fly becomes infected with bloodstreamtrypomastigotes when taking a blood meal on an infected mammalianhost.

In the fly’s midgut, the parasites transform into procyclic

trypomastigotes, multiply by binary fission, leave the midgut, andtransform into epimastigotes.

The epimastigotes rea ch the fly’s salivary glands and continmultiplication by binary fission.

The cycle in the fly takes approximately 3 weeks. Humans are the mainreservoir for Trypanosoma brucei gambiense but this species can alsobe found in animals. Wild game animals are the main reservoir ofTrypanosoma brucei rhodesiense.

IMPORTANT Infective stage to man - metacyclic trypomastigote Mode of transmission - inoculation of metacyclic trypomastigote

from a bite of a tsetse fly Diagnostic stage - trypomastigot e in blood or lymph



PARASITOLOGY 2.1

Figure 11. Domestic and Wild Cycles of Gambian and Rhodesian types of African Sleeping Sickness

) In West Africa, riverine tsetse flies (palpalis group) living in the bushnsmit the Gambian forms to humans (man-fly cycle) and sometimes tomestic animals, particularly pigs.) In East Africa, tsetse flies (morsitans group) of the open savannahnsmit the Rhodesian form to various mammals, mainly antelopes, and tomans. The Gambian cycle can result in an epidemic.

PATHOGENESIS of Trypanosoma brucei gambiense and Trypanosomabrucei rhodesiense

The clinical features of Gambian and Rhodesian disease are the same;however, they vary in severity and duration.Rhodesian disease progresses more rapidly and the symptoms areoften more pronounced.The symptoms of the two diseases are also more pronounced inCaucasians than in the local African population.Classically, the progression of African trypanosomiasis can be dividedinto three stages:1. The bite reaction (chancre)

A non-pustular, painful, itchy chancre forms 1-3 weeks afterthe bite and lasts for 1-2 weeks. It leaves no scar.

2. Parasitemia (blood and lymphoid tissues) Parasitemia is more prominent during the acute stage than

during the recurrence episodes. Parasitemia and lymph node invasion is marked by attacks of

fever which starts 2-3 weeks after the bite and isaccompanied by malaise, lassitude, insomnia, headache,lymphadenopathy, and edema.

Febrile episodes may last few months as in Rhodesian diseaseor several years as in Gambian disease.

Painful sensitivity of palms and ulnar region to pressure(Kerandel's sign ) may develop in some Caucasians.

Very characteristic of Gambian disease is visible enlargementof the glands of the posterior cervical region ( Winterbottom'ssign ).o Swollen nodes at the base of the skullo Lymph nodes become swollen and congested especially

in the neck, groin, and legso Named after a British officer who recognized the sign

among slaves bound for the Caribbean Marketo Symptoms are more marked in newcomers than in

people native to the area

Figure 12.(Left) Winterbottom’s sign - swollen nodes at base of skull

(Right) Kerandel’s sign - delayed sensation of pain after the release of pressure of the hands

3. CNS stage Marked by changes in character and personality Lack of interest and disinclination to work Avoidance of acquaintances Morose and melancholic attitude alternating with exaltation Mental retardation and lethargy Low and tremulous speech Tremors of tongue and limbs Slow and shuffling gait Altered reflexes

Males become impotent. There is a slow progressive involvement of cardiac tissue. The later stages are characterized by drowsiness and uncontrollable

urge to sleep. The terminal stage is marked by wasting and emaciation. Death results from coma, malnutrition, intercurrent infection or cardiac

failure, or a severe fall.

Some differences of T b rhodesiense from T b gambiense Rarely invades the nervous system Rapid weight loss and heart involvement No somnambulism or other protracted nervous disorders Patients die before CNS disorders develop Death within a few months of infection Causes a more rapid course toward death

Figure 13. Comparison of West and East African Sleeping Sickness



PARASITOLOGY 2.1

IMMUNOLOGYAfrican trypanosomes express a glycosylphosphatidyl inositol (GPI) -anchored variable surface glycoprotein (VSG) as a protective coat.

During infection, large amounts of VSG molecules are releasedinto the circulation.

Their interaction with various cells of the immune systemunderlies the severe infection-associated pathology.

Recent results have shown that anti-GPI vaccination can preventthe occurrence of this pathology.

The two properties of the VSG coat that allow immune evasion are: Shielding - the dense nature of the VSG coat prevents the immune

system of the mammalian host from accessing the plasmamembrane or any other invariant surface epitopes (such as ionchannels, transporters, receptors, etc.) of the parasite

Periodic antigenic variation - the VSG coat undergoes frequentgenetic modification, 'switching', allowing variants expressing anew VSG coat to escape the specific immune response raisedagainst the previous coat

Figure 14. VSG, the major surface component of Trypanosomes, is alsoleased in host fluids. VSG induce resistance to complement lysis, escape tospecific immune response, persistent cytokine production, autoantibody

synthesis by molecule mimicry with host tissues.

Infectivity of Trypanosoma brucei rhodesiense to humans is due to itsresistance to a lytic factor present in human serum.Host immune response

↑ Ig (host immune system greatly stimulated) ↑ complement → lysis of RBC → anemia

DIAGNOSISDemonstration of parasite in blood, bone marrow, CSF cardagglutination test (CATT) to detect antibodies in whole blood or serum

Figure 15. Diagnosis by demonstration of parasite in blood sample

TREATMENTArsenicals – eye damage; trypanosomes become tolerantOther drugs

Suramin Pentamidine Berenil Difluoromethylornithine (DFMO) – brain infections

EPIDEMIOLOGY AND PREVENTION Transmission of trypanosomiasis involves four interacting organisms:

The human host The insect vector The pathogenic parasite The domestic and wild animal reservoirs

Glossina are efficient vectors and are responsible for linking theseorganisms. Any reduction of their numbers should lead to significantlyreduced transmission and hence, contribute to elimination and thesustainability of control efforts.

Current vector control interventions involve the use of insecticideseither through:

Sequential aerosol spraying technique (SAT) Ground spraying Insecticide-treated targets or insecticide-treated animals

baits Use of traps Sterile insect technique (SIT)

Bush clearing (tsetse fly habitat destruction) or elimination of wildanimals (tsetse reservoir hosts) have been discarded for ecological andenvironmental concerns.

Odor baited traps and screen impregnated with insecticide andappropriate attractive colors have been used in many countries toeffectively suppress tsetse fly population by 99%.

These artificial bait methods are cheaper than ground and aerialspraying but communities and governments cannot deploy themon sustainable bases, as they are labor and managementintensive.

The Sterile Insect Technique (SIT) is another approach to reduce tsetsefly populations.

Females mate only once in their lifetime thus, any mating with asterile male will prevent females from giving birth to any offspring.

SIT consists in rearing a large numbers of laboratory male tsetseflies which are irradiated and subsequently released in the wild tocompete with wild (naturally occurring) males so that femalesinseminated by them produce no offspring.

SECTION STERCORARIA – TRYPANOSOMA CRUZI Depending on its host environment, the organism occurs in three

different forms. The trypanosomal (trypomastigote) form , foumammalian blood, is 15 to 20 microns long and morphologically similarto African trypanosomes.

The crithidial (epimastigote) form is found in the insect intestine. The leishmanial (amastigote) form , found intracellularly or

pseudocysts in mammalian viscera (particularly in myocardium andbrain), is round or oval in shape, measures 2-4 microns, and lacks aprominent flagellum.



PARASITOLOGY 2.1

Figure 16. Trypanosoma cruzi

American trypanosomiasis (Chagas Disease ) Widespread in the American continent chiefly among small wild

mammals (enzootic sylvatic cycle) Human Chagas Disease - bio-ecological and socioeconomic factors

leave rural poor populations of South and Central America incontact with the sylvatic cycle, where the parasite is transmittedby natural vectors of the infection

From the Public Health standpoint, the importance of ChagasDisease remains correlated to so called "domestic” cycle, not onlybecause millions of human beings are involved but also because allthe available control measures are directed against it.

The most important mechanism of transmission of T. cruzi tohumans and other mammals is the feces of infected triatomines.

The vectors of Chagas Disease are insects of the order Hemiptera,family Reduviidae, subfamily Triatominae.

These are species that colonize poorer quality rural houses, wherecolonies of hundreds of individuals (or even thousands) can befound.

The Triatominae (commonly known as kissing bugs) are defined assubfamily of Reduviidae (commonly known as assassin bugs) thatsuck vertebrate blood (strictly hematophagous) and are mainlyrestricted to the New World

Within the subfamily, genera Triatoma, Rhodnius, andPanstrongylus contain species of bugs that are especiallyimportant vectors of Trypanosoma cruzi, the agent of ChagasDisease in humans.

Figure 17. Kissing Bug (Rhodnius prolixus) Feeding on a Human

LIFE CYCLE The organism is transmitted to mammalian host by many species of

kissing (riduvid) bug. Transmission takes place during the feeding of the bug which normally

bites in the facial area (hence the name, kissing bug) and has the habitof defecating during feeding.

The metacyclic trypomastigotes, contained in the fecal material, gainaccess to the mammalian tissue through the wound which is oftenrubbed by the individual that is bit ten.

Subsequently, they enter various cells, including macrophages, wherethey differentiate into amastigotes and multiply by binary fission.

Figure 18. The amastigotes differentiate into non-replicatingtrypomastigotes and the cells rupture to release them into the bloodstream.

Additional host cells, of a variety of types, can become infected and thetrypomastigotes once again form amastigotes inside these cells.

Figure 19. Entry of metacyclic trypanomastigota via break on skin Uninfected insect vectors acquire the organism when they feed on

infected animals or people containing trypomastigotes circulating intheir blood.

Inside the alimentary tract of the insect vector, the trypomastigotesdifferentiate to form epimastigotes and divide longitudinally in themidgut and hindgut of the insect where they develop into infectivemetacyclic trypomastigotes.

Transmission may also occur from man to man by blood transfusionand by the transplacental route.

IMPORTANT Infective stage to man: metacycylic trypomastigote Mode of transmission: entry of metacyclic trypomastigote from a

break on the skin Diagnostic stage : trypomastigote in blood, CSF, fixed tissues, or



PARASITOLOGY 2.1

SYMPTOMSChagas Disease can be divided into three stages:

The primary lesion The primary lesion, chagoma, appearing at the site of

infection and within a few hours of a bite, consists of aslightly raised, flat, non-purulent erythematous plaquesurrounded by a variable area of hard edema.

It is usually found on the face, eyelids, cheek, lips or theconjunctiva, but may occur on the abdomen or limbs.

When the primary chagoma is on the face, there is anenlargement of the pre- and post- auricular and thesubmaxillary glands on the side of the bite.

Infection in the eyelid, resulting in a unilateral conjunctivitisand orbital edema ( Romaňa's sign), is the most commonfinding.

The acute stage The acute stage appears 7-14 days after infection. It is characterized by restlessness, sleeplessness, malaise,

increasing exhaustion, chills, fever, and bone and musclepains.

Other manifestations of the acute phase are cervical, axillaryand iliac adenitis, hepatomegaly, erythematous rash, andacute myocarditis. There is a general edematous reactionassociated with lymphadenopathy.

Diffuse myocarditis, sometimes accompanied by seriouspericarditis and endocarditis, is very frequent during theinitial stage of the disease.

In children, Chagas Disease may cause meningo-encephalitisand coma. Death occurs in 5-10 percent of infants.

Hematologic examination reveals lymphocytosis andparasitemia.

The chronic stage The acute stage is usually not recognized and often resolves

with little or no immediate damage and the infected hostremains an asymptomatic carrier. An unknown proportion(guessed at 10-20%) of victims develop a chronic disease.They alternate between asymptomatic remission periods andrelapses characterized by symptoms seen in the acute phase.

Cardiac arrhythmia is common. The chronic disease results in an abnormal function of the

hollow organs, particularly the heart, esophagus, and colon.The cardiac changes include myocardial insufficiency,cardiomegaly, disturbances of atrio-ventricular conduction,and the Adams-Stoke Syndrome. Disturbances of peristalsislead to megaesophagus and megacolon.

PATHOLOGY AND IMMUNOLOGYThe pathological effects of acute phase Chagas Disease largely resultfrom direct damage to infected cells.In later stages, the destruction of the autonomic nerve ganglions may

be of significance. Immune mechanisms, both cell mediated andhumoral involving reaction to the organism and to autologous tissues,have been implicated in pathogenesis.T. cruzi stimulates both humoral and cell mediated immune responses .The infection causes severe depression of both cell mediated andhumoral immune responses.Antibody has been shown to lyse the organism , but rarely causeseradication of the organism, perhaps due to its intracellularlocalization.Cell mediated immunity may be of significant value. While normalmacrophages are targeted by the organism for growth, activatedmacrophages can kill the organism .Unlike T. brucei, T. cruzi does not alter its antigenic coat.

Antibodies directed against heart and muscle cells have also detected in infected patients leading to the supposition that there is anelement of autoimmune reaction in the pathogenesis of ChagasDisease.

Immunosuppression may be due to induction of suppressor T-cand/or overstimulation of macrophages .

DIAGNOSIS Demonstration of the causal agent is the diagnostic procedure in acute

Chagas Disease. It almost always yields positive results and can beachieved by:

Microscopic examination of fresh anticoagulated blood or its buffycoat for motile parasites and of thin and thick blood smearsstained with Giemsa, for visualization of parasites.

Figure 20. The most reliable method for differentiating the trypomastigotes(motile blood stage forms) of T. brucei and T. cruzi is by the size of th

kinetoplast (see arrows). T. brucei has a relatively small kinetoplast , T. cruzi has a larger kinetoplast . T. cruzi trypomastigotes also commonly

form a "C" shape, although this is a less reliable feature. Finally, T. cruzi mayalso be found as a non-motile amastigote form in various tissues, while T.

brucei is only found in the trypomastigote form in humans. Isolation of the agent:

Inoculation in culture with specialized media (e.g. NNN, LIT) Inoculation into mice Xenodiagnosis where uninfected triatomine bugs are fed on the

patient's blood and their gut contents examined for parasites 4weeks later

TREATMENT The most effective drugs kill only extracellular protozoa. Nifurtimox and benznidazole

Somewhat effective in curing acute infections Require long treatment duration With significant side effects Patients remain seropositive even after disappearance of parasites

in the blood.

LEISHMANIA

Like trypanosomes – heteroxenous Part of life cycle in sandflies – promastigote Remainder of life cycle in vertebrate tissues

Only amastigotes are found Known as Leishmania-Donovan (L-D) bodies

Mammals most commonly affected - humans, dogs, and rodents

VECTORS: SANDFLIESFamily Psychodidae

Subfamily PhlebotominaeGenera: Phlebotomus (old world)

Sergentomyia (old world)Lutzomyia (new world)Bromptomyia (new world)



PARASITOLOGY 2.1

ETIOLOGYSeveral species of Leishmania are pathogenic for man:

L. donovani causes visceral leishmaniasis (Kala-azar, black disease,dumdum fever)

L. tropica (L. t. major, L. t. minor, and L. ethiopica ) causecutaneous leishmaniasis (oriental sore, Delhi ulcer, Aleppo, Delhior Baghdad boil)

L. braziliensis (also L. mexicana and L. peruviana ) are etiologicagents of mucocutaneous leishmaniasis (espundia, Uta, chiclero

ulcer)

VISCERAL LEISHMANIASISCaused by Leishmania donovaniDiscovered by William Leishman (1900) in spleen smears of a soldierwho died of a fever in Dum-Dum, IndiaDisease known as Dum-Dum fever or kala-azarLife Cycle

Female sand fly takes a blood meal from mammals and transferamastigotes in its mid gut

Amastigotes transform into procyclic promastigotes Procyclic promastigotes change into metacyclic promastigotes by

simple division Promastigotes migrate to the pharyngeal valve and are transferred

to mammals through blood meal as metacyclic promastigotes Metacyclic promastigotes actively invade macrophages,

granulocyctes or are phagocytosed Promastigotes transform into amastigotes and multiply by simple

division in the macrophages Amastigotes leave infected cells to infect new macrophages or

transferred to vector via blood meal Pathogenesis

Clinically, L. donovani infections may range from asymptomatic toprogressive to fully developed kala-azar

Incubation period – 2-4 months Begins slowly with low grade fever and malaise Followed by progressive wasting and anemia, protrusion of the

abdomen from enlarged liver and spleen Finally death in 2 -3 years

Treatment Antimony compounds applied to lesions or injected intravenously

or intramuscularly The immediate cause of death is often invasion of secondary

pathogen.

Figure 21. Amastigotes in macrophage (?? Unlabeled in slide)

CUTANEOUS LEISHMANIASISCaused by: Leishmania tropica and Leishmania major Found in west central Africa, the Middle East, and Asia Minor into IndiaThe 2 species are found in different localities and have differentreservoir and intermediate hosts.

Figure 22. Sandflies of the Genus Phlebotomus - Intermediate Hosts andVectors

Life Cycle When a fly takes blood meal-containing amastigotes, parasites

multiply in the midgut and then move to the pharynx. Sandfly saliva contains low molecular weight compound and

peptides that serve as vasodilators and facilitate infection. Leishmaniasis is transmitted by the bite of infected female

phlebotomine sandflies. The sandflies inject the infective stage (i.e. promastigotes) from

their proboscis during blood meals. Promastigotes that reach the puncture wound are phagocytized by

macrophages and other types of mononuclear phagocytic cells. Progmastigotes transform in these cells into the tissue stage of the

parasite (i.e., amastigotes), which multiply by simple division andproceed to infect other mononuclear phagocytic cells.

Parasite, host, and other factors affect whether the infectionbecomes symptomatic and whether cutaneous or visceralleishmaniasis results. Sandflies become infected by ingestinginfected cells during blood meals.

In sandflies, amastigotes transform into promastigotes, develop inthe gut (in the hindgut for leishmanial organisms in the Vsubgenus; in the midgut for organisms in the Leishsubgenus), and migrate to the proboscis.

Figure 23. In cutaneous leishmaniasis, the organism (L. tropica) multiplieslocally, producing a papule 1-2 weeks (or as long as 1-2 months) after thebite. The papule gradually grows to form a relatively painless ulcer. Thecenter of the ulcer encrusts while satellite papules develop at the periphery.The ulcer heals in 2-10 months, even if untreated but leaves a disfiguringscar. The disease may disseminate in the case of depressed immune function.The sores can change in size and appearance over time. They often end uplooking somewhat like a volcano, with a raised edge and central crater. A


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ab covers some sores. The sores can be painless or painful. Some peopleve swollen glands near the sores (for example, in the armpit if the sorese on the arm or hand).

Diagnosis Scrapings from the side of an ulcer smeared on a slide and stained

with Wright’s or Giemsa stain will show parasites in endothelialcells and monocytes, even if they cannot be found in circulatingblood.

Amastigotes of Leishmania are spherical to ovoid and measure 1-5µm long by 1-2µm wide. They possess a large nucleus, aprominent kinetoplast, and a short axoneme, the last of which israrely visible by light microscopy. The organisms reside inmacrophages of the host and can be found throughout the body.

Figure 24. Parasites in endothelial cells and monocytes

MUCOCUTANEOUS LEISHMANIASIS Caused by Leishmania braziliensis Produces a disease known as espundia, uta, mucocutaneous

leishmaniasis Found in vast area between central Mexico and northern Argentina Life cycle and Pathogenesis

Similar to L. tropica except that the promastigotes reproduce inthe hindgut of the sandfly

Figure 25. Mucocutaneous Leishmaniasis

Symptoms Initial symptoms of mucocutaneous leishmaniasis are the same as

those of cutaneous leishmaniasis except that in this disease, theorganism can metastasize and the lesions can spread to mucoid(oral, pharyngeal, and nasal) tissues and lead to their destructionand hence, sever deformity.



PARASITOLOGY 2.1

APPENDIX

Figure 1. Tsetse Fly Stages (Trypanosoma brucei)

Figure 2. Triatomine Bug Stages (Trypanosoma cruzi)



PARASITOLOGY 2.1

Figure 3. Visceral Leishmaniasis

Figure 4. Sandfly Stages (Cutaneous Leishmaniasis)


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SUMMARY

CHARACTERISTIC STAGES OF SPECIES OF LEISHMANIA AND TRYPANOSOMA IN MAN AND IN THE INSECT HOST (FROM PARA MANUALStages of Parasite

Parasite Leishmania Leptomonad Crithidial Trypanosomaltropica In macrophages of skin &

subcutaneous tissuesIn midgut, proboscis ofsandfly

Absent Absent

braziliensis Same as L. tropica Same as L. tropica Absent Absentdonovani In macrophages of liver,

spleen, bone marrow andlymph nodes

Same as L. tropica Absent Absent

rhodesiense Absent Absent In salivary glands of tsetse fly In proboscis of tsetse fly; inbloodstream and lynode

gambiense Absent Absent Same as T. rhodesiense Same as T. rhodesiensecruzi In macrophages of skin,

lymph node, liver, spleen,brain etc.

Transitional stage only In midgut of triatomid bug In hindgut, feces of bug; inblood stream during acattacks

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