PARASITOLOGY

INTRODUCTION TO PARASITOLOGY

INTRODUCTION

I. SYMBIOSIS (Sym = Together; Bio = Living  “Living Together”) -an association between two different species of organisms

-primary aim: to obtain food and seek shelter and protection from the bigger organism -commonly known as Living Together

-there are three types of Symbiotic Relationships: Commensalism + Mutualism + Parasitism

ORGANISM HOST

Commensalism Favorable Not Affected

Mutualism Favorable Favorable

Parasitism Very Favorable Not Favorable

II. PARASITOLOGY

-the study of the parasites

-the science that deals with a specialized group of animals which are physiologically dependent on other organisms for their survival

-Neva&Brown: science that deals with organisms that take up their abodes, temporarily or permanently, on or within other living organisms for food, with the relationship of these organisms to their host

**IMPORTANT NOTE:

o The key in studying Parasitology is in the Life Cycle

o If we know the life cycle and its important components, we would understand why it is the key

III. DEFINITION OF TERMS A. Parasites

-organisms ranging from microscopic to macroscopic which are known to inflict disease in man -exhibit great diversity in morphology and physiology

-animals depending on bigger organisms for food and shelter

-has intimate almost always obligatory relationship with the host (ALWAYS harms the host) **Ascaris lumbricoides

-compete for available nutrients (sugars, proteins, fats) in the food we had already eaten -tangled worms may obstruct the gut

-may damage vital organs during erratic migration **Description:

 Harms its Host

 Associated closely with host and derive nutrition from host

 May kill the host B. Hosts

-the bigger organisms in which the parasite lives

-provides the nourishment and protection needed by the parasites **Description:

 Does not harm the parasites

 Provides shelter to its parasite (parasites need the host)

PARASITES

I. CLASSIFICATION OF PARASITES

-Parasites: organism or animal that depends upon another bigger organism for food

-parasites can be classified according to location in their hosts, degree of association, and taxonomic groups A. According to Location

1. Ectoparasites

-found outside or on the surface of the body of the host -may cause INFESTATION

-ex) Scabies (Sarcoptes scabiei – human itch mite) 2. Endoparasites

-found living within the host in any internal organs (GIT, Respiratory Tract, RBCs) -may cause INFECTION

-examples of Endoparasites :

• Ascaris (GIT Lumen)

• Plasmodium (RBC) – Malarial Parasite

• Schistosoma (Mesenteric Veins) B. According to Degree of Association

1. Obligate Parasite

-those who cannot survive or reproduce outside the host

-always parasitic (must always be in contact with a susceptible organism) -ex) Trichomonas vaginalis

2. Facultative Parasite

-those that may exist in a free living state or as a commensal -if opportunity presents itself, they may become Parasitic -they can both live as free living or parasitic parasites -ex) Strongyloides stercoralis

3. Permanent Parasite

-if the live continuously in the host, and remain in the body of the host – from the early stage until maturity

-requires continuous contact with the host -ex) Pediculus, Trichomonas vaginalis 4. Intermittent (Temporary) Parasites

-contact with the host is only temporarily – closely associated with its host temporarily to feed -ex) Anopheles mosquitoes (they only bite when they want your blood)

5. Accidental Parasites

-enters the host that is not normally its host

-does not develop to full maturity since the host is not physiologically compatible -association lasts only for a short time

-ex) Echinococcus, Gnathostoma, Toxocara 6. Aberrant Parasite

-moves out of the normal location within its host -its Migration to ectopic sites may lead to complications -also known as erratic

-ex) Ascaris lumbricoides (intestines, then they migrate to bile duct, liver, or appendix) 7. Spurious Parasite

-organisms taken in by accident and then passed out without causing any harm to unsuitable host -example of Commnesalism (ex- Fruit Fly larvae or Plant Nematode)

II. TYPES OF HOST

-Host: organism in which a parasites live -classified according to the following: A. Definitive Host

-harbors the sexual or the mature stage of the parasite (site where sexual reproduction occurs, if present) -ex) Man, Pig, Dog, Mosquitoes

 Mosquitoes: definitive host in Malaria

 Ascaris: man is the definitive host B. Intermediate Host

-host that harbors the asexual or the immature / larval forms of the parasites -site where the asexual reproduction occurs

-Paratenic Host: host in which the parasite does not undergo any development C. Reservoir Host

-these are the animals that harbor the parasite that are also parasitic to man -site where the asexual reproduction occurs

-ex) Dog may carry hookworm eggs from one place to another, but eggs do not hatch or pass through any development in these animals

D. Accidental (Incidental) Host

-these are the hosts that accidentally ingested the Parasites (that usually affect the animal kingdom) -infection occurs in a host other than the normal host species

-Zoonosis: condition wherein animal parasites accidentally infect man -ex) Hyatid Tapeworms are usually found in dogs and sheep  Zoonosis E. Vector

-an invertebrate animal that transmits a parasitic organism from one host to another -has two types: Mechanical Vector + Biological Host

1. Mechanical Vector -an invertebrate animal which merely transfer parasitic organisms from one host to another (by carrying feces on their feet)

-ex) Flies, Cockroaches

2. Biological Vector -there is a development of the parasite inside the host

-harbor the parasite internally in which it may undergo further development, multiplication, or both

III. TAXONOMIC GROUPINGS OF PARASITES A. Protozoans (Unicellular Eukaryotic Parasites)

o Phylum Sarcomastigophora o Phylum Apicomplexa o Phylum Microspora o Phylum Ciliophora

PHYLUM REPRODUCTION LOCOMOTION SPECIES

Sarcomastigophora: Sarcodina

Asexual Pseudopodia Entamoeba histolytica

Sarcomastigophora: Mastigophora

Asexual Flagella Giardia lamblia

Dientamoeba fragilis Trypnasoma spp Loishmania spp

Apicomplexa Asexual & Sexual None Plasmodium spp

Toxoplasma Sarcocystis

Ciliophora Cilia Balantidium coli

1. Phylum Sarcomastigophora

-has Subphylum: Sarcodina and Mastigophora -both reproduce Asexually

a. Subphylum Sarcodina -move by Pseudopodia (can only move in one direction) -Pseudopodia: cytoplasmic protrusions

-ex) Amoeba, Entamoeba (pathogen) b. Subphylum Mastigophora -move by flagella (Flagellates)

-Flagellum: long threadlike extension of cytoplasm -ex) Giardia, Trypanosoma, Leishmania, Trichomonas 2. Phylum Apicomplexa

-no locomatory organ

-have a complex life cycle with alternating sexual and asexual generations

-Blood Parasites that cause Malaria: Isospora, Cyclospora, Cryptosporidium, Sarcocystis -Other Parasites: Taxoplasma, Sarcocystis

3. Phylum Ciliophora

-move by beating of many Cilia

-mostly free living and symbiotic species

-ex) Balantidium coli (the only pathogenic ciliate) 4. Phylum Microspora

-no locomatory organ

-intracellular parasite of many kinds of vertebrates and invertebrates -rarely cause disease in immunocompetent persons

B. Helminths (Metazoa; Wormlike Invertebrates)

PHYLUM ASCHELMINTHES PHYLUM PLATYHELMINTHES

CLASS NEMATODA CLASS CESTODA CLASS TREMATODA

AKA Round worms Flatworms Flukes

Shape Elongated, Cylindrical Elongated, Ribbon Like Leaf-shaped, Elongated

Segmentation Unegmented Segmented Unsegmented

Sexes Separate Hermaphrodite Hermaphrodite

1. Phylum Aschelminthes

a. Class Nematoda -Nematodes or Roundworms

-elongated, cylindrical worms attenuated at both ends -unsegmented worms; sexes are separate

-reproduction by:

• Oviparous (ex. Ascaris, Enterobius)

• Viviparous (ex. Trichinella, Filarial Worms) **Infection by:

• Ingestion of Eggs (ex. Ascaris, Trichuris)

• Skin penetration by Larvae

• Vector Borne

• Ingestion of Encysted Larvae 2. Phylum Platyhelminthes

-also called Flatworms – multicellular animals characterized by flat, bilaterally symmetric body -most Flatworms are Hermaphroditic, having both male and female reproductive systems a. Class Trematoda -majority are transmitted by ingestion (except – Schistosoma)

-unsegmented -Hermaphroditic

-flattened, leaf-shaped or cylindrical

-Order Digenea: contains all species that are parasitic to humans -Infection by: Metacercariae ORSkin penetration by Cercariae b. Class Cestoda -segmented

-Hermaphroditic, ribbon like

-reproduction by: Oviparous (Ex. Taenia); multiplication of larval forms -Infection by: Ingestion of Encysted Larvae

C. Arthropods

1. Phylum Arthropoda

-have segmented body parts: Head Thorax and Abdomen

-have three to four pairs of jointed appendages and covered by hard chitinuous skeleton -bilaterally symmetrical body

CLASS DESCRIPTION EXAMPLE Crustacea Some serve as intermediate hosts Crabs, shrimps, copepods

Chilopoda Centipedes

Arachnida spiderlike Scorpions, spiders, ticks, mites Insecta Includes most important of arthropods

**Class Insecta • Order Anoplura • Order Hemiptera • Order Coleoptera • Order Hymenoptera • Order Siphonaptera • Order Diptera

LIFE CYCLES

I. TWO TYPES OF LIFE CYCLES:

-Parasites undergo a Life Cycle during its existence

-it involves survival and development in the external environment and in one or more hosts -life cycle consists of the various morphologic forms and developmental stages

-life cycle summarizes development of the parasite from the time it leaves the host to start a new infection in a new susceptible host

-knowledge of life cycles = preventive measures A. Direct Life Cycle

-there is no intermediate host required to transmit the parasite from one host to another

-parasites are transmitted from an infected individual to a susceptible host without requiring an intermediate host

**Ex1) Trichomonas vaginalis

-direct cycle where the parasite is immediately infective when it leaves the host -Man  Man

**Ex2) Ascaris lumbricoides

-direct life cycle where the parasite develops into the Infective Stage in the external environment

-Man  Development in External Environment  Vectors (optional)  Man

B. Indirect Life Cycle

-at least one intermediate host / vector is required for the parasite to complete its transmission from the originally infected host to the susceptible potential new host

-the infective stage of the parasite develops in the Intermediate Host and the Susceptible Host acquires the infection through contact or exposure to the infected intermediate host

**Ex) Schistosoma japonicum, Capillaria phiippinensis

II. COMPONENTS OF THE LIFE CYCLE

• Infective Stage

• Portal of Entry

• Mode of Transmission

• Path or Route of Migration

• Developmental Stages

• Habitat

• Portal of Exit

• Diagnostic Stage A. Infective Stage

-stage of development of the parasite which when ingested to the definitive host will result to infection -in the direct life cycle: Infective Stage comes from original infected host

-in the indirect life cycle: it originates from the Intermediate Host or Vector -ex) Ascaris – Infected Stage is the Embryonated Egg

B. Portal of Entry

-refers to site of opening in the susceptible host through which the infective stage enters -most common portal of entry: Mouth, Skin, Nose

-ex) Ascaris – Portal of Entry: Mouth C. Mode of Transmission

-refers to the process on how a new host acquires the infective stage of the parasite

-man could acquire it by: Ingestion, Inhalation, Infected Intermediate Host, Skin penetration, Inoculation -ex) in Ascaris – Ingestion of Embryonated Egg; Hookworm by Skin penetration of Larva

D. Path or Route of Migration

-when the infective stage of parasite enters the new host, it usually undergoes without maturation stages -different developmental stages of parasites may occur in different parts or organs

E. Developmental Stages

-the parasite undergoes different developmental stages as it moves from the original infected hosts to the environment, inside the intermediate hosts & even w/in the new susceptible host before settling down -ex) For Helminths, developmental stage is Egg, Larva, Immature Adult and Adult

F. Habitat

-refers to the specific organ or site in the definitive host’s body where the sexually mature parasite resides -ex) Habitat of Ascaris is in the Lumen of Small Intestines

G. Portal of Exit

-opening or site in body wherein parasites leave the infected hosts to find a new susceptible host -ex) Eggs of Ascaris leave the host through the Anus

H. Diagnostic Stage

-stage passed out by the host or taken up by the vector or intermediate host

-serves as a basis for establishing presence of the parasite in host or diagnosis of infection

EXPERIMENT 24: LUNG FLUKES

INTRODUCTION

I. SLIDES TO VIEW:

• Paragonimus westermani

o Adult o Egg

II. PARAGONIMUS WESTERMANI

•

Paragonimus westermani = Affects the Human Lungs (Lung Fluke)

•

Transmission = Ingestion of Insufficiently cooked Crabs or Cray Fish infected with Metacercariae

•

Infective Stage = Metacercaria

•

Diagnosis = Finding Medium Sized Operculated Ova in the Sputum

•

Pathology = Acute, Subacute Inflammations of Lung Tissue; it may also wander erratically to Brain and Skin

**IMPORTANT Note:

o Clinically, it is usually Mistaken with Tuberculosis Symptoms due to Hemoptysis o TO Differentiate, we find Operculated Ova in the Sputum, instead of Acid Fast Bacilli

III. LIFE CYCLE OF P. WESTERMANI

• Type = Indirect Life Cycle (One or Two Intermediate Hosts and One Definitive Host) • Immature Larvae develops inside the Intermediate Host and Matures into Adult Stage inside Man

• Mode of Transmission = Ingestion of Raw Infected Crabs / Crayfish

• Diagnostic Stage = Eggs in Sputum / Stool

• Infective Stage = Metacercariae from Crabs

• Definitive Host = Man

•

1st _{Intermediate Host = Snails}

DEFINITIVE HOST = MAN SNAILS CRABS / CRAYFISH

Ingestion of Raw Infected Miracidium Cercariae

Crabs / Crayfish Hatches & Penetrates Snail Enter the Crabs

Immature Worm Sporocyst Encysts to form Metacercaria

Migrate into Peritoneal Cavity,

Diaphragm and Lungs Radiae

Adult Worm Cercariae Metacercariae

Encysted in Lung Tissue (Infective Stage)

(Lungs have Cavitary Lung Lesions,

Cough, Pleuritic Pain, Hemoptysis) Cercariae is released in the

Water

Eggs in Sputum or Stool

III. DESCRIPTION OF THE SLIDES: A. Paragonimus westermani Adult

-Plump, Ovoid

-Reddish-Brown Fluke (8-20mm in length; 5-9mm in breadth) **Diagnostic Features of this Fluke

 Scale-Like Integumental Spines

 Two Equal-Sized Ventral & Oral Suckers located in the Anterior Half **Other Structures:

 Two Lobate Testes situated Side-by-Side

 Centrally Located Lobate Ovary Anterior to the Testes

 Laterally, the entire body is occupied by the Highly Branched Vitellaria

 Excretory Bladder

 Coiled Uterus B. Paragonimus westermani Egg

-Medium Sized, Oval Shaped Egg (85 x 55u)

-Operculated: has a characteristic Broader Operculated Anterior End and a Thicker Posterior End -expectorated in Sputum or when swallowed, it can be detected in Feces in Unembryonated Stage

IV. ANSWERS TO QUESTIONS

❖ Discuss the Specimens of Choice in confirming the Diagnosis of Paragonmiasis o Stool

o Sputum

❖ Give the Characteristic Morphological Feature of Paragonimus westermani Ova o Broader Operculated Anterior End

o Thickened Posterior End

EXPERIMENT 27: SPOROZOANS

INTRODUCTION

• Sporozoans = Group of Unicellular Parasites with a characteristic Apical Complex

• Has NO Locomotory Organelles (except for Flagellated Microgametes in some groups)

•

Plasmodium = Commonly seen in Man which can cause Malaria

• Classification of Sporozoans (Based on Affected Organ)

o Blood Sporozoans o Tissue Sporozoans

I. SLIDES TO VIEW:

• P. falciparum = Trophozoites, Schizonts, Gametocytes

• P. vivax = Trophozoites, Schizonts, Gametocytes

• P. malariae = Trophozoites, Schizonts, Gametocytes

• P. ovale = Trophozoites, Schizonts, Gametocytes II. PROCEDURE

•

Stained Blood Smears are shown

• Pay attention to the Size of the Normal and Parasitized RBC, number of Chromatin Dots, Amount of Hemozoin Pigments, Appearance of the Cytoplasm, and presence / absence of Stippling on the Infected RBC’s Cytoplasm

III. STRUCTURE OF THE PARASITE (PLASMODIUM)

Host RBC Stippling

Cytoplasm

Chromatin Vacuole

IV. LIFE CYCLE OF MALARIA PARASITES

•

Type of Life Cycle = Indirect Cycle

•

Infective Stage = Sporozoites

•

Diagnostic Stage = Gametocytes

•

Biologic Vector = Female Anopheles Mosquito

•

Definitive Host = Female Anopheles Mosquito

•

Intermediate Host = Human

•

Two Phases = Human Phase + Mosquito Phase

V. OTHER IMPORTANT SPOROZOANS:

• Cryptosporidium

• Isospora

• Blastocystis

• Pneumocystis carinii

• Toxoplasma gondii V. STAGES OF THE PARASITE

• Asexual Stages = Trophozoites + Schizonts

• Immature Sexual Stages = Gametocyte

• NOTE: Man is just an Intermediate Host in Malarial Infections because they harbor only the Immature Forms of the Parasite (the Mature forms are found in the Mosquito)

A. Trophozoites (Ring Form) -it is the earliest stage

-they develop from Sporozoites in the Pre-Erythrocytic (Hepatic Schizogony) Cycle; in the Blood Cycle, it develops from Merozoites – therefore, we see Throphozoites in BOTH Cycles of Human Phases -with ONE or TWO Chromatin Dots

-ring-like structures with Few Malarial Pigment **Chromatin Dots:

 Usually only ONE!

 P.falciparum (exception) = More than One in Trophozoite Stage (thin ring like cytoplasm) **Cytoplasms:

 Thin Delicate Cytoplasm = P. falciparum

 Amoeboid Cytoplasm = P.vivax

 Band form Cytoplasm = P.malariae

B. Schizonts

-once the Chromatin Dot divides by Binary Fission, Parasite is now a Schizont

-with Two or More chromatin Dots (Chromatin Dots now develop to form Merozoites) -with plenty or increasing amount of Malarial Pigment (Hemozoin Pigment) in Cytoplasm

-can be divided into Growing / Immature and Mature (Cryptozoites) in the Pre-Erythrocytic Cycle **Merozoites  P.falciparum = 12-32  P.vivax = 12-24  P.malariae = 6-12  P.ovale = 4-12 **Tissue Schizonts:

 Primary Tissue Schizonts

 Secondary Tissue Schizonts C. Gametocytes

-it is only an Immature form of the Sexual Forms of the Parasite

-Banana Shaped in P.falciparum or special with definite Chromatin material -develops from Merozoites

DECSRIPTION OF THE SPECIES

I. MORPHOLOGY (From Lab Manual)

P. falciparum P. vivax P. malariae P. ovale

Trophozoite Size: Small-Medium Number: Numerous Shape: Ring&Comma Form Chromatin: Two Dots Cytoplasm: Regular Mature Forms: Compact Pigment: Coarse Grains

Size: Small-Large Number: Few-Moderate Shape: Broken Ring Chromatin: Single Cytoplasm: Irregular Mature Forms: Compact Pigment: Fine Scattered

Size: Small Number: Few Shape: Ring to Round Chromatin: Single Cytoplasm: Regular Pigment: Scattered

Size: Smaller than vivax Number: Few

Shape: Ring to Round Chromatin: Single Cytoplasm: Regular Pigment: Course, Scatter

Schizont Associate w/ Ring Forms Size: Small, Compact Number: Few

Mature Form:12-13 Merozoites Pigment: Single Dark Mass

Size: Large

Number: Few-Moderate Mature Form:12-24 Merozoite Pigment: Loose Mass

Size: Small, Compact Number: Few

Mature Form:6-12 Merozoite Pigment: Concentrated Daisy Flower Like

Size: like malariae Number: Few Mature: 4-12 Merozoite Pigment: Concentrated

Gametocyte Mature: Banana Shaped Chromatin: Single Pigment: Scattered Mature: Round Pigment: Scattered Mature: Round Chromatin: Single Pigment: Scattered Peripherally Mature: Round Chromatin: Single Pigment: Scattered

II. GENERAL CHARACTERISTICS (From Notes)

FORM P. falciparum P. vivax P. malariae P. ovale

Infected RBC Size Not Enlarged Enlarged Not Enlarged Enlarged

Shape Round but may be Crenated Round Round Oval; Fimbriated

Stipplings Maurer’s Dots Shuffner’s Dots Zieman’s or James Dots Shuffner’s Dots Trophozoites Small Rings

Multiple w/ Acole Forms

Small & Large Rings Amoeboid

Small Compact Rings Band Forms

Small; Compact Rings

Schizont Rare (Seen in Severe Cases) Irregular Contours Compact; Round Compact

# of Merozoites 12-32 12-24 6-12

Forms Regular Rosette-Like Clusters

4-12

Forms Irregular Rosette-Like Clusters

Gametocyte Banana / Sausage Shape Round-Large Round, Compact Round, Small

ANSWERS TO QUESTIONS

❖ Plasmodium falciparum Schizont VS Plasmodium vivax Schizont

FEATURES P. falciparum P. vivax

Size Small & Compact Large

Number Few, Uncommon Few to Moderate

Mature Forms 12-32 Merozoites 12-24 or More Merozoites

Cluster Compact Clusters Irregular Clusters

Pigment Single Dark Mass Loose Mass

EXPERIMENT 28: BLOOD & TISSUE FLAGELLATES

INTRODUCTION

• Trypanosoma and Leishmania Flagellates belong to Order Kinetoplastida of Superclass Mastigophora

• They Multiply in the Blood (Hemoflagellates) or Tissue of Humans

• All Species require an Arthropod Intermediate Host I. SLIDES TO VIEW

• Trypanosoma brucei gambiense (Trypomastigote)

• Leishmania tropica (in Culture)

• Leishmania tropica (Hamster) II. MORPHOLOGIC DIFFERENCES

AKA Leishmania Leptomonas Crithidia Trypanosoma Morphology Ovoidal / Spherical Spindle Shaped Spindle Shaped Spindle Shaped Flagellum Intracellular Axoneme Free Flagellum Free Flagellum Free Flagellum Course of

Flagellum

Intracellular (Not Free) Arise from Kinetoplast in the Anterior End

Ends in Kinetoplast Anterior to Nucleus

Ends in Kinetoplast Posterior to the Nucleus Found in Macrophages

Reticuloendothelials

Vector

Stage Leishmania Diagnostic Stage Leishmania Infective Stage _Typanosoma Trypanosoma Infective Stage • Leishmania = Amastigote + Promastigote

• Trypanosoma = Trypomastigote + Epimastigote III. TRYPANOSOMA SPECIES

A. Comparison of Diseases and Vectors

SPECIES DISEASE VECTOR

T. brucei gambiense West African Sleeping Sickness Riverine Tsetse Fly

T. brucei rhodesiense East Sleeping Sickness Woodland Tsetse Fly

T. cruzi Chaga’s Disease, Megacolon Reduviid Bug (Triatoma)

B. Life Cycles:

1. T. brucei gambiense

 Infective Stage = Metacyclic Trypopmastigote  Diagnostic Stage = Trypomastigote

 Stage that develops in the Salivary Glands of the Vector (Tsetse Fly) = Epimastigote  Mammalian Hosts = Humans, Pigs, Wild Animals

 Vector = Riverine Tsetse fly (Glossina palpalis) = Day time biters

 NOTE: Epimastigotes are NOT found in the Human’s Blood (it develops in the Tsetse Fly) 2. T. brucei rhodesiense

 Infective Stage = Metacyclic Trypopmastigote  Diagnostic Stage = Trypomastigote

 Stage that develops in the Salivary Glands of the Vector (Tsetse Fly) = Epimastigote  NOTE: Epimastigotes are NOT found in the Human’s Blood (it develops in the Tsetse Fly) 3. T. cruzi

 Infective Stage = Metacyclic Trypomastigote

 Epimastigote Develops inside the Reduviid Bug (Vector)

 Diagnostic Phases:

• Blood = Trypomastigote

• Heart Tissues = Amastigote IV. LEISHMANIA SPECIES

A. Comparison of the Diseases Caused:

LEISHMANIA DESCRIPTION DISEASES

Leishmania tropica Old World Cutaneous Leishmaniasis

Dry, Chronic, Urban Cutaneous Leishmaniasis Oriental Sore

Aleppo or Baghad Boil

Recividans or Chronic Relapsing Cutaneous L. Leishmania major Old World Cutaneous

Leishmaniasis

Leishmania aethiopica Old World Cutaneous Leishmaniasis

Diffuse / Disseminated Cutaneous Leishmanisis Leishmania mexicana New World Cutaneous

Leishmaniasis

Diffuse / Disseminated Cutaneous Leishmaniasis Chiclero Ulcers

Leishmania braziliensis Mucocutaneous Leishmaniasis

Mucocutaneous Leishmaniasis Espundia (disfigures face) Leishmania donovani Visceral Leishmaniasis Visceral Leishmaniasis

Kala-Azar or Black Disease (Zoonotic)

B. Life Cycle

o Infective Stage = Metacyclic Promastigote

o Diagnostic Stage = Amastigote (inside Macrophages) o Vector = Sandflies (Phlebotomus or Lutzomyia)

o

Reservoir Hosts = Humans, Dogs, Wild Animals

o

Habitat = Reticular Endothelial System

EXPERIMENT 29: FILARIAL WORMS

INTRODUCTION

•

Filarial Worms = Slender Tissue dwelling Nematodes with Reduced Lips and Buccal Capsules

•

All Species employ Arthropods as Intermediate Host

•

Transmission = through Arthropod Vector Bite

•

Infective Stage = Third Stage Larva (L3)

I. SLIDES TO VIEW (Blood Smears with):

• Wuchereria bancrofti

• Brugia malayi II. PROCEDURE

• Blood Smears with Microfilariae

• Take note of Morphological Differences between the Two Species and Tabulate based on the following: Shape & Size of the Larvae relative to the Host Cells, presence / absence of a Membrane Sheath and Number of Nuclei at the Posterior or Tail End

III. FILARIAL WORMS OF MEDICAL IMPORTANCE

FILARIASIS VECTOR DEFINITIVE HOST DISTRIBUTION

Wuchureria bancrofti Lymphatic Mosquitoes Man Philippines

Brugia malayi Lymphatic Mosquitoes Man Philippines

Brugia timori Lymphatic Mosquitoes Man Indonesia

Onchoceria volvulus Subcutaneous Fly Man

Loa loa Subcutaneous Fly Man

Manzonella perstan Animal Midget Animal

Manzonella ozzardi Animal Midget Animal

IV. LIFE CYCLE

• Infective Stage = L3 (Third Stage Larvae)

• Diagnostic Stage = Microfilariae

• Definitive Host = Man, Chimpanzee, Gorilla

• Mosquito Borne

L3 (Infective Stage)  Migrates to Lymph Gland  L1  L2  L3

Human Phase Mosquito

EXAMINATION OF THE SLIDES

I. STAGES OF THE PARASITE

A. Adult Stage

-found in Lymph Glands (they can create Obstruction, leading to Edema) -long slender, creamy white roundworm, smooth cuticle

1. Viviparous Female

-Vulva opens to the Surface of the Worm near the middle of the Pharynx -gives birth to a Prelarval Form (Microfilariae)

-6-10cm in length; 0.3cm wide 2. Males

-possess Caudal Alae with two Spicules -40-50mm in length, 0.3wide

B. Microfilariae

-also known as the Pre-Larval Form (L1)

-it is NOT an Infective Stage – it is a Diagnostic Stage

-given birth by a Viviparous Female Worm (there is NO egg stage) -Body Nuclei = represents are Rudiments of Internal Organs **Morphology:

 Morphology varies with Species

 May contain sheath with or without striations  Body Nuclei represent Rudiment of Internal Organs  Stained with Giemsa

**Comparison of Microfilariae between the Two Species:

W. bancrofti B. malayi

Cuticular Sheath Present Present

Cephalic Space Short

Length = Width

Long

Length > Width Body Curves Regular, Large Irregular, Kinky, Small Body Nuclei Round Medium-Size

Well Separated

Small, Angular

Overlapping (not separated)

Tail No Terminal Nuclei

Pointed End

With Two Terminal Nuclei Blunt Tip

*NOTE: Microfilariae are found in the Blood only at Night Time

II. COMPARISON OF LYMPHATIC AND SUBCUTANEOUS WORMS (BASED ON MICROFILARIAE)

W. bancrofti B malayi Onchocerca volvulus Loa loa

Sheath Present Present Absent Absent

Cephalic Space Short Long Short Short

Body Curves Regular Irregular Rigid Irregular

Body Nuclei Separated Overlapping Separated Overlapping

Tail No Nuclei With Terminal Nucleus No Nucleus With Terminal Nuclei III. ENDEMIC PLACES OF FILARIASIS IN THE PHILIPPINES

• Luzon • Leyte • Marinduque • Mindoro • Palawan • Samar

EXPERIMENT 30: SCHISTOSOMA (BLOOD FLUKES)

INTRODUCTION

•

Schistosoma = Comprises the Blood Flukes or the Blood Trematodes • They only require ONE Intermediate Host – have NO Metacercarial Stage

• Adults have Separate Sexes I. SLIDES TO VIEW:

• Schistosoma japonicum = Egg, Cercaria, Adult

• Schistosoma mansoni = Egg, Adult

• Schistosoma mekongi = Egg, Adult II. THREE MAJOR SPECIES

MAJOR SPECIES

SNAIL

INTERMEDIATE HOST

HABITAT DISEASE DISTRIBUTION

Schistosoma japonicum

Oncomelania Hupensis-Quadrasi

Veins of the Small Intestines

Oriental Schistosomiasis Katayama Disease Schistosomiasis japonica

Philippines (Misamis Oriental) China, Sulawesi in Indonesia, Japan, Taiwan

Schistosoma mansoni

Biomphalaria spp (Biomphalaria glabrata)

Veins of the Large Intestines

Intestinal Bilharziasis Schistosomiasis mansoni

Africa, South America, West Indies, Puerto Rico

Schistosoma haematobium Bulinus spp (Bulinus 18truncates) Veins of Genitourinary Tract Schistosomal Hematuria Vesical Schistosomiasis Urinary Bilharziasis

Middle East, Africa, Southern Tip of Europe

**NOTE: Schistosoma japonicum: produces the Most Number of the Eggs

III. LIFE CYCLE

• Infective Stage = Cercaria • Diagnostic Stage = Eggs in Feces

• Form which Enter the Snails = Miracidia

• Intermediate Host = Snails (different species for specific species of parasites – see table) • Mode of Transmission = Skin Penetration

EXAMINATION OF THE SLIDES

I. PROCEDURE:

• Note Morphologic Features of the Parasites and the Striking Differences between them II. MORPHOLOGY

A. Adult Worms

-0.6-2.5cm in length with Oral and Ventral Suckers

-Narrow Elongated Shape; has separate Sexes; Integument is Smooth or Tuberculated depending upon species -number of Testes in Males and the length of the Uterus & Number of Eggs are Distinctive to the species -Excretory System = Flame Cells, Collecting Tubules leading into a Small Bladder with Terminal Excretory Pore

MALE ADULT WORM FEMALE ADULT WORM

Shorter by more Sturdy; Grayish Has a Cylindrical Anterior End

Body Folded to form a Gynecophoral Canal

Longer More Slender Darker

**Gynecophoral Canal = Found in Males in which the Female is embraced during copulation B. Ova / Egg

-with Spine or Knob-Like Projections

-depending on the Stage of Development at the time they are passed out with the Feces, one may find them from Multicellular Stage to full Embryonation with a developed Miracidium within the Shell

S. japonicum Egg S. mansoni Egg S. haematobium Egg

*Developed Miracidium *Lateral Knob

*Small Lateral Spine

*Developed Miracidium *Large Lateral Spine

*Developed Miracidium *Large Terminal Spine

C. Miracidium

-ciliated, pyriform in shape with a Primitive Gut D. Cercariae

-forked-tailed when Discharged from the Snail -fork situated at the Posterior-Third of the Tail -Oral and Ventral Suckers are developed

III. ANSWERS TO QUESTIONS

❖ Differentiate the Appearance of the Ova of the Different Species of Schistosoma

S. japonicum S. mansoni S. haematobium S. mekongi

Oval

With minute Spine or Knob

Light Yellow=Brown Elongate

Lateral Spine Acid Fast

Terminal Spine Oval

Lateral Spine or Knob

o Feces are Collected

o When eggs cannot be found in Feces, a Rectal Biopsy may be done

EXPERIMENT 36: INTESTINAL AMOEBA

INTRODUCTION

• Protozoans in the Intestines obtain nutrients from the Parasitized Host or Predigested Food in the GIT

•

Trophozoite = Active or Feeding Stage

•

Cysts = form when the Environment is Unfavorable for Survival (Not Active, Non-Feeding) I. SLIDES TO VIEW:

• Entamoeba histolytica = Cyst, Trophozoite

• Entamoeba coli = Cyst, Trophozoite

• Entamoeba nana = Cyst, Trophozoite II. ENTAMOEBA HISTOLYTICA

• The ONLY Pathogen (others are Commensals)

• It has the Potential to Invade the Intestinal Wall & other Organs

•

Entamoeba dispar = another Intestinal Commensal which is morphologically similar w/ E. histolytica

• They could be differentiated using Specific Tests (Isoenzyme Analysis, Immunoassays, PCR) **NOTE: LIFE CYCLE Discussed Below

EXAMINATION OF THE SLIDES

I. TROPHOZOITES AND CYSTS:

TROPHOZOITES CYSTS

Entamoeba histolytica *Karyosome = Center

*Nucleus = Eccentric w/ Fine Chromatin Granules *Directional Movements (Active Progressive) *Defined Ectoplasm

*Blade-Like Hyaline Pseudopodia *Indistinct Nucleus

*Ingested RBC

*1-4 Nuclei (Mature Cyst) *Diffuse Glycogen Mass

*Cigar-Shaped Chromatoidal Bars

Entamoeba coli *Karyosome = Eccentric Karyosome in Nucleus *Nucleus = w/ Course Chromatin Granules *More Sluggish Non-Directional Movements *More Granular Cytoplasm

*Contain Ingested Bacteria

*Narrower; Less Differentiated Ectoplasm *Blunter and Broader Pseudopodia *Heavier

*Irregular Peripheral Chromatin *NO RBC inside

*As many as 8-Nuclei (Mature Cyst) *Larger; Slender

*More Granular Cytoplasm

Endolimax nana *Karyosome = Eccentric, Large Irregular *No Peripheral Chromatin Granules

*Has 4-Nuclei

II. ANSWERS TO QUESTIONS

❖ What is the Morphologic Feature of Entamoeba histolytica that signifies Pathogenicity?

o Trophozoite of E. histolytica is Active, Motile Feeding Stage that causes Pathology in the Colon o It is Anaerobic with no mitochondria

o Erythrophagocytosis

EXPERIMENT 37: INTESTINAL FLAGELLATES & CILIATE

INTRODUCTION

I. SLIDES TO VIEW: • Giardia lamblia • Dientamoeba fragilis • Chilomastix mesnili • Balantidium coli

II. FLAGELLATES VS CILIATES A. Flagellates

o Possesses one or more Flagella – Long Thread-Like Extrusions of the Cytoplasm

o

The Pathogenic Intestinal Flagellate of Man is Giardia lamblia

o Classified into Two Groups (According to Habitat):  Intestinal, Oral, and Genital Flagellates

 Blood and Tissue Flagellates

**NOTE: ALL have Trophozoite and Cystic Stage (EXCEPT Trichomonas)



Dientamoeba fragilis was originally grouped under Amoebas (now it is a Flagellate)  Now, it is in the Order Trichomonadida

 Dientamoeba fragilis has Two Nuclei and NO Cystic Stage B. Ciliates

o Move by means of Cilia – Threads of Cytoplasm (shorter and more numerous than Flagella)

o

The only Pathogenic Ciliate of Man is Balantidium coli

III. REVIEW OF PROTOZOANS

Entamoeba histolytica Giardia lamblia Dientamoeba fragilis Balantidium coli Disease Amoebiasis Amoebic Dysentery Amoebic Hepatitis Giardiasis Lambiasis Traveller’s Diarrhea Balantidiasis Balantidosis Balantidial Dysentery Infective Stage Mature Cyst Mature Cyst Trophozoite Mature Cyst

Habitat Large Intestine (Cecum) Small Intestines Large Intestines (Cecum) Large Intestines Diagnosis Parasite in Feces Cyst & Trophozoites Binucleate Trophozoites Cysts & Trophozoites Pathology Flask Shaped Ulcer Malabsorption Syndrome

Steatorrheac Stool

Transmission Ingestion Ingestion Uncertain Ingestion

Trophozoite

Nucleus Single & Eccentric Two Nuclei (Binucleate) Two Nuclei (Binucleate) Macro / Micro

Karyosome Center Center

Movement Unidirectional 4-Flagella

Twisting Movements

Pseudopods Non-Directional Others Easily Destroyed Sucking Disk Ventrally

Pear Shaped (Old Man) 2-Parabasal Bodies 2-Axonemes

No Flagella Surrounded by Cili Cytosome & Cytopyge

Cyst

Description Cigar-Chromatoid Bodies 1-4 Nuclei

Ellipsoidal 2-4 Nuclei

NONE Thick Cyst Wall Round or Oval

EXAMINATION OF THE SLIDES

I. TROPHOZOITES AND CYSTS

TROPHOZOITE CYST

Giardia lamblia *Sucking Disk on Ventral Surface *Pear Shaped

*Old Man’s Face

*Two Nuclei with Large Central Karyosomes *Two Axonemes

*Two Blepharoplasts

*Two Deeply Staining Bars (Parabasal Bodies) *4-Pairs of Flagella

*2-4 Nuclei

*Surrounded by a Cyst Wall *Ellipsoidal (9-12um)

Dientamoeba fragilis *Sluggish Non-Directional Motility *Two Nuclei

*NO Flagella!!! *May Ingest RBC

NONE

Chilomastix mesnili *One Nucleus

*Four Anterior Flagella *Cytosomal Groove *Curved Posteriorly *One Nucleus *Lemon-Pear-Shaped *Clear Knob *Cytosome Balantidium coli *Surrounded by Cili

*Has Vacuoles and Inclusions in the Cytoplasm *Some unique structures:

• Cytostome = Primitive Mouth

• Cytopyge = Excretory Pore

• Macronucleus = Bean / Kidney Shaped

• Micronucleus = within the Macronucleus

*Almost similar: Round or Oval *Thick Cyst Wall

❖ Give the Characteristic Morphologic Features

FLAGELLATE / CILIATE LOCOMOTORY ORGANELLE DISTINCT FEATURES

Giardia lamblia 1-4 Flagella Two-Nuclei

Large Central Karyosome

Dientamoeba fragilis Amoebaflagellate Two-Nuclei (Binucleate)

No Cysts!

Chilomastix mesnii Flagella Trophozoite = Tear Drop Shape

Evident Nucleus

Has a Cystosome (Cell Mouth) Cysts = Lemon Shaped

Balantidium coli Cilia Trophozoites: Large & Oval

EXPERIMENT 38: INTESTINAL NEMATODES

INTRODUCTION

I. SLIDES TO VIEW A. Adult Forms: o Ascaris lumbricoides o Trichuris trichiura o Enterobius vermicularis

o Hookworms (Necator americanus, Ancylostoma ceylanicum, Ancylostoma caninum) o Capillaria philippinensis

o Trichinella spiralis

B. Larva

o Strongyloides stercoralis

o Hookworms (Necator americanus, Ancylostoma ceylanicum) o Trichinella spiralis

C. Ova

o Ascaris lumbricoides (Fertilized, Unfertilized, and Decorticated Ova) o Trichuris trichiura

o Hookworms (Necator americanus, Ancylostoma ceylanicum) o Capillaria philippinensis

o Enterobius vermicularis

II. NEMATODES

•

Nematodes / Roundworms = comprise a Large Group of Helminths of simple structure • Elongated, Unsegmented, Bilaterally Symmetrical

• Separate Sexes

• Males < Females

EXAMINATION OF THE SLIDES

I. OVERALL CHARACTERISTICS Ascaris lumbricoi des Trichuris trichiur a Enterobius vermic ularis Hookworms Strongyloides stercoralis Capillaria philippinensis General Characteristics Common Name Giant Round Worm Whipworm Pinworm / Seatworm Hookworms Threadworm Diseases Ascariasis Ascaris Infection Trichuriasis Trichocephaliasis Enterobiasis Oxyuriasis Ancylostomiasis Uncinariasis Necatoriasis Strongyloides Strongyloidosis Cochin-China Capillariasis Pudoc’s Disease

Habitat Small Intestines Heart-Lung Route

Large Intestines Large Intestines Small Intestines Heart-Lung Route Small Intestines Heart-Lung Route Small Intestines Infective Stage

Embryonated Egg Embryonated Egg Embryonated Egg Filariform Larva Filariform Larva Third Stage Larva Diagnosis Eggs in Stool Eggs in Stool Eggs in Perianal

Region

Eggs in Feces Rhabditiform Larva

Eggs in Feces Transmission Ingestion Ingestion Ingested Skin Penetration Skin Penetration Ingestion Definitive

Host

Man Man (Monkeys) Man ONLY Man Man Man

Intermediate Host

NONE NONE NONE NONE NONE Freshwater Fish

Pathology Loeffler’s Syndrome

Rectal Prolapse Pruritus Ani Creeping Eruptions MH-Anemia Pneumonia-Like

Larva Curens Borborygmi Gurgling Stomach Treatment Mebendazole Pyrantel Pamoate Albendazole Mebendazole Albendazole Mebendazole Pyrantel Pamoate Albendazole Mebendazole Pyrantel Pamoate Albendazole Albendazole Thiabendazole Mebendazole Albendazol Thiabendazole Morphology

Female Adult Pointed Ends Blunt Posterior Sharp Pointed End Parasitic Free-Living Atypical = 2-3 Rows Typical = 1-Row Male Adult Curved Poserior Curved Posterior Curved Posterior Bursa to hold No Parasitic Male Long Spicular

End Single Spicule Retractile Sheet

Single Spicule female Free-Living Sheath

Eggs Fertilized = Embryo Unfertilized = Granules Lemon /Barrel Shaped Bipolar Mucus Plug D-Shaped With Embryo

2-8 Cell Stage Peanut Shaped

Rhabditiform Larva -- -- -- Long Buccal Capsule Short Buccal Capsule Filariform Larva

-- -- -- Pointed Tail Forked Tail

Distinct Features

3-Buccal Lips Anterior Whip like Posterior Round

Cephalic Alae Rounded Ends

II. RHABDITIFORM & FILARIFORM LARVAE

HOOKWORMS STRONGYLOIDES

Rhabditiform Larva

Buccal Cavity Long Buccal Cavity Short Buccal Cavity

Genital Primordium Small Genital Primordium Large Genital Primodrium

Filariform Larva

Tail Pointed Tail Notched / Forked Tail

Esophagus Shorter Esophagus Longer Esophagus

EXPERIMENT 39: DIRECT FECAL SMEAR

INTRODUCTION

I. PRINCIPLES

•

Direct Fecal Smear (DFS) = Simplest and most Rapid of all Fecal Examination Techniques • Recommended for Identification of Protozoan Trophozoites and Detection of Helminthic Infection

•

One Direct Fecal Smear Preparation contains approximately 2mg of Feces

•

Saline and / or Lugol’s Iodine Solution can be used to Emulsify the Fecal Material

A. Unstained Film

-useful for the Study of Living Parasite Objects

-ex) Motile Protozoan Trophozoites, Helminth Eggs, and Nematode Larva B. Iodine Film

-employed to study the Diagnostic Features of Protozoan Cysts

II. PROCEDURE

• Place 1-2 Drops Saline at the Center of a Glass Slide

• With an Applicator Stick, poke at various portions of the Specimen (it with blood streaks, make sure you touch your applicator at this portion)

• Make a smooth uniform emulsion in the drop of saline by Rotatory Motion starting from the Center

• Place the edge of Cover Slip in the slide so that it touches the edge of the water

• Slowly lower the coverslip to prevent Air Bubbles

ANSWERS TO QUESTIONS

❖ Give the Advantage of using Logol’s Iodine over Saline Solution in Direct Fecal Smear

o Lugol’s Iodine or D’Antoni’s Iodine helps visualize internal structures of Trophozoites and Cysts

o Because Stains will kill Motile Trophozoites, it is recommended that a Saline Smear and an Iodine Smear

be prepared Separately

❖ Briefly Discuss the Advantages and Disadvantages of a Direct Fecal Smear o Advantages

 Allows the viewer to Detect Motile Protozoa

 Can view Helminth Eggs / Larvae, Protozoan Cysts, WBCs, Some Yeast

o Disadvantages

 Should NOT be performed on Preserved Specimens

 Should be used for Fresh Stool Specimens that are very Soft or Liquid

 Cannot be examined using Oil Immersion

EXPERIMENT 40: CELLULOSE TAPE PERIANAL SWAB

INTRODUCTION

I. PRINCIPLES

•

Pinworm Infection (Enterobius vermicularis) is suspected in children with Perianal Itching, Insomnia, and Restlessness

• Evidence depends on recovery of Adult Worms, Eggs, or Both

• They are RARELY Found in Stool Examination

•

Pinworm Infection can be best diagnosed by Swabbing Perianal Area using Graham’s Cellophane (Cellulose)

Tape Method

II. GRAHAM’S CELLOPHANE TAPE METHOD

• Highest Sensitivity and Specificity Results

• Best to Collect Specimens in the Morning before the Patient bathes or defecates

III. PROCEDURE

• Place a Strip of Cellulose Tape, Sticky Side Down, on a slide and on one end a Small Strip of Paper

• Hold a Slide Against a Tongue Depressor one inch from the end and lift the tape away from the Slide

Tab attached to its End Portion

• Hold the Tape and Slide against the Tongue Depressor

• Press the gummed surface of the tape against several areas of the Perianal Area

• Replace the tape on the slide and examine directly under the microscope for Pinworm Eggs or Taenia Eggs

ANSWERS TO QUESTIONS

❖ Why is Early Morning the BEST Time to collect Specimens?

o The highest Positive Results and Greatest Number of Eggs can be detected in the morning

o Specimens are best obtained a few hours after the Person has retired (10-11pm) or the first thing in the

morning before bowel movement or bath

EXPERIMENT 41: KATO THICK SMEAR AND KATO-KATZ THICK SMEAR

KATO THICK SMEAR

(CELLOPHANE THICK SMEAR)

I. PRINCIPLES

•

Kato Thick Smear = Qualitative Method to detect Helminthic Infections (better than Direct Fecal Smear)

•

Requires 50-60mg Fresh Fecal Material so that even Light Infection can be detected

•

Cellophane Strips used is Soaked in Glycerine-Malachite Green Solution for 24 hours before use

•

This method is very useful in Mass Examination of Common Soil-Transmitted Helminthic Infecitons **Glycerine-Malachite Green Solution

o

Glycerine = clears the Fecal Film to visualize the Helminth Eggs

o

Malachite Green = Dye used to protect the eyes from Intense Light Needed in examining Thick Smear

o

Preparation: 500ml Distilled Water + 500ml Glycerine + 5ml 3% Malachite Green Solution in H2O **NOTE: This Method is NOT Suitable for Diarrheic Stool and CANT Detect Protozoan Cysts and Trophozoites

II. PROCEDURE

•

Place 50-60mg of Stool at the Center of a Glass Slide and Cover with a Square Piece of Pre-Treated Cellophane

• Using a Rubber Stopper, press the Cellophane gently to spread the Stool Specimen evenly, approximating the Circumference of the Rubber Stopper

• Leave the prepared slide at room temperature for 10-20minutes – during this time, the Microscopic Field becomes clear due to the action of Glycerine on the Stool Constituents

• Examine the Slide (The slide should be examined after 10-20 minutes or within 1 hour after preparation

• Allowing the slide to stand for Long Periods of time will cause Drying and Shells of Hookworm Ova will become transparent and difficult to see

III. ADVANTAGES VS DISADVANTAGES: A. Advantage

o Time Saving, Simple, Economical

o Useful in Mass Examination of Common Soil-Transmitted Helminth Infections

B. Disadvantages:

o Not Suitable for Diarrheic Stool

o Cannot be used to detect Protozoan Cysts & Trophozoites

KATO-KATZ THICK SMEAR

(MODIFIED THICK SMEAR)

I. PRINCIPLES

• Kato-Katz Thick Smear is a Modification of Kato-Thick Smear Method

•

It is a Quantitative Method for counting Helminthic Eggs

• The Number of Eggs per gram (NPEG) of Feces can be computed by Multiplying the Number of Eggs Observed per Thick Smear by 24

II. PROCEDURE

•

Place about Half a Gram of Fecal Sample on Filter Paper

• Place the wire net or screen on top of the fecal sample

• Using a Flat-Sided Applicator Stick, scrape across the upper surface of the screen to sieve the Fecal Material

• Transfer a small amount of Sieved Fecal Material into the hole of the Template and carefully fill the hole

• Remove the Template carefully so that all the Fecal Material is left on the slide

• Cover the Fecal Sample on the slide with a Glycerine-Malachite Soaked Cellophane Strip

• Invert the Slide and Press the Fecal Sample against the Cellophane gently on a Smooth Surface to spread evenly

• Let stand for about 20 minutes and examine

• Count all eggs seen in the whole preparation

• Multiply the Total Eggs counted by Factor 24 to express the Count as Eggs per Gram Feces (EPG)

**IMPORTANT Notes:

o The Wire-Mesh Net is used to Separate Fecal Material from the Large Debris which may affect the

Approximate Fecal Weight and Clearing of Fecal Film by Glycerol

ANSWERS TO QUESTIONS

❖ Discuss the Advantages of Thick Smear over the Direct Fecal Smear

o

Thick Smear can be used to Obtain an Accurate measure of the Number of Eggs in a given amount of

Sample so that the Worm Burden can be inferred

o However, Adult Schistosomes take weeks to months to pass eggs into the Feces ❖ Give the Rationale of using Glycerine-Malachite Green Solution in these two procedures

o It is a dye used to Dye the Smear and Protect the Eyes from the Intense Light that is required for the

Microscopic Examination of the Specimen

EXERCISE 42: INTESTINAL TAPEWORMS AND FLUKES

INTRODUCTION

• Cestoda (Tapeworms) & Trematoda (Flukes) of Phylum Platyhelminthes (or Flatworms) are Exclusively Parasitic I. SLIDES TO VIEW

A. Trematodes 1. Adult, Egg

 Echinostoma ilocanum

 Clonorchis sinensis

 Opisthorchis sp

 Fasciola hepatica / gigantica 2. Adult  Fasciola hepatica  Metagonimus yokogaeai  Haplorchis taichui 3. Metacercaria B. Cestodes 1. Adult  Taenia solium  Taenia saginata

2. Egg, Scolex, Mature / Gravid Proglottid of:

 Taenia solium  Taenia saginata  Diphyllobothrium latum  Hymenolepis nana  Hymenolepis diminuta  Dipylidium caninum 3. Cysticercus cellulosae II. TREMATODES (Flukes)

• Have Conspicuous Suckers

• Leaf-Shaped, Unsegmented, and Dorsoventrally Flattened

• External Cuticle which most species may be covered with Scales, Spines, Tubercles, Ridges

• They have Complex Life Cycles, involving one or more Intermediate Hosts

•

Transmission (Majority) = Eating Inadequately Cooked Second Intermediate Host containing the Infective Metacercariae

III. CESTODES (Tapeworms)

• Endoparasitic Worms with Elongated, Segmented, and Flat Body devoid of a Body Cavity

• Adult Tapeworm = Scolex (Head) + Neck + Proglottids that comprise the Strobila or Body of the Tapeworm

• Each proglottid contains one or two sets of Male and Female Reproductive Organs

EXAMINATION OF SLIDES:

I. TREMATODES (FLUKES)

A. Intestinal Flukes (Trematodes)

Fasciolopsis buski Echinostoma ilocanum Heterophyes heterophyes Metagonimus yokogawai Disease Fasciolopsiasis Echinostomiasis Heterophyiasis Mentagonimiasis Common Name Giant Intestinal Fluke Garrison’s Fluke None None

Life Cycle Indirect Indirect Indirect Indirect

Definitive Host Humans, Hogs, Dogs Humans, Mammals, Birds

Humans, Fish-Eating Mammals

Humans, Fish-Eating Mammals

1st _{Intermediate Planorbid Snails Planorbid Snails Brakish-Water Snails Snails}

2nd _{Intermediate Water Plants Snails (Pila luzonica) Freshwater Fish Freshwater Fish} Habitat Small Intestines Small Intestines Small Intestines Small Intestines Infective Stage Metacercaria Metacercaria Metacercaria Metacercaria Diagnostic Stage Eggs in Feces Eggs in Feces Eggs in Feces Eggs in Feces Morphology Testes Dendritic Tandem Lobed Tandem Lobate

Side by Side (Opposite)

Oval

Obliquely Side by Side Suckers Oral + Ventral Oral + Ventral Oral + Ventral + Genital Oral + Ventral + Genital Egg Unembryonated Operculated Unembryonated Operculated Embryonated Operculated Embryonated Operculated Unique Char. Vitellaria Lateral to

the Ceca

Surrounded by Crown of Spines

Fan-Shaped Vitellaria

B. Liver Flukes (Trematodes)

Fasciola spp Clonorchis sinensis Opisthorchis spp General Characteristics Disease Fascioliasis Liver Rot Pharyngeal Fascioliasis Halzoun Clonorchiasis Opisthorchiasis

Common Name Sheep Liver Fluke (F. hepatica) Liver Fluke (F. gigantica)

Chinese / Oriental Liver Fluke Cat Liver Fluke

Life Cycle Indirect Indirect Indirect

Infective Stage Metacercariae Metaceracariae Metaceracariae Definitive Host Man, Sheep, Cow, Deer, etc Man and Dog Man and Dog Diagnostic Stage Ova in Feces Ova in Feces Ova in Feces 1st _{Intermediate Lymneid Snail Planorbid (Operculate) Snails Planorbid Snails}

2nd _{Intermediate Water Plants Freshwater Fish (Cyprinoid) Freshwater Fish (Cyprinoid)}

Reservoir Host None None

Habitat Bile Ducts & Biliary Passages Bile Ducts & Biliary Passages Bile Ducts & Biliary Passages Morphology

Shape Leaf Shape Oblong, Lanceolate Shaped Oblong, Lanceolate Shaped Suckers Oral < Ventral Oral > Ventral Oral = Ventral

Testes Dendritic; Tandem Branched; Tandem Lobate; Oblique

Ovary Fan-Shaped Oval Oval

Egg Unembryonated Embryonated Embryonated

Intestinal Ceca Branched Simple Simple

Vitellaria Branched Diffuse, Irregularly Distributed Cluster / Compressed Follicles Unique Feature Cephalic Cone / Shoulder Eggs smaller than C. sinensis

Taenia solium Taenia saginata Diphyllobothrium latum

Hymenolepis nana Hymenolepis diminu

ta

Dipylidium caninum

General Characteristics

AKA Pork Tapeworm Beef Tapeworm Broad-Fish-Tapeworm

Dwarf Tapeworm Rat Tapeworm Dog Tapeworm Disease Taeniasis (Adult) Cysticercosis (Larva) Taeniasis Diphyllobothriasis (Adult) Sparganosis (Larva)

Hymenolepiasis Hymenoleipasis Dipylidiasis

Habitat Small Intestines Small Intestines Small Intestines Small Intestines Small Intestines Small Intestines Larval Form Cysticercus

cellulosae

Cysticercus bovis Plerocercoid Larva Cysticercoid Cysticercoid Cysticercoid Infective

Stage

Cysticercus Egg

Cysticercus Plerocercoid Larva Procercoid Larva

Embryonated Egg Cysticercoid Cysticercoid Diagnostic Stage Gravid Proglottids Embryonated Egg Gravid Proglottids Embryonated Egg

Unembryonated Egg Embryonated Egg Embryonated Egg Embryonated (Packets) Definitive

Host

Man Only Man Only Man, Dog, Cat, etc Man, Mice, Rats Rat, Mouse, Man Dogs, Cats, Man Intermediate

Host

Pig Cattle First: Copepod Second: Fish

NONE Required (Rat Fleas)

Fleas, Beetle Fleas, Louse Transmission Ingestion Ingestion Ingestion Ingestion Ingestion Ingestion Morphology Scolex Globular Armed Rostellum Four Suckers Pyriform No Rostellum Four Suckers Spatulate / Almond Bothria (Grooves) Grooves Globular Retractile.Rostellu m Four Suckers Club-Shaped Unarmed / Rudiment Rostellum Rows of Spine Four Suckers Mature Segment

Trilobed Ovary Bilobed Ovary More Testes

Bilobed Ovary Broader > Long Rosette Like Uterus

Bilobed Ovary 3-Testes Bilobed Ovary 3-Testes Pumpkin-Seed Like 2-Sets of Repro. Organ Gravid Segment

<13 Branches >13 Branches Saccular Uterus Egg Masses

Membranous Capsules Egg Embryonated Embryonated Unembryonated Embryonated Embryonated Embryonated

(Packets) Distinct

Feature

Longest Tapeworm Egg Bigger than H.nana

Rice Grain Appearance

ANSWERS TO QUESTIONS

I. CLASSIFICATION OF FLUKES

A. Classification of Flukes According to Size (Those in Bold Letters are the Intestinal Flukes) 1. Large Flukes (2-8cm)



Fasciolopsis buski (largest)  Fasciola spp 2. Medium Fluke (1-2cm)  Echinostoma ilocanum  Clonorchis sinensis  Opisthorchis spp  Paragonimus westermanii 3. Small Flukes (<1cm)  Heterophyes heterophyes  Metagonimus yokogawai  Phanerophsolus bonnie  Haplorchis spp

B. Classification of Flukes According to Second Intermediate Host 1. Water Plant-Borne  Fasciolopsis buski  Fasciola spp. 2. Snail-Borne  Echinostoma ilocanum 3. Fish-Borne  Heterophyes heterophyes  Metagonimus yokogawai  Clonorchis sinensis  Opisthorchis spp C. Classification of Trematode Eggs

1. Large Unembryonated Eggs (100-160u)  Fasciolopsis buski

 Echinostoma ilocanum  Fasciola spp

2. Medium Unembryonated Eggs (70-90u)  Paragonimus westermanii 3. Small Embryonated Eggs (23-32u)

 Heterophyes heterophyes  Metagonimus yokogawai  Clonorchis spp

II. SCOLICES OF THE DIFFERENT SPECIES:

CESTODE TYPE OF SCOLEX SCOLEX

Taenia solium Globular Sucking Disks (4)

Rostellum with Chitinous Hooks

Taenia saginata Pyriform Sucking Disks (4)

Diphyllobothrium latum Elongated / Spatulate 2-Elongated Suctorial Grooves = Bothria

MICROBIOLOGY PRACTICAL EXAM: PARASITOLOGY

INTRODUCTION: INTRODUCTION TO PARASITOLOGY EXPERIMENT 24: LUNG FLUKES EXPERIMENT 27: SPOROZOANS EXPERIMENT 28:

BLOOD & TISSUE FLAGELLATES EXPERIMENT 29:

FILARIAL WORMS EXPERIMENT 30:

SCHISTOSOMA (BLOOD FLUKES) EXPERIMENT 36:

INTESTINAL AMOEBA EXPERIMENT 37:

INTESTINAL FLAGELLATES & CILIATE EXPERIMENT 38:

INTESTINAL NEMATODES EXPERIMENT 39: DIRECT FECAL SMEAR

CELLULOSE TAPE PERIANAL SWAB EXPERIMENT 41:

KATO THICK SMEAR AND KATO-KATZ THICK SMEAR EXERCISE 42: