Introduction

to Protozoa:

Lecture 2
 Introduction to Protozoa: Learning
Objectives
(no test questions directly from these Introduction slides)

1. Introduction to the 5 general traits shared by all protozoa

2. Introduction to protozoa terminology and the pathogens we will cover.

3. Introduction to the different topics we will cover for each pathogen and why it is important
to know, as veterinarians.

4. Introduction to the 4 primary mechanisms of protozoa pathology.

 Protozoa Traits
1. Single cell, eukaryotes

2. Most protozoa are not pathogenic nor require a host

3. Most reproduce by binary fission (asexual multiplication)

-some reproduce sexually and asexually (Apicomplexa group)

4. Asexual multiplication provides the mechanism for

developing pathogenic protozoan populations.
Protozoa Taxonomy changes

Illustration by Allie Brosh, http://hyperboleandahalf.blogspot.com/

PROTOZOA FYI: Taxonomy
This is an older classification but organization/terms still relevant

APICOMPLEXA (phylum) SARCOMASTIGOPHORA (phylum)

CONOIDASIDA (class) MASTIGOPHORA (subphylum)
COCCIDIA (subclass) ZOOMASTIGOPHORA (class)
CRYPTOSPORIDIIDAE (family) KINETOPLASTIDA (order)
CRYTOSPORIDIUM (genus) TRYPANOSOMATIDAE (family)
EIMERIIDAE (family) TRYPANOSOMA (genus)
EIMERIA (genus) LEISHMANIA (genus)
SARCOCYSTIDAE (family)
DIPLOMONADIDA (order)
TOXOPLASMA (genus) GIARDIA (genus)
NEOSPORA (genus)
SARCOCYSTIS (genus)
PARABASALIA (phylum)
CYSTOISOPORA (genus)
TRITRICHOMONADIDA (order)
ACONOIDASIDA (class)
TRITRICHOMONADIDAE (family)
PIROPLASMIDA (order)
TRICHOMONAS (genus)
BABESIIDAE (family)
BABESIA (genus)
THEILERIIDAE (family)
Levine et al. A Newly Revised Classification of the Protozoa. J. Protozoo/
CYTAUXZOON (genus)
27(1), 1960, pp. 37-58
New Classification: FYI: Taxonomy
Eukaryote
Supergroups

Alveolates
Excavates

1. Adl SM, et al. The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol (2005)52:399-451
2. Keeling, Patrick J., et al. "The tree of eukaryotes." Trends in ecology & evolution 20.12 (2005): 670-676.
3. Burki, Fabien. "The eukaryotic tree of life from a global phylogenomic perspective." Cold Spring Harbor perspectives in biology 6.5 (2014): a016147.
Alveolates (supergroup) Excavates (supergroup)
Apicomplexa Kinetoplastids
BABESIA (genus) TRYPANOSOMA (genus)
CYTAUXZOON (genus) LEISHMANIA (genus)
TOXOPLASMA (genus)
NEOSPORA (genus) Parabasalians
SARCOCYSTIS (genus) TRITRICHOMONAS (genus)
CYSTOISOPORA (genus) Fornicatas
CRYTOSPORIDIUM (genus) GIARDIA (genus)
EIMERIA (genus)
Protozoa: topics we will discuss for each pathogen
1. Life cycle strategies fecal-oral
a. Transmission
b. Stages
c. Reproduction: sexual and asexual
d. Hosts

2. Pathology
3. Host clinical signs from infection
4. Diagnosis
5. Treatment / Control
6. Geographic location / Epidemiology

Fecal-oral diagram: Salak JS, Shirey JL, Strickl GT. "Successful treatment of symptomatic entamoeba polecki infection". Am J Trop Med Hyg 1979;28(2):190-3
 Protozoa: Life Cycle Strategies
◦ Direct Life cycle -- uses only a single host species (e.g. Eimeria)
◦ Indirect/complex Life cycle -- requires an intermediate host (e.g. Sarcocystis, Trypanosoma)

◦ Asexual stages only – thus “clonal” (e.g. Giardia)

◦ Sexual and asexual stages (all of the apicomplexans)

◦ Continuous life cycle

◦ Without host immunity, organism would continue multiplying (e.g. Plasmodium, Trypanosoma)
◦ Single direction life cycle
◦ Once the life cycle is completed then all organisms are gone (except in the case of re-infection) “all in – all out” (e.g.
Eimeria)

◦ High Host specificity (e.g., Sarcocystis, Toxoplasma – sexual stages only)

◦ Low Host Specificity (Cryptosporidium, Toxoplasma – asexual stages only).

◦ Infection strategies
◦ Infectious when passed (Giardia)
◦ Requires time in environment to become infectious (Eimeria)
 Protozoa: Pathology (how
the pathogen causes disease)

1. Direct destruction of the host cells

2. Indirect destruction of host cells

3. Changes in host immune system

4. Excretion of toxins (most all parasitic protozoa)

 Protozoa: Pathology (how
the pathogen causes disease)

1. Direct destruction of the host cells

Reproduces inside cells → Cellular trauma →

Organ dysfunction

e.g. Coccidiosis,
Babesia,
Cytauxzoon, T.
cruzi
 Protozoa: Pathology (how
the pathogen causes disease)

2. Indirect destruction of host cells

Causes cell death
Tritrichomonas foetus Pathology
without having to
penetrate the cell wall
or grow inside the host
cells. e.g. causes host
cell apoptosis

e.g. T. foetus, Giardia

 Protozoa: Pathology (how
the pathogen causes disease)

3. Changes in host immune system

Autoimmune Reactions
• Autoantibodies directed against host cells
(e.g. hemolytic anemia)

• Antibody-Antigen complexes in kidney or other tissues

Immunomodulation/suppression
• Stimulate antibody-producing B-cells that are not antiparasitic
• Proliferation of suppressor T-cells → release immune inhibitory cytokines
• Protozoa produce specific immune suppressing substances

e.g. Babesia, Leishmania, Trypanosoma

(know terms on this slide)

Grouped by Infection Site and Motility

Parasitic
Protozoa Apicomplexa Flagellates
(Alveolates) (Excavates)

Blood apicomplexa (piroplasms) Hemoflagellates

Blood/ Babesia spp.
tissue Trypanosoma cruzi
Cytauxzoon felis Leishmania infantum

systemic Systemic apicomplexa

Toxoplasma gondii Trypanosoma cruzi
Neospora caninum Leishmania infantum
Sarcocystis spp.

intestines Intestinal apicomplexan (coccidia) Mucoflagellates

Cryptosporidium parvum Tritrichomonas foetus
Eimeria spp. Tritrichomonas blagburni
Cystoisospora spp.
Giardia spp.
Hemoflagellates

Leishmania infantum
Trypanosoma cruzi

Vector-borne pathogens
Hemoflagellates
FYI: other terms include Trypanosomastids or Kinetoplastida

erythrocyte

flagellum

Trypanosoma cruzi Leishmania infantum

Hemoflagellates
Exist in different forms depending on the hemoflagellate, host and life stage

Amastigotes in lymph node aspirate

Trypanosoma cruzi Leishmania infantum

Hemoflagellates
Leishmania
infantum
 Learning Objectives: Leishmania infantum
1. Life cycle: know that it is an indirect life cycle, the different forms of Leishmania in the 2
hosts, and specified life cycle details.

2. Transmission: know the 3 main routes of transmission

3. Pathogenesis: know the primary cell infected in the host and the effect a strong or weak
cell-mediated immune response has on disease progression.

4. Clinical signs: know the 4 specified common clinical signs of canine leishmaniasis

5. Diagnosis: know the 3 main ways we can diagnose Leishmania

6. Epidemiology: know risk factors for canine leishmaniasis for dogs in the US and that
leishmaniasis is zoonotic in people via sandflies in other parts of the world.
Indirect Life Cycle

Promastigote
insect (vector) form
sandflies (transient in mammal)

Amastigote
mammalian form
 infective promastigotes are salivarian
Replicates in fly midgut regurgitated just before a
blood meal
transmission

Promastigote phagocytized by
Sandfly takes up host macrophage
macrophages in
blood meal and
amastigote
transforms into Disseminates throughout
promastigote in host
the sandfly gut
macrophage

Asexual
Replication
Transmission
Leishmania

1. Vector-borne
New World Old World
◦ female sandflies
(Americas) (Europe/Middle East/Africa)
◦ only 2 genera proven to transmit (Lutzomyia and Phlebotomus)
◦ salivarian

2. Blood transfusion
3. Transplacental (vertical)
◦ e.g. American Foxhounds

4. Others? (direct or perinatal)

◦ e.g. American Foxhounds

Incubation: ~3 months to 7 years

 Pathogenesis: Leishmania 1. direct damage
2. immune modulation
FYI: image details
• tissue damage by sandfly → recruit phagocytic
immune cells

• phagocytosed by macrophages and other

immune cells

cell-mediated
CD4+
immune response

• production of cytokines by CD4+ (TH1) cells leads to

enhanced killing by macrophages
(no or poor
cell-mediated response)

more severe disease mild disease

persistent infection immune clearance
 Pathogenesis: Leishmania

POOR STRONG
cell-mediated cell-mediated
immunitry Range of disease severity immunity

FYI: High
serology!
It will be
important when
you are trying
to diagnose
leishmaniasis

Solano-Gallego, Laia, et al. "LeishVet guidelines for the practical management of canine leishmaniosis." Parasites & vectors 4.1 (2011): 86.
This is a great reference if you ever have to manage a Leishmaniasis case
 Clinical Disease Dogs can be reservoirs, living
asymptomatically for years until
Leishmania change in immune system

Incubation period from 3 months to years

Clinical findings (stage disease from I – IV)

◦ Dermal lesions (dry exfoliative lesions, ulcers, alopecia),
severity

◦ Lymphadenopathy, fever, splenomegaly, ocular dz (uveitis)

◦ Hyperglobulinemia, hypoalbuminemia, anemia (non-regenerative
anemia)
◦ Kidney dz (azotemia, proteinuria) → poor prognosis
FYI: Factors that dictate clinical disease severity
Immune system
Leishmania
TH1,
spp.
cell-mediated
parasite
killing
L. mexicana

Disease
Manifestation

L. infantum

severity TH2 co-infxs pathogen

Humoral load
 Figure 1
New Advances in the Diagnosis of Canine Visceral Leishmaniasis. Patrícia Sampaio Tavares Veras, Deborah Bittencourt Mothé Fraga, Manuela da Silva Solcà
Figure 1. Major clinical signs associated with Canine
Leishmaniasis.
A: alopecia on the muzzle
B: periocular dermatitis with keratoconjunctivitis and hyperkeratosis;
C: hyperkeratosis of the nasal mucosa;
D: generalized non-pruritic exfoliative dermatitis;
E: ulcerated lesion in the ear;
F: crust with vascular injury on the tip of the ear;
G: lymphadenomegaly of the popliteal lymph node;
H: cachexia (wasting syndrome); (ka-kex-ea)
I: onychogryphosis (hypertrophy of claws). (on-i-ko-gri-fo-sis)
Photos of animals infected by L. infantum belong to archives from Laboratory of Pathology and Biointervention (LPBI -
CPqGM).
https://www.intechopen.com/books/leishmaniasis-trends-in-epidemiology-diagnosis-and-treatment/new-advances-in-the-diagnosis-of-canine-visceral-leishmaniasis
 Figure 2
Laia Solano-Gallego. LeishVet guidelines for the practical management of canine leishmaniosis. Parasites & Vectors20114:86 (https://doi.org/10.1186/1756-3305-4-86)
Figure 2: Different patterns of cutaneous lesions in Canine
Leishmaniasis.

A) Exfoliative periocular alopecia and blepharitis;

B) Ulcerative nasal mucocutaneous lesions;
C) Papular dermatitis in the inguinal region;
D) Nodular crateriform lesions bordering the muzzle;
E) Ulcerative erythematous lesions on the plantar surface of the paw and
between pads;
F) Onychogryphosis. (on-i-ko-gri-fo-sis)

https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-4-86
Figure 3
Figure 3: Some clinical signs found in Canine Leishmaniasis:

A) Epistaxis (nosebleed);
B) Bilateral uveitis and corneal opacity;
C) Purulent conjunctivitis and blepharitis;
D) Exfoliative alopecia in the rear leg and popliteal lymphadenomegaly;
E) Marked cachexia (wasting) and generalized exfoliative alopecia.

https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-4-86
Diagnosis Leishmania
◦ Serology: Immunofluorescence (IFA), ELISA
- high serology titers in leishmaniasis indicate a low cell-mediated immune response and
likely more severe disease
-antibodies may cross-react with T. cruzi
◦ PCR (lymph node, blood, bone marrow, spleen, conjunctiva, cutaneous lesions)
◦ Amastigotes in cytology specimens
- lymph nodes, skin, spleen, etc.
- not very sensitive

Rescue groups bringing

ASK ABOUT in dogs from
TRAVEL Leishmania endemic
countries
HISTORY!
Serology: Immunofluorescence (IFA)
FYI only
Interpretation
of cytology

https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-4-86
 FYI only Interpretation of cytology

https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-4-86
 FYI: Leishmaniasis Treatment
Combination therapy
◦ Meglumine antimoniate (not available in US)
◦ Miltefosine
◦ Liposomal amphotericin B
◦ Allopurinol –initial tx and use long-term alone

1. Combine allopurinol with meglumine antimoniate or miltefosine followed by

long-term allopurinol.

2. Temporary clinical improvement; difficult to clear infection

FYI: Control Canine Leishmaniasis

Vector Control
◦ Insect(sandflies) repellents; collars, spot-on’s, etc.

Breeding control
◦ prevent transplacental transmission

Screen blood donors

◦ prevent transfusion transmission

Vaccines have been developed in Brazil & Europe (70-80% efficacy)

◦ (Leishmune, Leish-Tec, CaniLeish, Letifend)
 FYI: Geographic Distribution
Southern Europe, Africa, Asia, Caribbean, Central & South America
 Leishmania infantum in Hounds in the US
• Foxhound cases identified in1980/90s

• Outbreak of canine leishmaniasis in

Foxhound kennel in NY in 1999

• Vertical transmission over many

generations

• ~ 10 % of American Foxhounds are

infected with L. infantum
- many are asymptomatic
• Consider Bassets and Beagles too
Gaskin, Amanda A., et al. "Visceral leishmaniasis in a New York foxhound kennel." JVIM16.1 (2002): 34-44.
 Leishmaniasis in the United States
Risk of contracting leismaniasis in the US (autochthonous) is very low!
We are NOT endemic for L. infantum, but we do support L. mexicana transmission cycle
(sandflies and reservoirs in southern US)

RISK FACTORS for canine leishmaniasis in the US

1. Dogs that were imported from or travel history to Europe, Mediterranean, Middle
East (usually L. infantum endemic areas)
2. US Foxhounds with L. infantum transmitted vertically (Bassets and Beagles)

3. Dogs (and cats) from TX can become infected with L. mexicana (causes skin lesions)

autochthonous = aw-tok-tha-nus = contracting dz in the same place

 Zoonosis Leishmania Organomegaly in a
child highlighted with
marker
• 12 million people infected via sandfly
transmission
• Dogs are important reservoirs for human
infections around the world http://www.enetmd.com/content/leishmaniasis

Kalman Watsky, MD
https://clinicalgate.com/protozoa-and-
worms

Cats
Mucocutaneous Leishmaniasis - L. infantum
Cutaneous – L. mexicana
Overall, few Leishmania cases in cats in endemic regions
Hemoflagellates
Trypanosoma cruzi
 Learning Objectives: Trypanosoma cruzi
1. Life cycle: know that it is an indirect life cycle, the 2 different forms of T. cruzi in the
mammal host, and specified life cycle details.

2. Transmission: know the 4 main routes of T. cruzi transmission

3. Pathogenesis: know primary pathology is direct destruction of host cells. T. cruzi can
infect any nucleated cell but we often see disease when cardiac cells are destroyed.

4. Clinical signs: know the different phases of disease and the main specified clinical signs
associated with each phase.

5. Diagnosis: know the 2 specified methods and which to use for acute or chronic phases of
the disease.

6. Epidemiology: know dogs in the southern US are more likely to be exposed to T. cruzi and
it is a zoonotic disease, transmitted to people by the vector
 Trypanosoma cruzi

FYI: Epimastigote Trypomastigote Amastigote intracellular

Insect blood

-Replicate asex in bug -Infective stage cardiac macrophage/

midgut
-Mammalian blood (doesn’t divide) monocyte
-Transforms into infective
metacyclic trypomastigote -Transforms into amastigotes (divide)
-Found in mammalian tissue
and is excreted -vector ingests trypomastigote form -Replicates by binary fission
Trypomastigote

stercorarian
 Transmission T. cruzi
1. Vector-borne -- Triatomid bugs via
stercorarian
2. Transplacental
3. Blood Transfusion
4. Ingestion of infected bugs or animals
(contact with mucous membranes)
 Pathology Cardiac Histology
T. cruzi (Chagas Disease)

1. Infect and destroy any nucleated cell –

primarily cardiac cells
2. Intracellular multiplication of parasites with
direct destruction of cells (cardiac cells).
3. Immune modulation and autoimmune
destruction of host tissues
Clinical Disease: T. cruzi
Acute Phase (1st month)
◦ Lymphadenopathy, febrile disease
◦ diarrhea, vomiting, anorexia, lethargy
◦ Severe mycocarditis and death has been described → young
chronic

Latent Phase (months to years post-infection)

◦ usually asymptomatic; often for years

Chronic Phase (maybe years post-infection)

◦ Gradual decline to death, usually about 2 years after dx
◦ Signs related to heart damage
Diagnosis T. cruzi
1. Parasite detection (better for acute)
◦ PCR: blood or tissue
◦ Blood smear -- trypomastigotes
◦ Lymph node aspirate -- amasitgotes
◦ Cardiac biopsy / histology -amastigotes in pseudocysts
◦ Xenodiagnosis

2. Antibody detection (better for chronic)

◦ Serology (IFA or ELISA) (may cross-react with Leishmania)
◦ Better for testing animals in chronic or latent stage of disease
 T. cruzi amastigotes in LN
aspirate

http://www.capcvet.org/capc-recommendations/trypanosomiasis
FYI: Treatment T. cruzi
1. No approved treatments for Vet med

2. Mainly symptomatic treatments

-management of heart disease
3. Human approved antitrypanosomal drugs
-Benznidazole
-Nifurtimox
 FYI: Control T. cruzi
No vaccination
Vector control
◦ identify triatomids & their habitat
◦ avoid outdoor lights
◦ control sylvatic reservoirs (don’t let dogs
eat other mammals)
Breeding control
◦ prevent transplacental transmission
Screen blood donors -prevent transfusion transmission
Geographic Distribution T. cruzi
Central & South America

Canine cases in southern USA

◦ Texas, Louisiana, Oklahoma, Tennessee,
Virginia, California, Georgia, and South
Carolina
Zoonosis T. cruzi
Zoonotic Human Chagas Disease
◦ Cardiomyopathy, megaesophogus, megacolon
◦ Endemic areas:
◦ Mexico, Central America, South America

◦ Triatomid transmission, thrive in poor housing

conditions
◦ Mud Walls, Thatched Roofs
◦ Dogs and other small mammals are
important reservoirs for human infections
 Case-based Questions
4 yr old MN Golden Retriever presents with
exfoliative dermatitis, alopecia and crusting on
pinna.
History: was adopted a year ago from a rescue group
Physical exam: skin lesions and popliteal
lymphadenomegaly

What are some follow-up questions you might ask the owner?

What are good samples to collet for diagnostic testing and which tests would you use?
Case-based Questions
A 6 yr old MN mixed breed from Michigan presents with increasing
exercise intolerance.
History: Moved from southern Texas a year ago.
Imaging: Thoracic rads show enlarged heart
Labs:
• HW tests – negative

What’s an infectious disease you should check for?

You can only perform serology or PCR to support your infection suspicion, which do
you choose?
Illustration by Allie Brosh, http://hyperboleandahalf.blogspot.com/