Principles of Parasite Identification: Worms

Why is diagnosis important?

Allows proper treatment

Understand where the infection came from

May help limit spread of infection (particularly in zoonotic cases)

Basis of diagnosis: Parasitological

Based on morphological characteristics of the parasite

Can be specific in some cases

Basis of diagnosis: Immunological

Based on presence of Ab or Ag

Antibodies are ________ specific; long lasting so do not indicate ___________

Less; infection, but rather exposure

Antigen tends to be ________ specific; indicates ___________

More ; active infection

Basis of diagnosis: Molecular

Based on PCR

Very specific!

Principles of diagnosis: Ideally a test should be:

Sensitive, specific, reliable

Samples for diagnosis that can be obtained from a live animal include

Blood, stool

Eggs may be concentrated from feces via

Flotation or sedimentation

Blood films are usually stained for what purpose?

To ID parasites

What is this an example of?

Direct nematode life cycle

Many gastro-intestinal worms are diagnosed via

detection of eggs in stool

Some worms are very fecund and eggs can be detected

In direct smear

Under some circumstances, may need to

Concentrate the eggs

What type of egg is shown?

Smooth egg shell (Toxascaris leonina)

What type of egg is shown?

Rough pitted egg shell (Toxocara canis/cati)

What type of egg is shown?

Strongyle egg (Hookworm, either Ancylostoma or Uncinaria)

What type of egg is shown?

Bipolar plugs (Trichuris egg)

Treatment options are often _____ for nematodes

The same

Easy to distinguish hookworms from ascarids via

Their eggs

Clinical signs of A. caninum infection

Anemic puppies

Clinical signs of T. canis

puppies with ill-thrift and swollen bellies

What is needed for concentration and quantification of eggs?

McMaster slide

Flotation of nematode eggs in saturated salt solution

Known volume added to each well on the slide

Why would we potentially need to culture nematode larvae from fecal samples?

If two species of eggs are identical

What is an example of two nematodes who may need culturing to distinguish?

Ancylostoma caninum and Uncinaria stenocephala

Nematode larvae found in feces: L1 of Angiostrongylus vasorum can be

detected in feces

Nematode larvae found in feces: Female worm in heart produce

Eggs, hatch rapidly, coughed and swallowed

What does this image show?

Tapeworm eggs (Taenia)

Echinococcus granulosus is

zoonotic

very similar egg to Taenia

Describe Taenia eggs

thick egg shell

radial striations

6-hooked (hexacanth) embryo inside

Antigen detection of parasites

ELISA test

Dipstick

Antigen detection relies on

good antibodies

DNA detection of parasites

PCR

PCR relies on

primers specific to parasite DNA

extremely sensitive → specific equipment

prone to contamination

many factors in stool can interfere

What is a staining profile of parasites used for

To distinguish pathogenic and nonpathogenic species of Dirofilaria

Serological tests for worms

Antibody is a measure of exposure and can be useful under some circumstances

Antigen detection in blood is very useful as indicated current infection

Ag detection test for heartworm

relies on detection of antigens release by adult female worms in the heart using a specific antibody to capture the Ag. color change denotes the presence of antigen in the blood

Not quantitative - need ELISA

Why is antigen testing for heartworm alone not useful in cats?

Only detects sexually mature female worms

level of antigen relates to number of worms

often contain few worms and not sexually mature

What in addition to antigen testing should you use for heartworm testing cats?

Antibody test

Antigen test for A. vasorum

detects antigen in the blood stream of infected dog

improvement over L1 in feces as these are shed intermittently

McMaster method: Large Animal

measure eggs per gram of feces (EPG)

McMaster method useful in

FECRT (fecal egg count reduction test) which measure the level of anthelmintic resistance in a worm population

Quantify eggs before vs eggs after treatment

Baermann test is used for

detecting L1 of cattle lungworm, D. viviparus

D. viviparus Baermann test

nematode larvae migrate from the fecal sample downward in water through the gauze and collect in the tubing above the clamp

What does this image show?

Cattle lungworm, D. viviparus

Protozoan - Feco-oral tranmission/GI of host examples

Toxoplasma gondii

Giardia spp.

Cryptosporidium parvum

Protozoan - Vector borne examples

Babesia

Leishmania

What does this image show?

Dirofilaria

Diagnosis of toxoplasmosis (Ovine): grossly

bright red placenta cotyledons

Diagnosis of toxoplasmosis (Ovine): microscopically

AB in fetal fluids/precolostral lamb serum

Ewe; AB test with paired samples or IgM detection

Immunohistochemcial/PCR detection of parasite (tissue cyst) in aborted (placenta) tissue (expensive)

Bradyzoite tissue cysts are

too small to be picked up in meat inspection

T. gondii - Diagnosis (Human): Pregnant mother

Serology - IgM detection + high igG titre (acute infection within las 3 months)

T. gondii - Diagnosis (Human): Congenital infection

prenatal - PCR of amniotic fluid/ultrasound

postnatal - CAT or MRI scan for brain lesions

T. gondii - Diagnosis (Human): immunocompromised (AIDS)

PCR on blod, cerebrospinal fluid and brain scans

Giardia clinical signs

Strong smelling, watery diarrhea/vomiting, weight loss

Giardia transmission

feco-oral via ingested cyst form

What does this image show?

Giardia ovoid cysts 10-14 um

fecal smear for diagnosis

test x3 - 3-5days

firm stool

What does this image show?

Giardia trophozoites - loss stools

Giardia is elusive parasite to test use

SNAP (ELISA) plus flotation

if both +, 95% correct diagnosis

Cryptosporidium spp. clincally

zoonotic - acute gastroenteritis

infection from public water sources, contact with livestock

faulty water treatment

Cryptosporidium oocytes detection

difficult in unstained fecal samples due to small size

What does the white circle show?

Cryptosporidium parvum

What does the black arrow show?

Toxoplasma gondii

What does the white arrow show?

Isospora felis

Cryptosporidium detection Common

modified Ziehl-Nielsen (acid-fast) staining technique

poor sensitivity - but will detect acute cases

detection limi 5×10⁵ oocysts/g feces following concentration of fecal sample

What does this image show?

Ziehl-Nielsen stain for detection of Cyptosporidium

Cryptosporidium detection Other

Immunofluorescent AB test (IFAT)

Antigens in feces detected by enzyme immunoassays

PCR

Cryptosporidium detection: Immunofluorescent antibody test (IFAT)

Microscopy based, fluorescently labeled Mab Ab

combined tests detect giardia

Sensitivity; 1x10⁴ – 1x10⁵ oocysts/g faeces (50x more than stained smears)

Municipal water monitored/filtered – Ab based oocyst capture

Cryptosporidium detection: Enzyme immunoassays

results inconsistent when applies to small animals

Cryptosporidium detection: PCR based assays

used for genotyping species ID

10x more cases positive

What parasites are detected via blood smear?

Trypanosomes, Leishmania, Theileria, Babesia

What does this image show?

Leishmania amastigotes in macrophages

What does this image show?

Theileria macroschizonts in lymphocytes

What does this image show?

Babesia piroplasms in RBC

What does this image show?

Trypamastigote of Trypanosoma

What does this image show?

Red water

Detection of blood-born protozoa excluding blood smear

Immunofluorescence

ELISA

PCR

PCR positives

very specific

very sensitive - needs small amounts of material

can differentiate mixed infections

PCR negatives

not that rapid/lab based

easy to contaminate, so need controls for false +s

for quantification need sophisticated thermal cycler

Antigen and PCR will

NOT always confirm the infection is viable

CAN generate over-estimation of risk