Parasitic Protozoa

ONE CELL MENACES

Mucoflagellates

Grouped by Infection Site and Motility

systemic

intestines

blood/tissue

Flagellates(sg = Excavates)

HemoflagellatesTrypanosoma cruzi

Tritrichomonas foetus

Leishmania infantum

Giardia spp.

Mucoflagellates

Trypanosoma cruziLeishmania infantum

Intestinal apicomplexan (coccidia)

Blood apicomplexa (piroplasms)

Cryptosporidium parvum

Eimeria spp.Cystoisospora spp.

Cytauxzoon felisBabesia spp.

Toxoplasma gondiiNeospora caninumSarcocystis spp.

Systemic apicomplexa

Parasitic Protozoa Apicomplexa

(sg =Alveolates)

MucoflagellatesTritrichomonas spp. Giardia spp.

Credit: David Gregory & Debbie Marshall, WellcomeImages

• Pear or Spindle-shaped• Flagellum (multiple)• Reside in mucous membrane-lined

anaerobic-to-microaerophilic, non-sterile organ cavities

Tritrichomonas foetus and blagburni

• Specific host-pathogen interactions• Bovine Tritrichomoniasis• Feline Tritrichomoniasis

• Obligate parasite; exists only as trophozoite• Urogenital and Gastrointestinal tracts

Cytological preparation of cultured felineTritrichomonas

(a) posterior flagellum(b) undulating membrane(c) anterior flagella(d) axostyle(e) nucleus

Wright-Giemsa, x100 magnificationGookin et al. The Conundrum of Feline Trichomonosis The more we learn the “trickier” it gets. JFMS (2017) 19, 261-274

T. foetus – feline genotype → GI dzT. foetus – bovine genotype → venereal dzT. suis – porcine commensal

Are these the same organism?

~1% difference genomelik

ely

the

sam

e

T. blagburniT. foetus

T. suis (FYI)

T. blagburni infects cats

Tritrichomonas

Bovine Genital TrichomoniasisLarge Bowel Feline Trichomoniasis

• Large bowel diarrhea• Venereal disease, causes

infertility,abortions in cows and heifers

• Colonize large intestine• Colonize urogenital tract

• Economic loss

(T. blagburni) (T. foetus)

T. foetus: Bovine Genital Trichomoniasis Direct Life Cycle

Primary Hosts1. cows/bulls

Transmission1. sexual2. artificial insemination

Stages1. Trophozoite2. No cyst stage

Reproduction1. binary fission in

urogenital tract

T. foetus-bovine Pathology – not well understood

Cows/Heifers

- autoimmune/inflammatory response/indirect → contact with the trophozoite and excreted enzymes

• infection is self-limiting; clearance ~20 weeks post-infection

Bulls • trophozoites attach to cells lining the penis, prepuce, and distal portion of the urethra

• no damage to cells, no lesions, no change in semen quality or sexual behavior

• vaginitis and endometritis → abortion, pyometra, or infertility

• trophozoites attach to epithelial cells in reproductive tract

• essentially serve as reservoirs (persistently infected)

Clinical Disease: Bovine Genital TrichomoniasisMain complaint:◦abortions (early to mid-term)◦ failed pregnancy, infertility suspect◦more open and late cows

(cows in heat when they should be pregnant)

Pathological findings• Vaginitis, cervicitis, pyometra, endometritis, mummified fetus• No clinical signs in bulls

Diagnosis: Bovine Genital TrichomoniasisCow – History of abortions. You can test cervical mucus, uterine fluid, fetal tissue; however…

Bull -- Preputial wash or scrapings

◦ Microscopic examination of of fresh wet-mounts for trophozoite◦ culture kits (~95% SE); successive cultures(~99.5% SE)◦ PCR

Testing method based on state requirementsPositive results - REPORTABLE

Most diagnostics are used to detect T. foetus in bulls

Diagnosis: Bovine Genital TrichomoniasisPreputal washings for diagnostic culture

https://www.merckvetmanual.com/reproductive-system/trichomoniasis/overview-of-trichomoniasis-in-cattle

Differential Diagnosisabortion in cattleBovine tritrichomonashistory and clinical signs are ~ bovine genital campylobacteriosis

FYI: this chart

Control: Bovine Genital Trichomoniasis

• Strict surveillance of bulls; sexual rest for exposed cows• Cull infected bulls, replace with young (virgin) bulls• Replace bulls >4 yrs• Use hygienic AI• T. foetus can survive the process used for freezing semen

• Vaccines (not-complete protection)[Trichguard, TrichGuard V5L]

Good Farm Management and Biosecurity!

Treatment: none

Geography: Bovine Genital TrichomoniasisStates with bovine trichomoniasis regulations

Yao C, Bardsley KD, Litzman EA, Hall ML, Davidson MR (2011) Tritrichomonas foetus Infection in Beef Bull Populations in Wyoming. J. Bacteriol Parasitol 2:117. doi:10.4172/2155-9597.1000117

Western US – more challengeswith management

FYI

• Bulls – permanently infected / infective; prevalent in >4 yrs age• Cows – immune clearance if left unbred for 3-4 months with partial, temporary immunity against reinfection

Epidemiology:

Bovine Genital Trichomoniasis

A beef farmer complains of having too many “opencows” at the end of the breeding season.

What laboratory diagnostics do you want to perform?

What are some questions you might ask the farmer?

Tritrichomonas blagburni

Feline Trichomonosis

The conundrum of feline trichomonosis – The more we learn the ‘trickier’ it getsGookin, Hanrahan, Levy. 2017. J. Feline Medicine & Surgery. 19: 261-274.

A cause of persistent Large Bowel Diarrhea in Cats

https://veteriankey.com/trichomoniasis/

Primary Host1. cats, large bowel

Transmission1. Fecal-oral –ingest trophozoites

Stages1. Trophozoite2. No cyst stage (why is this important?)

Reproduction1. binary fission in feline GI tract

T. foetus: Large bowel Feline Trichomoniasis Direct Life Cycle

Illustration by Alice MacGregor Harvey © North Carolina State University

Pathology: Large bowel Feline TrichomoniasisIndirect tissue destruction: extracellular, has a cytotoxic effect when interacting with the surfaces of host cells.

-Interacts with bacterial flora

-adherence to host mucusand epithelium

-cytotoxins, enzymes

-activation of host inflammation

FYI: this illustration

Not completely understood → Theorized pathogenic mechanisms of T. foetus

Contributing factors:

(rare)

Clinical Disease: Large Bowel Feline Trichomoniasis• Waxing and waning, chronic foul-smelling diarrhea• Semi-formed to “cow-pie” diarrhea

• +/- mucus; +/- fresh blood• Urgency and straining (tenesmus)

• frequent defecation with small volume of feces.• red, swollen, painful anus

• Usually BAR, good BCS, normal appetite• Worse in kittens and younger cats

• fecal incontinence• increased inflammation of anal region “fecal scalding”

• Some cats are subclinical

Clinical Disease: Large Bowel Feline Trichomoniasis

messy butt

Semi-formed to cow-pie

fresh blood or mucusfrequent dribbling

Diagnosis: Large Bowel Feline Trichomoniasis

1. Fresh wet-mounts and light microscopy to view motile trophozoites • don’t confuse with Giardia• low sensitivity (≤14%)

2. In-vitro culture kit (InPouch TF Feline for culturing)• perform in clinic; use with wet mount

3. PCR: T. foetus Diagnostic Laboratory at NC State

• Fecal sample (pathogen dead or alive)• high sensitivity

https://cvm.ncsu.edu/research/labs/clinical-sciences/tfoetus/#tabsPnl1-tab-1

Diagnosis: Large Bowel Feline Trichomoniasis

http://www.JodyGookin.com

GiardiaPetal shape

Falling leaf motility

T. foetusSpindle-shapedForward motility

wet mount

Treatment: Large Bowel Feline Trichomoniasis

Ronidazole {Tricho Plus}

◦30 to 50 mg/kg every 12 hrs. for 14 days (note: you won’t be tested on drug dosages)

◦Be alert for neurotoxicity◦Narrow Safety Margin◦Resistant populations increasing

Unresponsive to metronidazole

The FDA prohibits this drug for use in food animals.

Control: Large Bowel Feline Trichomoniasis

• Strict hygiene in group housing, cattery, shelter, and cat shows.

• No cyst form, so the organism does not persist for more than a few hours in clean, dry, aerobic environments

• It can survive for several days in feces and 1 day in water or urine

1. Young catsOlder cats may be infected and asymptomatic

2. Cats from high density populations, group housingCatteries – Breeding and boardingPure-breed show catsHoarding situation

Epidemiology: Large bowel Feline Trichomoniasis

Most prevalent in:Occurs worldwide

FYI Zoonosis: T. foetus –maybe?

Maritz, Julia M., et al. "What is the importance of zoonotic trichomonadsfor human health?." Trends in parasitology 30.7 (2014): 333-341.

known transmissiontheorized transmission

key

GiardiasisCauses small bowel diarrhea in avariety of hosts

Giardia duodenalis• G. duodenalis is part of a a species

complex including G. intestinalis, G. lamblia.

Credit: David Gregory & Debbie Marshall, WellcomeImages

• Can cause asymptomatic colonization or acute or chronic diarrhea

FYI: species complex = a group of closely related organisms that are so similar, the differences are unclear

Morphology

Direct Life Cycle: GiardiaPrimary Host

1. many species2. colonizes small intestines

on mucosal surfaceTransmission

1. fecal-oral, ingest cystsa. from fecesb. from contaminated water,

food, fomites, self-grooming2. ingested trophozoites will not survive

Stages1. trophozoite (active, noninfective)2. cysts (dormant, infective)

Reproduction1. longitudinal binary fission of trophozoites

Image: https://veteriankey.com/common-protozoans-that-infect-domestic-animals/

Pathology: GiardiaIndirect destruction: trophozoite attaches to small intestinal epithelial cells◦ damages epithelial cells, blunts intestinal villi → reduced surface area◦ Dysfunctional enterocytes → maldigestion, malabsorption, diarrhea

Normal duodenumChronic giardiasis:blunted villi; reduced surface area of enterocyte

Image: Troeger, Hanno, et al. "Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum." Gut 56.3 (2007): 328-335.

Clinical Disease: GiardiaComplaint◦Persistent Diarrhea: soft, loose, strongly malodorous◦+/- vomiting, anorexia, dehydration◦develop ~ 2 - 3 weeks post infection◦ Some animals are subclinical but shed giardia

Pathological findings◦ Fatty diarrhea (light-colored, foul smelling)◦Malabsorption syndrome◦ signs of poor nutrition – lethargy, weight-loss, etc.

Diagnosis: Giardia

Antigen (cysts) detection kits, ELISA (SNAP Tests, VetScan)Rapid in-house; ↑ SP

Direct microscopic fecal analysisb/c of intermittent shedding → may need to do several over time

Loose stool: motile trophozoites on fresh wet-mounts (use saline)(don’t confuse with Tritrichomonas in cats)

Solid stool: cyst stage (don’t confuse with yeast)use fecal float centrifugation with zinc sulfate solution

combine↑ SE

Saleh, Meriam N., et al. "Comparison of diagnostic techniques for detection of Giardia duodenalis in dogs and cats." JVIM (2019).

Uiterwijk, Mathilde, et al. "Comparing four diagnostic tests for Giardia duodenalis in dogs using latent class analysis." Parasites & vectors 11.1 (2018): 439.

Microscopic Diagnosis: GiardiaCyst Trophozoite

More solid the stool the more cysts More fluid the stool the more trophs

Giardia

Yeast

40x objective(mag. 400x)

Zinc sulfate fecal flotation showing Giardia cysts and yeast

Diagnosis: Wet mount Giardia

http://www.JodyGookin.com

GiardiaPetal shape

Falling leaf motilityForms cysts

T. foetusSpindle-shapedForward motility

Does NOT form cysts

Treatment: GiardiaDog: Metronidazole [Flagyl], Fenbendazole [Panacur], Febental-pyrantel-praziquantel [Drontal plus]

Cat: Metronidazole [Flagyl], Fenbendazole [Panacur], Febental-pyrantel-praziquantel [Drontal plus]

Calves: Fenbendazole [Panacur], Albendazole [Valbazen]

Companion Animal Parasite Council (CAPC) recommends treating only symptomatic dogs & cats to decrease development of resistance to antiprotozoal drugs.

Control: Giardia• Strict prevention of fecal contamination• Outdoor environment: pick up, dispose of feces• Indoor environments: clean (soap/water) followed by

disinfection with diluted bleach (~1/20 = 5% bleach)• Giardia cysts can survive: FYI

• soil → cool (<39.2 °F) ~ 7 weeks; room temp ~1 week• dry, warm surface with direct sunlight ~2 days• moist, cool surface ~ 2 weeks• water → <50 °F ~3 months; >50 °F, less time, summer ~4 days

https://www.cdc.gov/parasites/giardia/prevention-control-pets.htmlRefer clients to CDC website

Epidemiology: Giardia

More common in pets in high density situations ◦catteries, kennels, shelters, dog parks

More likely to spread Giardiato same species “house mates”.

FYI: In the USDog: 7.4%Cat: 3.7%

https://capcvet.org/maps/#2019/all/giardia/dog/united-states/

Zoonosis: Giardia•Giardia spp. contain molecular assemblages (differences in the genome) A-H•Zoonotic potential varies based on molecular assemblages •Most of the molecular assemblages seem to be host specific –zoonosis rare.

◦ Assemblage A-I – Humans, dogs, cats, others◦ Assemblage C & D – Canines◦ Assemblage F – Felines

◦ Some concern for immunodeficient patients

High host specificity

A cat owner complaining because of apparent fecal incontinence.

In-Class Discussion

Infectious differentials?

What are some additional questions you might ask?

Diagnostics?

Cross species or Zoonosis ?

See Review Table:Small Animal Diarrhea ProtozoaPosted on-line at Parasitology Website: https://parasitology.cvm.ncsu.edu/vmp930/lecture.html

The information in the review tables is basic information that you should know. You should also be able to use that information via critical thinking to answer more complex case-based questions.

What you’re expected to know

Have Questions?barbara_qurollo@ncsu.edu

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