Onchocerca lupi

Canid Falariasis in NM

BY AUDREY MELTON

“Coco”, inside/outside dogJemez mountains NM

Red inflamed eyesVeterinary treatmentBlindnessEuthanasia

AN EMERGING INFECTIOUS DISEASE IN DOGSNEW MEXICO

Veterinarians encounter“dog eye worms”

Bilateral Conjunctival swellingRetinal detachment

Surgical removal long thread-like worms

Filaria, NematodaOnchocerca sp.

Commonly O. lupi is mistaken as Thelazia callipaeda. Which is another “eyeworm” found in Canines in NM. They can look similar and cause a similar cyst/mass in the eye.

Adult worms can appear similar as well as the mass/cyst that forms in the eye

Life cycle of Thelazia callipaeda

Deposited in parasite collection ofMuseum for Southwestern Biology

(MSB)Perpendicular cuticular ridges(EtOH fixed specimen)

Morphology, ND5, CO1, obligate Wolbachia endosymbiont, Europe, (North America)

>lcl|OlupiNMCO1 Onchocerca lupiTTGGATGTTGCCTTTGATGTTGGGGGCTCCAGAGATGGCTTTTCCTCGAATTAACGCTTTGTCTTTTTGGTTTACTTTTGTATCTTTGTTGATGGTTTATCAGTCTTTTTTTATTGGAGGCGGTCCTGGTAGTAGTTGAACTTTTTATCCTCCTCTTAGAGTAGAGGGTCAGCCTGAGTTATCTTTGGATACTATAGTTTTGGGTTTGCATACTGTTGGTGTTGGTTCTTTGTTGGGTGCTATTAATTTTATGGTTACTACTCAAAATATGCGTTCTACTGCTGTGACTTTAGATCAAATTAGTATGTTTGTCTGGACTTCTTATTTAACTTCTTTTTTATTAGTATTATCTGTTCCTGTTTTAGCTGGGTCTTTGTTATTTTTGTTGTTGGATCGTAATTTTAGTACTTCTTTTTATGATACTAAAAAAGGTGGTAATCCTTTGTTGTATCAGCACTTGTTTTGGTTTTTCGGTCATCCCGAGGTTTATGTTATTATTTTACCGGTTTTTGGTATTATTAGGGAGGCAGTTTTATTTTTGACTGATAAGGATCGTTTGTTTGGTCAAACTAGGATGACTTTTGCTTCTATTTGGATTGCTGTTTTGGGGACTTCTGTTTGGGGTCACCATATGTATACGGCTGGTTTGGA

>lcl|OlupiND5 Onchocerca lupiCTGCTCCTACTCCTGTTAGTTGTTTGGTTCATAGTAGGACTTTAGTTACCGCGGGAGTTATGTTAATAGATTGTTATGTTTATGTTTCTTTGAATTCTGATGTTTTGTCTTTTGTTTTTTATGTTGGTTTTTTTACTATGGTTTTTTCTGGTTTTTGTTCTTTAGTTGAGCAGGATGCTAAAAAGATTGTTGCTTTGAGTACTATGTCTCAGATTGGTTTTTGTTTTTTGGCTATTGGTAGGGGTTTACATTATTTATCTTATGTTCATATGATTGGGCATTCTTTTTTTAAGAGTTTGTTATTTATGCAAATGGGTTATTTAATTTTTATTAATTTTGGTCAGCAGGATTGTCGTGGTTATTCTTTCTTTGGTTTTTGTGCCCCTGTTTTGGTTCAGTTACAGATTTTTTTATGTGTTTTTAGTT

SEQUENCES FROM COCO WORM SAMPLES

>lcl|Wolb_Olupi_NM Wolbachia endosymbiont of Onchocerca lupi from New Mexico, USA, 16S ribosomal RNA gene, partial sequenceTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCTAGTAGTACGGAACAATTGCTGGAAACGACAACTAATACCGTATACGCCCTACGGGGGAAAGATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTAGGGTAATGGCCTGCCAAGGCTATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGGAAGCTTGATCCAGCCATGCCGCGTGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTCAGTGAGGAAGATAATGACGGTACCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGAGCACGTAGGCTGGTTAGTAAGTTAAAAGTGAAATTCCAAAGCTTAACTTTGGAATTGCTTTTAAAACTGCTGATCTAGAGGTTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGGAGATTACTTTCTGTGTTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTG

nematodes I 20 leavesOnchocerca from Portugal cytochrome oxidase subunit (COI) gene, partial cds, mitochondrial

Onchocerca from Turkey cytochrome oxidase subunit (COI) gene, partial cds, mitochondrialOnchocerca lupi isolate 05RD‐0652 cytochrome oxidase subunit (COI) gene, partial cds, mitochondrial

nematodes I 8 leaves

nematodes I 14 leaves

nematodes I 6 leaves

nematodes I 3 leaves

nematodes I 3 leaves

nematodes I 13 leaves

nematodes I 17 leaves

nematodes I 7 leaves

nematodes I 3 leaves

nematodes I2l leaves

Dipetolonema gracile mitochondrial partial COI gene for cytochrome oxidase subunits

COCO WORM CO1, ND5 GROUP WITH Onchocerca lupiAND YIELDS Wolbachia ENDOSYMBIONT 16S SEQUENCE

CO1,Distance-tree through BLAST,(100-98% identity)

PROPOSED LIFE CYCLE Onchocerca lupi

Microfilariae penetrate vector’s gut and migrate to thoracic muscles

Adults in subcutaneous nodulespresent in periocular tissues 

Migrate to head and vector’s  proboscis

Vector insect takes a bloodmeal L3 larvae enter bite wound

Subcutaneous tissues

L2 Larvae

L3 Larvae

Vector insect 

(as yet unidentified)takes a bloodmealIngests microfilariae

Skin and subcutaneous tissues

Vector?/hosts

EARLY DIAGNOSIS OCULAR FILARIASIS IN DOGS

•Transmission dynamics, vector, habitat?• Prevention, Control?• How many dogs, prevalence?• Worm genetic diversity, origin?

•SKIN SNIP•EtOH FIXED•EXTRACT DNA•PCR DETECTION OFMICROFILARIA(DEAD, IVERMECTIN)•CytB (+) CONTROL

ND5 CO1

1 2 3 4 1 2 3 4Coco SKINSNIPS

Sample SKINSNIP to capture microfilaria

Chris Bauer, Kellie WillitsChris Bauer, Kellie WillitsNancy McLean

“Blanca”

CytB ND5

Worm fragment

PREVALENCE, INTERVIEW VETS, OWNERS, GENETIC DIVERSITYFOR CIRCUMSTANCES

HABITAT, VECTOR

CytB CytBND5 ND5

POUND Skinsnips

TRACKING FILARIASIS IN DOGS, NM

Onchocerca lupi, insights/conclusions

Morphology, DNA, Morphology, DNA, WolbachiaWolbachia identify identify Onchocerca lupiOnchocerca lupi

DNA extraction EtOH skinsnips challenging (inhibitors?)DNA extraction EtOH skinsnips challenging (inhibitors?) PCR protocol labor intensive, considering qPCR protocol labor intensive, considering q--PCRPCR

Transmission dynamics, vector, habitat?Transmission dynamics, vector, habitat?Also in arid conditions (drought) , less likely blackflies, mosqAlso in arid conditions (drought) , less likely blackflies, mosquitoes, perhaps ticks or biting uitoes, perhaps ticks or biting midges.midges.

Prevention, Control?Prevention, Control?Ivermectin treatment (heartworm) seems to kill microfilariaIvermectin treatment (heartworm) seems to kill microfilariaNovel detection of (obligate endosymbiont) Novel detection of (obligate endosymbiont) WolbachiaWolbachia USA) use antibiotics against adult USA) use antibiotics against adult wormsworms

How many dogs, prevalence? How many dogs, prevalence? >6 specific dogs, Aztec skin snips 3/53 (5%) since Fall 2012>6 specific dogs, Aztec skin snips 3/53 (5%) since Fall 2012

Worm genetic diversity? origin? high sequence similarity/identiWorm genetic diversity? origin? high sequence similarity/identity to other ty to other O. lupiO. lupi is USA, is USA, Europe.Europe.

Resources

Canid Ocular Filariasis in NM Davila KA, Jones C, McLean NJ, Adema CM*, Melton, AL