Biting midges (Diptera: Ceratopogonidae) as putative vectors of zoonotic Onchocerca lupi (Nematoda: Onchocercidae) in northern Arizona and New Mexico, southwestern United States.

Onchocerca lupi (Rodonaja, 1967) is an understudied, vector-borne, filarioid nematode that causes ocular onchocercosis in dogs, cats, coyotes, wolves, and is also capable of infecting humans. Onchocercosis in dogs has been reported with increasing incidence worldwide. However, despite the growing number of reports describing canine O. lupi cases as well as zoonotic infections globally, the disease prevalence in endemic areas and vector species of this parasite remains largely unknown. Here, our study aimed to identify the occurrence of O. lupi infected dogs in northern Arizona, New Mexico, and Utah, United States and identify the vector of this nematode. A total of 532 skin samples from randomly selected companion animals with known geographic locations within the Navajo Reservation were collected and molecularly surveyed by PCR for the presence of O. lupi DNA (September 2019-June 2022) using previously published nematode primers (COI) and DNA sequencing. O. lupi DNA was detected in 50 (9.4%) sampled animals throughout the reservation. Using positive animal samples to target geographic locations, pointed hematophagous insect trapping was performed to identify potential O. lupi vectors. Out of 1,922 insects screened, 38 individual insects and 19 insect pools tested positive for the presence of O. lupi, all of which belong to the Diptera family. This increased surveillance of definitive host and biological vector/intermediate host is the first large scale prevalence study of O. lupi in companion animals in an endemic area of the United States, and identified an overall prevalence of 9.4% in companion animals as well as multiple likely biological vector and putative vector species in the southwestern United States. Furthermore, the identification of these putative vectors in close proximity to human populations coupled with multiple, local zoonotic cases highlight the One Health importance of O. lupi.

LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples.

Onchocerca lupi is a filarial nematode that causes ocular onchocercosis in canines globally including North America and areas of Europe, North Africa, and the Middle East. Reported incidence of this parasite in canines has continued to steadily escalate since the early 21st century and was more recently documented in humans. Whole genome sequencing (WGS) of this parasite can provide insight into gene content, provide novel surveillance targets, and elucidate the origin and range expansion. However, past attempts of whole genome sequencing of other Onchocerca species reported a substantial portion of their data unusable due to the variable over-abundance of host DNA in samples. Here, we have developed a method to determine the host-to-parasite DNA ratio using a quantitative PCR (qPCR) approach that relies on two standard plasmids each of which contains a single copy gene specific to the parasite genus Onchocerca (major body wall myosin gene, myosin) or a single copy gene specific to the canine host (polycystin-1 precursor, pkd1). These plasmid standards were used to determine the copy number of the myosin and pkd1 genes within a sample to calculate the ratio of parasite and host DNA. Furthermore, whole genome sequence (WGS) data for three O. lupi isolates were consistent with our host-to-parasite DNA ratio results. Our study demonstrates, despite unified DNA extraction methods, variable quantities of host DNA within any one sample which will likely affect downstream WGS applications. Our quantification assay of host-to-parasite genome copy number provides a robust and accurate method of assessing canine host DNA load in an O. lupi specimen that will allow informed sample selection for WGS. This study has also provided the first whole genome draft sequence for this species. This approach is also useful for future focused WGS studies of other parasites.

Complete mitochondrial genome of Onchocerca lupi (Nematoda, Onchocercidae).

Onchocerca lupi, Rodonaja 1967, is an emerging, zoonotic filarial nematode parasite that causes ocular disease in dogs, cats, wild canids, and humans. It is the causative agent of ocular onchocercosis in canines with increasing incidence in both North America and the Old World during the early twenty-first century. We report the complete mitochondrial genome of an O. lupi isolate from a dog from Arizona, southwestern USA, and its genetic differentiation from related Onchocerca species. The whole mitochondrial genome was obtained from whole genome sequencing of genomic DNA isolated from an adult worm. This mitogenome is 13,766 bp in size and contains 36 genes and a control region. This mitogenome provides a valuable resource for future studies involving epidemiological surveillance, population genetics, phylogeography, and comparative mitogenomics of this emerging pathogen and other parasitic nematodes.

Coyotes as Reservoirs for Onchocerca lupi, United States, 2015-2018.

The Onchocerca lupi nematode infects dogs, cats, and humans, but whether it can be spread by coyotes has been unknown. We conducted surveillance for O. lupi nematode infection in coyotes in the southwestern United States. We identified multiple coyote populations in Arizona and New Mexico as probable reservoirs for this species.

Comparison of bacterial culture results collected via direct corneal ulcer vs conjunctival fornix sampling in canine eyes with presumed bacterial ulcerative keratitis.

OBJECTIVE: To compare aerobic bacterial culture results between samples obtained from the corneal ulcer versus lower conjunctival fornix in eyes with presumed bacterial ulcerative keratitis. ANIMALS STUDIED: Fifty five client-owned dogs diagnosed with ulcerative keratitis. PROCEDURES: Ophthalmic examinations were performed on each dog including slit-lamp biomicroscopy and indirect ophthalmoscopy. Microbial swabs were collected by direct sampling of the infected corneal ulcer as well as the lower conjunctival fornix, of the same eye, using a sterile rayon-tipped swab. Samples were submitted to an outside reference laboratory for aerobic bacterial culture and sensitivity. RESULTS: One hundred twelve samples were obtained from 56 eyes (55 dogs). Sixty-eight samples yielded bacterial growth. Positive growth from both sites was obtained in 31 eyes (55%). Six eyes yielded bacterial growth from the conjunctival fornix but not from the cornea. No bacterial growth was obtained from either sampling site in 19 eyes. Overall, 31/56 (55%) corneal samples were positive and 37/56 (66%) conjunctival fornix samples were positive. Comparison of organisms isolated from the two collection sites of the same eye revealed an exact correlation in 42/56 (75%) eyes and differed in 14/56 (25%) eyes. Twenty different bacterial isolates were obtained from 68 positive samples. Gram-positive (71%) organisms were more common than Gram-negative (29%). The most commonly isolated organisms were Staphylococcus pseudintermedius (25%), beta-hemolytic Streptococcus spp. (23%), and Pseudomonas aeruginosa (12%). Methicillin-resistant organisms were isolated in 9% of samples. CONCLUSION: Sampling from the conjunctival fornix may be a suitable alternative to direct ulcer sampling in eyes with compromised corneal structural integrity.

Penetrating keratoscleroplasty and bimodal grafting for treatment of limbal melanocytoma in a dog.

A 6-year-old, male neutered, German Shepherd dog presented for evaluation of a dark limbal mass OS. Based on the signalment and clinical findings, a presumptive diagnosis of limbal melanocytoma was made. Additionally, OU demonstrated corneal changes consistent with chronic superficial keratitis. Full thickness en bloc resection of the limbal mass was followed with reconstruction and grafting using both frozen cornea and bulbar conjunctiva. Nitrous oxide cryotherapy was utilized as an adjunctive modality for residual neoplastic cell destruction. Histopathologic evaluation confirmed the presumptive diagnosis. At 18 months postoperatively, there was no evidence of recurrence of the limbal melanocytoma. The dog was treated long-term with both topical steroid and tacrolimus 0.03% for control of the chronic superficial keratitis.