Figure 1

Figure 1. Subconjunctival nodule on the medial canthus of the right eye of dog 2 (Table), Minnesota, USA. This dog was found to be infected with Onchocerca lupi nematodes.

During April 2011–August 2014, a total of 8 privately owned dogs of various ages and sexes were referred to clinical practices in Minnesota (n = 1), New Mexico (n = 4), Colorado (n = 2), and Florida (n = 1) because of different degrees of ocular alterations (Table). At physical examination, nodules were detected in different areas of the eye (Figure 1) and associated with inflammatory reactions ranging from mild scleritis to episcleral swelling and vascular congestion (Table).

All nodules were surgically removed from bulbar conjunctiva or sclera, and white filaria-like parasites were collected and stored in 70% ethanol for morphologic identification. In addition, specimens extracted from 2 dogs (dogs 2 and 3) (Table) were characterized by using molecular techniques. All dogs were treated with macrofilaricides, microfilaricides, antimicrobial drugs, and corticosteroids, which lead to complete resolution of ocular conditions in all except 3 animals (dogs 1, 7, and 8). These 3 dogs had relapses 2, 6, and 12 months, respectively, after surgery.

Nematodes had external, round, transverse ridges and 2 transverse striae per each outer ridge interval, which suggested that they were filarial worms of the genus Onchocerca. The ratio between body diameter and distances between ridges (7–10:1) was specific for O. lupi nematodes (15). A small piece of nematode was used for molecular identification. Genomic DNA was extracted and partial cox1 genes were amplified and sequenced as described (13).

In accordance with clinical signs of nodular ocular lesions and morphologic identification, partial cox1 gene sequence analysis (GenBank accession nos. KP283476 and KP283477) confirmed the identity of the nematode as O. lupi, showing 98% nt homology with other sequences of O. lupi nematodes in GenBank (KC686701 from Portugal and KC686702 from Greece) and 100% with those derived from dogs and cats from the United States, as well as with a sequence from Greece (EF521409).

Figure 2

Figure 2. Phylogeny of Onchocerca lupi and other filarial nematode species based on partial sequences of the cytochrome c oxidase subunit 1 gene. Thelazia callipaeda was used as an outgroup. Bootstrap confidence values...

Phylogenetic analysis of partial cox1 gene sequences was performed by using the neighbor-joining method and the Kimura 2-parameter model in MEGA5 (http://www.megasoftware.net/). This analysis confirmed that sequences from nematodes examined clustered with O. lupi sequences from different areas of the United States (Nevada, California, Colorado, Utah) and with a sequence from Greece (Figure 2). In addition, these sequences were grouped with others from Greece, Hungary and Portugal and formed a paraphyletic clade with other Onchocerca species available in GenBank.

Our results indicate that a unique haplotype of O. lupi nematodes is circulating in the United States and is endemic to the canine population in this country. Although this onchocercid has been implicated as the causative agent of canine onchocercosis in the United States only recently (11), previous cases attributed to Onchocerca spp. have been described in dogs from Arizona, California, and Utah (5,8–10). The cases herein reported from Florida, New Mexico, and Minnesota suggest that the distribution of this nematode is probably wider than previously believed. Detection of O. lupi nematodes in Englewood, Colorado, confirms a previous report of infection in a dog from Mancos (11).

We identified 1 cox1 haplotype and found that it was identical to all sequences in GenBank from the United States and 1 from Greece. Conversely, up to 5 haplotypes were detected in Greece, Turkey, Iran, and Hungary (7). Genetic variation detected in O. lupi nematodes from Europe, Turkey, and Iran, along with isolation of this parasite from the Caucasian wolf, suggests that the infection probably originated in the Old World and was imported into the United States.

The low genetic distance detected for the cox1 gene is evidence of a substantially reduced evolutionary rate, which supports relatively recent divergence among specimens found in the Old World and New World. In addition to recent detection of O. lupi nematode infections in the United States, circulation of 1 haplotype could also suggest that a unique vector species occurs in areas of the Old World and New World where the infection has been diagnosed.

Given that all reports above are based on clinical signs, the epidemiology of O. lupi nematodes in the United States deserves to be thoroughly investigated. In particular, dogs relocated from disease-endemic areas to new areas should be routinely screened for skin-dwelling microfilariae because these parasites might represent a risk for other animals. In addition, because O. lupi nematodes circulate among canine populations, the potential role of dogs as reservoirs for human infection should not be underestimated, as also inferred by zoonotic cases reported in the United States (12). Finally, further studies are urgently warranted toward improving the diagnosis of O. lupi nematode infections, which will lead to a better appreciation of its distribution and potential risk for human populations.