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\begin{document}

\title{Autochthonous, zoonotic \emph{Onchocerca lupi} in a South Texas dog,
United States}



\author[1]{Guilherme Verocai}%
\author[1]{Caroline Sobotyk}%
\author[2]{Allegra Lamison}%
\author[3]{Mindy Borst}%
\author[3]{Erin Edwards}%
\affil[1]{Texas A\&M University College of Veterinary Medicine and Biomedical Sciences}%
\affil[2]{North 10th Street Animal Hospital}%
\affil[3]{Texas A\&M Veterinary Medical Diagnostic Laboratory}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
\emph{Onchocerca lupi} is an emerging, zoonotic filarioid nematode
associated with ocular disease in companion animals. We report the first
autochthonous case of \emph{O. lupi} in a dog from Hidalgo County,
southern Texas, USA. An 11-year-old, castrated male, Pitbull dog from
McAllen, Hidalgo County, southern Texas with no travel history, was
diagnosed with a perforating corneal ulceration of the right eye.
Enucleation was performed, and tissues submitted for histopathology.
Sections of two filarioid nematodes were observed. Parasite
identification relied on morphological features seen on histopathology
and molecular confirmation by PCR of the cox1 gene of the mitochondrial
DNA, followed by sequencing and phylogenetic analysis. Our finding
suggests Texas as an additional endemic state for this zoonotic
nematode. Further investigations are required to understand the
epidemiology of this parasite along the United States/Mexico border.%
\end{abstract}%



\sloppy


\textbf{Title:} Autochthonous, zoonotic \emph{Onchocerca lupi} in a
South Texas dog, United States

\textbf{Running Title:}Autochthonous \emph{Onchocerca lupi} , Texas, USA

\textbf{Authors:} Guilherme G. Verocai \textsuperscript{1}, Caroline
Sobotyk\textsuperscript{1}, Allegra Lamison \textsuperscript{2}, Mindy
M. Borst \textsuperscript{3}, and Erin E. Edwards\textsuperscript{3}

\textbf{Affiliations:}

1. Department of Veterinary Pathobiology, College of Veterinary Medicine
and Biological Sciences, Texas A\&M University, College Station, Texas,
United States (G. Verocai, C. Sobotyk) 77843 USA.

2. North 10\textsuperscript{th} Street Animal Hospital, McAllen, Texas,
United States (A. Lamison)

3. Texas A\&M Veterinary Medical Diagnostic Laboratory, College Station,
TX, USA (M. Borst, E. Edwards)

\par\null

\textbf{Corresponding author:}

Department of Veterinary Pathobiology, College of Veterinary Medicine
and Biological Sciences, Texas A\&M University, 668 Raymond Stotzer
Pkwy, VIDI Building 1813, 4467 TAMU, College Station, TX 77843, USA.
Phone: +1 979-862-4999; gverocai@cvm.tamu.edu

\textbf{Summary}

\emph{Onchocerca lupi} is an emerging, zoonotic filarioid nematode
associated with ocular disease in companion animals. We report the first
autochthonous case of \emph{O. lupi} in a dog from Hidalgo County,
southern Texas, USA. An 11-year-old, castrated male, Pitbull dog from
McAllen, Hidalgo County, southern Texas with no travel history, was
diagnosed with a perforating corneal ulceration of the right eye.
Enucleation was performed, and tissues submitted for histopathology.
Sections of two filarioid nematodes were observed. Parasite
identification relied on morphological features seen on histopathology
and molecular confirmation by PCR of the \emph{cox1} gene of the
mitochondrial DNA, followed by sequencing and phylogenetic analysis. Our
finding suggests Texas as an additional endemic state for this zoonotic
nematode. Further investigations are required to understand the
epidemiology of this parasite along the United States/Mexico border.

\textbf{Key words:} Filarioidea, Ocular onchocercosis, Vector-borne
diseases, Zoonotic onchocerciasis

\textbf{1. Introduction}

\emph{Onchocerca lupi} (Nematoda: Onchocercidae) is an emerging,
zoonotic parasite in North America and areas of the Old World (Cantey et
al., 2016; Eberhard et al., 2013; Gr\selectlanguage{ngerman}ácio, Richter, Komnenou, \& Grácio,
2015; Otranto et al., 2015). Infection in dogs and cats are usually
associated with episcleral and periocular tissues causing minor to
severe ocular lesions, including conjunctivitis, third eyelid prolapse,
exophthalmos, uveitis, and retinal detachment (McLean, Newkirk, \&
Adema, 2017; Otranto et al., 2015). However, the presentation of
zoonotic onchocerciasis by \emph{O. lupi} in humans may be variable
according to the site of infection.

To date, there have been seven confirmed zoonotic onchocerciasis cases
by \emph{O. lupi} in the United States. Six of these cases involved
children (Cantey et al., 2016; Eberhard et al., 2013). An additional
medical concern is that three of these cases were associated with
nodules at the cervical spinal cord; two of these cases had gravid adult
female specimens, suggestive that these were patent infections. These
North American human \emph{O. lupi} infections have been reported from
Arizona, New Mexico, and Texas. While, Arizona and New Mexico are
considered endemic for this parasite, with multiple cases reported from
companion animals, humans and wildlife (Cantey et al., 2016; McLean et
al., 2017; Roe et al., 2020); the origin of the single human case in
southern Texas remains uncertain. Thus far, all canine cases in North
America reported outside of the presumed endemic southwestern United
States (e.g., Minnesota, Florida, New York) have been associated with
travel (Edelmann, Jager, Espinheira, \& Ledbetter, 2018; Otranto et al.,
2015; Verocai et al., 2016). Similarly, some European cases in dogs and
humans have been linked to translocation or travel to endemic regions of
the Mediterranean (Colella et al., 2018; Hod\selectlanguage{polish}ž\selectlanguage{english}i\selectlanguage{polish}ć \selectlanguage{english}et al., 2018; Sandell,
Skogen, Lier, \& Pettersen, 2020).

\textbf{2. Materials and Methods}

In August 2020, an 11-year-old, castrated male, Pitbull dog from
McAllen, Hidalgo County, southern Texas, was presented to the
veterinarian with a history of ocular irritation for approximately 2-3
weeks. It was diagnosed with a perforating corneal ulceration of the
right eye. The dog was born in McAllen and had never left the Rio Grande
River Valley, Texas. The animal tested antigen-positive for
heartworm,\emph{Dirofilaria immitis} , during pre-anesthetic work-up,
despite somewhat compliant chemoprophylaxis with an ivermectin-based
monthly product. An enucleation was performed, and the eye was fixed in
10\% neutral buffered formalin and submitted to the Texas A\&M
Veterinary Medical Diagnostic Laboratory for histologic processing and
examination.

Genomic DNA was extracted using a Qiagen FFPE Tissue DNA extraction kit
(Qiagen, USA) according to manufacturer's instructions. Polymerase chain
reaction (PCR) was performed in 25 \selectlanguage{greek}µ\selectlanguage{english}L reactions containing 0.25\selectlanguage{greek}μ\selectlanguage{english}M of
each primer, 1x GoTaq\selectlanguage{ngerman}\textsuperscript{®} Green Master Mix (Promega
Corporation, Madison, Wisconsin, United States) and 2.5 \selectlanguage{greek}µ\selectlanguage{english}L of DNA
template. The cytochrome oxidase c subunit 1 (\emph{cox1} ) gene was
amplified using the forward primer COIintF: 5'-TGA TTG GTG GTT TTG GTA
A-3' and reverse primer COIintR: 5'-ATA AGT ACG AGT ATC AAT ATC- 3'
(Casiraghi, Anderson, Bandi, Bazzocchi, \& Genchi, 2001; Otranto et al.,
2015; Otranto et al., 2011),. The cycling conditions included an initial
denaturation step at 95 for 2 minutes, followed by 40 cycles at 95\selectlanguage{ngerman}°C
for 45 seconds, 52\selectlanguage{english} at 45 seconds, and 72\selectlanguage{english} for 90 seconds, and a final
extension step at 72degC for 5 minutes. PCR products were purified using
E.Z.N.A.\textsuperscript{(r)} Cycle Pure Kit (Omega Bio-tek, Norcross, GA,
USA) according to the manufacturer's instructions, followed by Sanger
sequencing. Phylogenetic analysis was performed in MEGA X using the
Maximum Likelihood method and General Time Reversible, Gamma distributed
as the best fit model (Kumar, Stecher, Li, Knyaz, \& Tamura, 2018).

\textbf{3. Results and Discussion}

Our report represents the first unequivocally autochthonous case
of\emph{O. lupi} , an agent of zoonotic onchocerciasis, in Texas, near
the United States/Mexico border based on integrated histopathological,
parasitological and molecular data.

Histologically, the most significant finding was the presence of corneal
perforation with severe keratitis and anterior iris synechiae.
Additionally, two long, filarioid nematodes were discovered embedded in
the episcleral tissues. One of these parasites was degenerated and
surrounded by granulomatous inflammation, while the other was intact and
lacked surrounding inflammation. This specimen was a gravid, female
nematode, suggesting a patent infection. The pattern of cuticular ridges
of two inner striae within the space between two outer cuticular ridges
observed on the specimens were morphologically consistent with \emph{O.
lupi} (Verocai et al., 2016) (Figure 1). Most likely, the dog was
co-infected with heartworm, \emph{D. immitis} , as a recent study, has
shown that \emph{O. lupi} infections are unlikely to generate
false-positive results in commercial heartworm antigen tests (Sobotyk
Oliveira, Savadelis, McLean, \& Verocai, 2020).

The generated \emph{cox1} sequence was accessioned in GenBank
(MW577256), and showed 99.9 -- 100\% maximum identity with \emph{O.
lupi} sequences available in GenBank. Phylogenetic analysis clustered
the Texas isolate with all previous isolates from North America, and
some European isolates, belonging to \emph{O. lupi} ``genotype 1'' (99\%
bootstrap support; Figure 2) (Rojas, Salant, Yasur-Landau, Tsarfati, \&
Baneth, 2020).

Zoonotic onchocerciasis has been previously reported from a 10-year-old
boy from Mission, Texas also in the Hidalgo County, who traveled to
South Dakota, and slept in tents and cabins in New Mexico and Colorado,
reported fishing in fresh water lakes near home, and had a pet dog with
history of conjunctivitis and eye lesion of unknown etiology (Cantey et
al., 2016). Altogether, the present canine case may suggest that this
human infection may have been, in fact, acquired in Texas.

It is necessary to better understand the epidemiology of \emph{O.
lupi}in this newly recognized endemic area by screening dogs and cats
from shelters using classical and molecular methods. In addition to
companion animals and humans, coyotes (\emph{Canis latrans} ) were
reported infected and may serve as wild reservoirs in the western United
States (Roe et al., 2020), and may also contribute to the epidemiology
of\emph{O. lupi} in southern Texas, and neighboring Mexico. Other wild
carnivores that are known or have postulated as \emph{O. lupi} hosts
such as wolves (\emph{Canis lupus} ) and certain foxes (red
fox,\emph{Vulpes vulpes} ; swift fox, \emph{Vulpes velox} ; kit
fox,\emph{Vulpes macrotis} ) are not present in southern Texas. However,
the widely distributed gray fox (\emph{Urocyon cinereoargenteus} ), is
reported in this region, and across \emph{O. lupi} endemic areas in
North America, and therefore, should be assessed as potential reservoir
host. Regarding the dipteran vectors, black flies (Simuliidae) have been
considered the putative intermediate host for \emph{O. lupi} (Hassan et
al., 2015); however, biological confirmation remains necessary.
Therefore, xenomonitoring in Texas could also involve screening of other
dipterans, such as biting midges (Ceratopogonidae: \emph{Culicoides} )
and sand flies (Psychodidae: Phlebotominae).

This unequivocal autochthonous \emph{O. lupi} case in southern Texas
suggests that this parasite is endemic to the region, and transmission
may occur locally. In this case, it is postulated that \emph{O.
lupi}could have caused exophthalmos with secondary traumatic corneal
ulceration and subsequent perforation. There is a need for
epidemiological surveillance of companion animals, wildlife, and
dipteran vectors along the United States/Mexico border. It is important
to raise awareness of public health and medical specialists and
authorities as humans in this region might be at risk of infection.

\textbf{Acknowledgements}

Dr. Sobotyk is the NCVP Merck Parasitology Resident at Texas A\&M
University.

\textbf{Conflict of interest statement}

The authors declare no conflict of interest.

\textbf{References}

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(2020). First report of \emph{Onchocerca lupi} from Israel and
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\textbf{Figure legends}

\textbf{Figure 1:} Histologic section with a gravid,
female\emph{Onchocerca lupi} specimen embedded in the episclera of a dog
from South Texas. The cuticular pattern of two inner striae (dashed
arrows) within the space between two outer cuticular ridges (solid
arrows) aided in the species---level identification. (H\&E, 40X, Bar =
50\selectlanguage{greek}µ\selectlanguage{english}m).

\textbf{Figure 2:} Phylogenetic relationship of based on \emph{cox1}gene
of \emph{Onchocerca lupi} nematode isolate from a dog in South Texas,
United States (GenBank accession no. MW577256), and
other\emph{Onchocerca} spp. isolates. Analysis was performed using the
Maximum Likelihood method (1,000 bootstrap replicates). Nodes with less
than 90\% support were condensed.\selectlanguage{english}
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