Oxyspirura petrowi infection leads to pathological consequences in Northern Bobwhite (Colinus virginianus)

Abstract

Debilitating ocular diseases are often reported in avian species. By and large, helminth parasites have been overlooked in avian diseases and regarded as inconsequential. The decline of Northern bobwhite quail (Colinus virginianus) in the Rolling Plains ecoregion of Texas has prompted an investigation of the factors influencing their disappearance. Infection by the eyeworm (Oxyspirura petrowi) has been documented in many avian species; however, the effect it has on its host is not well understood. Heavy eyeworm infection has been documented in Northern bobwhites throughout this ecoregion, leading to eye pathology in this host species. The present study further documents and supports the pathological changes associated with O. petrowi in bobwhites.

Full text

Oxyspirura petrowi infection leads to pathological consequences in
Northern bobwhite (Colinus virginianus)
Nicholas R. Dunham
a
, Scott Reed
b
, Dale Rollins
c
, Ronald J. Kendall
a
,
*
a
The Wildlife Toxicology Laboratory, The Institute of Environmental and Human Health, Texas Tech University, Box 43290, Lubbock, TX, 79409-3290, USA
b
Texas A&M Veterinary Medical Diagnostic Laboratory, 6610 W. Amarillo Blvd, Amarillo, TX, 79106, USA
c
Rolling Plains Quail Research Ranch, 1262 U.S. Highway 180 W., Rotan, Texas, 79546, USA
article info
Article history:
Received 2 September 2016
Received in revised form
22 September 2016
Accepted 27 September 2016
Keywords:
Colinus virginianus
Eyeworm
Northern bobwhite
Oxyspirura petrowi
Parasitism
Pathology
abstract
Debilitating ocular diseases are often reported in avian species. By and large, helminth parasites have
been overlooked in avian diseases and regarded as inconsequential. The decline of Northern bobwhite
quail (Colinus virginianus) in the Rolling Plains ecoregion of Texas has prompted an investigation of the
factors influencing their disappearance. Infection by the eyeworm (Oxyspirura petrowi) has been docu-
mented in many avian species; however, the effect it has on its host is not well understood. Heavy
eyeworm infection has been documented in Northern bobwhites throughout this ecoregion, leading to
eye pathology in this host species. The present study further documents and supports the pathological
changes associated with O. petrowi in bobwhites.
©2016 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Ocular diseases are commonly reported in avian species with
one of the most common being primary or secondary inflammatory
diseases of the eyelids and conjunctiva (Bay
on et al., 2007). Para-
sitic infection of the eye may lead to ocular disease which may be
very debilitating to the host. Visual function in birds is essential for
flying, surviving in the wild, and reproduction (Jezler et al., 2010;
Korbel and Habil, 2011). Even partial impairment of vision caused
by eye disease may have far-reaching consequences because
compensation by other senses is mostly insufficient and/or
impossible (Korbel and Habil, 2011).
Recently, a parasitic eyeworm (Oxyspirura petrowi) has received
increased attention due to the high prevalence detected in native
galliforme populations in the United States (Robel et al., 2003;
Villarreal et al., 2012; Dunham et al., 2016). Specifically, the
decline of the Northern bobwhite (Colinus virginianus)inwest
Texas has prompted an investigation of the factors influencing the
disappearance of these birds. Quail populations are a substantial
part of west Texas hunting culture, and consequently are of eco-
nomic importance for many rural communities throughout the
Rolling Plains (Johnson et al., 2012). With the quail decline
impacting many communities in Texas, researchers began looking
at every possible factor that could be influencing these populations.
Helminth parasites have been overlooked historically (Lehman,
1984), but eyeworm infections are now considered a possible factor
contributing to the decline of Northern bobwhites in Texas
(Dunham et al., 2014, 2016; Bruno et al., 2015). Oxyspiurura petrowi
is a heteroxenous parasitic nematode reported to infect the eyes of
many avian hosts (Dunham and Kendall, 2016) with species of this
genus being documented in >80 avian species worldwide (Addison
and Anderson, 1969). Eyeworms inhabit the eyelids, nictitating
membrane, nasolacrimal duct, lacrimal gland, Harderian gland, and
intraorbital tissues of its host (Cram, 1937; Addison and Anderson,
1969; Robel et al., 2003; Dunham et al., 2014; Bruno et al., 2015).
There has been interest in studying eyeworm infection in
Northern bobwhite (Colinus virginianus) since recent research
discovered that this parasite is endemic to the Rolling Plains ecor-
egion of Texas and Oklahoma (Dunham et al., 2016). Over the past
few years, eyeworm infection has been documented extensively in
bobwhites throughout the ecoregion and led researchers to
examine the potential influence that parasites have on their decline
(Villarreal et al., 2012; Xiang et al., 2013; Dunham et al., 2014). The
impact(s) of eyeworms on the host itself is unclear. A few studies
have shown that these parasites do not cause pathological effects or
*Corresponding author.
E-mail address: ron.kendall@ttu.edu (R.J. Kendall).
Contents lists available at ScienceDirect
International Journal for Parasitology:
Parasites and Wildlife
journal homepage: www.elsevier.com/locate/ijppaw
http://dx.doi.org/10.1016/j.ijppaw.2016.09.004
2213-2244/©2016 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
International Journal for Parasitology: Parasites and Wildlife 5 (2016) 273e276

gross lesions (McClure, 1949; Pence, 1972; Ruff and Norton, 1997);
however, Saunders (1935) observed ocular irritation caused by
eyeworm infections. Recently, O. petrowi infections were reported
to cause severe inflammation and edema (Dunham et al., 2014,
2015). Bruno et al. (2015) first reported lesions associated with
eyeworm infection in Northern bobwhites found in the Rolling
Plains of Texas. Eye inflammation progressing to destruction of the
eye has been noted in a related eyeworm species (Oxyspirura
mansoni) found commonly in poultry (Ruff and Norton, 1997).
The purpose of this study was to (1) examine the host response
to infection with O. petrowi, (2) determine the extent and incidence
of lesions associated with O. petrowi and (3) discuss the possible
contribution that O. petrowi-associated pathology has on the
decline of Northern bobwhites in the Rolling Plains ecoregion of
Texas and Oklahoma.
2. Materials and methods
2.1. Ethics statement
Animal experiments were approved by Texas Tech University
Animal Care and Use Committee under protocol 13066-08. All quail
were trapped and handled according to Texas Parks and Wildlife
permit SRP-1098-984 and SRP-0715-095.
2.2. Study area
The experimental study area of the present manuscript is
consistent with the study area described in Dunham et al. (2014).
2.3. Quail trapping
All Northern bobwhites were collected from the same trapping
location, in the same manner, and using the same techniques pre-
viously described by Dunham et al. (2014).
2.4. Histological techniques
After euthanasia, the head was removed from the body of each
sample. While holding the head in hand, the lower mandible, neck,
and additional tissues were gently removed. Next the skin and
feathers were carefully excised leaving only the skull, upper
mandible, and eyelids. Heads were fixed in 10% neutral buffered
formalin. Each head was fixed for a minimum of 4 days. Skulls were
decalcified with 23% w/w hydrochloric acid for 12e16 h. Decalcified
fixed heads were then sectioned in 3 mm intervals prior to pro-
cessing. All tissues subsequently were processed routinely in an
automated Shandon Pathcentre
®
histology processor (Thermo
Scientific, Waltham, MA), which dehydrated tissues in progres-
sively increased concentrations of ethanol and cleared in xylene
prior to paraffin embedding. Processed tissue was then embedded
in Paraplast™paraffin wax (VWR, Radnor, PA) to create tissue
blocks for microtomy. Tissue was sectioned at 4
m
thickness with a
microtome and mounted on glass slides for staining. Routine
staining with hematoxylin and eosin (VWR Premium Histology
Stains) was performed, slides were cover-slipped, and specimens
were examined microscopically by a board-certified veterinary
pathologist. Imaging was performed with a Nikon Digital Sight DS-
SM camera (Nikon Instruments Inc., Melville, NY) connected to an
Olympus BX-51 bright field microscope (Olympus America Inc.,
Center Valley, PA). The Harderian gland, lacrimal gland, cornea,
eyelids, nictitating membrane, and other eye-associated tissues
were adequately sampled for each eye of each sample.
2.5. Pre-preparation for scanning electron microscopy
Eyeworms were removed using techniques described in
Dunham et al. (2015). To prepare eyeworms for scanning electron
microscopy photographs, each eyeworm was dehydrated. The
dehydration started by placing the designated eyeworms in a
deionized water bath for 15 min. Next, eyeworms were placed into
each ethanol solution (30%, 50%, 80%, 90%, and 100%) for 15 min
followed by a 15 min acetone bath. Once finished, each eyeworm
was individually stored in a 5 ml tube with 100% ethanol and sent to
the Tulane Coordinated Instrumentation Facility (New Orleans, LA),
where scanning electron microscopy was performed. Voucher
specimens of O. petrowi (107282) were deposited in the U.S. Na-
tional Parasite Collection, Beltsville, Maryland.
2.6. Parasite identification
Identification of O. petrowi was based on histological and
morphological characteristics, such as spicule and esophagus
length, as described by Addison and Anderson (1969) and Pence
(1972).
3. Results
A total of 25 of the 28 (89.3%) Northern bobwhites were infected
with O. petrowi. Fourteen of 15 males, 11 of 13 females, 12 of 12
adults, and 13 of 16 juveniles were found to be infected. Eyeworms
were found in the lacrimal gland, Harderian gland, nictitating
membrane, bulbar conjunctive, fornix of the conjunctiva, and
nasolacrimal duct. Histological sections of the Harderian gland
demonstrated lesions, with a varying degree of presumed severity,
associated with the presence of O. petrowi (Figs. 1 and 2). O. petrowi
presence was associated with lymphoplasmacytic Harderian gland
adenitis in all cases. In addition to increasing inflammatory cell
infiltration associated with infection, there was also increased at-
rophy and corresponding duct dilation.
The intraluminal presence of the O. petrowi was associated with
a moderate to marked Harderian gland adenitis and fibrosis. In this
study, several birds had corneal epithelial erosions and edema
Fig. 1. Histological section of a Northern bobwhite (Colinus virginianus) Harderian
gland with intraluminal Oxyspirura petrowi parasites in transverse section (indicated
by arrows) and marked heterophilic Harderian adenitis. Hematoxylin and eosin
staining at 100, scale ¼100
m
m*¼marked lymphocyte and heterophilic inflam-
matory cell infiltrate; C ¼cuticle; HD ¼hypodermis; SM ¼somatic musculature;
LC ¼lateral cords; PC ¼pseudocoelom; A ¼alimentary tract; U ¼uterus containing
embryonated eggs.
N.R. Dunham et al. / International Journal for Parasitology: Parasites and Wildlife 5 (2016) 273e276274

causing corneal edema. Corneas of many infected bobwhites
appeared to be cloudy and have early ulcerative erosions. Eye tis-
sues from the three uninfected quail lacked gland atrophy when
compared to infected birds.
4. Discussion
While many ocular conditions are reported in avian species, the
impact that ocular parasites have on their hosts are not fully un-
derstood. Our results suggest that eyeworms, such as O. petrowi,
negatively impact ocular tissues by causing inflammation, fibrosis,
and adenitis to the host. Past studies with Oxyspirura spp. observed
ocular irritation with no signs of damage in other galliforme spe-
cies, but histological techniques were not implemented (Saunders,
1935; McClure, 1949; Pence, 1972). A closely related eyeworm,
Oxyspirura mansoni, has been associated with damage to the con-
junctiva and lacrimal ducts in poultry (Kobayashi, 1927) and re-
searchers hypothesized that severe infection, coupled with
inflammation, would likely lead to blindness (Sanders, 1929). Bruno
et al. (2015) first documented eye pathology in bobwhites associ-
ated with O. petrowi and infection within the Harderian gland.
Additionally, Dunham et al. (2014, 2015) revealed significant
inflammation and petechial hemorrhaging associated with
O. petrowi infection. This research, along with the present study,
describe a series of detrimental consequences within the Northern
bobwhite host when infected with eyeworms.
Visual acuity is not only necessary for finding/securing food but
also for identifying mates and escaping predators (Jezler et al.,
2010). In the present study, eyeworm infection caused adenitis
(glandular inflammation) in both juvenile and adult Northern
bobwhite. Inflammation of any kind is commonly associated with
pain due to swelling. The inflammation and gland destruction
associated with O. petrowi infection, if allowed to progress, would
lead to gland destruction and functional compromise. Swelling in/
around the eye causes impingement on stretch receptors which
releases cytokines followed by the release of stress hormones. Add
the irritation caused by oscillating and migrating filarids, these
quail would be suspected to be significantly compromised in terms
of foraging and escaping predation.
All avian species possess intraorbital gland which consist of the
lacrimal and Harderian gland (Dimitrov and Genchev, 2011). The
eye requires secretions from the lacrimal and Harderian gland for
moistening, nutrition, and controlling orbital and ocular defense
(Knop and Knop, 2005; Kozlu and Altunay, 2011). In time, the
adenitis would likely result in gland atrophy and fibrosis, a condi-
tion that was observed in several of the samples in this study. These
conditions cause a deficiency in tear production called kerato-
conjuctivitis sicca (KCS), which is commonly known as “dry eye”.
This KCS condition is likely to lead to corneal ulcerations and a
reduction in vision. In addition the inflammatory condition itself
would be expected to cause significant morbidity resulting from
pressure, swelling, and likely lead to pain associated with inflamed
glands. Inflammation was witnessed in all quail samples that were
infected.
Several cornea samples appeared cloudy and had signs of early
erosions. Corneal damage is often very painful and debilitating
when compromised in any manner. The cornea is the most
important structure of the ocular surface for the maintenance of
visual function (Knop and Knop, 2005) hence, corneal damage may
be painful and debilitating. With the Northern bobwhites having an
average life span of approximately six months (Hern
andez and
Peterson, 2007) and with the negative impacts of eyeworm infec-
tion, it is likely that bobwhites that survive longer harboring these
parasites have more inflammation and eye pathology. Additional
studies are warranted to determine if pain is associated with
inflammation and swelling in bobwhites infected with eyeworms.
Dunham et al. (2015) revealed that eyeworms have a unique
mouth structure that we speculate likely enables them to attach
Fig. 2. Histological section of Northern bobwhite (Colinus virginianus) Harderian glands with a varying degree of pathological response associated with Oxyspirura petrowi infection.
Scale bar ¼200
m
m.
N.R. Dunham et al. / International Journal for Parasitology: Parasites and Wildlife 5 (2016) 273e276 275

and potentially feed. The potential implications of attaching eye-
worms, along with a high prevalence of infection found within
individual quail, would be expected to cause localized tissue
trauma and inflammation. To strengthen this hypothesis, Fig. 3
shows the first ever image of an O. petrowi mouth structure. If
eyeworms do attach to tissues it is likely detrimental to the host;
however, determining damage caused by mouth parts was out of
the scope of the present study and additional research is needed.
The present study supports the recent data suggesting that
O. petrowi infection may impact the Northern bobwhite host.
Although cause and effect are not proved, we saw a varying degree
of lesion severity within the Harderian gland associated with
O. petrowi infection suggesting a causal relationship, which wasn't
previously documented in related studies. While the results of this
study cannot determine if O. petrowi infection leads to visual
impairment, it is likely that the pathological damage associated
with infection negatively impacts the eye function and reduces
survival. Additional research is needed to determine if O. petrowi
infections decrease vision and play a role in the fitness, hence
survival, of Northern bobwhites.
Acknowledgments
We thank Park Cities Quail and the Rolling Plains Quail Research
Foundation for their continued financial support of our quail
research. We thank the Texas A&M Veterinary Medical Diagnostic
Laboratory (Amarillo, TX) and Comparative Ocular Pathology Lab-
oratory of Wisconsin (Madison, WI) for performing the pathology
evaluation on our quail samples. We thank the owners and em-
ployees of our study ranch for allowing access and providing lod-
ging. Lastly we thank members of the Wildlife Toxicology
Laboratory for their field and laboratory assistance.
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