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Live Eyeworm (Oxyspirura petrowi) Extraction, In Vitro Culture, and Transfer for
Experimental Studies
Author(s): N. R. Dunham, L. A. Soliz, A. Brightman, D. Rollins, A. M. Fedynich, and R. J. Kendall
Source: Journal of Parasitology, 101(1):98-101.
Published By: American Society of Parasitologists
DOI: http://dx.doi.org/10.1645/13-448.1
URL: http://www.bioone.org/doi/full/10.1645/13-448.1
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J. Parasitol., 101(1), 2015, pp. 98–101
ÓAmerican Society of Parasitologists 2015
Live Eyeworm (Oxyspirura petrowi ) Extraction, In Vitro Culture, and Transfer for
Experimental Studies
N. R. Dunham*, L. A. Soliz*†, A. Brightman‡, D. Rollins§, A. M. Fedynich†, and R. J. Kendall*,*The Institute of Environmental and Human Health,
Texas Tech University, Box 41163, Lubbock, Texas 79409-1163; †Caesar Kleberg Wildlife Research Institute, Texas A&M University–Kingsville, 700 University
Boulevard, Kingsville, Texas 78363; ‡College of Veterinary Medicine, Kansas State University, 101 Trotter Hall, Manhattan, Kansas 66506; §Texas AgriLife
Research, Texas A&M University System, San Angelo, Texas 76901. Correspondence should be sent to: ron.kendall@ttu.edu
ABSTRACT: Northern bobwhite (Colinus virginianus) have experienced a
dramatic decline in West Texas over the last 3 yr, and investigations are
underway to evaluate the role of parasites in this decline. One of the key
parasites being investigated is the eyeworm (Oxyspirura petrowi). Live
eyeworms were extracted from both live and dead northern bobwhites,
and in vitro survival was tested using 10 liquid media. Eyeworms placed in
an egg white and physiological saline solution lived for at least 36 days.
Live O. petrowi placed into the eyes of uninfected pen-raised bobwhites
were monitored for 21 days to demonstrate successful transfer. Eyeworm
behavior during feeding, mating, and development were monitored. This
study is important to research that requires ‘‘banking’’ of live O. petrowi
from wild-captured definitive hosts for life history studies and assessing
the impact of O. petrowi on host individuals.
The long-term decline in northern bobwhite (Colinus virginianus)
populations throughout North America has prompted interest in re-
examining population regulating factors that have been previously
considered inconsequential. One of the factors that is often overlooked or
not thoroughly examined is the role that parasitic helminths may play in
negatively impacting host populations. The eyeworm (Oxyspirura petrowi)
has recently received attention from researchers concerned about the decline
of the bobwhite (Villarreal et al., 2012; Xiang et al., 2013). With concerns
rising, this study provides important behavioral characteristics and life cycle
information of the eyeworm that were previously unknown.
Although there is a substantial volume of literature on the eyeworm
Oxyspirura mansoni from the early 20th century (Ransom, 1904; Fielding,
1926; Sanders, 1928; Schwabe, 1951), likely due to its negative impact on
the poultry industry, little is known about the life history or potential
negative impacts of O. petrowi in wild birds. Ruff (1984) suggested that the
life cycle of O. petrowi is likely similar to that of O. mansoni. However, it
has yet to be described as to which arthropod species serve as intermediate
hosts, how infective larvae are maintained in intermediate hosts, how
larvae exit the intermediate host when ingested by the definitive host, and
where larval and adult stages occur in the definitive host for O. petrowi.
Studies based on visual observation have found little evidence of
pathological responses caused by O. petrowi (McClure, 1949; Pence, 1972),
whereas ophthalmia or eye inflammation progressing to destruction of the
eyeball has been noted for O. mansoni in poultry (Ruff and Norton, 1997).
Presently, no in-depth histological examinations have been performed. In
addition, there have been no studies examining the potential host
behavioral or physiological impacts caused by O. petrowi in bobwhites.
However, Robel et al. (2003) reported lesser prairie-chickens (Tympanu-
chus pallidicinctus) that had elevated numbers of O. petrowi tended to be
slightly underweight. Consequently, there is a need to better understand
the life history of O. petrowi and to determine its potential impact on its
definitive host through controlled experimental studies. The objectives of
this study were to determine whether live O. petrowi can be successfully
removed from northern bobwhites (definitive host), maintained in vitro,
and transplanted into uninfected bobwhites.
Northern bobwhites were live-trapped from June 2013 to September
2013 on a 120,000-ha cattle ranch (32810.280N, 10180.910W) in Mitchell
County, Texas, and transported to The Institute of Environmental and
Human Health Aviary at Texas Tech University. Each bobwhite was
individually housed in 25 361-cm quail breeding battery (G.Q.F
Manufacturing Co., Savannah, Georgia). Bobwhites were trapped and
handled under Texas Parks and Wildlife permit SRP-1098-984, Texas
A&M University AUP 2011-193, Texas Tech University ACUC 11049-07,
and Texas Tech University ACUC 13027-03. Pen-raised adult northern
bobwhites purchased from The Quail Ranch of Oklahoma (Wardville,
Oklahoma), a USDA and National Poultry Improvement Plan–approved
breeder, were used in the live eyeworm transfer experiment. Thirty days
before O. petrowi transfers, pen-raised bobwhites were gently restrained
manually and medicated with the topical parasiticide VetRx (Goodwinol
Products Corp., Pierce, Colorado), using the manufacturer’s dosing
recommendations, to ensure each bird was infection free. Voucher
specimens of O. petrowi (107283) were deposited in the U.S. National
Parasite Collection, Beltsville, Maryland.
The extraction of live O. petrowi from wild-captured bobwhites was
conducted by manually restraining and giving a topical anesthetic (0.5%
Proparacaine HCl Ophthalmic solution; Akorn Inc., Lake Forest, Illinois)
to reduce potential discomfort or irritation. A paintbrush hair was laid
over the eye, and reactivity was monitored to ensure that the bobwhite’s
eyes were fully anesthetized. Once the eye was fully anesthetized, forceps
(Sontec 14–4340; Sontec Instruments, Centennial, Colorado) were lightly
lubricated with GenTeal gel (clear soothing eye drops; Novartis
Ophthalmics, St. Louis, Missouri) to minimize any chance of corneal
ulcerations and then used to examine the eyes. Eyelids, upper and lower,
were gently lifted to examine for the presence of eyeworms. Next, the
nictitating membrane was gently pulled over the eye, and slowly
manipulated up and down, to find and/or initiate movement of any
eyeworms that were not previously found. Since eyeworms evade forceps,
the use of a magnifying ocular headset (DA-5 OptiVisor headband
magnifier, 32.5 magnification, 20-cm focal length; Donegan Optical,
Lenexa, Kansas) and a light source helped aid in eyeworm collection. Eyes
were flushed with balanced salt solution (Alcon Laboratories, Fort Worth,
Texas) using a 22-gauge irrigation cannula if eyeworms could not be
extracted using forceps. Eyeworms were placed in physiological saline
solution at 37 C (Schwabe, 1951) until they were divided into their
respective media. Bobwhite eyes were examined for 3 consecutive days
after eyeworm removal by applying 2 drops of fluorescein eye stain (Ful-
Glo, NDC-17478-404-01; Akorn Inc.) to each eye to determine whether
the procedure produced lacerations or lesions to the eyelid, eye surface,
and nictitating membrane. The extracted eyeworms were used for both the
transfer of eyeworms to 9 uninfected pen-raised northern bobwhites and
to study their survivability to various media.
The transfer of 54 eyeworms to 9 pen-raised northern bobwhites happened
within 30 min after removing the eyeworms from wild-captured bobwhites.
Uninfected bobwhites were implanted with live eyeworms and monitored for
21 days to determine whether eyeworms could be transferred and to gather
information about the life history of the eyeworm. Restraint and anesthesia
procedures previously described were used just before eyeworm transfer.
Once the eye was anesthetized, the upper eyelid was lifted to expose the eye,
DOI: 10.1645/13-448.1
98
and eyes were infected with eyeworms. The infection level for each of 3
bobwhites per treatment group was 1 O. petrowi per eye.Infection levels were
as follows for each of 2 bobwhites per treatment group: 3 O. petrowiper eye, 6
O. petrowi per eye, and 6 O. petrowi placed in only 1 eye. Juvenile O. petrowi
were identified and placed in the bobwhites that had only 1 worm in each eye.
The remaining birds had both juvenile and adult O. petrowi.Bobwhiteswere
monitoredtwice a day (morning and night) for signs of eye irritation, redness,
inflammation, and excessive scratching. In addition, 2 drops of fluorescein
eye stain were applied to each of bobwhite’s eyes to determine whether the
transfer procedure produced any lacerations or lesions to the eyelid, eye
surface, and nictitatingmembrane. Tissues where O. petrowi were found were
macroscopically examined for damage caused by infection.
During the extraction process, O. petrowi were observed evading
forceps. Evading was also seen during the extraction process after
bobwhites were euthanized. Eyeworms were observed evading and moving
from eye to eye via the nasal cavity and its associated ducts. With the
bobwhite beak removed and nasal cavity fully exposed during removal,
eyeworms were seen moving from the eye that was being examined to the
other eye. When we switched to examine the other eye, eyeworms were
seen moving back to the other eye via the nasal cavity. This behavior was
observed until all of the eyeworms were extracted.
Excised O. petrowi that were placed in room temperature physiological
saline were noticeably sluggish and slow moving. However, when
transferred to a solution closer to live bird temperature (37 C), worms
began to become more active and oscillate back and forth. This heightened
level of activity was also observed when O. petrowi were exposed to a light
source. When O. petrowi were initially placed into the 10% saline solution
held at 37 C, males and females were observed migrating across the petri
dish toward each other, attaching for several minutes, and exhibiting
mating behavior (Fig. 1A). Mating behavior was observed multiple times
on different occasions within the same petri dish.
Gravid female egg development was documented throughout the
experiment. Eggs were densely packed within the body. The anterior portion
of gravid females had primarily undeveloped eggs, but posteriorly the eggs
appeared to be more developed and were excreted out the vaginal pore.
When a blood meal was introduced into the saline holding solution,
several O. petrowi quickly attaching themselves to the blood clot and/or
piece of bobwhite tissue, whereas the rest of their body actively oscillated
back and forth. They remained attached for several hours despite being
prodded with forceps. Also, microscopic observation of a few recently
collected O. petrowi from necropsied bobwhites revealed a red-brown
coloration within the esophagus, suggesting blood ingestion (Fig. 1B).
Results of the live O. petrowi transfers to pen-raised bobwhites are
presented in Table I. When the transferred eyeworms were placed into the
treatment bobwhite’s eyes, they quickly self-orientated and moved under
FIGURE 1. (A) Adult male and female Oxyspirura petrowi that migrated toward each other in the physiological saline solution and began mating for
several minutes. (B) Adult male O. petrowi that recently ingested a blood meal from a northern bobwhite (Colinus virginianus). Dark portion in the
esophagus is the ingested blood. (C) Juvenile and adult O. petrowi that were removed from northern bobwhites captured in Mitchell County, Texas, from
June 2013 to September 2013. (D) Head structure of an adult female O. petrowi.
TABLE I. Results of 21-day experimental transfer of eyeworms
(Oxyspirura petrowi) from wild-captive adult northern bobwhites
(Colinus virginianus) captured in Mitchell County, Texas, to uninfected
pen-raised adult northern bobwhites during June 2013.
Infection type
No. of
bobwhites
per treatment
No. of
eyeworms
transferred
No. of
eyeworms
recovered
1 eyeworm per eye 3 6 2
3 eyeworms per eye 2 12 2
6 eyeworms per eye 2 24 7
6 eyeworms in 1 eye 2 12 3
* Note: Eyeworms were recovered in both eyes, despite being transferred
into only one eye.
SHORT COMMUNICATIONS 99
the eyelid or nictitating membrane out of sight. Of the 54 eyeworms placed
into uninfected pen-raised bobwhites during the experimental transfer,
26% of them were recovered 21 days later. By the end of the 21-day
exposure period, juvenile O. petrowi were noticeably larger and appeared
to be adults. Collection of O. petrowi showed distinct differences in size as
well as coloration throughout the duration of the field trapping activities
(Fig. 1C).
One hundred and sixty-eight live O. petrowi were cultured in vitro and
tested to determine their ability to survive outside of the host for long
periods. The first 7 types of liquid culture media were placed in standard
petri dishes (VWR 25384-062) with covers and maintained on a 40 C hot
plate: 10% saline solution, 10% saline solution with bobwhite blood/
tissue, Equate artificial tears (Equate Brands), equate artificial tears with
bobwhite blood/tissue, BioTrue artificial tears (Bausch & Lomb, Ro-
chester, New York), BioTrue artificial tears with bobwhite blood/tissue,
and 10% sucrose solution. An additional 3 liquid culture media were
tested to determine their ability to maintain live O. petrowi: physiological
saline recipe (Corba et al., 1969), phosphate buffered saline, and egg white
with physiological saline solution. Each was placed in a multiwelle6-well
culture plate (Falcon 35–3224) and maintained at 32 C with a 5% CO
2
intake in an Isotemp CO
2
incubator (model 3550; Fisher Scientific,
Pittsburgh, Pennsylvania). Four live O. petrowi were placed in each of the
media culture plates or media plates and monitored twice daily until all
worms were dead. Each worm was examined every 12 hr for the signs of
movement using a magnifying ocular headset and a light until all
eyeworms were dead. Several weeks after death, a gravid female
decomposed sufficiently for the eggs to be released, and the eggs dispersed
throughout the well. The well plate was then placed back inside the
incubator because O. petrowi in the 5 remaining wells were still alive. The
physiological saline recipe and egg white mixture was later repeated using
8 live O. petrowi per 6-well culture plate.
The survival experiment results using the 10 liquid media are presented
in Table II. Oxyspirura petrowi placed in egg white and physiological
saline recipe were sustained and kept alive for approximately 17 days. The
same media were then replicated again, and O. petrowi were sustained for
36 days, with 23 of 24 surviving to 15 days, compared with those in other
solutions that died within 48 hr (Table II). All other O. petrowi survived
no more than 5 days in their respective media (Table II).
There was no macroscopic evidence of eye surface or nictitating
membrane damage associated with O. petrowi infections. However, upon
examination of the lacrimal duct, visible inflammation and petechial
hemorrhaging were observed. In addition, upon necropsy, hemorrhaging
was noted in the sinus mucosal tissues when O. petrowi were present.
Examination of the head of O. petrowi suggests that this nematode has the
necessary structures for tissue attachment and lysis of sinus tissues to feed
on blood (Fig. 1D). Oxyspirura petrowi were also observed with bobwhite
tissue still attached upon being excised from the quail.
Most studies on O. petrowi have found 40–60% prevalence and 6–15
worms per infected host (Jackson, 1969; Robel et al., 2005; Villarreal et al.,
2012), which is problematic for researchers wishing to conduct pen studies
requiring sufficient numbers of worms to examine intensity-dependent
influences on infected hosts. Sustained maintenance of O. petrowi in egg
white and physiological saline media allows researchers to ‘‘bank’’ live
worms extracted from infected, wild-captured hosts for 5–15 days without
substantial worm mortality. It is likely that increased O. petrowi mortality
after 15 days in the egg white and physiological saline media resulted from
depletion of the nutrients within the culture plate and needs further study
to see whether O. petrowi can be successfully maintained in vitro for longer
periods.
We also demonstrated that O. petrowi can successfully be removed from
infected bobwhites without harming either live hosts or eyeworms. In
addition, both juvenile and adult O. petrowi individuals were successfully
transferred to uninfected bobwhites. These extraction and transplantation
procedures allow for the use of live O. petrowi in laboratory studies
examining life history and facilitates pen studies of captive quail
examining potential negative impacts of O. petrowi on their hosts.
Additional insight regarding O. petrowi life history was documented
throughout the course of this study. Oxyspirura petrowi was observed
feeding on host blood and tissues associated with the sinuses, suggesting
that high protein food resources are used instead of host tears. Searching
of the nasolacrimal duct for O. petrowi has been previously reported for
wild birds (Robel et al., 2003, 2005), but references to pathological
responses to infection are lacking. The present study represents the first
report of inflammation and petechial hemorrhaging of the nasolacrimal
duct in association with O. petrowi infections.
Based on our findings of O. petrowi in the nasolacrimal duct, nasal
sinuses, and eye surface under the nictitating membrane, infection could
result in multiple impacts to infected hosts, including impaired respiratory
capacity, visual obstruction, hemorrhage, increased energy expenditures,
and increased susceptibility to secondary infections. Consequently,
negative impacts of O. petrowi may be more extensive than previously
believed. In addition, findings underscore the importance of examination
for O. petrowi in multiple locations within the host for better assessment of
prevalence and intensity of infection. Given the interest in learning more
about O. petrowi in wild quail, our study provides the framework for
researchers wishing to conduct in-depth investigations within a laboratory
or aviary setting to elucidate life history attributes of O. petrowi and to
assess the consequences of infections within their hosts.
TABLE II. Eyeworm (Oxyspirura petrowi) survival experiment using 10 types of liquid media. Eyeworms cultured in media 1–7 were maintained in petri
dishes at 40 C for 35 days. Eyeworms cultured in media 8–11 were incubated in 6-well culture plates in an Isotemp CO
2
incubator maintained at 32 C
with a constant 5% CO
2
intake.
Medium
No. live
worms
Day
1
Day
2
Day
5
Day
10
Day
15
Day
20
Day
25
Day
30
Day
35
1. 10% saline solution 4 4 1 0 0 0 0 0 0 0
2. 10% saline solution with bobwhite blood/tissue 4 4 3 0 0 0 0 0 0 0
3. Equate artificial tears 4 4 0 0 0 0 0 0 0 0
4. Equate artificial tears with bobwhite blood/tissue 4 4 1 0 0 0 0 0 0 0
5. BioTrue artificial tears 4 4 1 0 0 0 0 0 0 0
6. BioTrue artificial tears with bobwhite blood/tissue 4 4 1 0 0 0 0 0 0 0
7. 10% sucrose solution 24 24 10 0 0 0 0 0 0 0
8. Phosphate buffered saline solution 24 24 24 5 0 0 0 0 0 0
9. Physiological saline recipe solution 24 24 24 4 0 0 0 0 0 0
10. 5 ml of physiological saline recipe solution with 5 ml of egg whites 24 24 24 20 10 4 0 0 0 0
11. 5 ml of physiological saline recipe solution with 5 ml of egg whites
cultured under sterile conditions 48 48 48 48 36 23 11 6 3 3
* Note: Four eye worms were placed in each of the 6-well culture plates in media 8–10 and 8 per well in media 11.
100 THE JOURNAL OF PARASITOLOGY, VOL. 101, NO. 1, FEBRUARY 2015
Funding was provided by the Rolling Plains Quail Research Founda-
tion. We thank Dr. Ernest Smith and Jibao He for microscopy and picture
help and Steve White and Randy Bullard for property access.
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SHORT COMMUNICATIONS 101
